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MilindMohanNaik SantoshKumarDubey


Pollution and
Marine Pollution and Microbial
Milind Mohan Naik
Santosh Kumar Dubey

Marine Pollution and

Microbial Remediation
Milind Mohan Naik Santosh Kumar Dubey
Department of Microbiology Department of Microbiology
Goa University Goa University
Taleigao Plateau, Goa, India Taleigao Plateau, Goa, India

ISBN 978-981-10-1042-2 ISBN 978-981-10-1044-6 (eBook)

DOI 10.1007/978-981-10-1044-6

Library of Congress Control Number: 2016948708

Springer Science+Business Media Singapore 2017

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This book is dedicated to
Late Shri Mohan Ladu Naik
Retired Police Sub-inspector
Menkurem Bicholim, Goa

No good work is created without joining hands, and it will be definitely

deluding and improper to say that we could have completed this study single-
handed. This effort was not carried out solely by us but was aided by a quite
few people whom we wish to thank now because this is the best time and
We are thankful to Dr. Satish Shetye, V.C. of Goa University, and
V.P. Kamat, Registrar, Goa University, for providing necessary facilities and
Prof. Sanjeev Ghadi and Prof. Savita Kerkar for their support. Our sincere
thanks go to Prof. Saroj Bhosle, Prof. Irene Furtado, Prof. Sarita Nazareth,
Dr. Sandeep Garg, Dr. Lakshangy S. Charya, Dr. Priya D Costa, Dr. Vishwas
Khodse, Dr. Sanika Samant, Dr. Gauri Achari, Dr. Varada Damare, Dr. Trelita
de Sousa, Dr. Swapnil Doijad, Dr. S. B. Barbuddhe, Dr. Flory Pereira, Dr.
Mahesh Majik, Dr. Teja Gaonkar and Dr. Pranay Morajkar. I express my sin-
cere gratitude to the entire non-teaching staff including Mr. Shashikant Parab,
Budhaji, Dominic, Saraswati, Deepa, Narayan and Rudransh for their con-
stant help and support in various ways. We owe our deepest sense of gratitude
to our friends Mr. Uday Naik, Amar Degvekar, Kashinath Shetgaonkar and
Mrs Pranaya Naik. We also thank Government of India for financial support
as SERB-DST young scientist project. We have successfully compiled our
creative and thoughtful research work due to genuine concern and painstak-
ing effort of many more well-wishers whose names are not mentioned, but
they are still in our heart. So, reward is surely worth for their efforts.
We want to dedicate this book to the late Shri Mohan Ladu Naik (retired
PSI Menkurem, Goa) and Mrs Manisha Mohan Naik (Menkurem).


1 Assessing Metal Contamination in Recent Creek

Sediments Using Fractionation Technique Along
Mumbai Coast, India ................................................................... 1
Lina L. Fernandes and G.N. Nayak
2 Bioremediation of Heavy Metals from Saline
Water Using Hypersaline Dissimilatory
Sulfate-Reducing Bacteria........................................................... 15
Savita Kerkar and Kirti Ranjan Das
3 Lead- and Mercury-Resistant Marine Bacteria
and Their Application in Lead and Mercury
Bioremediation ............................................................................. 29
Milind M. Naik and S.K. Dubey
4 Microbial Remediation of Organometals
and Oil Hydrocarbons in the Marine Environment ................. 41
Andreia Cruz, Ana Julia Cavaleiro, Ana M.S. Paulo,
Antnio Louvado, M. Madalena Alves, Adelaide Almeida,
and ngela Cunha
5 Extracellular Polysaccharide Production by Bacteria
as a Mechanism of Toxic Heavy Metal Biosorption
and Biosequestration in the Marine Environment.................... 67
P.V. Bramhachari and Ganji Purnachandra Nagaraju
6 Biosurfactant: A Promising Approach Toward
the Remediation of Xenobiotics, a Way to Rejuvenate
the Marine Ecosystem.................................................................. 87
Siddhardha Busi and Jobina Rajkumari
7 Optimization of Cultural Conditions for Marine
Microbial Biosurfactant Production: Future Prospects
from Untapped Marine Resources ............................................. 105
K.V. Deepika, Ganji Purnachandra Nagaraju,
and P.V. Bramhachari

x Contents

8 Biosurfactant-Producing Denitrifying Bacteria

in Marine Petroleum-Contaminated
Environmental Sites ..................................................................... 129
Trelita de Sousa
9 Interaction of Haloarchaea with Metals .................................... 143
Sanika Shivdas Naik and Irene Furtado
10 Manganese-Tolerant Bacteria from the Estuarine
Environment and Their Importance in Bioremediation
of Contaminated Estuarine Sites ................................................ 153
Flory Pereira
11 Applications of Siderophore Producing Marine
Bacteria in Bioremediation of Metals and
Organic Compounds .................................................................... 177
Teja Gaonkar and Sunita Borkar
12 Bacterial Degradation of Algal Polysaccharides
in Marine Ecosystem.................................................................... 189
Md Imran, Preethi B. Poduval, and Sanjeev C. Ghadi
13 Impact of Pollution on Phytoplankton and Implications
for Marine Econiches ................................................................... 205
Priya Mallika DCosta, Maria Shamina DSilva,
and Ravidas Krishna Naik
14 Selenium Pollution in the Marine Environment
and Marine Bacteria in Selenium Bioremediation.................... 223
Lakshangy S. Charya
15 Pathogenic Bacteria of Public Health Significance
in Estuarine Mangrove Ecosystem ............................................. 239
Krupali Poharkar, Swapnil Doijad, Savita Kerkar,
and Sukhadeo Barbuddhe
16 Global Concerns of Ships Ballast Water Mediated
Translocation of Bacteria ............................................................ 255
Lidita Khandeparker and A.C. Anil
17 Genotoxic Biomarkers as Indicators
of Marine Pollution ...................................................................... 263
M.K. Praveen Kumar, Avelyno Dcosta, and S.K. Shyama
About the Editors

Dr. Milind Mohan Naik has a PhD in Microbiology from Goa University.
He joined Department of Microbiology in Goa University as assistant profes-
sor of marine microbiology in the year 2013. His research efforts have been
focused on understanding the metal-resistance mechanism in bacteria from
marine and terrestrial environments and their potential applications in biore-
mediation of polluted environmental sites. He has published over 20 research
papers. He is a recipient of SERB-DST Young Scientist project award. He has
guided eight postgraduate dissertation projects. He has also worked as scien-
tist C on the Malaria Evolution in South Asia project funded by NIH and
University of Washington, USA, in the National Institute of Malaria Research
(ICMR). He has worked as scientist in Molbio Diagnostics Pvt. Ltd. in the
Research and Development Department in 2012. His research interests are
environmental microbiology and nanobiotechnology. He aims to gain knowl-
edge in the research field of marine microbiology and biotechnology with the
intent of learning new concepts.

Prof. Santosh Kumar Dubey has a PhD in botany from BHU, Varanasi,
Uttar Pradesh. He joined the Department of Microbiology, Goa University, in
the year 1996. His research efforts have been focused on understanding the
metal and organometal resistance mechanisms in bacteria from marine and
terrestrial environments, metagenomics, agriculture microbiology, genetic
engineering and molecular biology. He has published 32 research papers in
international journals, 9 in national journals and 8 chapters in edited books
published by international and national publishers. He has guided 9 PhDs and
30 M. Sc. dissertations. He has five postdoctoral research experiences in sev-
eral universities of the USA (Clarkson University; University at Buffalo;
Roswell Park Cancer Institute, Buffalo, New York; University of Nevada Las
Vegas, Las Vegas) and also is a recipient of JSPS fellowship in Japan. He has
served as Head, Department of Microbiology, and Chairman, Board of
Studies in Microbiology, at Goa University twice. He is programme coordi-
nator of the UGC sponsored innovative programme of M.Sc. in Marine
Microbiology in the Department of Microbiology, Goa University. He was
awarded a visiting scientistship from the Centre of Marine Environmental
Studies, Ehime University, Matsuyama, Japan, and a National Overseas
Scholarship (PDF) from the Department of Education M/O Human Resource

xii About the Editors

Development, Govt. of India. He is a member of the Institutional Biosafety

Committee, Goa University, and completed two major projects funded by
DBT and DOD, Govt. of India. Currently he is member of the advisory board
of UGC for the SAP-DRS programme of the Department of Microbiology
and Biotechnology, M.S. University, Baroda, Gujarat.
Assessing Metal Contamination
inRecent Creek Sediments Using 1
Fractionation Technique Along
Mumbai Coast, India

LinaL.Fernandes andG.N.Nayak

Three sediment cores from a creek environment in Mumbai, extending
from the head to the creek mouth, were studied with respect to elements
(Fe, Mn, Cu, Pb, Co, Zn and Cr) along with organic matter (total organic
carbon, total phosphorus, total nitrogen) and sediment components (sand,
silt, clay). A sequential extraction procedure was also applied to understand
the partitioning of trace metals among the different fractions of the
sediment. Together with this data, pollution indices were also computed
and comparison with numerical sediment quality guidelines carried out.
Correlation analysis among the different variables displayed weaker
relations of metals with the sediment components, while organic matter
and FeMn oxyhydroxides were found to act as important substrates for
metal sequestration in the creek region. The fractionation results reveal
almost all the elements were associated with the residual fraction, while
Mn was high in the bioavailable fraction.

1.1 Introduction Considering that a metals availability is affected

by its association with one or more of the differ-
Concerns regarding the trace metals in estuarine ent sediment components (organic matter, car-
environments have changed from the focus of bonates, sulphides, oxides), total metal
just measuring total metal levels to understanding concentration provides partial information about
the processes and controls on metal behaviour. its potential interaction between the biotic and
abiotic environments. Therefore, to better esti-
mate metal bioavailability, an understanding of
L.L. Fernandes (*) the partitioning of a particular metal among these
CSIR- National Institute of Oceanography, sediment components is necessary. This study
Dona Paula, Goa 403 004, India addresses the processes and controls on metal
e-mail: bioavailability so that further insight can be
G.N. Nayak gained in the prediction of trace metal uptake in
Department of Marine Sciences, Goa University, organisms.
Taleigo, Goa 403 206, India

Springer Science+Business Media Singapore 2017 1

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
2 L.L. Fernandes and G.N. Nayak

Thane Creek, in western Mumbai, being sediment transport and accumulation in this
surrounded by highly urbanised and industrialised urban-industrial creek setting.
region forms an integral part of Mumbai,
subjected to the effects and influences of these
developments. Total metal concentrations in 1.2 Materials andMethods
sediments of the region have been reported earlier
(Sharma etal. 1994; Zingde and Desai 1981; 1.2.1 Study Area
Patel etal. 1975, 1985; Bhosale and Sahu 1991),
which found increased level of contaminants Thane Creek is bound by the Ulhas River to the
such as Cu, Co, Zn, Cr and Ni. In recent years, north and Mumbai harbour bay towards the south
studies on quality of the creek sediments have (Fig.1.1). The creek lies at longitudes 7255 E to
been undertaken, and the results indicate that 7300 E and latitudes 1900 N to 1915
contamination problems concerning heavy metal N.Dense mangroves occur along both the banks
pollution still exist (Jha etal. 2003; Ram etal. of the creek, coupled with heavy urbanisation
1998, 2003, 2009). Study carried out by Krishna and industrialisation. Geologically, Mumbai
and Govil (2005) reported the concentration (Thane) region is part of the Deccan Trap that
ranges were Cr 177.91039mg/kg (average was formed by volcanic effusions at the end of
521.3mg/kg), Ni 64.4537.8mg/kg (average the Cretaceous period (Blasco 1975). The riverine
183.6mg/kg), Co 44.8101.6mg/kg (average end of the creek is narrow and shallow which
68.7mg/kg), Cu 3.10271.2mg/kg (average gradually broadens and deepens towards the sea.
104.6mg/kg) and Zn 96.6763.2mg/kg (average The industrial and domestic waste contributes
191.3mg/kg). The WHO standard for aquatic life about 294mld (million litres per day) and 145
with respect to metals is Fe 200ppb, Cu 5ppb, 260mld, respectively, within Thane City limits
Pb 25ppb, Zn 30ppb and Cr 100ppb. Thus, it is (TMC-ES 2000). The diversified industries
important to understand the geochemical (chemical, pharmaceutical, textile, engineering
dynamics for better assessing metal transport and and major fertiliser complexes), present along the
its ultimate fate in the creek environment. The creek banks, release high levels of nitrates and
total metal concentration in sediments is useful phosphates through their effluents into the creek
as an indicator of contamination of aquatic (Quadros etal. 2009).
environments but inadequate to understand
bioavailability, mobility or toxicity of metals to a
larger extent (Hooda 2010). Also, as the chemical 1.2.2 Sampling
form determines the toxicity and fate of
waterborne metals, it is more appropriate to Three sediment cores were collected, using a
quantify the different forms of metals than to hand driven PVC tube of 5.5cm diameter, from
estimate its total metal concentrations (Davidson the intertidal regions of Thane Creek (Fig.1.1),
etal. 1994; Lima etal. 2001; Jain 2004). namely, near the creek head (core TC1), middle
Therefore, the overall objectives of the present creek region (core TC5) and near the creek mouth
study are to (i) assess the extent of heavy metal (core TC4). In the laboratory, the cores were
(Fe, Mn, Cu, Pb, Co, Zn and Cr) pollution in the subsampled at 2cm intervals, transferred to
creek sediments and (ii) employ sequential labelled polyethylene bags and stored frozen to
extraction procedure to determine the potential 20C till further analysis.
mobility and risk to biota from sediment- For sedimentological and geochemical
associated metals. The present metals were parameters, the sediment samples were dried in
chosen because of their known abundance and an oven at 60C.Sediment components were
toxic effects in environment of highly urbanised analysed using standard sieve and pipette
and industrialised regions. The research will help technique after destruction of organic matter with
to better understand the patterns and pathways of H2O2 (Folk 1974). For geochemical analysis, the
1 Assessing Metal Contamination inRecent Creek Sediments Using Fractionation 3

Fig. 1.1 Map showing the

sampling locations

sediment samples were finely ground with mortar reference material (BCSS-1). Percentage
and pestle. Total organic carbon (TOC) content recoveries were always over 95%. For speciation
was determined employing a rapid titration study Tessiers sequential extraction procedure
method (in chromic acid environment) (Walkley was adopted (Tessier etal. 1979) and the differ-
and Black 1934), while a standard procedure ent steps involved are shown in Table1.1. The
(Grasshoff 1999) was used for total phosphorus five fractions, viz. exchangeable, carbonate
(TP) and total nitrogen (TN) in sediment. bound, FeMn oxide, organic and residual, were
Mineralogical phases of the sediments (<63m determined for Fe, Zn, Mn, Co, Cr, Cu and Pb.
fraction) were determined by X-ray diffraction The analytical results were statistically processed
(XRD). Sediment samples for trace metal with correlation analysis to group metals with
analysis were digested using a HFHClO4HNO3 similar behaviour using the computer software
total dissolution technique (Jarvis and Jarvis STATISTICA 6.
1985). The digested samples were aspirated for
Fe, Al, Zn, Mn, Co, Cr, Cu and Pb with the help
of Varian AA 240 FS flame atomic absorption 1.2.3 Enrichment Factor
spectrometry (AAS) with an airacetylene flame
for all of the above elements except for Al for An enrichment factor was used to differentiate
which nitrous oxideacetylene flame was metals from anthropogenic (non-crustal) and
employed at specific wavelengths. Each sample geogenic (crustal) sources and to further evaluate
was analysed in triplicate with a relative standard the magnitude of contamination in the
deviation of <5% for all the metals. Recovery environment (Feng etal. 2004). Al was used as
after digestion was checked using a certified the reference element:
4 L.L. Fernandes and G.N. Nayak

Table 1.1 Sequential extraction scheme of Tessier etal. Table 1.2 Geoaccumulation index proposed by Muller
(1979) (1979)
Extraction reagents/ Sediment Igeo
Step Fraction conditions Pollution intensity accumulation class
1. Exchangeable 8ml MgCl2, pH7, 1h, Very strongly polluted >5 6
(F1) room temperature, Strongly to very 45 5
continuous agitation strongly polluted
2. Acid soluble 8ml NaOAc, pH5, 5h, Strongly polluted 34 4
(carbonates) (F2) room temperature, Moderately to strongly 23 3
continuous agitation polluted
3. Reducible 20ml of 0.04M NH2OH. Moderately polluted 12 2
(FeMn oxide HCl in 25% HOAc, 6h,
Unpolluted to 01 1
bound) (F3) 963 C, occasional
moderately polluted
Practically unpolluted <0 0
4. Oxidisable 3ml of 0.02M HNO3 +
(organically 5ml of 30% H2O2, pH 2,
bound + sulphide 2h, 852C, occasional
bound) (F4) agitation. Add 3ml of
30% H2O2, repeat 3h, cool
estimate the metal pollution status of the creek
and then add 5ml 3.2M sediments. The Igeo is given by the following
NH4OAc in 20% HNO3, equation:
0.5h, room temperature,
continuous agitation Igeo = log 2 (C n / 1.5Bn )

5. Residual (F5) HF:HNO3:HClO4 = 7:3:1,
dryness, again
where Cn = the measured concentration of the
HF:HNO3:HClO4, 1h, element n in the sediment fraction and Bn =
2ml conc. HCl, dryness, geochemical background of element n, taken
10ml 50% HNO3, make from literature (average global shale). The factor
up to 50ml with distilled
1.5 is used to compensate for possible variations
of the reference data due to lithogenic effects.
The classification of pollution intensity is shown
EF = (C n / C Al )sediment / (C n / C Al )background in Table1.2.

where (Cn/CAl)sample is the ratio of concentration of
the element (Cn) to that of Al; (CAl) in the sediment 1.3 Results andDiscussion
sample and (Cn/CAl)background is the same ratio with
background value taken of average shale 1.3.1 Sediment Components
(Turekian and Wedepohl 1961). EF values
between 0.5 and 1.5 indicate the metal originates In the entire creek region, sand ranges from
from crustal material or natural processes, 0.16% to 4.77%, while silt and clay vary from
whereas EF values greater than 1.5 suggest the 18.24% to 44.82% and 52.80 % to 81.20% with
presence of an anthropogenic source (Zhang and averages of 0.96%, 32.44% and 66.60%, respec-
Liu 2002). tively. From the head to the creek mouth, a grad-
ual decrease in sand and clay percentage and an
increase in silt percentage are observed
1.2.4 I ndex ofGeoaccumulation (Table1.3).

Igeo, proposed by Muller (1979), to calculate 1.3.2 Clay Mineralogy

metal concentration in sediments by comparing
current concentration with undisturbed or crustal Clay mineral analysis was performed on selected
sediments levels, was used to quantitatively subsamples of two cores, namely, from the head
1 Assessing Metal Contamination inRecent Creek Sediments Using Fractionation 5

Table 1.3 Average concentrations of the different sedi- 1.3.3 O

 rganic Matter (TOC, TP
ment parameters analysed in the cores of the Thane
Variables Core TC1 Core TC5 Core TC4
Organic matter concentration, in general, shows a
Sand (%) 1.52 0.53 0.83
gradual decrease from the head towards the
Silt (%) 29.54 33.43 34.34
mouth of the creek. TOC ranges from 0.78 to
Clay (%) 68.94 66.04 64.82
5.00% and TP from 0.49 to 1.64mg/g and TN
TOC (%) 3.32 1.8 1.12
varies from 0.31 to 2.89mg/g for the entire creek
TP (mg/g) 1.11 0.98 0.79
TN (mg/g) 1.46 0.7 1.4
basin. The average for TOC is 2.08% and for TP
Al (%) 10.65 11.19 11.51 is 0.96mg/g, while for TN it is 1.19mg/g. The
Fe (%) 9.09 8.06 7.06 organic matter content of the cores studied
Mn (ppm) 1518 2025 1884 decreases with depth, as would be expected in
Cu (ppm) 235 227 242 any soil or sediment profile due to natural
Pb (ppm) 107 81 99 decomposition processes.
Co (ppm) 78 44 63
Zn (ppm) 424 138 170
Cr (ppm) 159 201 195 1.3.4 Metal Geochemistry

In general, of the metals analysed in the

(core TC1) and middle region (core TC5) of the sediments, the individual metals followed vary-
creek. Sample composition was found to be ing patterns (Table1.3). In the creek region, Al
consistent among the selected cores, with displays the highest average concentration (avg.
smectite (avg. 62.85%) as the major constituent 11.12%), while Co has the lowest average
clay mineral followed by illite (avg. 16.91%), concentration (avg. 62ppm). The creek head
kaolinite (avg. 10.59 %) and chlorite (avg. exhibits maximum average concentrations of Fe,
9.65%). The results are consistent with the study Pb, Co and Zn with low average values for Mn
of Bhosale and Sahu (1991), who carried out clay and Al. The middle region (TC5) displays highest
mineral analysis in the creek region and reported values of Mn and Cr, along with lowest average
the sediment mineralogy was dominated by values of Pb, Cu, Zn and Co. At the creek mouth
montmorillonite followed by degraded chlorite, (TC4), maximum average concentration of Al
illite and silica to a minor extent. In the creek and Cu occurs with low average values for Fe. In
region, the percentage of smectite ranges from general, the Al % distribution over the entire
38.59 to 79.73%, 7.48 to 28.26% for illite, 0.94 creek is found to remain constant. The order of
to 19.97% for kaolinite and 4.7720.48% for total metal concentrations of sediment samples
chlorite. Thus, the clay fraction is found to be was found to be Al > Fe > Mn > Zn > Cu > Cr >
characterised by its mineralogical simplicity, Pb > Co.
with an absolute predominance of smectite, Core TC1, near the creek head, contains more
derived from the weathering of the basalt rocks of than twice the concentration of metals as
the basin. Deccan basalts are the major source for compared to the other cores and a reduction in
smectite along the western continental shelf of metal content occurs with increasing distance
India (Rao and Rao 1995). Clay minerals in the from the creek head. Most of the industries are
marine environment are found to be largely housed near the head region of the creek.
detrital and widely dispersed (Biscaye 1965). Therefore, during periods of high rainfall and
However, in the present case, the bulk flooding, the core location at TC1 is bound to
mineralogical analyses, in general, show a receive the sediment-loaded water from the
remarkable consistency in the sediment industrial and urban areas first and would
mineralogy among the sites. therefore receive the larger proportion of
6 L.L. Fernandes and G.N. Nayak

contamination. The reduced tidal influence and positive correlation between the organic matter
decreased current speed in the inner creek region content and adsorption affinity of heavy metals in
favour conditions for the settlement of suspended sediments (Soares etal. 1999; Lin and Chen
load (NIO 1998). Sediments collected near the 1998). FeMn oxides, on the other hand, seem to
mouth of the creek (TC4) reveal a substantial associate with most of the metals in core TC5
reduction in concentrations of most of the metals than cores TC1 and TC4in which such
as compared to core TC1, as the location of core correlations are seen to a lesser extent. Among
TC4 is more prone to wave action from the sea, the metals, good inter-elemental correlations are
causing the sediments to be more uniformly observed in all the cores suggesting of similar
distributed. Therefore, there is evidence for a terrigenous sources or similar mechanisms of
distanceconcentration relationship in the transport and accumulation within the sediments.
sediment record, with concentration of metals It appears from the correlation analysis that in the
decreasing with greater distance from the head/ creek region, the sediment geochemistry may be
input. characterised by strong associations of metals
(M) with three matrices (MM, Morganic
carbon and MFeMn oxides). Therefore,
1.3.5 Correlation Analysis statistical analysis in general shows that there are
three different processes operating in the basin,
In order to understand the processes involved in which help us in understanding the metal
the distribution of sediment and associated metal behaviour and distribution.
in the creek environment, Pearsons correlation
analysis at p <0.05 was carried out on the
different variables studied for all the cores sam- 1.3.6 Enrichment Factor andIgeo
pled from the creek (Tables1.4a, 1.4b, and 1.4c).
Important factors controlling the abundance of Near the head region of the creek (core TC1), Cu,
metals in natural environment include organic Pb and Co are found to be highly enriched along
matter contents and granulometry (Zhang etal. the entire core length, while the remaining
2007). However, the sediment components are elements show an increase at the upper few cm of
found to exhibit weak or negative correlation as the core (Table1.5). On the other hand in core
compared to the organic matter which shows TC5, sampled from middle region on the eastern
better correlations with the metals in the creek bank of the creek, Fe and Zn are below the
region. Many studies have reported a significant enriched level, while the remaining elements

Table 1.4a Pearsons correlation coefficients for organic matter and metals in core TI (near creek head) (p<0.05,
TOC TP TN Sand Silt Clay Fe Mn Cu Pb Co Zn
TP 0.14 1.00
TN 0.30 0.39 1.00
Sand 0.25 0.26 0.20 1.00
Silt 0.29 0.23 0.04 0.56 1.00
Clay 0.30 0.25 0.06 0.66 0.99 1.00
Fe 0.47 0.36 0.29 0.05 0.09 0.09 1.00
Mn 0.35 0.01 0.48 0.17 0.38 0.37 0.30 1.00
Cu 0.35 0.32 0.43 0.10 0.16 0.13 0.11 0.44 1.00
Pb 0.49 0.22 0.35 0.05 0.09 0.09 0.30 0.14 0.47 1.00
Co 0.25 0.05 0.54 0.07 0.04 0.05 0.17 0.39 0.05 0.16 1.00
Zn 0.28 0.19 0.35 0.01 0.15 0.13 0.07 0.37 0.01 0.02 0.60 1.00
Cr 0.35 0.28 0.01 0.18 0.27 0.27 0.29 0.09 0.60 0.54 0.51 0.36
1 Assessing Metal Contamination inRecent Creek Sediments Using Fractionation 7

Table 1.4b Pearsons correlation coefficients for sediment component, organic matter and metals in core TC5 (lower
middle creek region) (p<0.05, n=31)
TOC TP TN Sand Silt Clay Fe Mn Cu Pb Co Zn
TP 0.25 1.00
TN 0.45 0.53 1.00
Sand 0.21 0.07 0.02 1.00
Silt 0.20 0.31 0.02 0.04 1.00
Clay 0.19 0.32 0.02 0.00 1.00 1.00
Fe 0.23 0.05 0.25 0.34 0.25 0.24 1.00
Mn 0.12 0.00 0.07 0.36 0.15 0.13 0.06 1.00
Cu 0.02 0.20 0.45 0.13 0.19 0.19 0.69 0.42 1.00
Pb 0.43 0.35 0.20 0.28 0.49 0.48 0.67 0.13 0.50 1.00
Co 0.02 0.43 0.02 0.16 0.14 0.15 0.24 0.46 0.30 0.32 1.00
Zn 0.45 0.44 0.74 0.28 0.12 0.14 0.45 0.10 0.63 0.04 0.04 1.00
Cr 0.35 0.35 0.22 0.01 0.39 0.39 0.42 0.15 0.31 0.77 0.39 0.06

Table 1.4c Pearsons correlation coefficients for sediment component, organic matter and metals in core TC4 (near
creek mouth) (p<0.05, n=23)
TOC TP TN Sand Silt Clay Fe Mn Cu Pb Co Zn
TP 0.00 1.00
TN 0.41 0.40 1.00
Sand 0.09 0.37 0.17 1.00
Silt 0.21 0.33 0.00 0.43 1.00
Clay 0.21 0.35 0.01 0.48 1.00 1.00
Fe 0.38 0.19 0.05 0.21 0.21 0.22 1.00
Mn 0.16 0.09 0.07 0.15 0.01 0.00 0.14 1.00
Cu 0.01 0.51 0.62 0.15 0.08 0.09 0.04 0.10 1.00
Pb 0.38 0.52 0.12 0.27 0.15 0.16 0.52 0.18 0.28 1.00
Co 0.11 0.25 0.28 0.19 0.09 0.10 0.12 0.28 0.26 0.04 1.00
Zn 0.28 0.01 0.31 0.01 0.08 0.07 0.15 0.14 0.33 0.05 0.08 1.00
Cr 0.35 0.00 0.36 0.16 0.21 0.21 0.31 0.48 0.14 0.37 0.70 0.11

Table 1.5 Range of enrichment factor for metals from the creek region
EF Fe Mn Cu Pb Co Zn Cr
TC1 1.201.75 1.512.33 2.894.85 2.864.85 2.683.85 2.104.55 1.151.86
TC5 1.021.43 1.633.19 2.824.15 2.243.61 1.012.57 0.841.26 1.262.18
TC4 0.791.44 1.692.97 2.874.94 2.345.67 1.713.28 0.931.82 1.262.27

point towards an anthropogenic source. Near the The core sampled near the creek head (core
creek mouth (TC4), the EF values of Fe and Zn TC1) exhibits unpolluted to moderately polluted
are low, while enrichment is observed for the Igeo class with respect to Pb, Cu, Zn and Co,
remaining elements. The distribution obtained while Mn, Fe and Cr remain unpolluted
suggests that the trace metals may be derived (Table1.6b). The middle creek region (core TC5)
from diverse sources including natural and displays moderate pollution with respect to Pb
anthropogenic inputs. Potential sources such as and Mn. In addition to this, in core TC5, moder-
agricultural activities, corrosion of building ate to strong pollution is observed with respect to
materials, urban and industrial sewages and Cu. The remaining elements fall in unpolluted to
atmospheric deposition are probable contributors moderately polluted class. In the core retrieved
for the elemental enrichment. near the creek mouth (core TC4), Cu, Pb and Co
8 L.L. Fernandes and G.N. Nayak

Table 1.6 Igeo values for Thane cores of heavy metals (Table1.7) and SQGs of USEPA
Stations Igeo class Elements (Pedersen etal. 1998) when compared indicate
TC1 1 Cu, Pb, Zn and Co that in all the cores, Cu and Cr values are above
0 Mn, Cr, Fe TEL, PEL and ERL values. For Pb, all the cores
TC5 3 Cu exhibit values above TEL but below PEL.In the
2 Pb and Mn case of Zn, cores TC1 and TC4 have values
1 Fe, Co, Zn and Cr higher than TEL and ERL but below PEL, while
TC4 2 Cu, Pb and Co core TC5 shows values above TEL but below
1 Fe, Mn, Zn and Cr PEL and ERL.All the elements in the study area
have values below ERM indicating low risk of
adverse effects on organisms. In general, in all
fall in moderately polluted class, while the the cores collected from the creek region, almost
remaining elements are in unpolluted to moder- all the elements are found to be above TEL, PEL
ately polluted class. In general, it is seen that the and ERL but below ERM.
creek exhibits moderate pollution with respect to
Cu and Pb. The principal anthropogenic sources
of Cu include municipal waste and sewage and 1.3.8 Speciation
corrosion of Cu-containing pipelines or fittings.
Further Cu salts are used in water supply systems Speciation analysis can provide information on
to control biological growth in reservoirs and dis- heavy metal origin, its form and its biological and
tribution pipes. The most probable sources of Pb physicochemical availability (Das etal. 1995).
are vehicle exhausts and leaded gasoline. Mn, in Speciation analysis was carried out in all the
the middle creek region, and also Co near creek cores. The percentage of metal extracted was
mouth are observed to fall in moderately polluted calculated from the ratio between the element
class suggesting there might be a risk to the biota. concentration in each fraction and the sum of
concentrations in all fractions. For most of the
samples, the sums of the extracted fractions agree
1.3.7 S
 ediment Quality Guidelines within 10%, with the independently determined
(SQGs) total metal concentrations, supporting the overall
accuracy of the extraction procedure. The plots of
Some researchers have used numerical sediment average metal concentration in the different
quality guidelines as predictors of contaminants sediment fractions are given in Fig.1.2.
in aquatic sediments (Muwanga 1997). For The distribution of Fe is similar in all the
assessment of ecotoxicological implications of three cores, being dominated by the residual
the total metal concentrations in the sediments, (>80%) phase (F5), with minor FeMn oxides
SQGs (two sets) developed for aquatic ecosys- (F3) and organic matter (F4)-bound fractions.
tems (MacDonald etal. 1996; Long etal. 1998) Mn in the all the cores is found to be associated
are considered in this study. These sets are to a considerable extent (70%) with bioavailable
defined as (i) the threshold effect level (TEL) and phases. The exchangeable, carbonate, FeMn
the probable effect level (PEL) and (ii) effect oxides and organic matter fractions contain
range low (ERL) and effect range median (ERM). 19%, 1831%, 3444% and 212%, respec-
ERL and TEL include concentrations below tively. Most Cu in the sediments is present in the
which adverse effects upon sediment-dwelling residual fraction, which amount to 8085%.
fauna are unlikely and occur only infrequently. In Also, some part was extracted mainly in organic
contrast, at concentrations above ERM and PEL, and sulphide bound (about 10%). These results
adverse effects are more likely to occur (Long are in accordance to previous results wherein the
etal. 1998). SQGs for elements such as Mn, Fe residual fraction is the most important fraction
and Co are not available. The mean concentration for Cu, followed by organic fraction (Li etal.
1 Assessing Metal Contamination inRecent Creek Sediments Using Fractionation 9

Table 1.7 Screening quick reference table for heavy metals in marine sediment (Buchman 1999)
Near creek Middle creek Near creek
Element head (TC1) (TC5) mouth (TC4) TEL PEL ERL ERM
Fe (%) 9.09 8.06 7.06
Mn (ppm) 1518 2025 1884
Cu (ppm) 235 228 243 31.6 149 34 270
Pb (ppm) 107 82 100 35.8 128 46.7 218
Co (ppm) 78 44 64
Zn (ppm) 424 139 170 121 459 150 410
Cr (ppm) 159 202 196 31.6 111 81 370

Fig. 1.2 Distribution of Core TC1

metals in the different a
sediment fractions of 100 F5
(a) core TC1, (b) core
TC5 and (c) core TC4 80 F4
60 F3

40 F2
20 F1
Fe Mn Cu Pb Co Zn Cr

Core TC5
80 F4
60 F3

40 F2

20 F1

Fe Mn Cu Pb Co Zn Cr

Core TC4

80 F5
60 F3


Fe Mn Cu Pb Co Zn Cr
10 L.L. Fernandes and G.N. Nayak

2000, 2001; Ip etal. 2007). Oxidisable fraction, parts of metals introduced by human activity are
which is the second most dominant Cu host in present in the exchangeable and the carbonate
the sediments of cores TC1 and TC4, consists fractions. With respect to the reducible Fe
mainly of organic- and sulphide-bound metals Mn-associated phase (F3), core TC1 (36.30%),
(Tessier etal. 1979; Kersten and Forstner 1989). core TC5 (41.85%) and core TC4 (38.10%)
Although chemical discrimination between show highest amounts of Mn. In the organic
these two phases is difficult (Kersten and fraction (F4), the highest percentage is seen for
Forstner 1989), the affinity of Cu for organic Cr in cores TC1 (14.46%) and TC4 (15.98%),
particles and coatings is well known (Comber while core TC5 exhibits the presence of Pb
and Gunn 1995). (10.42%). Organic matter plays an important
The highest percentages of Pb contents are in part in controlling the physicochemical
the F5 residual fraction followed by the carbonate- behaviours of metals at solidwater interface
bound fraction (F2) in core TC1 and the (Davis 1984; Stumm 1992; Schmitt etal. 2002),
exchangeable fractions in cores TC5 and TC4. with significant influence on the bioavailability,
The distribution patterns of Pb in the samples reactivity and mobility of metals in sediments.
collected from the study sites are similar. For Co, Metals bound to the reducible phase (FeMn
residual fraction (>60%) is found dominant. The oxides) and organic matter and sulphides are
remaining Co is almost evenly distributed in the better held by a scavenging effect (i.e. this
other fractions. For Zn, the highest content is in fraction acts as a sink of metals). Calmano etal.
the residual fraction, with nearly comparable (1993) reported a high proportion of metals
percentages in the carbonate and the FeMn strongly bound to the organicsulphide fraction.
oxide fractions. Some authors have proposed that Cu (80.05%) in core TC1 and Fe in core TC5
Zn due to its strong bonding with the Fe oxide (93.59%) and core TC4 (93.12%) display highest
occurs mainly in the reducible fraction (FeMn percentage in the residual fraction. Metals
oxide) (Carrol etal. 1998; ODay etal. 1998; associated with residual fractions, being part of
Harrington etal. 1998). Further, high stability the minerals crystalline structure, remain
constant for Zn when combined with FeMn relatively stable and inert and hence are not easily
oxides in sediments (Gonzalez etal. 1994) released into the mobile and bioavailable phases
favours its occurrence in the residual and FeMn (Tessier etal. 1979; Wong etal. 2007).
oxide fractions. For Cr, the residual fraction is The spatio-temporal metal variation results
always dominant, with the proportion decreasing show that at all the sites, the residual fraction is
from 75% at the inner creek end (TC1) to 60% found to be dominant fraction, with contribution
near the creek mouth (TC4). of the other fractions to the total concentration
From the results it is evident that different only minimal, except for Mn. Therefore, the
forms of the metals are bound with different relative content of the metal in the residual phase
strengths. In the three cores, among the metals can be used as a measure of the contribution of
analysed, the element which showed the highest natural sources, and also of the degree of
average percentages in the F1 phase is Pb in core contamination, with a higher percentage
TC1 (8.66%), core TC5 (14.47%) and core TC4 indicative of low pollution levels (Singh etal.
(18.21%). The F2 fraction was highest for Mn in 2005). For Fe, over 90% contents are found in
core TC1 (25.19%), core TC5 (26.33%) and core the residual fraction. As Fe concentration in the
TC4 (26.96%). Higher percentages of Pb and Mn mobile fraction is very low, the sediment may be
in the exchangeable and carbonate fractions unpolluted with regard to this metal. The metal
suggest that the metals in the creek sediments are distributions in the non-residual phases
readily more available for exchange and/or (F1+F2+F3+F4) are found to vary with the
release into the creek environment. Singh etal. element analysed and the sampling site. The
(2005) and Jain (2004) reported that significant order of mobility (from most to least bioavailable)
1 Assessing Metal Contamination inRecent Creek Sediments Using Fractionation 11

in metals extracted in the most labile fractions 1.4 Conclusions

(F1+F2+F3+F4) is Mn (75.05%) > Zn (59.77%)
> Pb (37.05%) > Co (29.45%) > Fe (22.77) > Cr Sediment cores analysed, from a creek in
(22.60%) > Cu (19.95) for core TC1 and Mn Mumbai, showed higher metal concentration
(74.43%) > Pb (43.64%) > Co (42.78%) > Zn near the creek head with decreasing trend towards
(31.38%) > Cu (19.01%) > Cr (13.01%) > Fe the creek mouth. Organic matter along with Fe
(6.41%) for core TC5, while in core TC4, the Mn oxides was found to be the main metal carri-
trend seen is Mn (77.02%) > Cr (39.40%) > Pb ers in the region. EF and Igeo pointed towards
(38.42%) > Zn (33.04%) > Co (28.18%) > Cu pollution with respect to Cu and Pb. Based on
(10.65%) > Fe (6.88%). Mn and Zn are present speciation, most of the elements analysed were
in greater percentage (>50 %) in the labile found to be in the residual fraction which clearly
fractions in samples collected near the creek indicated that these metals were primarily immo-
mouth (TC1) as compared to the other two bile and had or bore the least bioavailability
regions (TC5 and TC4) wherein only Mn is except for Mn. These findings are of great inter-
present in higher amounts in the labile fractions. est since the sediments studied showed relatively
Core TC1 being closer to the industrialised areas high Mn concentrations which can be a highly
is more prone to contaminant input. In general, toxic element for aquatic organisms and fish.
Mn is found to be the most bioavailable element
in the creek region, while Fe is observed to be the Acknowledgements The first author expresses her sin-
least bioavailable element. cere thanks to the Department of Science and Technology-
SERB, New Delhi for the award of fast track Research
Since the chemical form and concentration of Fellowship (SR/FTP/ES-26/2013).
the metal govern its bioavailability and toxicity in
sediments (Kwon etal. 2001), the high EF values
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Bioremediation ofHeavy Metals
fromSaline Water Using 2
Hypersaline Dissimilatory Sulfate-
Reducing Bacteria

SavitaKerkar andKirtiRanjan Das

Salt pans are man-made ecosystems which are fed by the tidal influx of
seawater through the estuaries. Most heavy metal contaminants from
industries and anthropogenic processes dissolve in water and thus gain
entry into the sea. Heavy metals are high-density metallic chemicals that
are potentially toxic at low concentrations and present a danger to human
and environmental health. The removal of these metals by general physical
separation techniques is a crucial issue and chemical treatment is not
always environmentally friendly. Biological methods provide an alterna-
tive to heavy metal remediation. In the present study, hypersaline dissimi-
latory sulfate-reducing bacteria (SRB) were found to remediate barium,
calcium, cadmium, cobalt, copper, iron, magnesium, molybdenum, zinc,
mercury, nickel, and lead metals from saline waters. SRB produce H2S by
utilizing sulfate as electron acceptor, which helps in oxidizing organic
matter, and reactive H2S precipitates dissolved heavy metals as their metal
sulfides and thus play an important role in detoxifying saline waters.
Among the 11 heavy metals found in the adjoining estuarine seawater,
9metals were detected in the salt pan water of Ribandar, Goa. Fe, Mn, and
Pb were observed in dissolved and particulate form, whereas Hg and Sb
were absent. In the salt manufacturing process, the brine starts crystalliz-
ing the salt and metal concentrations increase by 103 fold in brine and 104
in salt crystals. SRB precipitate almost 50% concentrations of the dis-
solved metals (from the overlying salt pan water) as their metal sulfides,
which gradually get deposited in the underlying salt pan sediments.
Hypersaline SRB show optimal sulfate-reducing activity from 80 to 115
psu and are thus potential bioremediators in salt pan ecosystems and in
turn have an application in detoxifying industrial effluents containing

S. Kerkar (*) K. Ranjan Das

Department of Biotechnology, Goa University,
Goa 403206, India

Springer Science+Business Media Singapore 2017 15

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
16 S. Kerkar and K. Ranjan Das

heavy metals. This study assesses the role of hypersaline SRB strains iso-
lated from salt pans in remediating heavy metal containing saline waters.

2.1  eavy Metal Influx inSaline

H posal, affecting the water and sediment quality in
Waters the salt pan and its inhabiting organisms. Solar
salt pan is a niche for extremophiles thriving on
Coastal areas are sites of discharge and accumu- temperature variation, oxygen availability, solar
lation of a range of environmental contaminants radiations, pH, nutrient concentration, salinity
due to urbanization and industrialization, which variation, and water activity. Besides other func-
include mining, agriculture, and waste disposal, tions, these extremophilic microbes play a key
being the main contributors of metal pollution in role in modulating the metal concentrations in
estuaries and rivers (Tabak etal. 2005; Ross the salt produced.
1994) which elevate metal concentrations in estu- White etal. (1998) have reported comfortable
aries (Kumar etal. 2010) and salt crystallizer leaching of metal contaminants, viz., Zn, Ni, Mn,
ponds (Pereira etal. 2013). Cr, Co, and Cd, from artificially contaminated
Metals, at high concentrations, influence the soil. In wastewater treatment, physiochemical
biochemical activity, growth, and morphology of methods, viz, chemical precipitation, carbon
microbes(Pereira etal. 2012) even at low (510 absorption, ion exchange, and electrochemistry,
ppm) concentrations. Metals form complexes and are generally used, but still have some disadvan-
combine with inorganic or organic metals and get tages. When higher concentrations of heavy met-
accumulated in the sediments. Microorganisms als (1100 mg/l) are present, proportionately the
use various strategies, like complex formation treatment and material cost increases. In some
and extracellular precipitation, reducing metal treatment processes, poor selectivity was
transport across cell membranes or impermeabil- observed for competitive metal absorption.
ity (Atlas and Bartha 1997). In some studies, Biological treatment has an advantage over tradi-
microorganisms have been utilized to remove tional chemical treatment due to low operational
metal contamination from wastewaters, to sepa- cost, steady effect, and a smooth recovery of the
rate metals from sediments and soil or to foster desired metals (Wang etal. 2001; Rehmanand
metal solubilization for extraction (Lovleyand Shakoori 2001).
Coates 1997). Biosorption and bioaccumulation of metals by
The Mandovi estuary of Goa faces a threat of microorganisms are probably one of natures
anthropogenic pollution. Consequently the salt safeguards for reducing metal ion toxicity in the
pans fed by the estuary would get affected. In the surrounding microbial niche. Potential applica-
salt pans, metals get concentrated along with the tions of these phenomena, however, are governed
formation of brine. Organisms like Bacteria, by certain criteria or characteristics of the biosor-
Archaea, and Eukarya are known to inhabit and bent. These include metal affinity, rate of metal
influence the salt pan water and thus the quality uptake, selectivity, temperature tolerance, versa-
of the salt produced. Solar salt obtained from the tility, and robustness (Eccles 1995).
evaporation of seawater has 86% sodium chlo- Bacterial resistance to metals may be due to
ride and 14% other trace minerals, viz., SO4, Mg, properties like metal precipitation, metal detoxi-
Ca, K, HCO3, Br, Sr, and F(Kerkar and Fernandes fication, absorption, or accumulation. Bio-
2013). absorption involves the cell surface with a
The Ribandar solar salt pans are fed by the complex formation between functional groups
Mandovi estuary and in turn are vulnerable to like phosphoryl, carbonyl, and hydroxyl present
metal effluent influx from ferromanganese ore on the cell surface with metal ions.
mining activity, barge traffic, and sewage dis- Bioaccumulation involves transport systems and
2 Bioremediation of Heavy Metals from Saline Water Using Hypersaline Dissimilatory 17

depends on active metabolism of the cell (Mohan centrations in the Mandovi estuary are
and Pittman 2007). 18.31.9% Fe(Attri etal. 2011), 0.190.002%
In the Ribandar salt pans of Goa, due to con- Mn, 36.24.2 ppm Co, and 102.39.8 ppm Zn.
tinuous exposure of heavy metals, there is an Attempts were made to precipitate high levels
emergence of metal-tolerant bacterial strains. It of Co using hypersaline SRB which revealed
was seen that these tolerant bacteria employed 90% of Co was precipitated (where 3% was by
specific and multiple mechanisms for detoxifica- the SRB cells and 87% by SRA) and hence
tion of metals. These metals were thus removed established the role of SRB in bioremediating
from the overlying water of the salt pans and Co. It was observed that SRB cells could bio-
were found to accumulate in the sediment(Pereira adsorb cobalt minimally; however the actively
and Kerkar 2014). growing SRB cells actually mediate the biore-
The average ranges of metal concentrations mediation of Co by utilizing the sulfide metabo-
recorded in the Ribandar salt pan water, salt crys- lized to precipitate Co as cobalt sulfide in the
tals, and sediment were listed below in Table 2.1 medium.
which shows the concentration ranges of toxic
heavy metals such as cadmium, zinc, and lead
were well within the permissible limits of 0.001 2.2 Sulfate-Reducing Bacteria
0.05 ppm, 0.0055 ppm, and 220 ppm in water
and 0.030.3 ppm, 50300 ppm, and 220 ppm SRB are members of delta subdivision of
in the sediment, respectively (RSMENR 2002). Proteobacteria. They are strict anaerobes and
Our previous assessment of the concentration their permanent habitats being estuarine, marine
of metals in the Ribandar salt pan sediment for all and salt marsh sediments, saline and hypersaline
seasons revealed that the metal concentration ponds, and lakes. Due to the high and almost
increases by 52% during salt harvesting season. inexhaustible supply of sulfate, SRB are able to
Attri and Kerkar (2011) reported the metal con- produce sulfide in high concentrations which

Table 2.1 Metal concentration in salt pan water, salt crystals, and salt pan sediment
Dissolved form Particulate form in Metal concentration in
insalt pan water salt pan water 103 salt crystals 104 (in Metal concentration in
Metals (inppm) (in ppm) ppm) sediment 104 (in ppm)
Li 0.0044
Mg 1.109 4 1.400 1.3190
Al 0.062 0.359 4.400 3.5245
Ti 0.010 1.0 0.620 0.51
Sc 0.001
V 0.047 0.121 0.013
Cr 0.021 0.009 0.018 0.0089
Mn 0.255 0.882 0.064 0.0258
Fe 0.173 2.749 5.900 3.037
Co 0.024 0.003 0.002
Ni 0.005 0.041 0.007 0.005
Cu 0.058 0.021 0.005 0.003
Zn 0.136 0.030 0.067 0.004
Sr 0.046 0.120 0.013 0.027
Cd 0.002
Ba 0.095 0.005
Pb 0.116 0.294 0.002 6.750
18 S. Kerkar and K. Ranjan Das

p recipitate most of the metals present in ionic SO42 + 8 e + 8 H + ====

form into their corresponding metal sulfides. The H 2 S + 2 H 2 O + 2 OH
role of sulfate-reducing bacteria in coastal marine
sediments amounts to almost 50% of the organic The reaction proceeds through a number of inter-
material degradation (Jorgensen 1982), and also mediate stages. The stable sulfate ion is first acti-
their involvement in anaerobic turnover of certain vated by the enzyme adenosine triphosphate
metals makes them important as metal contami- (ATP) sulfurylase to give adenosine phosphosul-
nation detoxifiers. fate (APS). In dissimilatory sulfate reduction, the
SRB comprise of anaerobic bacteria that use sulfate in adenosine phosphosulfate is then
sulfate as their terminal electron acceptor form- reduced to sulfite releasing adenosine monophos-
ing a mixed group which is morphologically and phate (AMP). In assimilatory reduction another
nutritionally diverse. SRB oxidize a range of phosphate molecule adds to APS to form phos-
compounds including fatty acids, organic acids, phoadenosine phosphosulfate (PAPS), after
alcohols, and H2 as an electron donor and carbon which the sulfate is reduced. Sulfite is the first
sources. A symbolic expression of SRB metabo- product of sulfate reduction in both cases
lism characterized by production of a strong (Madigan etal. 1997). SRB are associated with
reducing agent, hydrogen sulfide, is able to the systems that are characterized from fouling
inhibit growth of other microorganisms present problems and a pungent smell of H2S.
in its environment (Gibsonand Suflita 1990). The Desulfotomaculum nigrificans and Desulfovibrio
SRB perform dissimilatory and assimilatory sul- desulfuricans are the two widely distributed,
fate reduction, with the dissimilatory process far most common SRB species found in the anaero-
exceeding the assimilatory reduction. bic environment. Though SRB favor anaerobic
The genera of SRB are generally defined in conditions, they still can grow in oxygenated
terms of their morphology rather than physiol- environments, even in slimy deposits where aero-
ogy. The majority of SRB are reported to stain bic conditions persist. They also establish them-
gram-negative with Desulfovibrio being the most selves in the well water beneath the aerobes like
encountered genus and Desulfotomaculum being IRB (iron-reducing bacteria) which form bio-
the sole gram-positive genus. However gram- films on the surface and use up oxygen, while
staining behavior of SRB is diagnostically unreli- SRB thrive in the anaerobic condition below the
able (Boopathy etal. 1998a; Zehnder 1988). IRB biofilm. The presence of SRB is detected by
Although morphologically diverse, SRB are con- visualizing yellowish or reddish nodules on metal
sidered to be physiologically unified. Currently surfaces and exhibits a black color due to the pro-
18 dissimilatory SRB genera are placed into two duction of iron sulfide when nodules are broken
physiological subgroups. The first group contains open. A bright metallic pit on the metal forms on
Desulfonema, Desulfovibrio, Desulfobulbus, and the removal of nodules and releases H2S when
Desulfotomaculum genera as they can utilize eth- hydrochloric acid is added to it which is charac-
anol, lactate, and pyruvate or certain fatty acids terized by a rotten egg smell.
as carbon and energy sources. The second group SRB reduce sulfate via dissimilatory pathway
contains genera that are specialized in oxidation to obtain its energy. It can grow by utilizing min-
of acetate and fatty acids such as Desulfobacter, iscule amounts of grease and oil as a nutrient
Desulfococcus, and Desulfosarcina (Madigan source. Low flow or stagnant water favors its
etal. 1997). chances of growing. SRB are also considered as
The reduction of sulfate to hydrogen sulfide is biocorrosion agents, as the produced hydroge-
an eight-electron reduction reaction: nase enzyme enables them to use elemental
2 Bioremediation of Heavy Metals from Saline Water Using Hypersaline Dissimilatory 19

Fig. 2.1 Role of SRB

hydrogen to reduce sulfate and generate H2S act the presence of heavy metals in its surround-
which triggers biocorrosion. Hence iron corro- ing to facilitate bioremediation:
sion through such biological processes occurs
very rapidly as compared to normal iron rusting (a) Biomineralization: Biomineralization is a
(Fig. 2.1). process of precipitation of insoluble metal
with the interaction of metabolic products of
microbes. Such processes result in mineral
2.3  ioremediation ofHeavy
B formation and geochemical depositions.
Metals Biomineralization adds value to metal
microbe interaction research (Ehrlich 1999;
Bioremediation is an ancient technology, dates Banfield etal. 2000; White and Gadd 2000).
back to 6000 BC, as evident from compost pile (b) Bioaccumulation: Bioaccumulation is a metal
and kitchen middens (NABIR primer 2003), and uptake process which requires external energy
demonstrates an ancient bioremediation practice to enter into the cells and get accumulated.
by human beings. Bioremediation techniques uti- Some physiologically essential metal ions,
lize microbes to remove or convert toxic contam- toxic metals, and radionuclides have been
inants present in the environments like water, reported to enter into the cell using the energy
sediments, soil, and air to a less toxic form. The transport system. For example, K+ ion uptake
sewage treatment plant in Sussex, UK, in 1891 is linked with H+ ion bound to the plasma
(considered to be the first biological treatment membrane and ATPase through membrane
system), demonstrates bioremediation process potential. Such processes get affected by the
application that started more than 100 years ago factors that inhibit energy metabolism of cells.
(NABIR Primer 2003). The wastewater treat- As explained by White and Gadd (1987),
ment, using microorganisms to remove heavy absence of substrates, anaerobiosis, low incu-
metals, is one of the most investigated research bation temperature, and respiratory inhibitors
areas in the current scenario (Leusch etal. 1995; like cyanide could affect this metabolism.
Kaewsarn 2002; Wu etal. 1996; Xu etal. 2005; (c) Biosorption: It is a widely used approach to
Adeniji 2004). There are various strategies being bioremediate metals and radionuclides,
adopted by microorganisms in order to counter- involving passive sequestration of metals by
20 S. Kerkar and K. Ranjan Das

their interaction with living or dead biologi- When metal biosorption strategies are not fea-
cal entities. It is effectively used in wastewa- sible, then a consortium of metal-resistant cells
ter treatment (Schiewer and Volesky 2000; can ensure better remediation, combining bio-
Jang etal. 2001). sorption, bioprecipitation, and bioaccumulation.
(d) Biotransformation: Microbes can transform This approach can be used simultaneously in the
toxic metals to less toxic forms by catalyzing removal of toxic metals and organic and inor-
them to more volatile or less soluble form. ganic pollutants from water. But these living cells
For example, metal precipitation achieved by have some constraints like sensitivity to extreme
the microbial reduction of Cr(VI) to Cr(III), pH, high metal concentration, and metabolic
Se(VI) to Se(0), V(V) to V(III), Au(III) to energy production requirements limiting their
Au(0), Pd(II) to Pd(0), and U(VI) to U(IV) use in bioremediation. Therefore efforts are being
has been reviewed by Lloyd (2003). made to meet such challenges by metal-resistant
(e) Microbially enhanced chemisorption of microbial strains.
heavy metals (MECHM): This is a class of
microbial cell-mediated reactions, which
involve the precipitation of metal biomineral 2.4  RB asBioremediators
called priming deposit acting as a nucle- ofHeavy Metals
ation center on which subsequently targeted
heavy metals get deposited and promote a The use of microorganisms in metal polluted
targeted metal precipitation reaction water remediation was successfully used for the
(Macaskie etal. 1996). Generally priming reduction and precipitation of soluble metal sul-
deposit is initiated by sulfide or phosphate fates as insoluble sulfides in liquid wastes by
biomineralization route. SRB adapt such a using SRB, as they use sulfate as their electron
strategy when Fe is present on a precipitant acceptor to produce H2S, binding with metals to
metal to H2S produced and then FeS acts as give their metal sulfide. Nowadays this procedure
primary deposit and as an adsorbent for tar- is effectively used for surface water treatment,
get metals (Ellwood etal. 1992; Watson and underground water cleaning up, and even in com-
Ellwood 1994, 1988). mercial treatment plants (Fig. 2.2).

Fig. 2.2 Mechanism of metal

precipitation by SRB
2 Bioremediation of Heavy Metals from Saline Water Using Hypersaline Dissimilatory 21

Microbiologically produced H2S is reported to ments, suggesting that they might be involved in
be an effective way to immobilize metals like ferric ion reduction in these environments
iron, cadmium, nickel, lead, copper, and zinc in (Coleman etal. 1993). The biosulfide process
bioprecipitating metals as their insoluble sul- described by Rowley etal. (1994) involved the
fides, e.g., FeS, CdS, NiS, CoS, ZnS, and CuS separation of chemical precipitation of sulfide
(White etal. 1998; White and Gadd 2000; metals from the biological conversion of sulfate
Labrenz etal. 2000; Wang etal. 2001; Drzyzga to sulfide. The advantage of this process is that
etal. 2002; Valls and Lorenzo 2002; Utgikar the SRB biomass is not exposed to the fluctuat-
etal. 2002; White etal. 2003; Krumholz etal. ing conditions of the wastewater effluent, which
2003). Precipitating toxic concentrations of met- means bacterial sensitivity to toxic compounds
als as metal sulfides adds value to anoxic wetland is eliminated.
and sediment bioremediation (White etal. 1998;
Kaksonen etal. 2003; Labrenz and Banfield
2004). Jalali and Baldwin (2000) were able to 2.5  ypersaline SRB andMetal
grow SRB in a solution up to 150mg L1 of cop- Bioremediation
per and remove copper to levels below 0.1
mg/L.Mining and mineral processing of polyme- Hypersaline environments are those which con-
tallic ore in Vromos bay area near the Black Sea tain high concentration of salt. These include
coast of southeastern Bulgaria have resulted in mainly inland lakes (the Dead Sea, Great Salt
the contamination of the surrounding agricultural Lake, etc.), soda brines, deep sea brines, polyn-
land with Th, Ra, and U radioactive elements. ias, and marine salt pans. SRB are omnipresent
Other toxic heavy metals (e.g. Co, Cd, and Pb) and are hyperactive in ecosystems with high
are also present. Laboratory experiments demon- sulfate concentrations and are considered to be
strated efficient treatment of the soils by using in one of the oldest forms of bacterial life on earth.
situ treatment method, where acidified water was Hypersaline environments support the growth
used to solubilize the metals, and the SRB in turn of salt-loving organisms and these halophilic
immobilized the metals. Real field application of organisms can tolerate such environments which
this process gave promising results (Groudev limit the growth of other organisms. Halophilic
etal. 2001). The sulfate-reducing bacterium, organisms produce a variety of stable and
Desulfovibrio desulfuricans, was found to be unique biomolecules during their growth that
capable in coupling the oxidation of an organic may be useful in various biotechnological appli-
compound with an enzymatic reduction of ura- cations (Kerkar 2004). Literature reveals that
nium (VI) to uranium (IV), which precipitated halophiles possess excellent metal-scavenging
out of solution from groundwater contaminated capability. Metal- tolerant strains of bacteria
with uranium (VI) as uraninite (Abdelouas etal. from hypersaline niches were found to tolerate
1999, 2000). SRB are a physiologically impor- various metals at higher concentrations and can
tant group of microbes that are used extensively be considered as a potential candidate for metal
in metal bioremediation of water and soil with removal from wastes. In hypersaline environ-
their produced sulfides to remediate toxic metal ments like solar lakes and salt pans, SRB com-
contaminants in soil. munities carry out extremely active sulfate
Many sulfate-reducing microorganisms can reduction with a temporal, spatial, and func-
also reduce ferric ions and may preferentially tional separation of activities among the species
reduce Fe(III) at low electron donor concentra- present. SRB maintain an osmotically isotonic
tions, typically in the sediments (Coleman etal. cytoplasm to cope up with the outside medium
1993; Lovley etal.1993). Analysis of microbial osmolarity, as their survival strategy in order to
lipids suggested that sulfate-reducing bacteria thrive in hypersaline environments (Kerkar
belonging to genus Desulfovibrio were enriched 2004). We have detected Desulfobacteraceae at
in zones of ferric reduction in salt pan sedi- salt concentrations of 475 g/l which indicates
22 S. Kerkar and K. Ranjan Das

the existence of a unique oxidizing SRB with an 1. Spectroscopic method for fatty acid-amended
inorganic osmoadaptation strategy or the pres- sediment
ence of spatial microniches of lower salt con- 2. Radio isotope 35S method
centrations in the salt pan. Harithsa etal. (2002) 3. Monthly measurement of increase in natural
assessed the tolerance of HgCl2 and Pb(NO3)2 sulfide content
salts using three mesohaline SRB (HSR1, 4. Monthly assessment of decrease in natural

HSR4, HSR14) at 95 psu with concentrations sulfate content
ranging from 50,100 to 500 g/ml. Growth and
SRA were the assessed parameters. Growth of All the estimations were carried out during the
HSR1 strain was 80% at 100200 g/ml of mer- peak salt manufacturing seasons from 0 to 2, 2 to
cury, while sulfate-reducing activity decreased 5, and 5 to 10cm depth of sediment cores and the
to 60% as compared to the control. HSR14 values were expressed as ng g1 h1.
could grow normally at 200 g/ml of HgCl2, but From fatty acid-amended sediment method,
SRA was inhibited by 60%. In the presence of SRA at three depths were 2929, 1379, and
500 g/ml of Pb(NO3)2, HSR4 growth was stim- 1342ng g1 h1, while 35S method at 85 psu was
ulated by 160% and SRA by 170% as compared found to be 3,713,721 and 200ng g1 h1 but 330
to the control. There is a possibility that some psu. SRA varied from 108, 25, and 13ng g1 h1,
hypersaline SRB strains can tolerate heavy met- respectively, along the depth. SRA based on
als more efficiently than their mesohaline coun- increase in natural sulfide was found to be 65, 9,
terparts and thus can be a better candidate for and 33ng g1 h1, while natural decrease in sul-
metal bioremediation (Kerkarand Lokabharathi fate was measured to be 6.2, 0.1, and 3.5ng g1
2007, 2011). h1, respectively, at the three depths. Results
showed a trend of depth-wise decrease in SRA
measured by all four methods and higher values
2.6  easurement ofSRA andIts
M were obtained for surficial sediment at 02cm
Variation withSalinity depth (Fig. 2.3).
SRA measurements by fatty acid amended
In hypersaline anoxic sediments (Oren 1999; and 35S methods were comparable, while values
Skyring 1987; Ollivier etal 1994) and hypersa- at 330 psu were very low. Thus 330 psu sediment
line microbial mats (Canfield and Des Marais was amended with 10 mM of fatty acid cocktail
1993; Caumette etal 1994), sulfate reduction which helped in measuring SRA by 35S within 24
governed by bacteria is of great ecological and h. To overcome auto-oxidation of sulfide and
biochemical importance. Changes in biological other artifacts due to long incubation time, short-
lability and the amount of organic matter that period incubation is generally recommended.
undergoes decomposition show a large variation SRA measuredbyfatty acid-amended technique
in the sulfate reduction rate in marine sediments (3190 nM cm3 day1) are comparable to the
(Goldhaber and Kaplan 1974). In the marine tracer technique (2050 nM cm3 day1) which was
coastal ecosystem, SRA contributes almost 50% 1.3 times higher in SRA measured by 35S method
of organic carbon turnover in the sediments, (85 psu) at surficial sediments, which were four
while the total sediment respiration rate is esti- orders higher from the values given by Li etal.
mated to be 2.55.5 g cm2 day1. Therefore SRB (1999) of 0.5 nM ml1 day1.
play an important role in hypersaline ecosystems The rise in sulfide concentration revealed an
like solar salt pans of Goa. SRA of 65ng g1 h1, which was two orders lower
To ascertain the sulfate reduction rate in this than the values of above two methods, probably
complex system, multiple methods were carried because of the generated sulfides that rapidly get
out to determine an integrated and comparative oxidized in the system chemically or biologically
estimate of SRA.Four different methods were in their natural conditions. Moreover H2S easily
followed: escapes out of the system. SRA based on decrease
2 Bioremediation of Heavy Metals from Saline Water Using Hypersaline Dissimilatory 23

Fig. 2.3 Effect of salinity on

sulfate reduction activity

in sulfate concentration level yielded a very low thus enabling them as a better candidate for saline
value of 6.2ng g1 h1, which is one order lower wastewater treatment.
than the sulfide increase value. Natural sulfide
and sulfate concentration analyses showed a 100-
fold difference between the sulfide formed and 2.7 Effect ofMetals onSRA
the increase in sulfate concentration. Such a vari-
ation in a chemically stable compound could be To access the impact of certain metals influencing
explained through biological oxidation in the activity of hypersaline SRB, lead (Pb) at concen-
natural system and is much faster than biological trations of 0, 100500 psu, was used at incubation
reduction. Salinity plays a key role in the process periods of 7 and 14 days with different carbon
as 85 psu salinity yielded higher SRA than 330 sources (individually) like formate, acetate, lac-
psu. Thus we measured SRA on different salinity tate, butyrate, etc. Formate was found to stimulate
gradients to obtain an optimal value for SRA.It the growth and respiration after 14 days. Similarly
was found that salinity enhances SRA as pre- when selected strains were grown with mercury
sented in Fig. 2.4. Increase in SRA was observed (Hg) at 0500 ppm. it was found that the activity
from 20 to 115 psu. Maximum activity was was inhibited. However formate and lactate stim-
observed at 80 psu; however the activity drasti- ulated the activity up to 100 ppm of Hg and SRA
cally decreased at 125 psu. Highest values of was observed up to 400 ppm of Hg. These experi-
29.96 and 25.87 g ml1day1 were obtained for ments indicated that different metals influence
80 and 115 psu suggesting it to be the optimal the SRA, whereas the presence of low concentra-
salinity to foster higher SRA.We observed the tions of specific carbon sources with the respec-
sulfate reduction rate is somewhat related to the tive metal stimulates the SRA.Therefore addition
optimal salinity range of 80115 psu as the val- or the presence of certain carbon sources in an
ues are significantly higher (Fig. 2.3). environment contaminated with metals stimulates
From the obtained data, it could be a promis- the SRA and thus could be used to enhance the
ing alternative to use a consortium of hypersaline rate of metal bioremediation, i.e., in the forma-
SRB from Ribandar salt pan in heavy metal tion of metal sulfides, and to increase the level of
remediation as they resist and precipitate more tolerance to higher concentrations of the metal.
heavy metals than its mesohaline counterpart, The higher the SRA, the higher the production of
24 S. Kerkar and K. Ranjan Das

Fig. 2.4 Metal remediation

from industrial effluent

H2S which ultimately acts upon the heavy metals Modified Hatchikians media (1972) of 100 psu
and precipitates them as metal sulfide, thus were prepared by dissolving NaCl in seawater to
increasing bioremediation efficiency. isolate hypersaline SRB from the Ribandar salt
pans. SRB tolerating higher concentration of Pb,
Hg, Ni, and Co were used in bioremediation result-
2.8  ioremediation ofIndustrial
B ing in almost 100% heavy metal precipitation
Effluent withSRB from industrial effluents with a salinity ranging
from 8 to 90 psu and containing various metals.
Organisms in hypersaline ecosystems can toler- Bioprecipitation of nine metals (viz., Mg, Ca,
ate high metal concentrations in their surround- Fe, Co, Cu, Zn, Mo, Ba, Pb) using mix consortia
ing and adapt various strategies for survival in (three strains) of potential SRB out of 15 differ-
such extreme conditions. Thus SRB from such ent metals in varying concentration (listed in
environments are considered to be ideal in miti- Table 2.2) from an industrial effluent was
gating problems with environments having high achieved over an incubation period of 17 days.
metal contaminations. This is due to their adapt- Experimental results show that the consortium
ability to extreme saline condition and metal pre- was capable of precipitating out nine metals at
cipitation capabilities. Researchers worldwide 5099% concentrations.
have surveyed the heavy metal contents of the Tolerance of SRB to environment stress fac-
sediments from rivers, salt pans, bays, lagoons, tors like salinity and temperature in the salt pans
and harbors and mostly detected sulfur in the and their capability of reducing toxic metals at
deposits of heavy metals and attributed it to the neutral to alkaline pH has an added advantage
role of SRB.H2S gets discharged into the envi- over traditional metal remediation techniques
ronment as the final product of sulfate respiration requiring acidic condition for metal removal.
via SRB which are responsible for precipitation This process of using hypersaline SRB for biore-
of metal ions as less soluble metal sulfides. The mediation of metals is clearly a more attractive
H2S production is generally considered to be the option as the anaerobic waste treatment systems
main reaction that governs the metal sulfide pre- are advantageous as they do not require oxygen
cipitation (Amacher etal. 1993). and mixing as the aerobic counterparts.
Table 2.2 Different metal concentrations in industrial effluent
Metals in effluent Mg Al Ca Ti V Cr Mn Fe Co Ni Cu Zn Cd Ba Pb
Concentration in 3.41 2.54 12.53 0.20 22.17 0.13 7.98 33.35 0.06 0.75 11.30 76.20 3.39 0.04 0.06
2 Bioremediation of Heavy Metals from Saline Water Using Hypersaline Dissimilatory
26 S. Kerkar and K. Ranjan Das

2.9 Conclusion Attri K, Kerkar S (2011) Seasonal assessment of heavy

metal pollution in tropical mangrove sediments (Goa,
India). JEcobiotechnol 3:915
Hypersaline SRB develop tolerance and their Attri K, Kerkar S, LokaBharathi PA (2011) Ambient iron
metabolic activity gets stimulated in the presence concentration regulates the sulfate reducing activity in
of metals in its surrounding environment by vir- the mangrove swamps of Divar, Goa, India. Estuar
Coast Shelf Sci 95:156164
tue of its survival strategy in this natural habitat
Banfield JF, Welch SA, Zhang H, Ebert TT, Penn RL
bearing higher metal concentrations. Since SRA (2000) Crystal growth and microstructural evolution
is the main functional process in SRB which con- of FeOOH biomineralization products. Science
trols the bioremediation of heavy metals from 289:751754
Boopathy R, Gurgas M, Ullian J, Manning JF (1998)
saline waters, stimulating the sulfate-reducing
Metabolism of explosive compounds by sulfate reduc-
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rapid precipitation of metal sulfides and thus cycles of carbon, sulfur, and free oxygen in a micro-
bial mat. Geochim Cosmochim Acta 57:39713984
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Caumette P, Matheron R, Raymond K, Relexans JC
waters. Saline waters have limited types and low (1994) Microbial mats in the hypersaline ponds of
concentrations of carbon sources. Hypersaline Mediterranean salterns (Salins-de-Giraud, France).
sulfate-reducing bacteria have a tremendous FEMS Microbiol Ecol 13:273286
Coleman ML, Hedrick BD etal (1993) Reduction of
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Ehrlich HL (1999) Microbes as geologic agents: their role
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Ellwood DC, Hill MJ, Watson JHP (1992) Pollution con-
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Lead- and Mercury-Resistant
Marine Bacteria and Their 3
Application in Lead and Mercury

Milind M. Naik and S.K. Dubey

With rapid industrialisation, enormous amounts of industrial waste includ-
ing heavy metals have accumulated in marine environments over several
decades and require special attention. Untreated wastes from mining,
metal rening industries, battery manufacturing industries, sewage sludge,
power plants and waste incineration plants often contain substantially high
levels of lead (Pb) and mercury (Hg); when dumped into marine and estua-
rine waters, these pose serious threat to environmental biota and urgently
need to be removed from polluted marine/estuarine sites. Lead and mer-
cury are non-bioessential, persistent and hazardous heavy metal pollutants
of environmental concern. Bioremediation of heavy metals using Pb- and
Hg-resistant bacteria has become a potential alternative to the existing
technologies for the removal and/or recovery of toxic Pb and Hg from
waste waters before releasing it into marine/estuarine water bodies for
environmental safety. Various strategies through which marine/estuarine
bacteria resist high concentrations of lead/mercury include efux mecha-
nisms, extracellular sequestration, biosorption, precipitation, reduction,
volatilisation, alteration in cell morphology, enhanced siderophore pro-
duction, altered permeability, demethylation and intracellular bioaccumu-
lation. These unique characteristics of marine/estuarine bacteria proved to
be an ideal tool in bioremediation of lead and mercury from contaminated
marine and estuarine environmental sites.

3.1 Introduction

Metals that have a specic gravity greater than 5

or density more than 5 g/cm3 are termed as heavy
M.M. Naik (*) S.K. Dubey metals, e.g. mercury, lead, copper and cadmium
Department of Microbiology, Goa University, (Gadd 1992; Dash and Das 2012). The
Taleigao Plateau, Goa, India International Union of Pure and Applied

Springer Science+Business Media Singapore 2017 29

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_3
30 M.M. Naik and S.K. Dubey

Chemistry (IUPAC) technical report has elimi- Nishimura 1978; Lam et al. 2007; Naik et al.
nated the term heavy metal, and the alternative 2012b; Naik and Dubey 2013b). Bioavailability
name proposed is toxic metal (Duffus 2002), of metals is an important factor for metal toxicity
but still the term heavy metal is used exten- since soluble metals can readily penetrate cell
sively in scientic world. Contamination of membranes (Roane 1999). Therefore, metal
marine and freshwater bodies due to release of immobilisation or detoxication strategies are
toxic metals lead and mercury poses serious applied by microbes to counteract toxic effect of
threat to natural biota including humans (Nies heavy metals. Some natural microbial population
1999; Fernandes and Beiras 2001; Dirilgen that possesses a variety of protective mechanisms
2011; Dash and Das 2012). Untreated wastes can survive and colonise at very high concentra-
from mining, metal rening industries, car bat- tions of toxic lead and mercury without any
tery manufacturing industries, sewage sludge, impact on their growth and metabolism. This
hydroelectric power plants, paper and pulp unique characteristic of heavy metal-resistant
industries and waste incineration plants often microbes including bacteria makes them an ideal
contain substantially high levels of Pb and Hg tool for bioremediation of metal-contaminated
when dumped into marine/estuarine waters, pose marine and terrestrial sites. Various strategies
serious threat to environmental biota (Naik et al. through which they resist high concentrations of
2012d, 2013; De et al. 2014). It is interesting to heavy metals include efux mechanism, altera-
mention that even Antarctic Ocean water, which tion in cell morphology, siderophore production,
is considered relatively more pristine than any biosorption, precipitation, volatilisation, reduc-
other ocean water, is also contaminated with tion, demethylation, oxidation, extracellular
heavy metals due to anthropogenic activities sequestration, reduced permeability and intracel-
(Bonner 1984). lular bioaccumulation (Borremans et al. 2001;
Lead and mercury do not have any biological Barkay et al. 2003; Naik et al. 2012a, 2013; Naik
function and are toxic to cells in a variety of ways and Dubey 2013b; De et al. 2014). Bioremediation
which include DNA damage, oxidative damage processes are cost effective, highly efcient and
to proteins and lipids and binding to essential environment friendly as compared to physico-
proteins and enzymes (Nies 1999; Asmub et al. chemical methods for removal of toxic metals;
2000; Hartwig et al. 2002; Naik et al. 2012a, therefore, over the last few decades, attention has
2013; De et al. 2014). However, the most com- been focused towards exploiting marine microbes
mon route of exposure to mercury and lead is by for heavy metal bioremediation. Understanding
eating marine sh containing methylmercury and the mechanism by which marine and estuarine
lead (Eisler 1988; Nascimento and Chartone- bacteria sequester/detoxify/biotransform lead
Souza 2003; De et al. 2014). Due to rapid indus- and mercury to protect themselves from their
trialisation, enormous amounts of industrial toxic effects on physiological processes is crucial
waste containing heavy metals such as (mercury to the development of microbial processes for
and lead) have accumulated in marine and terres- their concentration, removal and recovery from
trial environments over several decades and industrial efuents, sewage, marine sediments
require special attention. Therefore, the US and marine/estuarine waters.
Environmental Protection Agency (EPA) has In the present chapter, we are focusing on
included lead and mercury in the list of the most lead- and mercury-resistant marine and estuarine
hazardous inorganic wastes (Cameron 1992). bacteria and how we can employ them to biore-
Lead and mercury are mutagenic and terato- mediate marine and estuarine sites highly con-
genic metals causing severe deleterious effects taminated with lead and mercury. Here we will
on human beings such as neurodegenerative stress on various resistant mechanisms employed
impairment, renal failure, reproductive damage, by marine and estuarine bacteria to resist very
neurological diseases and cancer (Kumagai and high concentrations of lead and mercury and
3 Lead- and Mercury-Resistant Marine Bacteria and Their Application in Lead and Mercury 31

exploitation of these resistant mechanisms to sample from semi-enclosed bay,

clean up and biomonitor marine and estuarine Gunnekleivfjorden from Southwest Norway,
contaminated sites. contained mercury ranging from 90 to 350 ppm
(Skei 1978). The mercury concentrations anal-
ysed along Indian coasts at (i) Mormugao
3.2 Lead and Mercury Pollution (152435 N, 73482 E; Hg concentration
in Marine and Estuarine 152456 ng/l in water and 53194 ng/g dry sedi-
Environments ment), (ii) Gopalpur (191812 N, 845755 E;
Hg concentration 2117 ng/l in water and 72128
3.2.1 Mercury ng/g dry sediment) and (iii) Chennai (13640
N, 80183 E; Hg concentration 1002,100 ng/l
Mercury is the sixteenth rarest element on earth; in water and 237338 ng/g dry sediment) were
however, concentrations of global mercury has found to be very high (De et al. 2007).
increased approximately threefold due to various Methylmercury is the most toxic among all the
anthropogenic activities, and the worlds oceans forms of mercury, affecting the immune system,
are the major reservoirs for its deposition (Mason altering the genetic system and causing damage
et al. 2012; De et al. 2014). Various anthropo- to the nervous system including coordination and
genic sources of mercury are burning of coal and the senses of touch, taste and sight (De et al.
petroleum products, dental llings, use of mercu- 2014). Mercury causes toxicity by binding to the
rial fungicides in agriculture, paper making sulfhydryl groups of enzymes and proteins,
industry and mercury catalysts in industries. thereby inactivating crucial cell functions.
Inorganic and organic mercury compounds such Generally, mercury accumulates upwards through
as elemental mercury (Hg0), mercuric mercury aquatic food chains, so that organisms at higher
(Hg+2), methylmercuric chloride (MMC) and trophic levels have higher mercury concentra-
dimethylmercury are discharged into marine tions (Nascimento and Chartone-Souza 2003).
environment through untreated sewage and Mercury is of environmental concern because it
industrial efuents (Wang et al. 2004; Dash and biomagnies in the food chain by up to seven
Das 2012; De et al. 2014). orders of magnitude, resulting in high concentra-
According to some recent models on the ow tions in top predators such as sh and polar bears
of mercury through the environment, it is sug- (Hintelmann 2010; Sonne 2010). Europe and
gested that natural sources account for about North America are reducing their contribution
10 % of the estimated 55008900 tons of mer- towards the global mercury burden, but the emis-
cury currently being released to the atmosphere sion rates in Asia are increasing at frightening
from all sources (De et al. 2014). Minamata dis- rates (Dash and Das 2012) and need special
ease which causes severe neurological disorder attention.
and was discovered in Minamata Bay, Japan, in
1956 is the rst record of severe mercury poison-
ing in people who consumed marine sh and 3.2.2 Lead
shellsh from mercury-contaminated marine
waters. Thousands of people where affected and Lead (Pb), obtained mainly from galena ore
887 were killed (Nascimento and Chartone- (PbS), is known to mankind for last 7,000 years,
Souza 2003). In Minamata Bay, very high level and its poisoning has been reported for at least
of mercury was reported which caused a serious 2,500 years (Nriagu 1978; Eisler 1988). Lead is
neurological disorder in humans referred as well known to inhibit haem biosynthesis, causes
Minamata disease. The level of total mercury serious neurodegenerative diseases and repro-
in seawater of Minamata Bay ranged from 56 to ductive impairment, interferes with kidney func-
285 ng/L and 2.1506 ng/L (Kumagai and tion, possesses carcinogenic properties and, when
Nishimura 1978). Interestingly, surface sediment blood level exceeds 70 g/dl, results in coma and
32 M.M. Naik and S.K. Dubey

death (Naik et al. 2012b). Lead is known to cause enters marine food chains at the 108 g Pb/g
damage to DNA, protein and lipid and to also level by adsorption onto surfaces of algae which
replace essential metal ions such as Zn, Ca and are consumed by herbivores. Dissolved Pb and
Fe from enzymes (Nies 1999; Asmub et al. 2000; biological productivity tend to be inversely cor-
Naik et al. 2013). Lead has broad range of appli- related in surface ocean waters (Burnett et al.
cations in various industries viz. petroleum, elec- 1980). Sediment sample of Mandovi Estuary of
tronics, battery, paints, ceramics, stained glass, Goa revealed appreciable levels of lead ranging
biocide preparation and ammunitions with annual from 4.5 to 46.5 g g1 at different source points
global demand of rened lead exceeding 87 lakh (Alagarsamy 2006). The pollution load index
tonnes (Naik et al. 2012c, 2013). Lead is a non- (PLI) for Pb, Fe, Mn, Zn, Cu, Co and Cr for
bioessential persistent environmental pollutant Divar sediments (Mandovi Estuary) was far
with half-life of approximately 5,000 years and greater (i.e. 1.652.19) than that of Tuvem
biomagnies through the trophic levels and accu- (Chapora Estuary) (0.911.3) reecting the
mulates at high concentrations in top predators intensity of anthropogenic inputs into the eco-
such as sh and polar bears. Environmental lead system due to transport of ferromanganese ore
has increased more than 1,000-fold over the past along the Mandovi River (Attri and Kerkar
three decades as a result of extensive anthropo- 2011). Estuarine sediment around the North
genic activities (Naik et al. 2013). The WHO has Irish Sea, UK, was reported to be contaminated
recommended <10 g/L lead as safe permissible with 52207 g/g lead (Smith and Orford 1989).
level in drinking water (Watt et al. 2000). Wastes Lead concentrations in surface sediments in a
from industries, sewage sludge, power plants and near-shore environment, Jurujuba Sound,
incineration plants contain substantial amounts Southeast Brazil, were found to be contami-
of toxic lead which are nally discharged into nated with 64174 g/g lead (Neto et al. 2000).
marine and estuarine environment which cause Lead concentration in estuarine sediments along
serious damage to marine micro- and the western coast of Mauritius was accounted to
macrofauna. be 27 mg/Kg (Ramessur 2004). Lead in marine/
The analysis of heavy metals in coastal waters estuarine environment is a persistent environ-
and sediments from Tianjin Bohai Bay, China, mental pollutant, slowly accumulates and results
revealed that Pb and Zn were the main heavy in biomagnication in the food chain and is
metal pollutants in the coastal waters of the bay. referred as cumulative poison, and there is
High levels of Pb and Zn appeared especially pressing need to remove lead from marine con-
near the estuary, indicating that river discharge taminated sites.
was the main pollution source. Analysis of data
for the period 19872004 indicated that Pb pol-
lution in coastal waters was due to river dis- 3.3 Lead- and Mercury-Resistance
charge before 2001. Lead levels did not decrease Mechanisms in Marine
after 2001 when annual run-off levels declined, and Estuarine Bacteria
indicating that Pb pollution by atmospheric
deposition had increased due to the use of leaded 3.3.1 Mercury Resistance
petrol in motor cars. Pb, Zn and Cd were the Mechanisms
dominant polluting elements in supercial sedi-
ments from Tianjin Bohai Bay, with levels in Unusual rise in mercury-resistant bacteria (MRB)
excess of the corresponding upper limits of envi- in coastal waters and sediments of India from
ronmental background values (Meng et al. 2008; year 1997 to 2003 was reported by Ramaiah and
Naik et al. 2012d). Dissolved Pb, ranging from De (2003). The mercury-resistant bacteria (MRB)
515 ng/kg in open-ocean surface waters up to that belong to Pseudomonas, Proteus,
50 ng/kg in highly polluted coastal waters, Xanthomonas, Alteromonas, Aeromonas and
3 Lead- and Mercury-Resistant Marine Bacteria and Their Application in Lead and Mercury 33

Enterobacteriaceae were isolated from west (i) Reduced uptake of mercuric ions: In
coast of India and were also found to grow in the Enterobacter aerogenes, resistance to mer-
presence of different toxicants, viz. heavy metals cury is by reduction in the cellular permea-
(Pb, Cd), pesticides, phenol, formaldehyde, for- bility to Hg2+ ions which is believed eventual
mic acid and trichloroethane (De et al. 2003). due to the expression of a couple of plasmid-
Mercury-resistant marine bacteria are found to be encoded proteins (Phung 1996). Since bacte-
very promising in dealing with mercury and most ria have reduced permeability to mercury, it
of other highly toxic heavy metals and xenobiot- can tolerate very high concentration of mer-
ics (De et al. 2006). Occurrence of large fractions cury in contaminated environment.
of mercury-resistant bacteria in the Bay of Bengal (ii) Demethylation of methylmercury followed by
was reported by De and Ramaiah (2006). Bacillus conversion to mercuric sulfide compounds:
isolates from Minamata Bay sediment were The best studied resistance system in
selected for the ability to volatilise mercury from marine bacteria is primarily based on clus-
a range of organomercurials (including methyl- tered genes in an operon (i.e. mer). Genes
mercury) (Nakamura and Silver 1994). The conferring resistances to mercury com-
higher abundance of the Gram-negative merA pounds are clustered in an operon in most
gene in the Seine Estuary mudats indicates a known naturally occurring systems.
relationship between the degree of anthropogenic TnMERI1 was found as the rst mercury
pollution and the abundance of the merA gene in resistance transposon identied from
the mudat sediments (Ramond et al. 2008). Five Gram-positive bacteria and was harboured
different types of resistance mechanisms (Fig. by a Minamata Bay sediment-isolated bac-
3.1) have been reported in bacteria for mercury terial strain which was designated as
which include (i) reduced uptake of mercuric Bacillus megaterium MB1. The mer operon
ions, (ii) demethylation of methylmercury fol- encoded by TnMERI1 has merR, merT,
lowed by conversion to mercuric sulde com- merP, merA and merB genes, which code
pounds, (iii) enzymatic reduction of Hg+2 to Hg0, for metal-specic activator-repressor,
(iv) sequestration of methylmercury and transporting, extracellular metal ion bind-
(v) intracellular bioaccumulation of mercury by ing, mercuric reductase and organomercu-
bacteria (Barkay et al. 2003). rial lyase, respectively (Huang et al. 2010).

Fig. 3.1 Mercury resistance

mechanisms in marine/estuarine Hg+2 CH3Hg
bacteria. (i) Reduced uptake of
Hg+2 Hg+2
mercuric ions, (ii) demethylation of MerT (i) Reduced Hg uptake
methylmercury by enzyme
organomercurial lyase, (iii) enzyme
mercuric reductase responsible for (ii) (v)Bioaccumulation
reduction of Hg+2 to Hg0, (iv) MerB Hg
extracellular sequestration of Hg+2 Iyase enzymeCH3Hg Hg
methylmercury and Hg+2 and (v)
intracellular bioaccumulation of (iii) MerA
mercury (reductase enzyme) BACTERIA

Hg Hg Hg
(iv) Extracellular sequestration
Hg0 (Volatile)
34 M.M. Naik and S.K. Dubey

In mer operon, determinants RTPCDAB in merP: MerP protein acts as an extracellular metal
marine bacteria are located on plasmid or ion-binding protein and can also act as free
transposons and also can be found in chro- radical scavenger. MerP protein possesses a
mosomes (Nascimento and Chartone- highly conserved domain with two cysteine
Souza 2003; Barkay et al. 2003). The residues for mercuric ion binding.
mer operon of a mercury-resistant merC: The exact role of MerC protein is not
Pseudoalteromonas haloplanktis strain was known, but mutating merC does not have any
isolated from Minamata Bay, Japan (Lohara effect on mercury resistance.
et al. 2001). The mercury-resistant mer (iv) Extracellular sequestration of Hg+2 and
operon is also reported in marine gliding methylmercury: The exopolymer (EPS)
flavobacterium, Tenacibaculum discolor present in the biolm of marine bacteria has
9A5 (Allen et al. 2013), whereas a diversity a specic relation with heavy metals; mer-
of mercury resistance determinants among cury binds with EPS and gets entrapped
Bacillus strains were isolated from sedi- extracellularly. The immobilised mercury
ment of Minamata Bay (Narita et al. 2003). outside bacterial cell is relatively toxic, and
(iii) Enzymatic reduction of Hg+2 to Hg0 therefore bacteria producing EPS can toler-
mer Operon ate very high concentration of mercury
merR: merR is a regulatory gene in mer operon. present in its immediate environment.
Mer R, the metalloregulatory protein, has high Mercury entrapped into EPS can be detoxi-
afnity for Hg+2. In the absence of Hg+2, it ed by activity of mer operon (Anthony
binds to the promoter region of mer operon 2014), i.e. mer B encodes enzyme called
and represses the transcription of structural organomercurial lyase, which transforms
genes from mer operon (TPCDAB) responsi- CH3-Hg to less toxic Hg+2, and merA
ble for mercury resistance. Mer R protein acti- encodes enzyme mercuric reductase respon-
vates transcription of structural genes whose sible for reduction of Hg+2 (toxic) to less
product requires mercury resistance, in the volatile form Hg0.
presence of inducing concentration of Hg+2 (v) Mercury bioaccumulation by bacteria:
(Lohara et al. 2001; Allen et al. 2013; Although methylmercury is more toxic than
Nascimento and Chartone-Souza 2003; Hg2+, in some bacteria, methylmercury is
Barkay et al. 2003; Huang et al. 2010). characterised to be the less toxic form.
merD: A secondary regulatory protein also binds Methylation has been reported in bacteria
the same operator-promoter region as MerR, from water, soil and sediments and is both
although very weakly. plasmid and chromosomally encoded
merT: Product of merT gene is required for trans- (Barkay et al. 2003; Miller et al. 2005). In
port of Hg+2 inside bacterial cell where Hg+2 is Desulfovibrio desulfuricans, the methyla-
acted upon by mercuric reductase (encoded by tion of mercury exists as a duo-step process
merA) and reduced to Hg0. which elaborates the transfer of a methyl
merB: It confers resistance to organomercurials group from methyltetrahydrofolate to meth-
such as methylmercury (CH3-Hg). It hydroly- ylcobalamin to Hg.
ses C-H by coding an enzyme called organo- (vi) Mercury methylation: Marine mercury-
mercurial lyase, which transforms CH3-Hg to resistant bacteria (Gram negative) isolated
less toxic Hg+2. Hg+2 is acted upon by mercu- from Bay of Bengal along Odisha coast
ric reductase (encoded by merA) and reduced were found negative for mer operon. These
to Hg0 (volatile form). bacterial isolates were found to resist very
merA: It encodes enzyme mercuric reductase high amount of mercury (MIC 50 ppm) by
responsible for reduction of Hg+2 (toxic) to bioaccumulating mercury intracellularly
less toxic volatile form Hg0. (Sinha 2012).
3 Lead- and Mercury-Resistant Marine Bacteria and Their Application in Lead and Mercury 35 Detoxication of Toxic (iv) cell surface biosorption and (v) precipitation
Chemicals and Heavy Metals (Fig. 3.2).
by Mercury-Resistant Bacteria
De et al. (2007, 2008) have reported that marine (i) Efflux mechanism
mercury-resistant bacteria exposed to polluted Heavy metal-resistant marine bacteria pos-
environments such as coastal areas can tolerate, sess ATPase-mediated efux pump which
detoxify or biotransform a variety of other maintains intracellular heavy metal homeo-
toxicants. Several mercury-resistant marine bac- stasis by efuxing excessive heavy metals
teria (Bacillus pumilus, Alcaligenes faecalis, outside the cells (Naik et al. 2013; Naik
Brevibacterium iodinum and Pseudomonas et al. 2013). Soft metal transporting PIB-type
aeruginosa) from the coastal waters of India ATPases are group of proteins involved in
were found to biotransform heavy metals, viz. transport of heavy metals outside the cell
cadmium, lead and xenobiotics like polychlori- membrane and governing bacterial heavy
nated biphenyls and tributyltin. A Pseudomonas metal resistance (Nies and Silver 1995;
aeruginosa strain CH07 aerobically degraded 14 Rensing et al. 1999; Coombs and Barkay
toxic polychlorinated biphenyls including con- 2004). Lead-resistant bacterial isolates
geners with ve or more chlorine atoms on the Pseudomonas stutzeri M-9 and Vibrio har-
biphenyl ring and was also equally efcient in veyi M-11 isolated from Zuari Estuary
degrading more than 54 % tributyltin. These bac- showed efux-mediated lead resistance
teria offer great biotechnological opportunities (Naik et al. 2013). Pseudomonas stutzeri
in bioremediation of marine waters contami- M-9 and Vibrio harveyi M-11 exhibited
nated with different xenobiotics and heavy resistance up to 0.8 mM and 1.2 mM lead
metals. nitrate, respectively. Nested PCR clearly
demonstrated presence of pbrA gene (ampli-
con size, 750 bp) belonging to P-type
3.3.2 Lead-Resistance Mechanisms ATPase family on chromosomal genome,
and 5.4 0.7- and 7.9 0.9-fold expression
Various lead-resistant mechanisms employed by of pbrA gene in Pseudomonas stutzeri strain
marine/estuarine lead-resistant bacteria include M-9 and Vibrio harveyi strain M-11 when
(i) efux mechanism, (ii) intracellular lead grown in TMM amended with 0.5 mM lead
bioaccumulation, (iii) extracellular sequestration, nitrate conrmed efux-mediated lead resis-

Pb Pb (iii) Extracellular
Pb sequestration

(ii) BmtA
Pb Pb+2 Pb+2
Pb (i) PIB-type ATPases
Efflux pump ADP
(v) Phosphatase
Pb Pb
-2 Pb Pb
PO4 + Pb+2 (iv) Surface biosoption

Lead phosphate (lead precipitation)

Fig. 3.2 Lead resistance mechanisms in marine/estuarine sequestration in EPS, (iv) cell surface biosorption to nega-
bacteria. (i) Efux pump, (ii) intracellular lead bioaccu- tive groups (carboxyl, hydroxyl) and (v) lead precipitation
mulation by bacterial metallothionein, (iii) extracellular into insoluble form catalysed by phosphatase enzyme
36 M.M. Naik and S.K. Dubey

tance in both bacterial isolates. These estua- Cu and Pb in a marine food chain were
rine bacterial isolates possess pbrA gene investigated using a partially puried and
encoding P-type ATPase for lead resistance chemically characterised EPS isolated from
and mdrL gene for multidrug resistance via Marinobacter sp. (Bhaskar and Bhosle
efux pumps, suggesting possible contami- 2006). In the marine bacterium P. aerugi-
nation of Zuari Estuary with heavy metals/ nosa CH07, lead was entrapped in EPS indi-
antibiotics. The Ralstonia metallidurans cating it as a possible resistance mechanism
CH34 complete operon pbrUTRABCD, (De et al. 2007, 2008). Lead ions could
conferring efux-mediated lead resistance, interact with carboxyl, hydroxyl and amide
has already been sequenced (Borremans groups and glucuronic acid from different
et al. 2001). Lead(II) resistance in chains of the polyanionic EPS produced by
Cupriavidus metallidurans CH34: interplay bacteria which acted as an electrostatic
between plasmid and chromosomally bridge between them producing a mesh of
located functions (Taghavi et al. 2009). polymer large enough to sequester very high
pbrUTRABCD operon was found to be levels of lead. Therefore, exopolysaccharide
responsible for lead resistance in producing lead-resistant bacterial strains
Cupriavidus metallidurans CH34. may serve as a potential bioremediative
(ii) Intracellular bioaccumulation agent (lead biosorbent) in lead-contami-
Intracellular metal bioaccumulation and nated environmental sites (Naik et al. 2012b,
homeostasis in cell cytosol involve the low 2013).
molecular weight, cysteine-rich metallo- (iv) Surface biosorption
thioneins which range from 3.5 to 14 kDa This surface biosorption of lead is due to
(Hamer 1986). Metallothioneins play an various negatively charged chemical groups
important role in immobilisation of toxic present on the bacterial cell surface, viz. the
heavy metals, thereby protecting bacterial carboxyl group of the peptidoglycan serves
metabolic processes catalysed by enzymes as main metal binding site at the cell wall of
(Blindauer et al. 2002; Liu et al. 2003). P. Gram-positive bacteria, whereas phosphate
aeruginosa strain WI-1 isolated from groups contribute signicantly in the case of
Mandovi Estuary possesses bacterial Gram-negative bacteria (Gadd and White
metallothionein (BmtA) to alleviate lead 1993; Naik et al. 2013). Shamim et al.
(Pb+2) toxicity (Naik et al. 2012a). P. aerugi- (2013) reported that lead-resistant
nosa strain WI-1 resists 0.6 mM lead nitrate Aeromonas caviae strain KS-1 isolated from
by bioaccumulating 26.5 mg lead/g dry Mandovi Estuary,Goa, India, can resist lead
weight of cells intracellularly. SDS-PAGE up to 1.4 mM, and signicant biosorption of
analysis conrmed lead-induced bacterial lead (8 %) on the cell surface of this isolate
metallothionein with molecular weight 11 was clearly revealed by scanning electron
kDa, which corresponds to the predicted microscopy coupled with energy-dispersive
bmtA gene. Thus, estuarine bacteria pos- X-ray spectroscopy.
sessing metallothioneins are an ideal tool (v) Bioprecipitation
for bioremediation of heavy metal-contami- Soluble (bioavailable) lead is toxic to bac-
nated environmental sites. teria; therefore, resistant bacteria precipi-
(iii) Extracellular sequestration tate toxic lead as insoluble complexes to
A metal immobilisation strategy is applied reduce their bioavailability and toxicity
by microbes to counteract toxic effects of (Naik et al. 2013). Three pleiotropic, quo-
heavy metals by secreting extracellular high rum sensing-defective Vibrio harveyi
molecular weight biopolymers referred as mutants were observed to precipitate solu-
exopolysaccharides (EPSs). Bacterial EPS ble Pb +2 as an insoluble compound. The
and its possible role in bioaccumulation of compound was puried and subjected to
3 Lead- and Mercury-Resistant Marine Bacteria and Their Application in Lead and Mercury 37

X-ray diffraction and elemental analyses. zole) and 30 g/disc (cephalexin), whereas for
These assays identied the precipitated strain M-11 it was 30 g/disc (nalidixic acid),
compound as Pb9(PO4)6, an unusual and 50 g/disc (ampicillin), 15 g/disc (erythromy-
complex lead phosphate salt that is pro- cin), 30 g/disc (chloramphenicol), 30 g/disc
duced synthetically at 200 C (Mire et al. (cephalexin) and 25 g (co-trimoxazole).
2004). This reaction is catalysed by phos- Metal contamination has been reported to
phatase enzyme. Sulfate-reducing bacteria function as a selective agent in the proliferation
(SRBs) are anaerobic heterotrophic bacte- of antibiotic resistance (Baker-Austin et al.
ria in marine environment which also pre- 2006). The co-selection mechanisms of antibiotic
cipitate heavy metals as insoluble metal and metal resistance include co-resistance (dif-
suldes, e.g. ZnS, PbS, CdS and CuS ferent resistance determinants present on the
(Chamberlain et al. 1988). Lead-resistant same genetic element) and cross resistance (the
Bacillus iodinium GP13 and Bacillus pum- same genetic determinant responsible for resis-
ilus S3 precipitate lead as lead sulde tance to multiple antibiotics and heavy metals).
(PbS) (De et al. 2008). Co-resistance occurs when the genes specifying
resistant phenotypes are located together on the Co-resistance and Cross same genetic element such as plasmid transpo-
Resistance sons or integron (Chapman 2003). There is grow-
Several mercury-resistant marine bacteria iso- ing concern that metal contamination in marine
lated from the coastal waters of India were evalu- environment may co-select multiple drug-
ated for their ability to biotransform heavy metals resistant pathogens. Therefore biomonitoring and
like cadmium and lead, e.g. Pseudomonad aeru- bioremediation of marine and estuarine environ-
ginosa strain CH07 (De et al. 2007). Mercury- ment is a desperate need.
resistant marine bacterial isolates Pseudomonas,
Proteus, Xanthomonas, Alteromonas, Aeromonas
and Enterobacteriaceae were also found to toler- 3.4 Conclusion
ate very high concentrations of lead and cadmium
(De et al. 2003). Lead-resistant P. aeruginosa Over last three decades, attention has been
strain WI-1 isolated from Mandovi Estuary pos- focused towards exploiting marine/estuarine
sesses bacterial metallothionein (BmtA) to alle- microbes for lead and mercury bioremediation.
viate Pb+2 toxicity (Naik et al. 2012a). P. Marine and estuarine bacteria possess different
aeruginosa strain WI-1 resists 0.6 mM lead lead and mercury resistance mechanisms; there-
nitrate by bioaccumulating 26.5 mg lead/g dry fore, it is necessary and highly desirable to char-
weight of cells intracellularly. P. aeruginosa acterise such marine bacterial isolates with
strain WI-1 also showed cross-tolerance to cad- reference to their biochemical and genetic mecha-
mium, mercury and tributyltin chloride (TBTCl) nism of resistance. Lead- and mercury-resistant
along with resistance to multiple antibiotics bacterial strains possessing various lead-resistant
(Naik et al. 2012a). The two bacterial strains P. mechanisms such as efux mechanism, extracel-
stutzeri M-9 and V. harveyi M-11 isolated from lular sequestration, biosorption, precipitation,
Zuari Estuary resist lead nitrate up to 0.8 and 1.2 demethylation, reduction, volatilisation, demeth-
mM by efux mechanism (Naik et al. 2013). P. ylation and intracellular bioaccumulation dis-
stutzeri M-9 and V. harveyi M-11 were also found cussed in this chapter may serve as potential
resistant to cadmium and mercury along with biotechnological agents for bioremediation of
multiple antibiotics. MIC of antibiotics for lead- lead- and mercury-contaminated marine and estu-
resistant bacterial strain M-9 was 50 g/disc arine environmental sites. Valuable properties
(ampicillin), 30 g/disc (chloramphenicol), 10 already present in certain bacterial strains can be
g/disc (noroxacin), 25 g/disc (co-trimoxa- combined or improved through state-of-the-art
38 M.M. Naik and S.K. Dubey

genetic engineering tools. Marine/estuarine resistance (mer) operon in marine gliding avobacte-
rium, tenacibaculum discolor 9A5. FEMS Microbiol
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Acknowledgements The authors would like to acknowl- (Environmental Protection Agency EPA/600/
edge Dr. Satish Shetye, Vice Chancellor of Goa University, 491/029)
for providing necessary facilities. Dr. Milind Naik also Chamberlain AHL, Simmonds SE, Garn BJ (1988)
thank Government of India for nancial support as SERB- Marine copper-tolerant sulphate reducing bacteria
DST young scientist project. and their effects on 90/10 copper-nickel (CA 706). Int
Biodeter 24:213219
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cross resistance, and co-resistance. Int Biodeterior
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Microbial Remediation
ofOrganometals andOil 4
Hydrocarbons intheMarine

AndreiaCruz*, AnaJuliaCavaleiro*, Ana

M.S.Paulo, AntnioLouvado, M.MadalenaAlves,
AdelaideAlmeida, andngelaCunha

Marine environments are exposed to pollution that mostly results from
human activities. Organometals and oil hydrocarbons are among the most
hazardous pollutants. In surface waters and along the water column, these
compounds are more easily degraded than in sediments, especially under
anoxic conditions, where they are highly persistent. Due to their negative
impact in living organisms, decontamination of polluted marine sites with
minimum collateral impacts is imperative. Bioremediation strategies, ben-
efiting from the ability of aerobic and anaerobic microorganisms to
degrade organometals or oil hydrocarbons to simpler and less toxic deriva-
tives, represent an alternative to traditional physicochemical decontamina-
tion methods. Different bioremediation strategies have been applied in
marine environments, including monitored natural recovery, biostimula-
tion, bioaugmentation and phytoremediation. Individual microbial agents
or mixed microbial consortia able to remediate these pollutants in marine
environments have been identified, and the most relevant mechanisms of
biodegradation of pollutants are characterised.
This chapter provides an overview on microbial bioremediation of
organometals and oil hydrocarbons in marine environments, focusing on
the bioremediation concept, microbial aerobic/anaerobic agents, meta-
bolic pathways and genetic determinants involved in the degradation/
transformation processes while highlighting the importance of microbial
consortia and their applications. A critical analysis of the advantages and
limitations of microbial remediation and a perspective on future develop-
ments are also provided.

*Both authors contributed equally to this chapter.

A. Cruz (*) A. Louvado A. Almeida . Cunha
Biology Department & Centre for Environmental and A.J. Cavaleiro A.M.S. Paulo M.M. Alves
Marine Studies (CESAM), University of Aveiro, Centre of Biological Engineering, University of
Campus de Santiago, 3810-193 Aveiro, Portugal Minho, Campus de Gualtar, 4710-057 Braga,
e-mail: Portugal

Springer Science+Business Media Singapore 2017 41

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
42 A. Cruz et al.

4.1 Introduction incomplete combustion of fuels; post-depositional

transformation of biogenic precursors or diffus-
One of the major concerns of the industrialised ing from the mantle, oil spills, petroleum source
world is the high level of marine pollution with rocks or reservoirs; and circulation of ships and
hazardous compounds, such as organometals and tank washings (Perelo 2010).
oil hydrocarbons. These pollutants can reach the These contaminants may be discharged
marine environment by different sources, mostly directly in coastal waters or in open sea. In areas
associated with anthropogenic activities like where chronic inputs occur, pollutants can be
industrial and municipal wastewater disposal, the transferred to deeper areas, due to their adsorp-
use of pesticides or fertilisers, sewage sludge, tion to suspended particulates, and/or tend to
run-off of landfill leachates and harbour activities accumulate in bottom sediments (Gadd 2000;
(Hoch 2001; Du etal. 2014; Perelo 2010) Louvado etal. 2015; Rainbow 1995). Sediments
(Fig. 4.1). represent the major sink for these compounds
The main source of organometals, in particu- (Huang etal. 2013; Rahmanpour etal. 2014;
lar organotin compounds (OT), is their use as a Maanan etal. 2015; Antizar-Ladislao 2008;
component of antifouling paints employed as Oliveira etal. 2015). On the other hand, when
coating in the hull of ships and/or boats and in resuspended, particulate-bound contaminants
nautical equipment (Hoch 2001). Hydrocarbons may be remobilised into the water column,
reach marine environments as a consequence of becoming more bioavailable (Roberts 2012).
biosynthesis by aquatic or terrestrial organisms, Organometals and hydrocarbons have
but its main sources are petrochemical industries; immediate and long-term effects on marine

Fig. 4.1 Sources of organometals and oil hydrocarbons pollution into the marine environment
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 43

organisms, from bacteria to mammals and from indigenous microbial population in combination
the molecular to the community levels. Some of with naturally occurring physical and chemical
these compounds have the potential to be bioac- processes (Yu etal. 2005); (ii) biostimulation as
cumulated and biomagnified along aquatic tro- the metabolic activation of the indigenous popu-
phic webs (Cooney and Wuertz 1989; Cruz etal. lation by manipulating the factors that affect
2015; McGenity etal. 2012b; OBrien and microbial growth, as, for instance, the supply of
Keough 2014; Zhang etal. 2015). Considering nutrients (such as the addition of N and P to
the magnitude and the persistence of the ecotoxi- counteract nutrient limitation) or surfactants that
cological effects, efficient and sustainable enhance dispersion and bioavailability of the pol-
approaches for remediation are required. lutant (Zhang and Lo 2015; Yu etal. 2005); (iii)
Conventional methods have been used to remedi- bioaugmentation as the introduction of natural or
ate pollution in marine environments. The com- engineered species that enhance biodegradation/
mon approaches to remove organometals are transformation of specific contaminants (Cruz
sorption, ion exchange, precipitation and electro- etal. 2014); and (iv) phytoremediation as the use
chemical techniques (Du etal. 2014). In turn, the of plants and algae for the degradation and
remediation of hydrocarbons has been performed removal of contaminants from the environment
through physical adsorption with activated car- (Iwamoto and Nasu 2001; Czako etal. 2006;
bon, hydrophobic meshes and nanomaterials Cruz-Uribe and Rorrer 2006). Combinations of
(Han etal. 2015; Nafees etal. 2013; Lin etal. bioremediation strategies, for example, phytore-
2015; Sabir 2015), chemical dispersants mediation and biostimulation, may also be
(Kujawinski etal. 2011) and oxidants (Usman applied (Bianchi etal. 2010; Moreira etal. 2011;
etal. 2012). Most of these methods are expen- Oliveira etal. 2015).
sive, are labour demanding and can generate sec- Low cost is one of the major advantages of bio-
ondary materials or by-products that must often remediation relatively to conventional strategies.
be disposed as hazardous compounds (Lee etal. Additionally, it is a non-invasive approach with
2012; Du etal. 2014). Thus, as the use of biore- minimum undesirable effects in the ecosystem
mediation, other alternatives are being (Perelo 2010). However, bioremediation has some
considered. drawbacks that can limit the success of its appli-
The concept of bioremediation has been cation, such as the relatively long periods of time
described as the process of using the metabolic involved and the higher uncertainty of results, in
potential of microorganisms to immobilise, comparison with conventional methods, because
degrade, transform or remove hazardous compo- of the multiplicity of environmental factors affect-
nents from the environment (Watanabe 2001). In ing the biological processes (Perelo 2010;
fact, microorganisms play important roles in the Boopathy 2000). At the laboratory scale, biore-
environmental fate of toxic compounds through a mediation of organometals and hydrocarbons has
multiplicity of physical, chemical and biological shown to be efficient, even for cleaning up con-
mechanisms (Gadd 2004). taminated samples presenting low toxic concen-
Bioremediation techniques can be classified trations, which would be impracticable with
as ex situ and in situ. Ex situ approaches imply conventional remediation techniques. At environ-
the removal of the contaminated material from mental scales, few examples of hydrocarbon bio-
the natural site to be treated elsewhere. On the remediation were reported (Rocchetti etal. 2014).
other hand, in situ technologies are performed at This chapter provides an overview of the main
the contaminated site (Boopathy 2000). Strategies microbial agents involved in the bioremediation
for the in situ bioremediation of marine environ- of organometals and oil hydrocarbons in marine
ment include (i) monitored natural recovery environments, metabolic pathways, transforma-
(MNR) using the self-healing capacities of the tion processes and associated genes and presents
44 A. Cruz et al.

a critical analysis of the efficiency and constraints solubility, OT strongly bind to suspended mate-
to the practical implementation of bioremedia- rial and inorganic sediment particles (Laughlin Jr
tion protocols. etal. 1986).
Tributyltin (TBT) was, during decades, the
most used organometal. International Maritime
4.2 Organometals Organization (IMO) called for a worldwide treaty
that bans the application of TBT-based paints
4.2.1 Organometal Contamination starting 1 January 2003 and total prohibition by 1
inMarine Environments January 2008 (EC 2002; IMO 2001). As a conse-
quence of partially prohibiting the use of TBT in
Organometals are generally defined as com- several developed countries, the recovery of
pounds having at least one metalcarbon (nor- affected gastropod and oyster populations has
mally metalalkyl) polarised bond, i.e. where the been observed (Alzieu 1998; Evans etal. 2000;
carbon is more electronegative than the metal Evans 1999). However, the use of TBT as biocide
(Craig 2003). These compounds can occur in the in antifouling paints is still permitted in IMO
environment by being naturally formed there or non-member countries (Sousa etal. 2013). Also
associated to anthropogenic inputs (Gadd 1993). the ineffectiveness of the alternative products has
Organometals formed by arsenic, mercury, tin led to the illegal use of TBT, resulting in environ-
and lead are used in a wide variety of industrial mental concentrations that remain high enough to
processes due to their biocidal properties (Craig motivate concern (Okoro etal. 2011; Santillo
2003; Frache and Rivaro 2000). Within organo- etal. 2001; Barroso and Moreira 2002; Santos
metals, organotins (OT) are the most widely etal. 2002).
used, reaching a worldwide production estimated The major deleterious effect of TBT is
at 50,000 tons; thus, their impact in the marine imposex, a phenomenon characterised by the
environment is highly relevant (Cruz etal. 2015; superimposition of male sexual characteristics
Ayanda etal. 2012). OT conform to the general (vas deferens, penis) on female gastropods
formula R( 4 n ) SnX n with n = 0 3 , in which R is (Smith 1971; Barroso etal. 2002), caused by
any organic alkyl or aryl group and X is an anionic endocrine disruption. However, negative effects
species, namely, halide, oxide, chloride and of TBT are not restricted to invertebrates. TBT
hydroxide (Rudel 2003). The properties of OT inhibits the growth and metabolic activity of bac-
vary significantly, depending mainly upon the teria (Gadd 2000; Cruz etal. 2012) and marine
number and nature of the R groups, but also upon microalgae (Gadd 2000), causes apoptosis in
the type of the ligand (X) (Hoch 2001). The toxic- marine sponges (Batel etal. 1993), decreases
ity of the OT can be more influenced by the alkyl neonate survival in crustaceans (Takeuchi etal.
substitutes than the anionic substitutes (Antizar- 2001), causes alterations on shell growth in mol-
Ladislao 2008). In general, inorganic tin is non- luscs (Alzieu 2000) and causes masculinisation
toxic, whereas trisubstituted compounds have in fish females (Shimasaki etal. 2003) and inhi-
maximum toxicological activity (Sekizawa etal. bition of hepatic cytochrome P450 and
2003). ethoxyresorufin- O-deethylase (EROD) and
The solubility of OT in water decreases with penthoxyresorufin- O-depenthylase (PROD) in
increasing number and length of the organic sub- marine mammals (Kim etal. 1998). Additionally,
stitutes, but it also depends on the particular X TBT shows a substantial potential for bioaccu-
(Hoch 2001). Water solubility of most OT is low mulation and biomagnification through the food
and also dependent on pH, ionic strength and chains, thus being of particular concern in long-
temperature. Data on solubility for tributyltin lived biota (Murata etal. 2008).
chloride (TBT-Cl) range from 5 to 50mg L1, Dafforn etal. (2011) revised the global levels
whereas for dibutyltin chloride (DBT-Cl2) is as of TBT in marine environments and reported that
high as 92mg L1 (Rudel 2003). Due to low water in the water column, the maximum values (3.2 g
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 45

L1) were observed in Singapore in 2000, and in Aerobic conditions facilitate the degradation
sediments, the maximum value (89 g g1) was of TBT, which takes about 13 months to be
registered in Australia, in 2006. The hotspots of degraded, in comparison with anaerobic condi-
TBT are the marinas and commercial harbours in tions, that result in higher persistence of the com-
developed countries, where the contamination in pound (6 months to 8.7 years) (Sternberg etal.
the surface water and sediments is correlated 2010).
with the intensity of shipping or boating activities A microcosm set-up, simulating a temperate
(Dafforn etal. 2011). shallow estuarine, was used to determine the per-
In the aquatic environment, TBT is quickly sistence and behaviour of TBT.Radiolabelled
removed from the water column and accumu- TBT was introduced in a microcosm containing
lated in the sediment compartment due to the estuarine water and sediment. The experiment
high specific gravity (near 1.2kg L1 at 20 C) was conducted during 40 days, and TBT and its
(Landmeyer etal. 2004), low solubility (less degradation products were monitored during this
than 10mg L1 at 20 C and pH 7.0) (Fent 1996) period. TBT rapidly disseminated to the three
and log Kow values near 4.4 at pH 8. Additionally, compartments of the microcosm, being more
TBT is ionisable and exhibits a pKa acidity con- than 60% of the TBT and of its metabolites found
stant of 6.25 (Antizar-Ladislao 2008). TBT can in the sediment. In the water column, the TBT
be degraded by debutylation to form dibutyltin half-life was 2.55 days for the first 11 days and
(DBT), monobutyltin (MBT) and ultimately then slowed to 13.4 days. The concentration of
inorganic tin (Cooney 1995; Gadd 2000; Antizar- TBT adsorbed on suspended particles was three
Ladislao 2008). Considering that TBT and its orders of magnitude higher than that in the dis-
degradation products are frequently associated solved form (Dai etal. 1998).
with marine pollution, these compounds will be
addressed in this chapter as a model organometal
contaminant, and a particular focus will be given 4.2.2 B
 ioremediation ofOrganotin
to their properties and bioremediation Compounds (OT) inMarine
strategies. Environments
Rates of TBT degradation may be influenced
by several biotic and abiotic factors, such as con- In the water column as in sediments, OT degrada-
centration of dissolved/suspended organic mat- tion is predominantly performed by microorgan-
ter, salinity, temperature, pH, light and the nature isms such as bacteria, fungi and microalgae
and density of the microbial communities (Dubey (Table 4.1).
and Roy 2003). However, the degradation pro- Although the biodegradation of TBT in marine
cess occurs primarily through biotic processes, environments is long known, the mechanistic
and the efficiency of TBT biodegradation will reactions involved are still not completely under-
depend mainly on the bioavailability of this com- stood. It was suggested that the enzyme homo-
pound (Sakultantimetha etal. 2011). gentisate 1,2-dioxygenase is involved in the
In the marine environment, the half-life of aerobic degradation of butyltins in microalgae,
TBT can be different depending on the ecological via successive dealkylations (Tsang etal. 1999;
compartment. In the water column, TBT can be Lee etal. 1989).
rapidly degraded with a half-life of several days Recently, two strains with TBT-degradative
(Gadd 2000). In sediments, the rate of TBT debu- capability, Klebsiella pneumoniae SD9 and
tylation is dependent upon microbial activity Alcaligenes faecalis SD9, showed enhanced sid-
(Sternberg etal. 2010), and in deep sediments, erophore production, in the presence of TBT
the degradation rates are much lower than in the (Khanolkar etal. 2015a, b). Moreover,
water column, being the half-life within 1.93.8 Alcaligenes faecalis SD9 increased the produc-
years (Gadd 2000). tion of exopolysaccharide (EPS) when exposed
46 A. Cruz et al.

Table 4.1 OT-degrading microorganisms isolated from marine environment

Agent Reference
Fungi Tramates versicolor Barug (1981)
Chaetomium globosum
Coniophora puteana Dubey and Roy (2003)
Coriolus versicolor
Cunninghamella elegans Bernat and Dlugonski (2006)
Microalgae Pavlova lutheri Saint-Louis etal. (1994)
Skeletonema costatum Lee etal. (1989)
Nannochloropsis oculata Taha etal. (2009)
Dunaliella parva
Leptocylindrus danicus Xie etal. (2011)
Amphidinium carterae
Bacteria Pseudomonas aeruginosa Barug (1981)
Alcaligenes faecalis
Pseudomonas diminuta Kawai etal. (1998)
Pseudomonas aeruginosa USS25 Roy and Bhosle (2006)
Pseudomonas stutzeri DN2 Khanolkar etal. (2014)
Aeromonas molluscorum Av27 Cruz etal. (2007)
Enterobacter cloacae TISTR1971 Sakultantimetha etal. (2010)
Alcaligenes faecalis SD5 Khanolkar etal. (2015b)
Klebsiella pneumoniae SD9 Khanolkar etal. (2015a)

to TBT (Khanolkar etal. 2015b). Thus, in these contribute for TBT degradation (Suehiro etal.
studies, authors concluded that siderophore and 2006).
EPS might be involved in TBT degradation pro- At a larger scale, a mesocosm experiment dur-
cesses. Nonetheless, information on microbial ing which TBT and its degradation products were
degradation processes is still scarce. monitored was performed during 278 days.
Some few studies were performed with algae Radiolabelled TBT (59020ng L1) was added
and fungi (Barug 1981; Dubey and Roy 2003; to a 13m3 marine enclosure with near-natural
Bernat and Dlugonski 2006; Lee etal. 1989; water column and benthos. The degradation rate
Saint-Louis etal. 1994; Taha etal. 2009; Xie was estimated as 0.20 per day for 15 days,
etal. 2011), but the vast majority is focused on decreasing afterwards to 0.10 per day. Two thirds
bacteria. of the TBT present in the water was degraded to
TBT degradation rates and the changes in the DBT that was further converted to MBT, and one
bacterial community were evaluated during 150 third of the TBT was degraded directly to MBT
days in microcosm experiments with sediments (Adelman etal. 1990).
from the Mekong River in Vietnam (Suehiro A 150-day microcosm approach was used to
etal. 2006). The initial concentration of TBT evaluate the ability of Aeromonas molluscorum
was of 1.01.4 g g1 (dry weight) and decreased Av27, a TBT-resistant/degrading bacterium, to
to 0.6 g g1 (dry weight) at the end of the exper- enhance TBT degradation in estuarine sediments.
iment. The identifications of the TBT-degrading At the end of the experiment, 28% of the TBT
strains were not achieved, but denaturing gradi- had been degraded to DBT and MBT.TBT deg-
ent gel electrophoresis (DGGE) and bacterial radation was significantly enhanced by A. mol-
number profiles indicated that the community luscorum Av27. The characterisation of the
was well adapted to TBT pollution in the structural diversity of bacterial community indi-
Mekong River sediment and could efficiently cated that Proteobacteria was the predominant
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 47

phylum, followed by Bacteroidetes. At the begin- half-life of TBT was 578 days confirming its high
ning of the experiment, the relative abundance of persistence in the sediment. Different experimen-
different classes could be ordered as tal conditions consisting in the improvement of
Gammaproteobacteria (55%) > bioavailability (manipulation of salinity, surfac-
Deltaproteobacteria (34%) > Alphaproteobacteria tant addition and sonication), the stimulation of
(6%) > unclassified Proteobacteria (5%) (Cruz biological activity (aeration and temperature
etal. 2014). At the end of the experiment, the manipulation), the inoculation with a TBT-
abundance of Deltaproteobacteria and resistant bacterium (Enterobacter cloacae) and
Alphaproteobacteria increased. nutrient addition (succinate, glycerol and
Deltaproteobacteria includes sulphur- and metal- L-arginine) were tested. The shorter half-life esti-
reducing bacteria (Webster etal. 2010), and mates were 45 days, after bioavailability
Alphaproteobacteria includes members that are improvement and inoculation of E. cloacae, both
sensitive to hydrocarbons (LaMontagne etal. with or without nutrient addition (Sakultantimetha
2004; Polymenakou etal. 2005; Sun etal. 2013), etal. 2011).
which may have contributed to the degradation of As shown in the examples given above, some
pollutants over time. Although some changes in of the bioremediation processes described are
bacterial community structure were noticed, the attributed to bacteria consortia, usually associ-
relative abundance of the phylotypes was gener- ated with the indigenous bacterial community.
ally maintained (Cruz etal. 2014). By the many experiments performed, it has been
The biological degradation of TBT in harbour reinforced that the use of microbial consortia
sediments under aerobic and anaerobic condi- offers considerable advantages over the use of
tions and upon land deposition demonstrated that pure cultures.
TBT degradation was faster under aerobic condi-
tions, as above mentioned, and it increased with
increasing temperature. Using 320 g kg1 of 4.3 Oil Hydrocarbons
TBT, it was observed that TBT half-life decreased
from 90 to 4 weeks when temperature raised 4.3.1 Oil Hydrocarbon
from 5 to 55 C, in aerobic conditions. After 7 Contamination inMarine
months, at 55 C, in aerobic conditions, the deg- Environments
radation was completed, being the concentration
of TBT below the detection limit (1 g kg1 dw) Oil demand is increasing worldwide, largely due
(Brandsch etal. 2001). to development countries, averaging 94.0 million
Experiments with sediments from various barrels per day in 2015 (International Energy
Japanese coastal areas, involving successive Agency 2015). The intensive exploitation of
enrichments with 100 g L1 of TBT with 21-day petroleum sources, and the vast range of activi-
intervals, were conducted in order to select TBT- ties developed by the oil industry, increases the
degrading bacteria. The ratio of TBT degradation risk of accidental oil spills. Oil-derived pollutants
varied between 0 and 72.7%. Two microorgan- enter marine environments through leaks during
isms with high homology with Halomonas aqua- drilling, transport and storage, discharge of water
marina and Halomonas alimentaria were isolated at offshore oil exploration and combustion of fos-
and identified, and the ability to degrade 60 g sil fuels (Notar etal. 2001; Guitart etal. 2007,
L1 and 97 g L1 of TBT after 21 days of incuba- 2010). Chronic contamination of harbour sedi-
tion was demonstrated (Hamada-Sato etal. ments from shipping activities, fuel oil spills and
2002). run-offs is also frequent (Coates etal. 1997). In
The effect of bioaugmentation and the com- addition, oil hydrocarbons are introduced in the
bined action of coadjuvants to enhance bioavail- marine ecosystems by natural sources such as oil
ability was confirmed in microcosm experiments. seepages that represent 47% of the oil released to
Under natural attenuation control conditions, the marine environments (National Academy of
48 A. Cruz et al.

Science 2002; Kvenvolden and Cooper 2003). sess one or more organic rings with delocalised
Despite the significant contribution of natural electrons. The different physical and chemical
sources, anthropogenic oil spills are still the properties of the oil components, namely, solu-
major cause for the fast and uncontrolled release bility, volatility and tendency to adsorb to parti-
of high quantities of oil in specific areas, causing cles or sediments (Table 4.2), greatly influence
severe ecological and economical damages. In bioavailability, biodegradation and, subse-
1989, the Exxon Valdez oil spill in Alaska caused quently, oil composition after its release in the
the release of 41144,000m3 of crude throughout environment.
2100km of coastline, affecting surface water, Short-chain alkanes, monoaromatics and low
sediments, wetland, beaches and other coast fea- molecular polycyclic aromatic hydrocarbons
tures and caused the death of microorganisms, (PAHs), which have a higher solubility and
plants and animals, including birds, cetaceans, vapour pressure (Table 4.2), will dissolve and
sea otters and seals (Carson etal. 1992; Peterson evaporate easily. Afterwards, long-chain and
etal. 2003). During the Gulf War (19901991), alkylated alkanes will be biodegraded, leaving a
more than 10 million barrels (approx. 1.6 million remnant composed of high molecular weight
m3) of crude oil were spilled in the Arabian Gulf, (HMW) PAH, resins and other more recalcitrant
and the impacts on marshlands and mud tidal pollutants. The rate of biological degradation is
flats persisted for over a decade (Bejarano and lower for more complex hydrocarbons, which are
Michel 2010). In 2010, the Deepwater Horizon more resistant to biodegradation (Atlas 1995).
oil spill in the Gulf of Mexico released 4.9 mil-
lion barrels (approx. 0.8 million m3) of crude oil
that impacted salt marshes (Silliman etal. 2012) 4.3.2 Hydrocarbon Degradation
and animals (Barron 2012). byAerobic Marine Bacteria
At sublethal concentrations (chronic contami-
nation), hydrocarbons induce growth inhibition Marine oil-degrading bacteria tend to be meta-
in sensitive prokaryote groups (Vzquez and Rial bolically more specialised than their terrestrial
2014) and significant changes in the structure and counterparts (Yakimov etal. 2007). Some
function of bacterial and fungal communities hydrocarbon-degrading bacteria actually prefer
(Bik etal. 2012). Theyaffect embryogenesis and other carbon sources, but obligate hydrocarbon-
growth in invertebrates (Bellas etal. 2013; Vieira degrading bacteria, also designated as hydrocar-
and Guilhermino 2012), cause oxidative stress in bonoclastic bacteria, are also present in the
benthic communities (Coelho etal. 2015), environment and will flourish after pollution
inducephototoxicity and metabolic disorders, (Yakimov etal. 2007). Hydrocarbon-degrading
impair development in fish, particularly in early capacity is found in a wide range of taxa but is
growth stages (Incardona etal. 2013; Lucas etal. particularly frequent among and
2014), cause immunotoxicity and adrenal and -Proteobacteria (Yakimov etal. 2007). Aerobic
lung disease in marine vertebrates, including alkane biodegradation can be performed by fac-
mammals (Barron 2012; Schwacke etal. 2013), ultative anaerobic or obligate aerobic bacteria
and reduce growth and photosynthetic activity in (Yakimov etal. 2007). Facultative anaerobic bac-
algae and macrophytes (Lewis and Pryor 2013). teria have a versatile metabolism and will not
Crude oil is a very complex mixture of organic degrade alkanes if other more attractive carbon
compounds, mainly composed by aliphatic and sources are available (Rojo 2009). Some exam-
aromatic hydrocarbons, resins and asphaltenes. ples of alkane-degrading bacterial genus are
Aliphatic hydrocarbons may be saturated or Alcanivorax, Oleiphilus, Oleispira and
unsaturated (with double bonds in the carbon Thalassolituus (Yakimov etal. 2007). PAH-
chain) and can appear with linear, branched or degrading isolates are widespread among differ-
cyclic structures. Aromatic hydrocarbons pos- ent bacteria phyla, being related to Proteobacteria
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 49

Table 4.2 Physical and chemical properties of selected hydrocarbons

Molecular Vapour Log EPA
Molecular weight (g Solubility pressure Kowc carcinogenic
Groupa Name formula mol1) (mg L1)b (Pa)b () classificationd
Aliphatics Hexane C6H14 86.2 13.0 17,600 3.3 D
Hexadecane C16H34 226.4 4.5105 <10 8.3 D
Cyclohexane C6H12 84.2 43.0 10,300 3.2 D
BTEX Benzene C6H6 78.1 2.0102 12,700 2.1 A
Toluene C7H8 92.1 6.0103 2,920 2.7 D
Ethylbenzene C8H10 106.2 1.0103 950 3.2 D
Xylene C8H10 106.2 2.0103 787 3.0 D
PAH Naphthalene C10H8 128.2 31.7 11 3.3 C
Phenanthrene C14H10 178.2 1.3 2102 4.5 D
Pyrene C16H10 202.3 0.1 6104 5.3 D
Aliphatic hydrocarbons, monoaromatic (BTEX benzene, toluene, ethylbenzene and xylene) or polycyclic aromatic
hydrocarbons (PAH)
Aqueous solubility and vapour pressure at 20 C
Kow = octanolwater partition coefficient
EPA carcinogenic classification (all classes described): A-human carcinogenic, B1 and B2-probable human carcino-
genic, C-possible human carcinogenic, D-not classifiable as to human carcinogenicity, E-evidence of noncarcinogenic-
ity for humans
Adapted from Weelink etal. (2010), McGenity etal. (2012b)

(, and classes), Actinobacteria, immediately after the accident were dominated

Cyanobacteria, Bacteroidetes and Firmicutes. by Oceanospirillaceae and pseudomonads.
Bacteria belonging to the genera Cycloclasticus, However, in a subsequent phase, when the pro-
Pseudomonas (-Proteobacteria) and some gen- portion of alkanes and cycloalkanes was the
era of the Sphingomonadaceae family highest, the relative abundance of PAH degrad-
(-Proteobacteria, Novosphingomonas, ers like Colwellia, Cycloclasticus and
Sphingomonas and Sphingobium) are frequently Pseudoalteromonas increased. Methylomonas
referred to as PAH degraders (Dyksterhouse etal. and Methylophaga responding to methane in
1995; Kasai etal. 2002). the presence of high molecular weight organic
Before a pollution event, oil-degrading bac- compounds persisted for weeks after the acci-
teria have a low relative abundance in the dent (Dubinsky etal. 2013).
undisturbed community (Vila etal. 2010). In Pseudomonas species have widely docu-
response to an oil spill, the pioneer oil-degrad- mented as hydrocarbon degraders, and the genes
ing populations will bloom, and with time, the related with the capacity to degrade different
bacterial community will increase in diversity, hydrocarbons are associated with plasmids that
as secondary strains feed on bacterial metabo- may be transmitted between cells (Friello etal.
lites and on the less toxic oil intermediates 2001). This was the basis for the patented con-
(Vila etal. 2010; Yakimov etal. 2007). In the struction of super-degrader strains of P. aerugi-
aftermath, the community is reported to return nosa carrying multiple degradative plasmids
to its initial structure (Kasai etal. 2001; Yang (Chakrabarty 1992).
etal. 2014). Shifts in the metabolic and struc-
tural diversity of the community are directly Pathways ofAerobic
related with the pool of hydrocarbons Hydrocarbon Degradation
(Ringelberg etal. 2001). A field work con- Aerobic biodegradation of alkanes and aromatic
ducted after the Deepwater Horizon oil spill hydrocarbons can occur within the same bacte-
revealed that bacterial communities developing rial species, although some species are special-
50 A. Cruz et al.

ised in the utilisation of one or the other type of Jutkina etal. 2011; Ben Said etal. 2008). These
hydrocarbons (Rojo 2009; Yakimov etal. 2007). elements may remain after the recovery period
In both processes, the reactions occur intracellu- and will quickly respond to a new pollution event
larly, though the uptake of the pollutants is lim- (Yang etal. 2014).
ited by their low bioavailability. To overcome
this, PAH- and alkane-degrading bacteria adhere
to oil droplets and secrete biosurfactants that 4.3.3 Hydrocarbon Degradation
enhance emulsification and assist uptake (Ron byAnaerobic Marine Bacteria
and Rosenberg 2002). Alkanes and PAH are very
stable molecules that present a reduced reactiv- In the water column or in surface waters, oil bio-
ity. Thus, their biodegradation is unattractive remediation occurs mainly due the activity of
since a large amount of energy is needed to acti- aerobic microorganisms. However, a significant
vate these compounds (Rojo 2009). In aerobic amount of water-soluble or particle-associated
catalysis, alkanes and PAH activation occurs in hydrocarbons may settle on the seafloor, reaching
the first step of biodegradation through oxygen- deep-sea sediments where oxygen is scarce
ation of the compound by monooxygenases in (Kimes etal. 2014). Oil spills may also impact
the case of alkanes and by dioxygenases for PAH the shoreline, affecting beaches and salt marshes
(although monooxygenases are also reported). (Brundrett etal. 2015). In the fine-grained inter-
These reactions make the organics more reactive tidal sediments, oxygen diffusion is limited, and
in future biodegradation steps (Rojo 2009). these environments are generally anoxic (Head
Alkane oxygenation can occur at terminal or sub- and Swannell 1999; McGenity 2014).
terminal positions in the carbon chain. In both Anaerobic biodegradation of aliphatic and
cases, alkane will form a fatty acid that will be aromatic hydrocarbons has been demonstrated in
degraded by a -oxidation pathway. A schematic laboratory enrichment cultures developed from
representation of the aerobic pathway of degra- marine sediments and was reported to occur
dation PAH is presented in Fig. 4.2. The di- or in situ in several marine environments (Coates
monooxygenation of the molecule is catalysed etal. 1997; Weelink etal. 2010; Jaekel etal.
by the -subunit of an aromatic ring enzyme and 2013). Biodegradation at in situ conditions was
results in a metabolite with one or two hydroxyl also verified in mesocosms artificially contami-
radicals. This intermediate ring structure is nated with naphthalene (Massias etal. 2003;
cleaved by intradiol or extradiol oxygenases, Surez-Surez etal. 2011) or crude oil (Massias
yielding protocatechuates and catechol interme- etal. 2003; Surez-Surez etal. 2011) which
diates (Jimnez etal. 2004; Nzila 2013; Seo etal. were installed on the seabed of undisturbed areas.
2009). Frequently, PAH with benzenic rings Although anaerobic oil biodegradation is wide-
sequentially form dihydriols with n-1 aromatic spread, natural attenuation in anoxic marine sedi-
rings until reaching catechol that, by ring cleav- ments is slow, and oil persists for long periods of
age, is converted to tricarboxylic acid cycle time (Kolukirik etal. 2011).
(TCA) intermediates (Nzila 2013). In alkane and Pure cultures of anaerobic Archaea and Bacteria
PAH biodegradation, the downstream steps are able to couple hydrocarbon degradation to sul-
more common among bacteria, while the oxy- phate or Fe(III) reduction have been isolated from
genation reaction is less widespread and consid- hydrocarbon-rich marine environments, namely,
ered the rate-limiting step (de Lorenzo 2008). contaminated sediments and hydrocarbon seeps,
The genes that encode these essential enzymes but no isolate obtained from these environ-
are frequently located in mobile genetic elements mentswas reported using nitrate as electron accep-
(i.e. transposons and plasmids) that are prone to tor (Mbadinga etal. 2011; Weelink etal. 2010).
horizontal transfer, which, in some cases, may The majority of the isolated alkane-degrading
occur between strains phylogenetically distant anaerobic bacteria retrieved from marine envi-
(broad- host range) (van Beilen etal. 2001; ronmentsare sulphate reducers affiliated with the
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 51

Fig. 4.2 Schematic representation of pathways ofaerobic degradation of aromatic hydrocarbons

family Desulfobacteraceae within the pane- and butane-degrading enrichment cultures

Deltaproteobacteria (Table 4.3). Among them, were recently reported by Jaekel etal. (2013), all
the ability to grow on long-chain n-alkanes (C12 affiliated with the Desulfosarcina/Desulfococcus
C20) was described in Desulfococcus oleovorans cluster. The crucial role of this microbial clade on
Hxd3, Desulfatibacillum aliphaticivorans anaerobic alkane biodegradation was also high-
CV2803T, Desulfatibacillum alkenivorans lighted by Kleindienst etal. (2014) using stable
AK-01 and strain Pnd3, from the Desulfococcus/ isotope probing (SIP) techniques.
Desulfatibacillum cluster (Mbadinga etal. 2011). Only one thermophilic axenic culture capable
Strain BuS5, clustering within Desulfosarcina/ of alkane biodegradation was reported,
Desulfococcus clade, is only able to grow on Desulfothermus naphthae TD3T (Rueter etal.
hydrocarbon gases propane and butane 2004). This microorganism was retrieved from
(Kniemeyer etal. 2007). Novel cold-adapted pro- sediments of the hydrothermal vent Guaymas

Table 4.3 Overview of bacterial axenic cultures isolated from marine environments capable of hydrocarbon degradation coupled to sulphate reduction
Substrate Optimal T pH Salinity (g L1)
Class Source Alkanes Alkenes Aromatics Motility Spores (C) Optimum (Range/optimal) Ref.
Desulfococcus -prot Oilwater C12 C16 n.d N N 2830 n.d. 20a Aeckersberg
oleovorans Hxd3 separator C20 etal. (1991)
Desulfatibacillum -prot Contaminated C13 C7C23 N N 2835 7.5 645/24 Cravo-Laureau
aliphaticivorans marine sediment C18 etal. (2004)
Desulfatibacillum -prot Contaminated C13 C15, n.d N N 2628 6.97.0 160/10 So and Young
alkenivorans AK-01 estuarine C18 C16 (1999)
Strain Pnd3 -prot Marine sediments C14 C16 n.d N n.d. 30 n.d. 20b Aeckersberg
C17 etal. (1998)
Strain BuS5 -prot Marine C3C4 n.d. n.d n.r n.d. 28 6.9 26b Kniemeyer
hydrocarbon etal. (2007)
Desulfothermus -prot Sediments from C6C14 Y n.d. 6065 6.56.8 21 Rueter etal.
naphthae TD3T hydrothermal (2004)
Desulfobacula -prot Marine sediments n.d. To Y n.d. 28 7.07.1 20 Rabus etal.
toluolica Tol2 (1993)
Desulfotignum -prot Oil reservoir n.d. To N n.d. 34 7.2 5-55/15 Ommedal and
toluenicum H3 model column Torsvik (2007)
Strain oXyS1 -prot Oil-contaminated n.d. To, oXy n.r. n.d. 32 7.5 26b Harms etal.
sediments (1999)
Strain mXyS1 -prot Oil-contaminated n.d. To, mXy n.r. n.d. 30 7.2 26b Harms etal.
sediments (1999)
-prot Deltaproteobacteria, n.d. not determined, n.r. not reported, Y yes, N no, Be benzene, Nap naphthalene, To toluene, mXy m-xylene, oXy o-xylene, = no growth observed
No growth in freshwater;
Growth was observed at this value, but the range and optimum conditions were not evaluated
A. Cruz et al.
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 53

Basin and confirmed to use C6 to C14 alkanes as are endergonic under standard conditions, as
substrates (Table 4.3). Another group of microor- shown by the positive Gibbs free energy change
ganisms that degrade aliphatic hydrocarbons is (Dolfing etal. 2008), they become energetically
composed by Archaea from the genus favourable when the hydrogen partial pressure is
Archaeoglobus and the order Thermococcales. low, which is generally achieved by the activity
Archaeoglobus-related species are not known to of methanogenic or sulphidogenic partners.
utilise alkanes, but Archaeoglobus fulgidus strain Anaerobic degradation of benzene under metha-
VC-16 reduces thiosulphate coupled to the anaer- nogenic conditions was also demonstrated in
obic oxidation of n-alkenes (Beeder etal. 1994). microcosms (Weiner and Lovley 1998).
Among the monoaromatic hydrocarbons, ben-
zene is the most recalcitrant to anaerobic biodeg- Pathways ofAnaerobic
radation. Benzene oxidation has been observed in Hydrocarbon Degradation
sediments and in microcosms, but only recently Usually, the microorganisms involved in aro-
isolates capable of benzene biodegradation were matic hydrocarbons biodegradation are not able
described (e.g. Dechloromonas and Azoarcus to degrade aliphatics, and the reverse is also true.
spp.). However, these isolates use nitrate as ter- Several metabolic pathways of anaerobic hydro-
minal electron acceptor, and none was isolated carbon biodegradation have been proposed, all
from the marine environment (Weelink etal. involving biochemical reactions that differ com-
2010). Several sulphidogenic bacterial isolates pletely from the ones described for aerobic
belonging to the Deltaproteobacteria class were hydrocarbon catabolism.
reported as capable of growing on toluene. Four The first mechanism described was shown to
were retrieved from marine sediments: occur during toluene biodegradation. Activation
Desulfobacula toluolica, Desulfotignum tolueni- of the aromatic hydrocarbon occurs by addition
cum, strain oXyS1 (98.7 % similarity with of the methyl group to the double bond of fuma-
Desulfosarcina ovata) and strain mXyS1 (86.9% rate, yielding benzylsuccinate, which is then fur-
similarity with Desulfococcus multivorans) ther converted to benzoyl-CoA.This compound
(Table 4.3). was previously identified as an important inter-
Pure archaeal and bacterial cultures able to mediate in the degradation of several aromatic
use Fe(III) as electron acceptor in the degradation hydrocarbons, resulting from a carboncarbon
of aromatic hydrocarbons have also been reaction that does not require any co-substrate.
reported. Ferroglobus placidus is a hyperthermo- The enzyme involved in toluene activation, ben-
philic Archaea isolated from a hydrothermal vent zylsuccinate synthase (bssABC), presents a type
sediment, capable of benzene degradation at of glycine radical protein. The bss pathway was
85 C coupled to Fe(III) reduction (Holmes found in phylogenetically diverse bacteria-
etal. 2011). degrading toluene under nitrate-, Fe(III)- and
Oil biodegradation under methanogenic con- sulphate-reducing conditions, and bss genes were
ditions was first demonstrated in highly enriched also identified in a methanogenic toluene-
cultures able to convert long-chain alkanes to degrading culture, as well as in marine sediments
methane and CO2 (Zengler etal. 1999). Recent (Weelink etal. 2010; von Netzer etal. 2013).
studies provided strong evidence on the phylog- Other pathways for toluene activation have also
eny of the microorganisms involved in these been proposed, namely, hydroxylation of the
reactions. Smithella/Syntrophus spp. from the methyl group via a dehydrogenase to benzyl
Syntrophaceae family, within Deltaproteobacteria alcohol or hydroxylation to cresol, although the
class, have been associated to alkane biodegrada- latter is considered a minor pathway (Weelink
tion, in syntrophy with hydrogenotrophic metha- etal. 2010; Widdel and Rabus 2001).
nogens (Zengler etal. 1999; Gray etal. 2011; A mechanism similar to the bss pathway was
Siddique etal. 2012). Although these reactions proposed for the activation of alkanes by anaero-
54 A. Cruz et al.

bic bacteria, despite the higher energy required lation, by adding nutrients, alternative electron
for CH bond cleavage in alkanes comparatively acceptors or co-substrates, or through bioaug-
to toluene. In this case, fumarate addition at the mentation, using natural or engineered microbes.
subterminal position C-2 of alkanes yields an
alkyl-succinate and is catalysed by the glycyl Nutrient Supply
radical enzyme alky-succinate synthase, encoded Oil biodegradation depends on the growth of
by the assABC genes (Widdel and Rabus 2001; hydrocarbon-degrading microorganisms, in
Grossi etal. 2008). An alternative pathway for which N and P typically account for approxi-
anaerobic alkane biodegradation was suggested mately 15% and 1% of the biomass dry weight,
that involves carboxylation of the alkyl chain at respectively (Head etal. 2014). Due to the high
the C-3 position and removal of the C-1 and C-2 carbon content of hydrocarbons, N and P are gen-
carbon atoms, to produce a one carbon shorter erally limiting in oil-contaminated environments.
fatty acid (Grossi etal. 2008; Kimes etal. 2014). Thus, additional N and P supplies can improve
The possibility of different activation mecha- hydrocarbon biodegradation (Head etal. 2006).
nisms being performed by different microorgan- The addition of 23:2 N/P garden fertiliser signifi-
isms, or under different redox conditions (e.g. cantly enhanced the efficiency of bioremediation
sulphate-reducing or methanogenic), was pro- of coastal habitats following the Exxon Valdez
posed (Aitken etal. 2013). oil spill (Bragg etal. 1994). Biostimulation with
Due to the high stability of benzene, anaerobic the oleophilic fertiliser S200 after the Prestige oil
degradation of this compound is difficult, and the spill, in 2002, increased the bioavailability and
mechanisms of activation and further degradation stimulated the degradation of high molecular
of benzene are still unknown. Proposed pathways weight n-alkanes and alkylated PAH (Jimnez
for benzene activation are (i) anaerobic hydroxyl- etal. 2007). The requirements of N and P are
ation, with the formation of an alcohol; (ii) direct lower for anaerobic microorganisms, which typi-
carboxylation yielding benzoate; and (iii) meth- cally have lower biomass yields than aerobes,
ylation to toluene. These reactions proceed e.g. 210 % in methanogenic hydrocarbon-
towards the formation of central metabolites such degrading consortia compared to the 50% growth
as benzoyl-coA, which is further degraded to yield expected for aerobic heterotrophs (Gray
CO2 (Weelink etal. 2010). etal. 2011).
Kolukirik etal. (2011) assessed the
hydrocarbon-degrading activity in marine sedi-
4.3.4 B
 ioremediation ofOil ments from the Halic Bay (Istanbul), a highly
Hydrocarbons anaerobic deep sludge, with hydrocarbon con-
centrations in the range of 40006000 and
The capacity of microbes to remove large quanti- 15003000mg L1 for aromatics and aliphatics,
ties of oil is limited, even in areas with continu- respectively. These sediments were incubated in
ous natural seepage where hydrocarbon-degrading anaerobic microcosms prepared with the same
microbial populations are positively selected TOC/N/P ratio of the sediment porewater
(e.g. the Gulf of Mexico) (Ramirez-Llodra etal. (1000/5/1) and in microcosms where N and P
2011). Several environmental constraints affect concentrations were gradually increased. The
the activity of oil-degrading microorganisms, lowest TOC/N/P ratio studied (i.e. 1,000/40/6)
namely, temperature, pH, salinity, water activity, induced a 92% improvement in hydrocarbons
hydrostatic pressure and availability of resources removal, along with a ninefold increase in bio-
such as carbon and energy sources, electron gas production, comparatively to the natural
acceptors and inorganic nutrients (Head etal. Halic Bay conditions. This strategy resulted in a
2014). Bioremediation attempts to counter these hydrocarbon degradation rate around 700 g g1
limitations and enhance biodegradation of pollut- L1 day1 which, although still much lower than
ants are mainly accomplished through biostimu- the aerobic degradation rates, represents signifi-
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 55

cant enhancement of anaerobic microbial activ- tron acceptors can be used (e.g. manganese,
ity. In nutrient biostimulation, the low cost and iron(III) and sulphate). Sulphate, nitrate and a
stock availability of inorganic hydrophilic fertil- mixture of sulphate+nitrate enhanced biodegra-
isers favour their usage in emergency situations. dation of two-, three- and four-ring PAH from the
However, dispersion hinders their effectiveness 16 PAH listed as priority pollutants by the US
in open ocean and in tidal zones. Thus, the con- Environmental Protection Agency (Lu etal.
tinuous application of exogenous N is required 2012), although five- and six-ring PAH showed
in order to maintain a high biodegradation rate the lowest biodegradation rates under these redox
(Nikolopoulou etal. 2007). The quantity applied conditions. Successful improvement in anaerobic
should be balanced (Singh etal. 2014), since the bioremediation of phenanthrene was also accom-
excessive use of fertilisers worsens plished when gypsum (CaSO42H2O) and nitro-
eutrophication of aquatic ecosystems (Haehnel cellulose were used as slow-release electron
etal. 2014) and can reduce degradation due to acceptors (Tang etal. 2005).
microbial inhibition (Singh etal. 2014) and O2 Bacterial guilds able to use different electron
depletion (Macaulay and Rees 2014). Alternative acceptors are likely to co-occur within sediments.
strategies, such as the use of oleophilic and Thus, supplementation with a cocktail of electron
slow-release fertilisers (Nikolopoulou etal. acceptors may be an efficient strategy for enhanc-
2007) or the positive selection of endogenous ing oil metabolism in mixed microbial communi-
N2-fixing bacteria (Chronopoulou etal. 2013; ties. In laboratory microcosms inoculated with
Toccalino etal. 1993), have been conjectured. wetland sediments of BaratariaTerrebonne estu-
Water- soluble nutrients coated with an oleo- ary (the USA), a mixture of different electron
philic degradable compound will be less dis- acceptors, i.e. sulphate, nitrate and bicarbonate,
persed and maintain higher bioavailability to yielded extensive biodegradation of diesel fuel
attached oil-degrading bacteria (Nikolopoulou No. 2, relatively to the addition of single electron
etal. 2007), also providing an additional carbon acceptors (Boopathy 2003). Under mixed elec-
source. Dinitrogen fixation is encountered in tron acceptor conditions, 99% removal of diesel
many hydrocarbonoclastic bacteria in undis- fuel No. 2 was achieved within 510 days, while
turbed and polluted environments (Al-Mailem for the same period, only 62% and 27% were
etal. 2010, 2013;Dashti etal. 2015; Sorkhoh degraded upon of sulphate addition and by natu-
etal. 2010;Thavasi etal. 2006), and naphtha- ral attenuation, respectively. Siegert etal. (2011)
lene adduction has been shown to promote dini- reported an increase in the rate of methane pro-
trogen fixation (Hanson etal. 2012). In general, duction from hexadecane in microcosms
high carbon and low N conditions promote dini- amended with ferrihydrite, comparatively to the
trogen fixation (Karl etal. 2002; Sorkhoh etal. ones supplemented with sulphate (872 and
2010) in association with hydrocarbon 387 nmol cm3 day1, respectively). The posi-
biodegradation. tive effect of ferrihydrite was possibly related
with the growth of Methanosarcina-like Archaea, Electron Acceptors which, once triggered by the Fe(III) addition, can
When oil biodegradation is limited by poor oxy- boost methane production (Siegert etal. 2011).
gen mass transfer, mechanical aeration increases The potential of chlorate to support oil minerali-
hydrocarbon removal. Genovese etal. (2014) sation was also demonstrated by Brundrett etal.
reported improved hydrocarbon biodegradation (2015). In this study, mesocosms were prepared
(up to 98%) in mesocosm experiments in which with oiled salt marsh sediments from an area
anaerobic marine sediments contaminated with impacted by the Deepwater Horizon oil spill and
petroleum were oxygenated during 3 months. If studied over a 4-month period. Crude oil degra-
the use of mechanical techniques to enhance oxy- dation in mesocosms amended with chlorate was
gen mass transfer is not feasible, alternative elec- not significantly different (p>0.05) from that in
56 A. Cruz et al.

the aerated mesocosms, both presenting similar the use of fertilisers (Prince etal. 2013). A num-
reduction (662%) in the alkane/hopane ratio. ber of dispersants are listed in the National
Chlorate was rapidly exhausted in both replicates Contingency Plan produced by the EPA (United
during the first 30 days, and successive chlorate States Environmental Protection Agency 2015).
additions were consumed at similar rates. These The effect of a microemulsion composed by
authors suggest the use of chlorate to promote Tween 80, 1-pentanol, linseed oil and deionised
in situ oil mineralisation for the bioremediation water, on TPH biodegradation in nitrate-induced
of contaminated salt marshes. bioremediation, was investigated in sediment
column assays reported by Zhang etal. (2014). Addition ofCo-substrates After 6 weeks of treatment, higher TPH removal
Amendments with organic co-substrates have efficiencies were attained with the combined use
also been tested in laboratory batch assays. The of nitrate and microemulsion, compared with the
presence of co-substrates may enhance the use of nitrate alone (30% and 8%, respectively).
growth of hydrocarbon-degrading microorgan- The choice of surfactants should take into consid-
isms or stimulate complex microbial communi- eration the associated costs, effectiveness and
ties, facilitating the degradation of the more toxicity (Megharaj etal. 2011). The increase of
stable compounds present in the oil mixture. The pollutant dispersion by surfactant addition will
presence of an exploitable carbon and energy lead to higher mortality and higher bioconcentra-
source can also stimulate the degradation of a tion of PAH in macrofauna (Milinkovitch etal.
second nongrowth compound (e.g. hydrocar- 2011). Toxicity of some surfactants per se
bons) that otherwise would not be attacked by towards oil-degrading bacteria can actually
the microorganisms, in a process called inhibit natural biodegradation (Louvado etal.
co-metabolism. 2012). Additionally, surfactants can increase
Incubation of contaminated marine harbour eutrophication (Garca etal. 2009), and similarly
sediments with acetate or lactose under anaerobic to fertilisers, the use of chemical surfactants may
conditions significantly improved hydrocarbon be unacceptable in some sensitive environments
biodegradation (DellAnno etal. 2012), indicat- (Haehnel etal. 2014). Therefore, biosurfactants
ing that bioremediation strategies which sustain have been conjectured as environment-friendly
high bacterial diversity may be more efficient alternatives (Joo and Kim 2013). Surface-active
than those selecting specific taxa. The positive lipopeptide production was documented on
effects of acetate or methanol as co-substrates on marine bacteria, namely, strains of Bacillus subti-
the anaerobic biodegradation of total petroleum lis (Tareq etal. 2015; Dusane etal. 2011),
hydrocarbons (TPH, C10C40, >1000mg kg1 dry Bacillus mojavensis (Ma etal. 2012) and
weight) in marine sediments were also shown by Brevibacterium luteolum (Vilela etal. 2014).
Zhang and Lo (2015). Some marine bacteria also produce surface-active
glycolipids (Tareq etal. 2015). The production of Surfactants biosurfactants under anaerobic conditions by
A reduced oilwater interface limits oil biodegra- halotolerant facultative anaerobic microorgan-
dation. Through the application of surfactants, isms has been reported, e.g. Bacillus mojavensis
the interfacial tension is reduced, and oilwater strain JF-2 (former Bacillus licheniformis strain
emulsification increases, thereby increasing the JF-2) and Bacillus strain SP018 (McInerney etal.
cell-oil contact area and, if all other conditions 1990; Pfiffner etal. 1986), and presents high
permit, enhance biodegradation (Hazen etal. potential as a bioremediation strategy in anaero-
2010). Application of dispersants can reduce oil bic marine environments. However, the direct
droplets to 10100 m diameter and significantly application of semi-purified biosurfactants in
shorten biodegradation half-time, even without environmental remediation is extremely expen-
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 57

sive. The use of biosurfactant-producing oil- Mao etal. 2012; Nzila 2013.Vallero 2010). In a
degrading microorganisms or consortia could be synergistic consortium, metabolically comple-
a more sustainable approach. mentary bacteria work on different steps along
the degradation pathway, and this avoids the Bioaugmentation building-up of inhibiting metabolites (Bouchez
Bioaugmentation can be beneficial if the natural etal. 1999). While some strains are directly
community is incapable of biodegradation or if, involved in the biodegradation process, others are
even possessing that ability, biodegradation indirectly beneficial (Ron and Rosenberg 2014).
occurs slowly. However, this approach is not Secondary strains can increase PAH bioavailabil-
always successful, and in many cases it is also ity through cell hydrophobicity and biosurfactant
considered too expensive for field bioremediation production (Pedetta etal. 2013) and provide
of oil-impacted sites (Megharaj etal. 2011). The essential growth factors (Pedetta etal. 2013;
maladaptation of the bioaugmenting strains to the Sorensen etal. 2005; Singh etal. 2014). Consortia
new environment diminishes their capacity to can also involve microalgae (Tang etal. 2010)
outcompete the natural strains that initiate degra- and fungi (Boonchan etal. 2000). The microal-
dation immediately after spill (Ron and gae Scenedesmus obliquus is incapable of oil bio-
Rosenberg 2014). Often, logistics difficult a degradation but promoted it when added as an
prompt intervention and, in that case,natural axenic culture to a four-strain bacterial consor-
communities have a time advantage. Compared tium composed by obligate alkane and PAH
to the bioaugmentation performed with pure cul- degraders (Tang etal. 2010). Algae can indirectly
tures, the use of active hydrocarbon-degrading stimulate oil biodegradation through the release
sediments may be an interesting strategy (Head of oxygen and EPS that promote adsorption of
and Swannell 1999). hydrophobic pollutants and attachment of hydro-
carbonoclastic bacteria, besides providing an
additional carbon source (McGenity etal. 2012a).
4.3.5 Consortia ofMarine Bacteria Reciprocally, algae can benefit from CO2, dini-
forEnhanced Hydrocarbon trogen and iron produced by the bacteria, and
Bioremediation most importantly, they can benefit from removal
of toxic hydrocarbon (McGenity etal. 2012a).
Oil is a complex mixture of compounds, from In N-depleted microcosms, the PAH-
which almost all constituents are biodegradable. degrading genus Cycloclasticus was favoured,
Complete oil biodegradation can only be achieved while the alkane-degrading genus Alcanivorax
by mixed communities composed by microor- thrives under N supplementation (Singh etal.
ganisms specialised in the degradation of alkanes, 2014). In this case, nutrient supplementation may
monoaromatics and PAH, among other compo- favour the biodegradation of the more abundant
nents (Yakimov etal. 2007; Tang etal. 2010). and less toxic alkanes over the least abundant and
Furthermore, a single strain is often either inca- more toxic PAHs. The understanding of the com-
pable of completely degrading a specific pollut- plexity of interactions established within
ant or does it very slowly (Festa etal. 2013;Wang hydrocarbon-degrading microbial communities,
etal. 2008; Tixier etal. 2002). In the lack of the as well as the understanding of the benefits that
necessary enzymatic capacities, more toxic inter- can come from these interactions, is still in its
mediate metabolites may accumulate, and inhibi- infancy. The unveiling of the black box of micro-
tion of biodegradation will likely occur (Festa bial ecology of hydrocarbon-contaminated
etal. 2013;Tixier etal. 2002). In this situation, a marine habitats can help to optimise the applica-
more successful biodegradation is achieved by a tion of biostimulation strategies and fulfil absent
consortium of strains (Cui etal. 2014; Festa etal. metabolic roles by bioaugmentation (Rling and
2013; Gallego etal. 2013;HuiJie etal. 2011; van Bodegom 2014).
58 A. Cruz et al.

4.4  ritical andFuture

C cacy of bioremediation at field scales and the
Perspectives concerns raised by the introduction of allochtho-
nous microorganisms are also constraints to the
Bioremediation of hydrocarbons and organomet- practical application of these strategies. In fact, it
als is likely to gain applicability in the future, has been proposed that EPA should actually ban
since these pollutants can be biodegraded to sim- bioremediation agents that can result in the intro-
pler and, in general, less toxic compounds. A duction of non-native microorganisms (Prince
diversity of microorganisms has been identified William Sound Regional Citizens Advisory
as capable of degrading or removing these from Council 2015). Notwithstanding, bioremediation
different marine ecosystems, and a multiplicity can be highly efficient and sometimes the only
of metabolic pathways has been characterised. possible strategy for the removal of recalcitrant
Moreover, the high potential of microorganisms, pollutants.
either isolated or in consortia, for bioremediation Future research should consider the forms and
is currently accepted, and several bioremediation possible fate of each contaminant throughout the
agents are listed in the EPA National Contingency whole bioremediation process, by following the
Plan (United States Environmental Protection interactions between different contaminants and
Agency 2015). covering the biological processes occurring since
Despite the significant advances from the last the release from the original sources up to the
decades on the major mechanisms and microbial formation of end products and/or sinking in
agents of organometals and hydrocarbon biodeg- marine environmental compartments. This over-
radation, bioremediation of these compounds in all tracking should not disregard the evaluation of
marine environments is still in its infancy. impacts and risks of the contaminants, as well as
Laboratory-scale experiments, pilot-scale tests of their degradation/transformation products, on
and some few full-scale remediation trials have environment and human health.
been conducted as an attempt to demonstrate the Other approaches to optimise the bioremedia-
potential of bioremediation for the clean-up of tion processes are being pursued. Considering the
polluted marine environments and simultane- effectiveness of genetically engineered microor-
ously overcome the economic, technical and ganisms (GEM) in other biotechnological
logistic difficulties of this process. These goals research areas, the applicability and adaptability
are yet to be fully achieved and intensive research of GEM or designed consortia, able to cope with
on bioremediation is still needed. multiple adverse/stress conditions, may represent
Bioremediation is generally not adopted as the a significant development in bioremediation of
primary strategy for treating environmental con- pollutants in marine environments. For that,
tamination mainly because of the difficulties in GEM need be proven safe and the difficulties
predicting the outcome of bioremediation pro- with public acceptance should be overcome.
cesses. Physical, chemical and biological factors It is consensual that the design and imple-
significantly affect the results of bioremediation, mentation of bioremediation approaches and the
and for that reason, the extrapolations from predictive analysis of the process outcome rep-
laboratory-scale experiments to the field are still resent complex multidisciplinary tasks.
associated with a high degree of uncertainty. However, bioremediation is an indispensable
Moreover, the responses of complex microbial tool for preserving or restoring environmental
communities, which include several uncharacter- quality in the framework of sustainability, and
ised organisms, in natural environments or under the need for efficient protocols justifies keeping
different environmental conditions, are not easily bioremediation as a priority topic in the research
predictable. Difficulties in confirming the effi- agenda.
4 Microbial Remediation ofOrganometals andOil Hydrocarbons intheMarine Environment 59

Acknowledgments The authors thank the financial sup- marine environment. A review. Environ Int 34(2):
port from the European Regional Development Fund 292308
(ERDF), through the Operational Programme Thematic Atlas RM (1995) Petroleum biodegradation and oil spill
Factors of Competitiveness (COMPETE), and the bioremediation. Mar Pollut Bull 31(4):178182
Portuguese Foundation for Science and Technology Ayanda OS, Fatoki OS, Adekola FA, Ximba BJ (2012)
(FCT) in the frame of project FCOMP-010124- Fate and remediation of organotin compounds in sea-
FEDER-027917 (FCT: PTDC/AAG-TEC/3428/2012). waters and soils. Chem Sci Trans 1(3):470481
The authors also thank the FCT strategic funding of UID/ Barron MG (2012) Ecological impacts of the deepwater
BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145- horizon oil spill: implications for immunotoxicity.
FEDER-006684), and Project RECI/BBB-EBI/0179/2012 Toxicol Pathol 40(2):315320
(FCOMP-01-0124-FEDER-027462). Research of AC was Barroso CM, Moreira MH (2002) Spatial and temporal
funded by the postdoctoral grant (BPD/UI88/2886/2013), changes of TBT pollution along the Portuguese coast:
from the project Sustainable Use of Marine Resources inefficacy of the EEC directive 89/677. Mar Pollut
MARES (CENTRO-07-ST24-FEDER-002033), funded Bull 44(6):480486
by QREN, Mais Centro- Programa Operacional Regional Barroso C, Moreira M, Bebianno M (2002) Imposex,
do Centro e Unio Europeia/Fundo Europeu de female sterility and organotin contamination of the
Desenvolvimento Regional. AL was funded by a PhD prosobranch Nassarius reticulatus from the Portuguese
grant SFRH/BD/86447/2012 funded by FCT.Research of coast. Mar Ecol Prog Ser 230:127135
AJC was supported by ERC grant (project 323009). Barug D (1981) Microbial degradation of bis(tributyltin)
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Batel R, Bihari N, Rinkevich B, Dapper J, Schacke H,
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Extracellular Polysaccharide
Production by Bacteria 5
as a Mechanism of Toxic Heavy
Metal Biosorption
and Biosequestration
in the Marine Environment

P.V. Bramhachari
and Ganji Purnachandra Nagaraju

Marine environments are one of the most diverse environments owing to
their vast natural resource of imperative functional molecules. Interestingly,
marine bacteria offer a great diversity of polysaccharides which could play
an important role in biotechnology and industry. Among the various bioac-
tive compounds, marine exopolymers are attracting major interest and
attention due to their structural and functional diversity. Bacterial exo-
polysaccharides (EPSs) contain ionizable functional groups, which enable
them to bind and sequestrate toxic heavy metal ions. Due to their biode-
gradability and safety of the environment, biosorption of heavy metals by
these biopolymers has attracted considerable attention as promising alter-
natives capable to compete with expensive, inefcient and conventional
technologies, including chemical precipitation, adsorption on activated
carbon, membrane separations, ion exchange and solvent extraction meth-
ods. This review particularly emphasizes on utilization of marine bacteria
in the eld of bioremediation and understanding the mechanism behind
acquiring the characteristic feature of adaptive responses. Fundamental
insights regarding metals in relation to metal-binding proteins/peptides for
immobilization, information regarding genetic engineering for enzymes

P.V. Bramhachari (*)

Department of Biotechnology and Botany, Krishna
University, Machilipatnam 521001,
Andhra Pradesh, India
G.P. Nagaraju
Department of Hematology and Medical Oncology,
Winship Cancer Institute, Emory University,
Atlanta, GA 30322, USA

Springer Science+Business Media Singapore 2017 67

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_5
68 P.V. Bramhachari and G.P. Nagaraju

involved in metal transformation and strategies that can be employed to

overcome the bottlenecks associated with microbial-based remediation are
highlighted in this review. The important engineering properties based on
structural characteristics such as adsorption, biodegradability and hydro-
philicity/hydrophobicity of EPS matrix are also discussed. A thorough
understanding of microbes that produce exopolysaccharides for metal
biosequestration and biosorption would solve several problems in biore-
mediation process.

5.1 Introduction strongly bonded to many biochemicals inhibiting

their functions. Many heavy metal ions and met-
Majority of the marine microbial world remain alloids are well known to be toxic or otherwise
unexploited due to the enormous size of the carcinogenic to humans (Fu and Wang 2011).
marine biosphere which offers afuent ora and Heavy metals can contribute to degradation of
fauna, signifying a vast natural resource of essen- marine ecosystems by reducing species diversity
tial functional commercial grade products. and profusion through accumulation of metals in
Among the various bioactive compounds, marine living organisms and food chains (Hosono et al.
exopolymers are attracting principal interest 2011). Therefore, there is a critical need to study
attributable to their structural and functional the heavy metal resistance mechanism in marine
diversity which tends to nd abundant applica- bacteria with a comprehensive approach to com-
tions in bioremediation (Satpute et al. 2010). bat and manage heavy metal pollution in the
Marine environments are one of the most stress- marine environment.
ful environments due to their unstable nature of In recent years, marine microbial EPSs have
temperature, pH, salinity and sea surface tem- fascinated additional attentions, particularly for
perature and are more correctly adapted to the those originated from marine bacteria (Jouault
adverse conditions, hence possessing complex et al. 2004). Nevertheless, there is a growing
characteristic features of adaptation (Mancuso interest in isolating novel bacteria producing
Nichols et al. 2005). Therefore, the bacteria iso- EPSs from marine environments with potential
lated from the marine environments are believed applications (Arena 2004). Exopolysaccharides
to be better utilized in bioremediation of heavy (EPSs) excreted by majority of marine microor-
metals, hydrocarbons and many other recalcitrant ganisms are known to produce EPS for cryopro-
compounds and xenobiotics through production tection, halotolerance, attachment to substrate,
of extracellular polymeric substances (EPSs). nutrient uptake, and formation of colonies and
Heavy metal contamination in coastal and marine biolms (Decho 1990; Hoagland et al. 1993;
environments is receiving increasingly serious Decho et al. 2010). Due to scores of ecological
threat to naturally stressed marine ecosystems roles, EPSs are expected to be present at a signi-
and moreover humans directly rely on marine cant level in aquatic systems, primarily as col-
resources for food, industry and recreation. loidal organic matter. Exopolymeric substances
Heavy metals are introduced to coastal and (EPSs) are yet inadequately explored organic
marine environments through a variety of sources ligands that are heterogeneous, polyfunctional
and activities including sewage and industrial macromolecules containing uronic acids, neutral
efuents, brine discharges, coastal modications sugars, mono- and polysaccharides, amino acids,
and oil pollution. The seriousness of heavy metal and proteins (Mancuso Nichols et al. 2005;
contamination is further compounded by the fact Verdugo et al. 2004; Hassler et al. 2011).
that they are by and large water-soluble, nonde- Interestingly, these EPSs are primarily composed
gradable, vigorous-oxidizing agents and are of polysaccharides and glycoproteins, in addition
5 Extracellular Polysaccharide Production by Bacteria as a Mechanism of Toxic 69

to charged functional groups and amphiphilic, multiple complexes with metal ions. Since EPSs
adsorptive, and adhesive properties (Quigley are usually the primary barrier of microbial cells
et al. 2002; Gutirrez et al. 2007), and provide a that directly contact and interact with metals in
substantial pool of organic carbon available to aqueous environment, they are of vital impor-
serve as a natural ligand source for numerous tance through not only protecting the interior
other molecules, together with trace metals microbial cells (Ozturk et al. 2009) but also for
(Bhaskar et al. 2005). Extracellular polymeric geochemical cycling and remediation of metals
substances (EPSs) are produced by a wide range in natural environments (Wu et al. 2013). Some
of microorganisms from both terrestrial and metals are indispensable to many physiological
marine environments. EPSs differ among organ- processes (e.g., copper and iron), while others
isms and producing conditions in carbohydrate are nonessential to the cell life (e.g., cadmium
chain length and branching, composition, type of and lead). Conversely, in excess, all metals are
sugar linkages, and presence of additional chemi- deleterious to the cells (Ishaque et al. 2006; Valls
cal groups such as sulfates, proteins, lipids, and and de Lorenzo 2002; Lewinson et al. 2009).
even nucleic acids (Ruas-Madiedo and De Los Several protection mechanisms have already
Reyes-Gaviln 2005; Mancuso Nichols et al. been described for bacterial cells to avoid/mini-
2005). Different bacterial strains produce EPS of mize the toxicity of heavy metals. These strate-
diverse chemical composition and structure gies can crop up extracellularly, for example, the
(Lemoine et al. 1997; Mancuso Nichols et al. sequestration of metal ions on outer cell surface.
2005); nevertheless a single strain can also pro- However, these strategies are necessitated to be
duce more than one kind of EPS (Schiano pooled with other cellular and biochemical
Moriello et al. 2003). Similarly, the type and mechanisms to achieve homeostasis, such as
amount of EPS produced by a bacterial strain can exporting the metals through P-type ATPases
be customized by exposure to certain environ- and detoxify the metal cations by efux and/or
mental conditions such as salinity (Vyrides and the binding of these cations to metallothioneins,
Stuckey 2009), temperature (Mancuso Nichols preventing free toxic metals (Nies 2003; Baptista
et al. 2005a, b, c), or presence of heavy metals and Vasconcelos 2006).
(Guibaud et al. 2005; Naik et al. 2012). Therefore, Therefore, to better engineer such metal bio-
the term EPS not only refers to a single chemi- sorption processes, it is essential to have pro-
cally dened molecule but also encompasses a found understanding on the biological
complex mixture of diverse polysaccharides and mechanisms of metal adsorption by microbial
other ancillary compounds. In nature, bacterial EPS. The discovery of an effective EPS-
EPSs serve a range of biological functions, viz., producing marine microorganism could therefore
as a buffer material for biodegradable compounds enhance the use of environmentally biodegrad-
and trace metal nutrients or as part of a protective able EPS molecules in industry and could reduce
structures against predators, desiccation, salinity, the dependence on biohazardous, nondegradable
and cytotoxic compounds and toward high or low synthetic polymers. In these perspectives, marine
temperatures (Nicolaus et al. 2010; Poli et al. microbial polysaccharides show impending
2010). source of macromolecules with high-value addi-
The metal-binding properties of microbial tions from the marine environment (Pomin 2014).
EPS are well investigated (Geesey et al. 1988) Thus a thorough understanding of marine
and widely employed in bioremediation of heavy microbes that produce EPSs for metal sequestra-
metals (Loaec et al. 1997). In the marine envi- tion and detoxication would result in economic
ronment, binding of heavy metals by microbial production with several applications in bioreme-
EPS (dissolved or particulate) may comprise diation process. This review summarizes funda-
broad implications on its dynamics with superior mental insights regarding the marine microbial
binding capacity for metals than any other diversity, extracellular and intracellular metal
known sorbents (Quigley et al. 2002) and form detoxication strategies, and chemical,
70 P.V. Bramhachari and G.P. Nagaraju

enzymatic, and genetic modication of bacterial contains sulfate and phosphorus substituents
EPS. It also evaluates the biotechnological appli- (Rinker and Kelly 2000); the archaea Sulfolobus
cations of exopolysaccharides and future pros- solfataricus were shown to produce a sulfated
pects of both naturally occurring and genetically polysaccharide (Nicolaus et al. 1993).
modied bacterial strains in bioremediation and Geobacillus sp., Bacillus thermodenitrificans,
biomonitoring of hazardous metals present in and B. licheniformis thermophilic strains were
metal-polluted environment for improved detoxi- isolated from shallow marine hydrothermal vents
cation and degradation of toxic pollutants. of Vulcano Island (Italy) and the polysaccharides
they produced were analyzed (Poli et al. 2010).
Several bacteria belonging to gammaproteo-
5.2 Marine Microbial Diversity bacteria Alteromonadales or Vibrionales orders
Producing EPS isolated from the deep-sea Polychaeta annelids
Alvinella pompejana and A. caudata tissues
Several EPS-producing marine strains were were also shown to produce different EPS:
investigated, which led to the discovery and iso- HYD1545 (Vincent et al. 1994), HYD1644
lation of novel biomacromolecules (Finore et al. (Dubreucq et al. 1996), HYD721 (Rougeaux
2014). Marine bacterial exopolysaccharides have et al. 1999a), HYD657 (Cambon-Bonavita et al.
been the subject of interest in numerous reviews 2002) and HE800 (Raguenes et al. 1997;
(Guezennec 2002; Nazarenko et al. 2003; Rougeaux et al. 1999b). The most convincing
Mancuso Nichols et al. 2005b; Laurienzo 2010; evidence also came from another gammaproteo-
Satpute et al. 2010; Freitas et al. 2011; Jouault bacterium isolated from a deep-sea hydrother-
and Delbarre-Ladrat 2014; Pomin 2014). Most of mal vent shrimp (A. macleodii subsp. fijiensis
the marine derived EPS are bacterial (viz. meso- biovar medioatlantica) and was shown to pro-
philic, heterotrophic, psychrophilic), archaeal duce the EPS MS907 (Raguenes et al. 2003),
(thermophilic and halophilic) been shown to pro- whereas Alteromonas macleodii sp. fijiensis pro-
duce EPS (Rinker and Kelly 2000; Schiano ducing the pyruvated EPS ST716 (Raguenes
Moriello et al. 2003; Marx et al. 2009; Nicolaus et al. 1996; Rougeaux et al. 1998) and
et al. 2010; Poli et al. 2011; Finore et al. 2014). Alteromonas infernus producing the sulfated
Mancuso Nichols et al. (2004) depicted the EPS GY785 were isolated from hydrothermal
production of EPS by the marine strains uids (Raguenes et al. 1997; Roger et al. 2004).
Pseudoalteromonas CAM025 and CAM036 iso- In another study, Pseudoalteromonas strains
lated in Antarctica seawater and sea ice. Most produced diverse EPS: SM9913 isolated from
recently, few more strains from Arctic sea ice deep-sea sediments produces an acetylated EPS
were also demonstrated to produce EPS with (Qin et al. 2007) and SM20310 from the arctic
cryoprotective effect (Marx et al. 2009; Liu et al. sea ice produces a complex -mannan (Liu et al.
2013). The halophilic strains Halomonas maura 2013). Pseudomonas, Alteromonas, Paracoccus
(Arias et al. 2003), Halomonas ventosae and Vibrio sp. bacteria producing EPS were
(Martinez-Canovas et al. 2004; Mata et al. 2006), explored from an extreme marine ecosystem.
Halomonas alkaliantarctica (Poli et al. 2007), Among them, Paracoccus zeaxanthinifaciens
Hahella chejuensis (Poli et al. 2010) and the subsp. payriae and Vibrio sp. RA29 are shown to
archaeal halophilic Haloferax mediterranei produce sulfated polysaccharides and Vibrio sp.
(Parolis et al. 1996) isolated from hypersaline MO 245 produced a biopolymer very similar to
environments were shown to produce EPS; a few EPS produced by Vibrio diabolicus (Guzennec
of them are sulfated (Poli et al. 2010). Marine et al. 2011). Several researchers supported the
thermophilic anaerobes Sulfolobus, fact that chemical and structural diversity of
Thermococcus and Thermotoga were described these macromolecules conrms the high value of
to produce EPS (Vanfossen et al. 2008). marine environment as a source of exciting
Thermococcus litoralis produces an EPS which chimiodiversity.
5 Extracellular Polysaccharide Production by Bacteria as a Mechanism of Toxic 71

5.3 Extracellular boxyl groups of these anionic polysaccharides

Biosequestration of Metals (Gyurcsik and Nagy 2000; Nagy et al. 2003),
by Marine EPS with greater stability, while two or more of the
carboxyl groups are able to interact with a single
The adsorption of heavy metals by EPS is non- Fe (III) cation (Rendleman 1978). In this context,
metabolic and energy independent and can be sorption of heavy metals based on metal-binding
caused by interaction between metal cations and capacities of biological materials can be an alter-
negative charge of acidic functional groups native solution (Gadd 2009; Park et al. 2010;
(COOH, OH, C=O, PO4, NH2) of EPS as shown Vieira and Volesky 2010). Among numerous bio-
in Fig. 5.1. Here the EPSs are recommended as sorbents (e.g., yeast, seaweed, bacteria, fungi),
surface-active agents for heavy metal removal bacterial exopolysaccharides (EPSs) have been
because of their extensive capacity. Similarly, a effectively and successfully used in heavy metal
cell-bound polysaccharide produced by the removal studies (Comte et al. 2006; Loaec et al.
marine bacterium Zoogloea spp. is also known to 1997, 1998). Moreover, EPSs have ionizable
act as an adsorbent of metal ions like cadmium, functional groups like carboxyl, acetate,
ferrous, lead, and chromium (Kong et al. 1998). hydroxyl, amine, phosphate, and more rarely sul-
Anionic polysaccharides have a strong afnity fate groups, which are potential binding sites for
for metal cations (Rendleman 1978); for instance, the sequestration of heavy metals (Fig. 5.1) and
alginate, a naturally occurring polysaccharide show metal binding (Liu and Fang 2002). It is
invented solely of uronic acids, was shown to sta- admitted that metal sorption implies a physico-
bilize Fe (III) (Sreeram et al. 2004). For instance, chemical interaction between the metal cation
in seawater, the Fe (III) can coordinate with car- and functional groups based on physical sorption,

Fig. 5.1 Mechanism of metal biosorption and biosequestration in the marine bacteria
72 P.V. Bramhachari and G.P. Nagaraju

ion exchange, complexation, and/or precipitation metals like Ni(II), Cr(VI), and Mo(IV).
(Gadd and White 1989). Moreover, metal sorp- Interestingly, the predominance of uronic acid in
tion also depends on external factors like pH, EPS suggests that high molybdenum content was
metal concentration, or biomass concentration. detected in the EPS than other metals and it was
Extracellular high molecular weight biopoly- also assumed to form stable complex with uronic
mers secreted by marine bacterial cells, referred acids (Beech and Cheung 1995). Interestingly,
to as exopolysaccharides (EPS), consist of mac- Shah et al. (2000) reported that the extracellular
romolecules such as polysaccharides, proteins, polysaccharide produced by a marine Cyanothece
nucleic acids, humic substances, lipids, and other sp. ATCC 51142 contained 2-methylglucose,
non-polymeric constituents of low molecular mannuronic acid, and sulfated mannose, linked by
weight (Bramhachari et al. 2007). These exo- -1,4-linkage and 3,6-linkages, and was exploited
polysaccharides are chemically diverse and are toward metal removal from solutions. In another
mostly acidic heteropolysaccharides with func- study, Enterobacter cloacae, an
tional groups such as hydroxyl, carboxyl, amides, exopolysaccharide-producing marine bacterium,
and phosphoryl which exhibit high afnity was tested for its Cr (VI) tolerance and chelation.
toward heavy metals (Bramhachari and Dubey. X-ray uorescence (XRF) spectroscopy analysis
2006; Bramhachari et al. 2007; Bhaskar and of both the biomass and the exopolysaccharide
Bhosle 2006; Braissant et al. 2007). Bacterial revealed that 6070 % chromium was accumu-
EPSs play a key role in initial attachment of cells lated by this bacterium (Iyer et al. 2004). In the
to different substrata, cell-to-cell aggregation case of marine environments, several studies were
protection against desiccation, and resistance to also performed on macroscopic characterization
harmful exogenous materials (Decho 1990; Iyer of heavy metal uptake and concentration, reten-
et al. 2004; Pal and Paul 2008). It is generally tion, and release of radioactive materials by
assumed that diverse microbial biopolymers were aquatic organisms (Iyer et al. 2005; Sakamoto
shown to possess potential to bind heavy metals, et al. 2008). For instance, the exopolysaccharide
including lead, with different degree of specic- produced by the marine bacterium Enterobacter
ity and afnity (De et al. 2008; Bhaskar and cloacae was reported to have excellent chelating
Bhosle 2006; Pal and Paul 2008; Naik et al. properties with respect to 65 % of cadmium, 20 %
2012). Bacterial EPS and its possible role in bio- copper, and 8 % cobalt at 100 mg/L of metal con-
accumulation of Cu and Pb in marine food chains centration (Iyer et al. 2005). In the marine envi-
were investigated using a partially puried and ronment, the EPS is also capable of binding lead
chemically characterized EPS isolated from and could potentially be used in water treatment
Marinobacter sp. (Bhaskar and Bhosle 2006). plants (Mancuso Nichols et al. 2005). Interactions
The exopolymer binding process is important in between cations and polysaccharides also con-
the downward transport of metals in the ocean ferred both algal and bacterial EPS with the
environment (Decho 1990). In the marine bacte- potential to adsorb heavy metal contaminants
rium P. aeruginosa CH07, lead was entrapped in such as Cd2+ (Guibaud et al. 2005), Pb2+ (Comte
EPS indicating a possible resistance mechanism et al. 2008), and Hg2+ (Zhang et al. 2012), which
(De et al. 2007, 2008). EPSs are high molecular can then be incorporated into the food chain
weight polyanionic polymers which bind metals (Bhaskar and Bhosle 2006). Disconcerting con-
by electrostatic interaction between metal cation centrations of these heavy metals were found in
and negatively charged components of EPS the Arctic marine food web (Campbell et al.
resulting in metal immobilization within the exo- 2005); EPS from sea-ice organisms may play an
polymeric matrix (Roane 1999; van Hullebusch imperative role in the fate of these contaminants.
et al. 2003). Exceptional interest is the dependence of heavy
Marine sulfur-reducing bacteria were shown to metal adsorption to EPS on properties such as
produce EPS and formed complex with heavy salinity, pH, and Ca2+ concentrations (Comte et al.
5 Extracellular Polysaccharide Production by Bacteria as a Mechanism of Toxic 73

2008) that underwent seasonal changes in sea-ice cury efciently and also could resist higher con-
brines. The ecological implications of metal-bind- centrations of CdCl2, ZnSO4, PbNO3, and
ing properties of bacterial EPS and its role in the Na2HAsO4 (Dash et al. 2014). Removal of Na+ by
bioaccumulation of pollutants in the marine food binding with exopolymeric substances was dem-
chain were investigated using a partially puried onstrated in Rhodopseudomonas palustris strains
microbial EPS from Marinobacter sp. This EPS TN114 and PP803. Interestingly, the principal
was shown to selectively bind more amount of component from EPS of strain PP803 was identi-
Cu2+ per mg (182 nmol mg1) of EPS than Pb2+(13 ed as a polysaccharide (18 kDa) mainly com-
nmol mg1) (Bhaskar and Bhosle 2006). posed of galacturonic acid (Nunkaew et al. 2015).
Puried EPS from marine Pseudoalteromonas Strikingly, Alcaligenes faecalis was shown to
sp. was explored for its role in Fe chemical spe- enhance exopolysaccharides and siderophore
ciation, solubility, as well as bioavailability for production, when cells were exposed to toxic lev-
two keystone Southern Ocean phytoplankton els of organometal (TBTCl), suggesting their
microbial strains. Strikingly, the combined effect involvement in conferring resistance to this anti-
of EPS on Fe was shown to increase the seques- fouling biocide and degradative capability,
trating time of bioavailable Fe in the euphotic respectively (Khanolkar et al. 2015). Extracellular
zone, therefore possibly sustaining and control- polymeric substances (EPSs) produced by marine
ling primary productivity (Hassler et al. 2011). In bacteria play a fundamental role in different
a most recent report on the biolm-forming stages of biolm formation, maturation, and
marine bacterial isolates, Pseudomonas putida maintenance. The inuence of loosely bound and
SP-1, capable of volatilizing 89 % of mercury, tightly bound EPS, extracted from marine
has conrmed the efciency of mercury-resistant Pseudomonas NCIMB 2021, on the electrochem-
marine bacteria in bioremediation (Zhang et al. ical behavior of 70 Cu30 Ni (wt %) alloy in
2012). It is noteworthy that the proteomic static articial seawater (ASW) and on the chem-
responses of the marine bacterium Pseudomonas ical composition of oxide layers was studied.
fluorescens BA3SM1 revealed cellular adapta- Interestingly, Bautista and colleagues (2015)
tions to Cd, Zn, and Cu by inducing defense recently demonstrated a metal corrosion inhibi-
mechanisms, i.e., biolm formation and modi- tion effect and a low oxide layer thickness are
cation of envelope properties to increase the shown with loosely bound EPS.
extracellular metal biosorption (Poirier et al.
2013). More recently, Morcillo and colleagues
(2014) investigated the biosorption and biomin- 5.3.1 Marine Microbial Surface-
eralization of radioactive U(VI) associated with Active Molecules
the marine bacterium Idiomarina loihiensis
MAH1. Interestingly, the U(VI) speciation in Marine microbial surface-active molecules pos-
sea-water samples was shown to be more intri- sess several physiological roles of biosurfactants
cate, by forming different complexes; however, including the increasing of the surface area and
the removal of uranium was demonstrated due to bioavailability of hydrophobic water-insoluble
biosorption to extracellular polysaccharides substrates, heavy metal binding, bacterial patho-
(EPS) and cell wall components as evident from genesis, quorum sensing, and biolm formation
TEM analysis. Biomass and extracellular poly- (Ron and Rosenberg 1999; Flemming and
meric substances secreted by biolm-forming Wingender 2010). Microbial surface-active
marine bacterium Pseudomonas aeruginosa metabolites, called biosurfactants (polysaccha-
JP-11 effectively removed 58.760 10.62 % and ride moiety attached to lipid and/or protein), are
29.544 8.02 % of Cd, respectively (Chakraborty such metal-complexing agents that were previ-
and Das 2014). In another study, a marine ously reported to be successful in the bioremedi-
exopolysaccharide-producing bacteria Bacillus ation of heavy metal-contaminated environments
thuringiensis PW-05 was found to volatilize mer- (Mulligan et al. 2001; Singh and Cameotra 2004).
74 P.V. Bramhachari and G.P. Nagaraju

There are many reasons that make biosurfactants 5.4 Intracellular Metal
promising alternative agents for remediation pur- Sequestration
poses. These are their less toxic nature (Poremba
et al. 1991), better environmental compatibility, 5.4.1 Biosorption of Heavy Metals
and biodegradability (Georgiou et al. 1992).
Most biosurfactants reported till date are obtained Increasing contamination of marine environment
from microorganisms of terrestrial origin. with heavy metals has become a major global
Nonetheless, the marine environment that forms concern. Several techniques, such as adsorption,
the majority of the earths surface serves as a chemical precipitation, chemical oxidation and
huge catalog of microbial population producing a reduction, ion exchange, and evaporative recov-
variety of active metabolites and molecules. In ery, were used for removal and recovery of valu-
the quest of these molecules, Das et al. (2008) able or toxic metals from wastewater (Singh et al.
reported another marine bacterium with heavy 1998). Since these physicochemical methods are
metal sequestration potential and removal of lead incredibly expensive, the applications of biologi-
and cadmium (Das et al. 2009). There is a grow- cal methods are emerging as a viable alternative
ing evidence to implicate uronic acids of EPS in (Muraleedharan et al. 1991). Interestingly, sev-
conferring these macromolecules with an ability eral algae, bacteria, fungi, mosses and macro-
to interface with hydrophobic organic chemicals, phytes and several higher plants were previously
such as hydrocarbons (Gutierrez et al. 2008, employed for metal recovery from water systems
2009). Strikingly, Gutierrez and colleagues (Singh et al. 1998). In an attempt to nd more
(2008) demonstrated an emulsifying and metal economically viable biological methods, much
ion-binding activity of a glycoprotein exopoly- interest has been focused on nding novel marine
mer produced by marine Pseudoalteromonas sp. microbes able to bind large amounts of metals
strain TG12. Amino acids and peptides are also rapidly. It is interesting to note that Vibrio har-
frequently found associated with marine bacterial veyi, a normal inhabitant of the marine environ-
EPS, which conferred amphiphilic characteristics ment, is reported to possess the potential for
to these polymers (Gutierrez et al. 2009; bioaccumulation of cadmium up to 23.3 mg
Wolfaardt et al. 1999). Scores of marine microor- Cd2+/g of dry cells (Abd-Elnaby et al. 2013). In
ganisms such as Acinetobacter sp., Arthrobacter line with that, Von Canstein et al. (2002) reported
sp., Pseudomonas sp., Halomonas sp., Myroides a consortium of marine bacteria to efciently
sp., Corynebacteria sp., Bacillus sp., and remove mercury in a bioreactor in disturbance-
Alteromonas sp. were studied for production of independent mechanism. Likewise, a new combi-
bioemulsiers and exopolysaccharides nation of genetic systems in marine bacteria for
(Bouchotroch et al. 2000; Pepi et al. 2005; the potential degradation capability of phenol
Satpute et al. 2010; Iyer et al. 2006). Hexavalent and heavy metals was also described (El-Deeb
chromium reduction and trivalent chromium tol- 2009). The effect of cell wall-associated extracel-
erance of marine Bacillus sp. MTCC 5514 are lular polymeric substances (EPS) of a Gram-
known to be mediated by its extracellular enzyme, negative bacterium Shewanella oneidensis strain
chromate reductase, and its biosurfactant MR-1 on Zn(II) and Pb(II) adsorption was inves-
(Gnanamani et al. 2010). An excellent alternative tigated using an amalgamation of titration/batch
for enhanced metal bioremediation is the use of uptake studies, surface complexation modeling,
microbial biomolecules such as microbial surfac- and attenuated total reectanceFourier trans-
tants, and extracellular polymers suggested the form infrared (ATR-FTIR) spectroscopy.
increase in efciency of metal reduction/seques- Interestingly, this study demonstrated a greater
tration by microorganisms to achieve eld biore- amount of EPS in enhanced Zn(II) and Pb(II)
mediation of metal-contaminated sites (Singh uptake. Attributable to its dissimilatory metal-
and Cameotra 2004). reducing activity, S. oneidensis MR-1 was chosen
5 Extracellular Polysaccharide Production by Bacteria as a Mechanism of Toxic 75

as a target of marine bioremediation research (Ha Synechococcus PCC 7942 (SmtA), Anabaena
et al. 2010). Correspondingly, certain purple non- PCC 7120 (SmtA), Oscillatoria brevis (BmtA),
sulfur marine bacterial isolates, e.g., Rhodobium Pseudomonas aeruginosa (BmtA) and
marinum and Rhodobacter sphaeroides, have Pseudomonas putida (BmtA) (Blindauer et al.
also been found to possess the potential of remov- 2002). P. aeruginosa strain WI-1 isolated from
ing heavy metals like copper, zinc, cadmium, and Mandovi estuary possesses bacterial metallothio-
lead from the contaminated environments either nein (BmtA) to alleviate lead toxicity (Naik et al.
by biosorption or biotransformation (Panwichian 2012). Typically, the metals for which metallo-
et al. 2011). Cao and his colleagues (2011) com- thioneins have the highest afnity are Cd, Pb, Hg,
pared U(VI) immobilization using cells with Cu and Zn but also bind to Ni with lower afnity.
bound EPS (bEPS) and cells with minimal EPS The heterotrophic marine bacterium, Vibrio algi-
and demonstrated that (i) EPS from Shewanella nolyticus, interestingly showed production of
sp. HRCR-1 biolms contributed signicantly to extracellular copper-binding compounds when
U(VI) immobilization, particularly at low initial exposed to copper in a sea-water medium
U(VI) concentrations, through both sorption and (Schreiber et al. 1990). Proteomic responses of
reduction. Therefore, the marine bacteria have the marine oxic sediment bacterium Pseudomonas
been designated for assessing marine pollution fluorescens to Zn, Cu, and Cd metals demon-
through tolerance (Das et al. 2007) and biosorp- strated de novo synthesis of metal responsive
tion of heavy metals (Das et al. 2009). Biosorption proteins, indicative of metal-stimulated synthe-
capabilities and extracellular polysaccharides sis, particularly for Cu and Cd (Poirier et al.
(EPSs) were demonstrated in marine Bacillus 2008). Recent genome-mining studies suggest
cereus CURB-4 strain and were shown to pro- that marine Synechococcus sp. WH8102 have
duce more EPS (71 0.6 %) in the presence of Cu metal uptake systems for zinc and proteins that
(II) ions (Rajaram et al. 2013). utilize zinc as a cofactor. Interestingly, a mecha-
nism for zinc sequestration was particularly ef-
cient and the expression of SYNW2224, a
5.4.2 Small Metallothionein putative porin protein, was shown upregulated
Proteins during growth in zinc-depleted conditions
(Barnett et al. 2014).
Microorganisms have evolved several resistance
mechanisms to withstand the toxic effects of
heavy metals and organometals. One of the com- 5.5 Biotechnological
mon mechanisms is induction of specic metal- Applications of Marine
binding proteins facilitating the sequestration/ Exopolysaccharides
bioaccumulation of toxic metals inside the cell.
These well-studied metal-binding proteins are Microbial exopolysaccharides are of signicant
referred to as metallothioneins (MTs). commercial value, since they embody an interest-
Intracellular metal bioaccumulation and homeo- ing alternative to the plant and macro algal exo-
stasis in cell cytosol involves the low-molecular- polysaccharides traditionally used in the food,
weight, cystein-rich metallothioneins which textile, painting, cosmetic, paper, pharmaceutical
range from 3.5 to 14 kDa (Hamer 1986). and oil industries. Contemporary commercial
Metallothioneins play a signicant role in immo- production of microbial polysaccharides is built
bilization of toxic heavy metals in that way pro- on fermentation of heterotrophic bacteria. Marine
tecting bacterial metabolic processes catalyzed bacteria offer a great diversity of polysaccharides
by enzymes (Fig. 5.1) (Blindauer et al. 2002). which could play an important role in biotechnol-
Several cyanobacterial and bacterial strains were ogy and industry as well as in future development
reported to encode metallothioneins for main- of cell therapy and regenerative medicine among
taining cytosolic metal homeostasis such as other applications. Mancuso Nichols et al. (2005)
76 P.V. Bramhachari and G.P. Nagaraju

isolated several EPS-producing strains from the is a main component of many EPSs from cold
Antarctic sea ice and seawater and found that marine environment strains like
mannose represented the most abundant neutral Pseudoalteromonas arctica KOPRI 21653,
sugar in the EPSs. However, the ecological role Pseudoalteromonas strain SM20310 and
of mannose in these EPSs is still unknown. The Pseudoalteromonas haloplanktis TAC 125, iso-
structure of this EPS is different from that of lated from Antarctic seawater and has a mannan
EPSs secreted by other marine bacteria. For and phosphomannan structure (Corsaro et al.
instance, EPS produced by V. diabolicus contains 2004; Kim and Yim 2007; Liu et al. 2013).
equal amounts of uronic acids and hexosamines
(hyaluronic acid like) and was shown to enhance
bone healing in rats (Zanchetta et al. 2003). Kim 5.6 Strategies for Enzymatic
and Yim (2007) showed that the EPS produced by Modifications of EPS
the Antarctic bacterium Pseudoalteromonas arc-
tica KOPRI 21653 could improve the freeze- The marine biodiversity has previously shown a
thaw survival ratio of E. coli and suggested that it great potential in providing new biocatalysts
may have biotechnological potential as a cryo- (Trincone 2010). Quite a lot of enzymes have
protection agent. In another study by Liu et al. become promising biotechnological tools for
(2013), a Pseudoalteromonas strain SM20310, in vitro synthesis or in vivo biosynthesis engi-
screened from Arctic sea ice, produced a highly neering. Conversely, the in silico genomic data
complex mannan. This EPS had a cryoprotective would also consent to identify new enzymatic
effect and could also improve high-salinity toler- tools to modify microbial glycopolymers. Most
ance, a characteristic feature benecial for the importantly, the depolymerizing enzymes can be
strain to adapt to sea-ice environment. Although used in in vitro depolymerization process but also
several known marine bacteria were shown to as tools to study the chemical structure.
produce structural and functionally diverse EPSs, Carbohydrate sulfotransferases and other
few of the produced EPSs from Salipiger muco- enzymes grafting substituents such as acetate, as
sus strain A3T, a halophilic species belonging to well as enzymes catalyzing their removal, may be
Alphaproteobacteria, are of immense biotechno- used in vitro for binding or for elimination of
logical importance, so the investigation for novel substituents which are of great importance for the
EPSs that have innovative applications is still of nal bioactivity of the molecule. Identifying both
potential interest (Llamas et al. 2010). the polysaccharide chemical structure and genetic
Marine bacteria have become increasingly cluster for the biosynthesis would broaden the
popular for producing novel EPS molecules. The knowledge of the glycopolymer enzymatic func-
presence of charged EPS from the Arctic marine tion in biosynthetic pathway by means of genetic
psychrophile has intricate implications for the knockouts.
dynamics of carbonates in the sea-ice environ- Several research studies proved that exopoly-
ment given that these charged polysaccharides, saccharides can be modied by the enzymatic
unlike non-ionic polysaccharides, have well- action of transferases and hydrolases which pos-
known effects on the precipitation of carbonates sess the capability to add or remove functional
(Hardikar and Matijevi 2001). In the case of groups such as acetyls, pyruvyls (Marzocca et al.
cold-adapted marine organisms, EPS was shown 1991), glyceryls (Kuo et al. 1986), succinyls
to increase the stability and half-life of a cold- (Reuber and Walker 1993), lactyls (Maalej et al.
active extracellular aminopeptidase isolated from 2014), or a combination of these, leading to vari-
subzero Arctic marine sediment bacteria C. psy- ations in polymer surface electrostatics and solu-
chrerythraea 34H (Huston et al. 2004). Mannose bility. Additionally, epimerization can drastically
5 Extracellular Polysaccharide Production by Bacteria as a Mechanism of Toxic 77

alter the structural conformation of polysaccha- 5.7 Strategies for Chemical

rides, affecting polymer interactions within the Modifications of EPS
biolm (Steigedal et al. 2008). Some of these
modications were earlier studied with respect to Interestingly, chemical modications are widely
their importance in bacterial virulence, pathogen- demonstrated in terrestrial microbes (Dayala
esis, biolm formation, or symbiosis (Ridout et al. 2008; Laurienzo 2010; Senni et al. 2011):
et al. 1997), as well as their commercial utility in acid hydrolysis (Colliec et al. 1994), radical
the food and cosmetic industries. Despite this depolymerization (Nardella et al. 1996),
wealth of knowledge, there remain a number of N-deacetylation (Zou et al. 1998) and sulfation
unresolved questions regarding the biological (Guezennec et al. 1998). However, several draw-
implications of EPS modications in marine bac- backs such as lack of control and regioselectivity,
teria as well. It is interesting to note that glycos- use of organic solvents, and nonhomogeneous
aminoglycans (GAGs) are a group of polymers conditions are identied (Al-Horani and Desai
that are typically composed of a disaccharide 2010). More recently, ionic liquids were also
repeat unit containing an amino sugar and a hex- used for cellulose sulfation in homogeneous
uronic acid (Laurent and Fraser 1992; Esko and media (Gericke et al. 2011). Only few reports
Lindahl 2001; DeAngelis 2002). GAGs were ini- have been dealt with chemical modications on
tially thought to exist only in the animal king- marine EPS (Dayala et al. 2008). The most con-
dom, where they serve essential biological vincing evidence for the low molecular weight
functions; however, there was an emergence of over sulfated derivatives of the EPS GY785 from
GAG-like polymers among few prokaryote the deep-sea bacterium A. infernus and EPS
strains (DeAngelis et al. 2002). Prokaryotic HE800 from V. diabolicus has been obtained by
GAGs are typically less complex than their depolymerization, acid hydrolysis, or free-radical
eukaryotic counterparts due to an absence of reaction followed by sulfation with sulfur triox-
chemical modications such as sulfation on the ide pyridine complex (Jouault et al. 2001; Senni
structures (Raedts et al. 2011). The identication et al. 2011). Nevertheless, there are no reports of
of the bacteria glucuronyl C5-epimerase has chemically modied EPS used in
proven elusive (Raedts et al. 2011). However, an bioremediation.
enzyme (RED65_08024) from the marine bacte-
rium Bermanella marisrubi that shares 37 %
sequence similarity with the human glucuronyl 5.8 Genetic Manipulation
C5-epimerase was characterized and shown in Marine Bacteria
in vitro to convert GlcA to IdoA in de-sulfated to Enhance Metal
mouse HS (Raedts et al. 2013). This glucuronyl Detoxification
C5-epimerase represents the rst prokaryotic
protein capable of generating IdoA residues and In order to increase the bioremediation potential
only identied epimerase that can function on and/or metabolic activity of any bacteria, inser-
bacterial polysaccharides after post polymeriza- tions of certain functional genes are highly indis-
tion, besides AlgG and AlgE1-7. Unfortunately, pensable into their genome. This phenomenon
the EPS produced by B. marisrubi has not been can be achieved by insertion of new genes into
characterized, so its target remains unknown. the genomic complexion, insertion of new plas-
Therefore, increasing list of identied modica- mid, alteration of metabolic pathways like trans-
tions in marine bacteria will allow future efforts port and chemotaxis and, most importantly,
to focus on linking these modications to specic adaption of features toward the environmental
biosynthetic genes and biolm phenotypes. conditions (Pieper and Reineke 2000). However,
78 P.V. Bramhachari and G.P. Nagaraju

limited reports are available to date for the genetic 5.9 Future Prospects in Marine
manipulation of marine bacteria to achieve a goal EPS Research
of enhanced bioremediation (Valls and de
Lorenzo 2002). An interesting result came from a The important roles of marine microbial EPS in
study with the insertion of bmtA gene coding for metal biosorption have been widely documented.
metallothionein into suitable vector and its trans- However, the underlying mechanisms of the
formation into marine bacteria was conducted metalEPS interactions and engineering of prac-
and successfully employed in highly metal- tical metal biosorption processes are largely
contaminated environments (Chen et al. 1999). unclear. In fact, the complexity in the EPS com-
These genetically modied marine bacteria position and the diversity in metal species in the
expressing extremely high amount of vast marine biosphere make it difcult to explore
metallothionein can be employed to bioremediate into metalEPS interactions and microbial-based
marine sites highly contaminated with heavy metal biosorption. Furthermore, the details
metals. These hyper-metal accumulating bacteria regarding the genes and proteins involved in the
maintain metal homeostasis by reducing metal polysaccharide modications remain largely
bioavailability. Expression of metallothioneins unavailable. Characterization of these genes and
on the cell surface by fusion with cell surface proteins will probably provide details on to how
proteins improves the bioaccumulation capacity the levels and types of modications are regu-
of bacteria (Chen et al. 1999). Similarly, lated under different conditions. Therefore, to
Pseudoalteromonas haloplanktis, possessing a gain an in-depth understanding of the metal
shuttle plasmid-encoding suppressor for amber detoxication process and promote its practical
mutation, was used for genetic manipulation in implementation, future research efforts in the
bioremediation studies (Kivela et al. 2008). above directions are warranted.
Bacteria possessing plasmid with merA gene
responsible for converting toxic form of mercury
to nontoxic form may be transformed into marine 5.10 Concluding Remarks
bacteria for better application in eld conditions
of bioremediation of mercury (De Rore et al. Marine bacteria offer a great diversity of polysac-
1994). Deinococcus radiodurans, the most radio- charides which could play an important role in
resistant marine organism, was modied geneti- biotechnology and industry as well as in future
cally to metabolize toluene and ionic form of development. Fundamental studies on marine
mercury from nuclear wastes (Brim et al. 2000). bacterial exopolysaccharides are relatively few in
Cheung and Gu (2003) demonstrated chromate number compared to other polysaccharides.
(CrO42) reduction in marine sulfate-reducing There are indeed many gaps in our knowledge on
bacteria. In a recent report, Stahl et al. (2015) elu- fundamental questions such as the structure,
cidated the function of two CzcCBA pumps physicochemical properties and biosynthesis.
encoded by Marinobacter adhaerens HP15s Thus, research is urgently wanted on these
genome during exposure to cadmium, zinc and aspects. The data currently available indicate that
cobalt by generating single and double knockout marine bacterial exopolysaccharides have con-
mutants. Modication of bacterial isolates which siderable biotechnological importance; neverthe-
can overproduce EPS and biosurfactants may be less, further studies are highly indispensable to
a good strategy for bioremediation of signicant evaluate the feasibility of their practical applica-
amount of heavy metals. Microbial enzymes can tion. This may limit the practical application of
be modied to increase their kinetics to reduc- marine bacterial exopolysaccharides as a metal-
tively precipitate heavy metal ions or increase chelating agent. Studies on interactions between
efciency of enzyme to effectively detoxify met- metals and EPS produced by bacteria have estab-
als (Karigar and Rao 2011). lished the phenomenon of biosorption as one of
5 Extracellular Polysaccharide Production by Bacteria as a Mechanism of Toxic 79

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Acknowledgments Dr.P.V. Bramhachari gratefully alkaline pH and implication for formation of carbon-
acknowledges DST-SERB, Government of India for ate minerals. Geobiology 5:401411
nancial support under the Grant No: SR/FT/ Bramhachari PV, Dubey SK (2006) Isolation and charac-
LS-109-2011 terization of exopolysaccharide produced by Vibrio
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Biosurfactant: A Promising
Approach Toward the Remediation 6
of Xenobiotics, a Way
to Rejuvenate the Marine

Siddhardha Busi and Jobina Rajkumari

Xenobiotics petroleum hydrocarbons, dyes, heavy metals, pesticides,
and antifoulants like tributyltin (TBT) which are dispersed in the marine
environment through various activities have caused ecological and social
catastrophes. Pesticides, metals, dyes, etc. used in agriculture and other
industrial purposes eventually reach the sea through streams and rivers.
The presence of these xenobiotics in the water bodies may lead to many
serious health consequences as xenobiotics like heavy metals are muta-
genic and carcinogenic in nature. Heavy metals are usually discharged
from power generation industries, and some like mercury are an active
component in many pesticides. Synthetic dyes and dry cleaning uid like
tetrachloroethylene used largely in dyeing and printing industries consti-
tute one of the major organic pollutants. Polycyclic hydrocarbons and
crude oil sludge are discharged into the marine environment as a result of
offshore oil drilling, overow from oil tanker, and ship accidents and as
by-products of coal treatment. As a result of increasing public awareness
on environmental pollution and the need for sustainable development,
various research programs have been initiated for development of tech-
nologies to manage the xenobiotics. The use of surface-active biomole-
cules (biosurfactant) synthesized by microorganisms in bioremediation
has been proposed in recent years and is gaining prominence due to their
high potential in mobilization and solubilization of various pollutants.
Biosurfactants such as rhamnolipid, sophorolipid, surfactin, and alasan
produced by diverse species of Pseudomonas, Candida, Bacillus, and
Acinetobacter, respectively, have been widely studied and reported for the
remediation of hydrocarbons (e.g., phenanthrene, uoranthene, pyrene,
hexadecane, kerosene, and 1-methyl naphthalene) and heavy metals, e.g.,

S. Busi (*) J. Rajkumari

Department of Microbiology, Pondicherry University,
Puducherry 605014, India

Springer Science+Business Media Singapore 2017 87

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_6
88 S. Busi and J. Rajkumari

cadmium and pesticides (endosulfan and beta-cypermethrin). Moreover,

Bacillus subtilis and Bacillus licheniformis are well-known producers of
biosurfactant employed in oil recovery and bioremediation. The biosurfac-
tant seem to enhance the biodegradation of oils and lipids as a result of its
emulsifying property. Owing to its biodegradability and low toxicity, they
might serve as promising tool for the use in bioremediation of both organic
and inorganic pollutants. This chapter provides an overview on the
advances in the application of microbial biosurfactant in rejuvenating
marine ecosystem and in combating the issues of xenobiotics.

6.1 Introduction 6.2 Xenobiotics in Marine

Marine environment is the largest habitat cover-
ing approximately 70 % of the total earth sur- Xenobiotics refer to chemical compounds like
face. Oceans play a major role in regulating synthetic pesticides, herbicides, and industrial
earths climate and harbor a huge diversity of pollutants which are foreign to the environment.
living organisms. The marine ecosystems have Unlike the organic pollutants that are biodegrad-
been acting as a sink for large amounts of toxic able, these pollutants, e.g., heavy metals, dyes,
wastes/xenobiotics, which even in minute quan- nuclear wastes, etc. are extremely stable making
tities threatens the ocean communities. Rapid them persistent in the environment. Xenobiotics
industrialization and extraction of natural released mainly from industries like chemical
resources to feed the growing urbanization have and pharma, oil spills, nuclear explosions, and
resulted in large-scale environmental pollution. agricultural practices reach the marine environ-
Xenobiotics, chemicals which are foreign to life ment through land runoffs and as industrial efu-
and persistent organic pollutants, are now ubiq- ents. The major environmental threats include
uitous in the environment, raising its risk to residual compounds like pesticides, petroleum
public health and its surrounding environment hydrocarbons, different dyes, paints, heavy met-
at an alarming rate. Pollution of marine ecosys- als, radioactive substances, fertilizer, insecti-
tem has endangered the aquatic ora and fauna. cides, etc.
The uptake and accumulation of toxic chemicals
like pesticides in the food chain and its biomag- Petroleum Hydrocarbons Petroleum represents
nication lead to lethal behavioral effect on one of the most important industries today with-
animals and contribute to the rise in global epi- out which modern life will come to a halt. The
demic of cancer and other chronic diseases. world depends on petroleum and petroleum prod-
Environmental pollution of the marine environ- ucts for its economic development and techno-
ment is one of the most important problems in logical advances. Although the petroleum plants
the context of human health and havoc to the and crude oil reneries provide vital fuel like
ecosystem. The need of the hour is to develop gasoline and power to the society, they are also
novel, eco-friendly, and cost-effective way to responsible for the global pollution of oceans with
revive the polluted environment and restore the large amount of hazardous waste from reneries.
marine ecosystem. Poisoning of sea by man-made disasters is one of
6 Biosurfactant: A Promising Approach Toward the Remediation of Xenobiotics 89

the urgent and serious environmental problems all Elaziouti et al. 2011). Textile and paper printing
over the world. The pollutions are caused by spill- industries commonly use azo dyes, anthraqui-
ing of crude oil from tankers during transporta- none, and phthalocyanine dyes. The degradation
tion, collisions, and leakage of oil and gas on the of these dyes results in the production of aro-
ocean oor from underground pipelines. On April matic amines, which may also be carcinogenic
20, 2010, the world witnessed the largest acciden- and mutagenic. Anti-biofouling agents in paint
tal oil spill in the Gulf of Mexico, also known as like tributyltin (TBT) are also considered
BP oil spill. Following the explosion and sinking harmful.
of deep water horizon oil rig off the coast of
Louisiana (USA), the open ducts continued to dis- Insecticides and Pesticides A large number of
charge oil (estimated 3.19 million barrel) into the insecticides, nematocides, herbicides, fungicides,
ocean for 87 days until nally plugged on July and pesticides are used on a daily basis in agri-
2010 (British 2010). Another incident of such oil culture. Indiscriminate use of chemicals like
dispersing tragedy includes the Nigeria delta. In methyl bromide, chlorinated organics, endosul-
the hydrocarbon family, polycyclic aromatic fan, nitrophenols, organophosphorus compounds,
hydrocarbons (PAHs), e.g., naphthalene, phenan- synthetic pyrethroids, morpholine, carbamates,
threne, biphenyl, anthracene, and pyrene are the etc. severely contaminates the groundwater, and
most toxic pollutants and major constituent of the runoff ows to rivers and ultimately reaches
crude oil. PAHs are the large group of organic the ocean (Lalithakumari 2011). The demand and
compounds formed by the hydrogen and carbon use of beta-cypermethrin (beta-CP), a synthetic
atoms with two or more fused benzene rings. pyrethroid (SP) insecticide, have been consider-
These are the hydrophobic compounds and insol- ably increased due to its high insecticidal ef-
uble in water. More than 1000 types of PAHs ciency and with the restrictions and ban on import
compounds are present in the environment with and export of organophosphate and carbamate
differing in the number and position of aromatic pesticides. However, beta-CP poses serious threat
rings. PAH ingested by marine organisms passes to the survival of aquatic shes and invertebrates.
through food chain and ultimately reaches man. It may even cause serious health consequences
Most of the PAHs are carcinogenic, mutagenic, like reproductive, neurotoxicity, and develop-
and teratogenic to many organisms including mental disorders in humans (Zhang et al. 2011).
mammals. 3,4-Benzopyrene and its derivatives The use of high amount of nitrogen- and
are well-known carcinogen. Oil/hydrocarbon phosphorous-rich fertilizers causes algal bloom
waste being hydrophobic in nature forms slick and creates a zone of hypoxia with loss of species
layer on the surface of water bodies which blocks diversity in sea. The main problem associated
the passage of sunlight and prevents gas exchange, with these chemicals is bioconcentration and bio-
respiration, and photosynthesis of phytoplankton magnication. Lipophilic insecticides like
communities (Yeung et al. 2011; Asimiea and dichlorodiphenyltrichloroethane (DDT) accumu-
Sam-Wobo 2011). Marine organisms are affected late into the fatty tissues of shes and pass on to
due to the shortage of dissolved oxygen and fail to successive trophic levels. DDT causes birth
cope with the polluted environment. The oil spill defects and cancer in humans and also drastically
has the physical, psychological, genotoxic, and affects the bird population.
endocrine effects in human. It also causes disori-
entation, lesions, and deformities in shes, sea Heavy Metals Heavy metals like mercury, arse-
turtles, dolphins, and sea birds. nic, lead, cadmium, and zinc are highly toxic and
accumulate in the organisms leading to biomag-
Dyes and Paints Dyes are persistent xenobiotics, nications. This increases their toxicity to the
and their presence even in minute amount reduces subsequent tropic levels. These heavy metals are
penetration of sunlight, affecting photosynthetic highly carcinogenic. Arsenic and lead adminis-
activity in aquatic life (Abdelkader et al. 2011; tered above the permissible level in agriculture
90 S. Busi and J. Rajkumari

causes groundwater poisoning. Copper used in bioavailability of these hydrophobic compounds,

marine antifouling paints was found to be lethal thereby enhancing the solubility of pollutants and
to marine organisms. Chronic exposure to cad- making it susceptible to microbial attack. Some
mium is responsible for the Itai-itai disease in of the isolated biosurfactant increases the solubil-
Japan which is characterized by renal dysfunc- ity of hydrocarbons like pyrene while other con-
tion, severe osteoporosis, and anemia in women tributes to an enhanced solubilization of
(Mulligan et al. 2001). Mercury is a potent neuro- phenanthrene or uorene. Bordoloi and Konwar
toxin and is responsible for the Minamata disease (2009) reported enhanced solubility and metabo-
in Japan (Mulligan et al. 2001). In children, lism of petroleum hydrocarbons in presence of
Mercury and lead poisoning causes brain damage biosurfactant obtained from different P. aerugi-
and behavioral disturbances. For example, the nosa strains (Bordoloi and Konwar 2009). The
methylated mercury present in the sea organisms effect of biosurfactant on the biodegradation of
(sh, shellsh, etc.) enters into the human body crude oil by various marine bacterial isolates like
by food which adversely affects mankind. The Bacillus megaterium, Corynebacterium
effects of heavy metal toxicity are due to their kutscheri, and P. aeruginosa has been studied
ability in interrupting the process of transcription (Thavasi et al. 2011).
and translation of proteins, removal of metal ion
from biomolecules, and thus deactivating them
(Varsha et al. 2011). 6.4 Introduction
to Biosurfactant

6.3 Bioremediation Biosurfactants are surface-active compounds

that reduce the surface tension between interface
Several physical and chemical methods have of liquid- liquid or liquid-solid. Structurally, all
been employed in the treatment or removal of biosurfactant are amphiphilic in nature, i.e., they
xenobiotics. The physicochemical methods are consist of hydrophilic head and hydrophobic
expensive and often result in undesirable toxic tail. The hydrophilic moiety comprises of a car-
by-products. To overcome these problems, eco- bohydrate, amino acid, cyclic peptide, phos-
friendly technique like bioremediation has been phate, carboxylic acid, or alcohol with the
employed. Microbial bioremediation relies on hydrophobic group of long-chain fatty acids,
either removal or degradation of toxic pollutants hydroxyl fatty acids, or -alkyl--hydroxy fatty
present in environment into less toxic or nontoxic acids (Mulligan et al. 2001). Biosurfactant pos-
forms. The process takes place through intracel- sesses different chemical structures and exhibits
lular accumulation either by microbes or by different properties. They can be lipopeptides,
enzymatic transformation. Many microorgan- glycolipids, neutral lipids, or fatty acids. They
isms naturally possess the ability to degrade, are mainly classied into two classes: low-
transform, or chelate various toxic chemicals, but molecular-weight surface-active agents (biosur-
these processes are relatively slow. Polycyclic factant) and high-molecular-weight
aromatic hydrocarbons (PAHs), crude oil sludge, surface-active agents (bioemulsiers)
dyes, and pesticides that pollute the marine envi- (Rosenberg and Ron 1999). Microbial surfac-
ronment are toxigenic, mutagenic, and carcino- tants have been increasingly gaining attention
genic. The low hydrophobicity of these due to its interesting and diverse properties such
compounds limits their degradation by microor- as low toxicity, biodegradability, eco-friendly
ganisms, which is a common problem encoun- nature, possibility of large-scale production,
tered in bioremediation of contaminated sites. In selectivity, and optimal activity at extreme con-
such scenario, microbial surfactants increase the ditions of temperatures and pH.
6 Biosurfactant: A Promising Approach Toward the Remediation of Xenobiotics 91

6.5 Biosurfactant-Producing growth by facilitating the translocation of insolu-

Microorganisms ble substrates across cell membranes (Zinjarde
and Pant 2002). These amphiphilic compounds
A wide range of bacteria, fungi, and yeast pro- provide access to hydrophobic substrates by
duces biosurfactant either intracellularly or extra- reducing surface tension and increase in the area
cellularly. The genus Pseudomonas is well known of contact of insoluble compounds, thus enhanc-
for the production of rhamnolipids; Bacillus sub- ing the mobility, bioavailability, and biodegrada-
tilis produces a lipopeptide, surfactin (Ron and tion of xenobiotics (Abraham 1998).
Rosenberg 2001; Mulligan 2005). Nocardia The structure, size, and composition of bio-
amarae (Moussa et al. 2006), Saccharomyces surfactant synthesized are inuenced by the
lipolytica CCT- 0913 (Lima and Alegre 2009), microorganism, medium composition, and cul-
Candida bombicola (Casas and Garca-Ochoa ture conditions (Lang and Wullbrandt 1999;
1999; Casas et al. 1997; Williams 2009), Candida Franzetti et al. 2008). The carbon sources used
lipolytica (Sarubbo et al. 2007; Runo et al. for biosurfactant production include petroleum
2007), Candida ishiwadae (Thanomsub et al. hydrocarbons, carbohydrates, oil sludge, lactic
2004), and Candida batistae (Konishi et al. 2008) whey and distiller wastes, starchy substrates,
are among the most common biosurfactant- renewable resources, industrial or municipal
producing yeasts and fungi. wastewater, etc. (Lang and Wullbrandt 1999;
Marine microorganisms have gained attention Kosaric 1992; Gautam and Tyagi 2006) (Table
particularly for their biocompatibility and biore- 6.1).
mediation potential of the sea polluted by crude
oil. High diversity of biosurfactant-producing
marine microorganisms offers great potential in 6.6 Classication
bioremediation of petrochemical waste in oce- of Biosurfactants
anic environments. The marine microorganisms
produce four major classes of biosurfactants such Surfactants, diverse group of surface-active com-
as glycolipids; lipopeptides and lipoproteins; pounds, are mainly classied by their chemical
fatty acids, neutral lipids, and phospholipids; and structure and molecular weight. Depending on
polymeric biosurfactants (Desai and Banat 1997). the molecular weight, microbial surfactants can
Hassanshahian (2014) isolated biosurfactant- be classied into two main classes, biosurfactant
producing bacteria of genera Shewanella, Vibrio, and bioemulsans. Biosurfactants include mole-
Gallaecimonas, Brevibacterium, Psychrobacter, cules with low molecular mass, like surfactin and
and Pseudomonas from samples collected from rhamnolipid having a molecular weight of 1036
the coastline of the Persian Gulf. Maneerat and and 802 Da, respectively (Mulligan and Gibbs
Phetrong (2007) reported biosurfactant- 1990). On the other hand, bioemulsans include
producing marine bacteria, Vibrio parahaemo- high-molecular-weight polymers like emulsan
lyticus, Bacillus subtilis, Micrococcus luteus, and alasan with an average molecular weight of
Myroides sp., Acinetobacter anitratus, and B. about 1000 kDa and 1 MDa, respectively (Kim
pumilus, isolated from oil spill-polluted seawater et al. 1997; Navon-Venezia et al. 1995).
along Thailand coast. Marine bacteria Alcaligenes Due to their amphiphilic nature, the biosurfac-
sp. has been reported to produce glycolipid. tant and emulsiers alter the facial interface of
Zinjarde and Pant (2002) also reported a marine contaminant by different mechanisms, resulting
bacterium, Yarrowia lipolytica, for the produc- in enhanced bioremediation. High-molecular-
tion of polymeric biosurfactant with the ability to weight bioemulsiers are tested as enhancers of
emulsify alkanes and crude oil. hydrocarbon biodegradation in bioremediation of
It is believed that microbes secrete biosurfac- marine environment. These emulsiers have a
tant into the culture medium to support their great potential in stabilizing emulsions between
92 S. Busi and J. Rajkumari

Table 6.1 Different type of biosurfactant and their microbial source

Class/type of biosurfactant Microorganisms Reference(s)
Rhamnolipids Pseudomonas fluorescens, Pseudomonas Robert et al. (1989)
sp., Pseudomonas aeruginosa
Glucose lipids Alcanivorax borkumensis, Alcaligenes sp. (Zinjarde and Pant (2002), and
Maneerat (2005))
Sophorolipids Torulopsis bombicola, Candida Konishi et al. (2008), Maneerat
bombicola, Candida batistae, and Phetrong (2007). Inoue and
Trichosporon asahii, Candida apicola, Ito (1982), and Cavalero and
Candida lipolytica, Candida bogoriensis Cooper (2003)
Ornithine lipids Myroides sp. SM1
Trehalolipids Rhodococcus erythropolis, Arthrobacter Maneerat (2005), Shulga et al.
sp. Arthrobacter paraffineus, (1990), and Suzuki et al. (1974)
Corynebacterium spp., Mycobacterium
Lipopeptides and lipoproteins
Lipopeptide Bacillus licheniformis, Arthrobacter MIS Mclnerney et al. (1990),
38, Bacillus subtilis, Acinetobacter Morikawa et al. (1993), and
calcoaceticus, Candida petrophilum Horowitz et al. (1990)
Surfactin Bacillus sp. AB2 Morikawa et al. (1992)
Subtilisin Bacillus subtilis Al-Bahry et al. (2013)
Viscosin Pseudomonas fluorescens Alsohim et al. (2014)
Serrawettin Serratia marcescens Matsuyama et al. (2011)
Fatty acids, neutral lipids, and phospholipids
Fatty acid Corynebacterium lepus, Acinetobacter Akit et al. (1981)
Bile acids Myroides sp. SM1 Maneerat (2005)
Neutral lipids Nocardia erythropolis, Corynebacterium Cooper et al. (1979) and Lesik
hydrocarbolastus, Corynebacterium et al. (1989)
Phospholipids Thiobacillus thiooxidans, Zajic et al. (1977)
Corynebacterium lepus, Corynebacterium
Polymeric surfactants
Emulsan Acinetobacter calcoaceticus Goldman et al. (1982)
Lipid-carbohydrate-protein Yarrowia lipolytica, Pseudomonas Desai and Banat (1997)
Biodispersan Acinetobacter calcoaceticus Rosenberg et al. (1988a)
Liposan Candida lipolytica Cirigliano and Carman (1985)
Carbohydrate-lipid-protein Pseudomonas fluorescens, Candida Persson et al. (1988)

liquid hydrocarbons and water, thus increasing rhamnolipids have been shown to accelerate the
the surface area available for bacterial biodegradation of hydrocarbons when added to
biodegradation. Barkay et al. (1999) reported the contaminated soils at a concentration above the
signicant increase in the mineralization rate of critical micelle concentration (CMC). Surfactin
uoranthene and phenanthrene by Acinetobacter has been reported for the biodegradation of pesti-
radioresistens and Sphingomonas paucimobilis cide (Awashti et al. 1999).
EPA505 when supplemented with alasan, respec- On the basis of the chemical composition,
tively. Low-molecular-weight biosurfactant biosurfactant may be classied as glycolipids;
6 Biosurfactant: A Promising Approach Toward the Remediation of Xenobiotics 93

lipopeptides and lipoprotein; phospholipids, fatty and Asselineau 1978; Kretschmer et al.
acids, and neutral lipid; and polymeric and 1982).
particulate biosurfactant (Lang and Wullbrandt (c) Sophorolipids: These glycolipids are pro-
1999). duced mainly by yeast such as Torulopsis
bombicola, T. petrophilum, Candida bom-
1. Glycolipids: Glycolipids are the most studied bicola, T. petrophilum, and T. apicola.
microbial surfactants. They compose of car- Sophorolipids consist of a hydrophilic
bohydrates linked to long-chain aliphatic carbohydrate, sophorose with a hydro-
acids or hydroxyl aliphatic acids by an ester phobic long-chain hydroxy fatty acid. The
group. Rhamnolipids, trehalolipids, and soph- terminal carboxyl group can be in the lac-
orolipids are the most widely used tonic form or hydrolyzed to give an
glycolipids. anionic surfactant (Rosenberg and Ron
(a) Rhamnolipids: Rhamnolipids are the 1999; Cooper and Paddock 1984).
most studied glycolipids till date. These 2. Lipopeptides and Lipoproteins: Lipopeptides
are made up of L-rhamnose molecules compose of a lipid attached to a polypeptide
bonded to -hydroxy fatty acids. The pro- chain. Surfactin, subtilisin, viscosin, and ser-
duction of rhamnolipids was rst reported rawettin are produced by Bacillus sp. AB2,
and extensively studied in Pseudomonas Bacillus subtilis, Pseudomonas fluorescens,
aeruginosa. Rhamnolipid 1 (L-rhamnosyl- and Serratia marcescens, respectively. They
L-rhamnosyl--hydroxydecanoyl-- are again grouped to surfactin and lichenysin.
hydroxydecanoate) and rhamnolipid 2 (a) Surfactin: They are amphiphilic molecule
(L-rhamnosyl--hydroxydecanoyl-- - synthesized by B. subtilis that not only
hydroxydecanoate) are the main glycolip- lowers the interfacial tension but also
ids produced by P. aeruginosa (Edward brings about changes in interfacial rheo-
and Hayashi 1965). Bacillus and other logical properties of liquids. It is com-
Pseudomonas sp. like P. chlororaphis and posed of a seven-amino-acid ring structure
P. putida have also been reported for the coupled to a fatty-acid chain via lactone
production of rhamnolipids. Rhamnolipids linkage. They are used in oil recovery and
are fully commercialized for use in manu- remediation of nondispersive pollutants
facture of cosmetics and pharmaceuticals (Arima et al. 1968).
and for the bioremediation of heavy met- (b) Lichenysin: It is similar to surfactin in
als and hydrocarbons. terms of its structural complexity and
(b) Trehalolipids: Trehalolipids are glycolip- physicochemical properties. It is pro-
ids composed of disaccharide trehalose duced by Bacillus licheniformis strains
hydrophilic part. There exists a wide and exhibits excellent stability under wide
diversity of trehalolipids depending on ranges of temperature, pH, and salinity.
the hydrophobic part; it can be a short They may be used in cleaning of oil spills
fatty acid as found in Rhodococcus sp. or and enhanced oil recovery and bioreme-
a long-chain -branched-- hydroxy fatty diation of sites contaminated with pollut-
acids as found in Mycobacterium sp. ants like hydrocarbons and heavy metals.
Trehalolipids are produced by a number B. licheniformis lipopeptide biosurfactant
of different genera of microorganisms, has been reported to lower the interfacial
such as Mycobacterium, Arthrobacter, tension between water and n-hexadecane
Nocardia, Micrococcus, and (Rosenberg and Ron 1999).
Corynebacterium. However, trehalose 3. Fatty Acids, Phospholipids, and Neutral
dimycolates produced by Rhodococcus Lipids: Several bacteria and yeast utilize
erythropolis are the most extensively hydrocarbon and alkanes as carbon source and
studied class of trehalolipids (Asselineau produce a large quantity of fatty acid and phos-
94 S. Busi and J. Rajkumari

pholipid surfactants (Cirigliano and Carman (Cirigliano and Carman 1985), and manno-
1985). In Acinetobacter sp. strain HO1-N, protein obtained from Saccharomyces cerevi-
phosphatidyl ethanolamine-rich vesicles are siae (Cameron et al. 1988).
produced, which are able to produce optically
clear microemulsions of alkanes in water.
Thiobacillus thiooxidans, Corynebacterium 6.7 Properties of Biosurfactant
lepus, and Corynebacterium insidiosum are
some of the organisms reported for production Biosurfactants have advantages over chemical
of phospholipid biosurfactant. R. erythropolis surfactants because of various reasons. The most
when grown on alkane produces phosphatidyl important characteristic of biosurfactant is its
ethanolamine and decreases the interfacial ten- eco-friendly nature. They are biodegradable and
sion between water and hexadecane. have low toxicity, high specicity, and stability at
4. Particulate Biosurfactant: Microemulsions extreme temperature, pH, and salinity (Lang and
playing an important role in alkane uptake by Wullbrandt 1999; Kosaric 1992). These proper-
microbial cells are formed when extracellular ties increase the potential use of biosurfactant in
membrane vesicles partition hydrocarbons. the remediation of xenobiotics such as dyes,
Vesicles of Acinetobacter sp. strain HO1-N heavy metals, hydrocarbons, and other
are composed of protein, phospholipids, and recalcitrant.
lipopolysaccharide (Hommel et al. 1987). In
Sphingomonas sp., the cell surface gets cov- (a) Surface and Interface Activity: The ability to
ered with extracellular vesicles when grown emulsify, i.e., to reduce the interfacial ten-
on polyaromatic hydrocarbons. sion of oil in water, has favored the applica-
5. Polymeric Biosurfactants: Polymeric biosur- tion of biosurfactant for oil recovery from
factants like emulsan, biodispersan, liposan, water (Mulligan 2005). Sophorolipids from
alasan, and lipomanan produced by genera T. bombicola reduce surface and interfacial
Acinetobacter, Pseudomonas, Candida, etc. tension but are not efcient emulsier
are also termed as bioemulsiers. In 1997, (Cooper and Cavalero 2003). By contrast,
Husain et al. reported the production of poly- liposan is not efcient in reducing surface
meric biosurfactant composing of proteins, tension, but has been used successfully to
carbohydrates, and lipid at the ratio of 35:63:2, emulsify edible oils. Surfactin and
respectively, by Pseudomonas nautica. Rhamnolipids efciently reduce the surface
Zinjarde and Pant (2002) also reported the tension of water to 25 mN/m and interfacial
production of emulsier in the presence of tension of water: hexadecane to <1 mN/m.
alkanes or crude oil by marine fungi, Yarrowia The sophorolipids from T. bombicola have
lipolytica. Acinetobacter calcoaceticus been reported to reduce the surface tension to
RAG-1 also produces an extracellular polyan- 33 mN/m and the interfacial tension to 5
ionic amphipathic heteropolysaccharide bio- mN/m. Thus, biosurfactants are more ef-
emulsier (Lawniczak et al. 2013; Cameron cient emulsiers as their CMC is about
et al. 1988). Emulsan, a polyanionic amphi- 1040 times lower to that of chemical surfac-
philic heteropolysaccharide, is a very effec- tants which means less surfactant is required
tive emulsier even at low concentrations, to get a maximum decrease in surface tension
ranging from 0.01 to 0.001 % (Rosenberg (Lang and Wullbrandt 1999).
et al. 1979). Biodispersan, produced by A. cal- (b) Tolerance to Temperature and pH: The most
coaceticus A2, can bind to CaCO3 and TiO2 signicant property of the microbial surfac-
allowing their dispersion in water (Rosenberg tant is that these surface activities are not
et al. 1988b). Bioemulsiers obtained from much affected by environmental stress of
yeasts include the protein-polysaccharide temperature and pH. Lichenysin from B.
complex, liposan from Candida lipolytica licheniformis JF-2 could withstand the
6 Biosurfactant: A Promising Approach Toward the Remediation of Xenobiotics 95

temperature up to 50 C, pH of 4.59.0, and resources, and industrial or municipal

NaCl and calcium ion concentrations up to wastewater.
50 and 25 g/l, respectively. A lipopeptide (f) Specificity: The culture conditions and pres-
obtained from B. subtilis LB5a was found to ence of specic functional groups attribute to
be stable even after autoclaving at 121 C for the specicity in the properties of the biosur-
20 min and after storage for 6 months at 18 factant molecules. These properties are
C; the surface activity was stable at a pH exploited in detoxication of specic pollut-
range of 511 and NaCl concentrations up to ants and emulsication of industrial
20 % (Cooper et al. 1981). efuents.
(c) Biodegradability: Unlike synthetic or chemi-
cal surfactants, biosurfactant is degraded by
bacteria and other microorganisms easily, 6.8 Bioremediation
owing to their simple chemical structure and of Hydrocarbons Using
low toxicity. These compounds donot persist Biosurfactant
for longer time rendering them harmless to
the environment. As most biosurfactants are Biodegradation of a hydrocarbon depends on its
degraded faster than synthetic surfactants, bioavailability and state of dispersion. To maxi-
they are suitable for environmental applica- mize the biodegradation rate, the water-insoluble
tions such as bioremediation and dispersion substrate should either be solubilized or emulsi-
of oil spills. ed. Synthetic detergents used to clean up oil
(d) Low Toxicity: Microbial surfactants are gen- spillages have often led to more destruction of
erally considered nontoxic products, making the environment due to the release of toxic by-
it appropriate for pharmaceutical, cosmetic, products. Biosurfactants due to their emulsifying
food, and environmental applications. property, low toxicity, and high biodegradability
Glycolipid from Rhodococcus species was may play an important role in bioremediation
found to be 50 % less toxic as compared to process (Silva et al. 2014). Biosurfactant can be
Tween 80 (Kanga et al. 1997). A biosurfac- widely employed in petroleum industry where it
tant produced by P. aeruginosa when com- is used to the remediate oil spills and removal of
pared with Marlon A- 350, a synthetic oil residue from storage tanks and in microbial-
surfactant widely used in the industry, was enhanced oil recovery (Sobrinho et al. 2013). In
found to be nontoxic and non-mutagenic, microbes, the biosurfactant both enhances the
while the chemical-derived surfactant indi- bioavailability of inaccessible substrates and
cated higher toxicity and mutagenicity (Flasz helps in survival without desiccation in low
et al. 1998). moisture content. Biosurfactant production gen-
(e) Availability: Biosurfactants can be produced erally requires both a hydrophobic and hydro-
using relatively inexpensive raw materials philic carbon source in the culture medium.
available in abundance. The carbon source Hence, agro-based or industrial waste products
ranging from hydrocarbons, oils, to carbohy- can be used to achieve economically and envi-
drates may be used separately or in combina- ronmentally attractive way of production
tion for large-scale production. Biosurfactant (Makkar et al. 2011; Dziegielewska and
can also be produced by growing microor- Adamczak 2013).
ganisms on industrial wastes, starchy sub- Petroleum efuents mainly contain polycyclic
strates, oil mill efuent, industrial or PHAs (polynuclear aromatic hydrocarbons), sat-
municipal waste/by-products, petroleum urated hydrocarbons and nitrogen-sulfur-oxygen
hydrocarbons, carbohydrates, oil sludge, compounds. Benzene, ethylbenzene, toluene,
olive oil mill efuent, lactic whey and dis- styrene, and xylenes are extensively used as fuels
tiller wastes, starchy substrates, renewable and industrial solvents. Moreover, PHAs serve as
96 S. Busi and J. Rajkumari

the primary substrate for the production of hydrocarbon-degrading and heavy metal-tolerant
pharmaceuticals, polymers, agrochemicals, and bacterium which also had the ability to produce
many more. The low solubility of these high- lipopeptide biosurfactant (Sriram et al. 2011).
molecular-weight hydrophobic compounds leads Sobrinho et al. (2008) found that biosurfactant
to their prolonged persistence as these com- produced by Candida sphaerica exhibited an oil-
pounds are difcult to access by the biodegrading spreading efciency of 75 % when tested on
microorganisms. Biosurfactants can enhance motor oil containing seawater. Alasan was
microbial growth on bound substrates by desorb- recently shown to signicantly enhance the rate
ing them from surfaces or by increasing their of biodegradation of several PAHs (Rosenberg
solubility (Marcoux 2000). The most common and Ron 1999). Noordman et al. (2002) studied
role of biosurfactant in hydrocarbon bioremedia- hexadecane degradation by biosurfactant from P.
tion includes (a) increasing the substrate bio- aeruginosa, and Rahman et al. (2003) examined
availability to microorganisms and (b) interacting the bioremediation of n-alkanes present in petro-
with the cell surface which increases the hydro- leum sludge. Research with biosurfactants from
phobicity. Low-molecular-weight biosurfactant Pseudomonas marginalis indicated that biosur-
that has low critical micelle concentrations factants could solubilize polycyclic aromatic
(CMCs) increases the solubility and mobility of hydrocarbons (PAHs) such as phenanthrene and
hydrocarbons by incorporating them into the enhance biodegradation (Burd and Ward 1996).
hydrophobic cavities of micelles (Miller and Naphthalene and phenanthrene are degraded
Zhang 1997). by Pseudomonas alcaligenes PA-10. The strain
PAHs are one of the many products obtained uses these PAHs as carbon and energy sources
in petroleum rening process (Park et al. 1990). and also co-metabolizes uoranthene (Gordon
Some of the petroleum hydrocarbons are alkanes, and Dobson 2001). According to Nnamchi et al.
cycloalkanes, aromatics, polycyclic aromatic (2006), Pseudomonas aeruginosa and
hydrocarbons, asphaltenes, resins, etc. Saturated Burkholderia cepacia could degrade anthracene
hydrocarbons with straight chain (n-alkanes) are and carbazole. According to Garcia-Junco et al.
susceptible to microbial degradation, but the (2001), Pseudomonas aeruginosa 19SJ degraded
branched alkane and aromatics are difcult to the phenanthrene by producing rhamnolipid
degrade. Many PAHs are suspected to be which increases the bioavailability of phenan-
carcinogenic. threne. According to Arulazhagan and coworkers
Microorganisms with the ability to degrade (2010), Ochrobactrum sp., Enterobacter cloacae,
hydrocarbon compounds are particularly isolated and Stenotrophomonas maltophilia have showed
from petroleum-contaminated sites, and the bio- their potential in degradation of uorene and
surfactant plays a major role in providing the phenanthrene. Anand S. Nayak et al. (2009)
ability to the microorganisms to grow in such reported that the biosurfactant isolated from
extreme habitat and utilize hydrocarbon as car- Pseudoxanthomonas sp. PNK-04 aids in uptake
bon and energy source. According to Itoh and and solubilization of 2-chlorobenzoic acid,
Suzuki (1974), the hydrocarbon utilization abil- 3-chlorobenzoic acid, and 1-methyl naphthalene.
ity of a rhamnolipid-negative mutant strain of P. Obayori et al. (2009) reported 92.34 % degrada-
aeruginosa could only be restored when it is tion of crude oil and 95.29 % removal of diesel
grown in medium supplemented with rhamno- oil by using biosurfactant produced by
lipid. Since then, a large number of biosurfactant- Pseudomonas sp. LP1. Reddy et al. (2010) inves-
producing microorganisms with the ability to tigated the biodegradative properties of biosur-
degrade hydrocarbons have been isolated factant producing Brevibacterium sp. PDM-3
(Bordoloi and Konwar 2009). E. fergusonii strain. The strain could degrade 93.92 % of the
KLU01 was isolated and identied to be a phenanthrene and also had the ability to degrade
6 Biosurfactant: A Promising Approach Toward the Remediation of Xenobiotics 97

other polyaromatic hydrocarbons, anthracene, studied the removal of metals from water by sur-
and uorene. Barkay et al. (1999) tested the solu- factin, using 50,000 Da molecular weight cutoff
bilization of polyaromatic hydrocarbons (PAHs), ultraltration membrane. It was indicated that
phenanthrene (PHE), and uoranthene (FLA) metals, cadmium and zinc, became associated
using bioemulsier, alasan produced by with the surfactin micelles. In 2012, K. Ramani
Acinetobacter radioresistens KA53. et al. reported a lipoprotein biosurfactant isolated
from Pseudomonas gessardii grown on slaughter
house lipid waste and its ability to remove metal
6.9 Bioremediation of Heavy ions in aqueous solution. The lipoprotein biosur-
Metals Using Biosurfactant factant was found to have a high efciency for the
removal of heavy metal and ions in aqueous solu-
Heavy metals are utilized in various industrial tion followed by moderate absorption efciency
activities. These metal residues get mixed up for calcium, copper, and iron. Thus the ability of
with the industrial efuent and subsequently biosurfactant of marine origin to chelate toxic
released into the environment, contaminating heavy metals and form an insoluble precipitate
drinking water and food. These toxic heavy met- could be exploited in treatment of heavy metal-
als being nonbiodegradable accumulate in ani- contaminated water.
mals and humans undergoing a series of
Biosurfactants have the ability to form com- 6.10 Bioremediation
plexes with metals, i.e., the anionic biosurfactant of Insecticide and Pesticides
creates complexes with metals in a nonionic Using Biosurfactant
form, while cationic biosurfactant can replace the
same charged metal ions by ion exchange. Das Microbial remediation of pesticides has attracted
et al. (2009) investigated the ability of anionic increasing attention as a safe, effective, and cheap
biosurfactant produced by marine bacterium in biotechnological approach. Biosurfactants unlike
removing heavy metal from aqueous solutions. It synthetic surfactants have attracted much interest
was found that the percentage removal of lead in environmental remediation due to their excel-
and cadmium was inuenced by the concentra- lent biodegradability, low toxicity, and diverse
tions of metals and biosurfactant. functions (Van Dyke et al. 1991; Yamane et al.
Biosurfactants produced by microorganisms 2008). Biosurfactants have the ability to disperse
were found to enhance biodegradation when low-water-soluble hydrophobic organic pollut-
complexes with metals. Sandrin et al. (2000) ants such as beta-cypermethrin, making it bio-
reported that metal-complexed rhamnolipids available to the bacteria which break it down to
enhance metal biodegradation by Burkholderia nontoxic fragments. Zhang et al. (2011) reported
sp. by reducing metal toxicity to microbial con- Pseudomonas aeruginosa CH7 which produces
sortia. Mata-Sandoval et al. (2001) reported the rhamnolipid which enhances the isomerization
biodegradation of the pesticides by rhamnolipid and biodegradation of beta-cypermethrin by pro-
and Triton X-100, in liquid cultures. Neilson moting adsorption, absorption, and dissolution of
et al. (2003) reported the removal of lead using the hydrophobic compound. Two strains JC1 and
rhamnolipids. Surfactin constitutes two negative JCN13 of Serratia spp. have also been reported
charges, one on the aspartate and the other on the for their ability to degrade 92 % beta-CP within
glutamate residues which allow the binding of 10 days and 89 % within 4 days, respectively.
metals, magnesium, manganese, calcium, bar- Mata-Sandoval et al. (2000) compared the ability
ium, lithium, and rubidium onto the biosurfactant of biosurfactant, rhamnolipid and synthetic sur-
(Thimon et al. 1992). Mulligan et al. (1999b) factant, Triton X-100 in solubilizing pesticides:
98 S. Busi and J. Rajkumari

triuralin, coumaphos, and atrazine. It was found Another chemical that is widely used and
that synthetic surfactant was less effective when poses a great environmental threat is tributyltin
compared to the rhamnolipid. Awashti et al. (TBT). It is an active agent in the antifouling
(1999) evaluated the ability of surfactin produced paints applied to ships, boats, and nets. TBT
by Bacillus subtilis in bioremediation of pesti- which is used to check the growth and attachment
cide, endosulfan, which is reported to cause con- of marine micro- and macroorganisms onto the
genital anomalies, mental retardation, physical submerged objects when leached into the water
deformities, cerebral palsy, epilepsy, hydroceph- bodies could pose a serious threat to human
alus, etc. (Kerala et al. 2011). health. TBT is a biocide which has been banned
in many countries due to its toxic effects on the
targeted as well as nontargeted marine organ-
6.11 Bioremediation of Dyes isms. Sampath et al. (2012) reported biosurfac-
Using Biosurfactant tant rhamnolipid producing Pseudomonas sp.
with the ability to degrade TBT. The emulsifying
In textiles industries, the xation rate of the dyes activity of surfactant produced by the microor-
used is as low as 50 %, and a large quantity of ganisms resulted in the dissolution of the hydro-
unabsorbed toxic dyes are released through phobic compound in the aqueous phase, thus
wastewater discharge at an unprecedented scale. increasing their bioavailability for degradation
Many works have been done on microbial decol- (Table 6.2).
orization and degradation of dyes; however, a few
researches have been done and reported on the
use of microbial biosurfactant for dye degrada- 6.12 Conclusions and Future
tion/removal. According to Jadhav et al. (2011), Perspectives
rhamnolipid produced by Pseudomonas desmo-
lyticum and Bacillus sp. VUS could enhance the The implementation of biosurfactant and
ability of bacterial cells and the enzymes involved biosurfactant-producing marine bacteria in reme-
in the degradation of Brown 3REL dye. It was diating the environment, polluted by industrial
found that there was a decrease in half the time efuents and xenobiotics, has been studied.
required for the degradation of dye, Brown According to Burd and Ward (1996), a strain of
3REL, on permeabilization of Bacillus sp. VUS Pseudomonas marginalis produces high-
with 1 mg/ml rhamnolipid. It might be due to molecular-weight biosurfactant composed of
increase in permeability of cell membrane which protein and lipopolysaccharide which aids in the
in turn facilitates the release of enzyme. It is growth of the strain on PAHs. Cladosporium resi-
thought that surfactants stimulate the effects of nae produces fatty acids and phospholipids extra-
enzymes like -amylase, cellulases, xylanases, cellularly which enhance the alkane degradation.
and phytase. Liu et al. 2008 reported that in the Pyrene hydrocarbons were found to be success-
presence of rhamnolipid, there was 161.98 % fully degraded by different species of
increase in the activity of Lip by Phanerochaete Mycobacterium, Corynebacterium, Nocardia,
chrysosporium. Liang et al. 2010 found that with Pseudomonas, Rhodococcus, and Micrococcus.
increase in activity of enzymes involved in dye Mycobacterium sp. and Corynebacterium sp.
degradation, Lip by 86 %, there was simultane- showed a good degradation rate of pyrene mak-
ous increase in the biodegradation rate of Brown ing them potential agent to remediate pyrene-
3REL by 50 %. Lakshmipathy et al. 2010 reported polluted area (Choi et al. 1996). Garcia-Junco
biosurfactant producing marine actinomycetes, et al. (2001) reported Pseudomonas aeruginosa
Streptomyces spp. VITDDK3, with the ability to 19SJ with the ability to degrade the phenanthrene
decolorize nearly 98 % of the sulfonated azo dye, by the aid of rhamnolipid which increases the
RR5B, in static condition. bioavailability of phenanthrene. Both organic
6 Biosurfactant: A Promising Approach Toward the Remediation of Xenobiotics 99

Table 6.2 Biosurfactant and their applications

Biosurfactant Microorganism(s) Action Reference(s)
Rhamnolipids Pseudomonas cepacia, Bioremediation of marine Jadhav et al. (2011),
Pseudomonas sp., Candida and soil environments Pei et al. (2010),
tropicalis, Vibrio fischeri, Phenanthrene Cassia et al. (2014),
P. fluorescens, P. Zeng et al. (2011),
aeruginosa, Escherichia Liu et al. (2010),
coli, Bacillus subtilis, P. Hexadecane Bondarenko et al.
aeruginosa and P. Phenol (2010), and Das and
desmolyticum Cadmium Mukherjee (2007)
Dye, Brown3REL
Sophorolipids Torulopsis bombicola, Emulsication of oil; Whang et al. (2008),
Torulopsis petrophilum, alkane dissimilation Pesce (2002), and
Torulopsis apicola, C. Baviere et al. (1994)
Trehalose lipids Rhodococcus spp. Enhance bioavailability of Muthusamy et al.
Arthrobacter sp., Nocardia hydrocarbons (2008) and Franzetti
erythropolis, et al. (2010)
Corynebacterium sp.,
Mycobacterium sp
Other glycolipid Pseudozyma hubeiensis Bioremediation of marine Pei et al. (2010)
oil pollution
Fatty acids, phospholipids and neutral lipids
Spiculisporic acid Penicillium spiculisporum Dispersion of hydrophilic Ishigami et al.
pigments; heavy metal (1983), (2000), and
sequestration Hong et al. (1998)
Mannosylerythritol lipid Calyptogena soyoae Bioremediation of marine Pei et al. (2010)
Nocardiopsis lucentensis Bioremediation of marine Pei et al. (2010)
MSA04 environment
Surfactin Bacillus subtilis Enhance solubility of Awashti et al.
hydrocarbons; removal of (1999), Arima et al.
heavy metals and (1968), and Jennema
chlorinated pesticides from et al. (1983)
Lichenysin B. licheniformis, B. subtilis MEOR enhancement of oil Yakimov et al.
recovery (1997) and Thomas
et al. (1993)
Serrawettin Serratia marcescens Enhance bioavailability of Lai et al. (2009)
Phospholipids Acinetobacter sp. Bioremediation Pei et al. (2010)
Lipopeptide Rhodococcus sp. TW53 remediation of oil pollution Pei et al. (2010)
in marine environment
Polymeric biosurfactants
Emulsan Acinetobacter Hydrocarbon emulsier Choi et al. (1996)
calcoaceticus RAG 1
Alasan Acinetobacter Degradation of Barkay et al. (1999)
radioresistens polyaromatic compounds
Liposan Candida lipolytica Bioemulsication Cirigliano and
hydrocarbon in water Carman (1985)
100 S. Busi and J. Rajkumari

and inorganic contaminants can be treated using spreading motility and plant growth promotion.
Environ Microbiol 16(7):22672281
biosurfactants through different processes. Lu
Arima K, Kaknuma A, Tamura G (1968) Surfactin, a crys-
et al. (2003) isolated biosurfactant producing two talline peptide lipid surfactant produced by Bacillus
bacterial species which can effectively degrade subtilis: isolation, characterization and its inhibition of
petroleum hydrocarbons, from the oil- brin clot formation. Biochem Biophys Res Commun
contaminated site of Dawu water source area in
Arulazhagan P, Vasudevan N, Yeom IT (2010)
Zibo City, China. Biodegradation of polycyclic aromatic hydrocarbon by
The property of biodegradability and low tox- a halotolerant bacterial consortium isolated from marine
icity of biosurfactants makes them very promis- environment. Int J Environ Sci Tech 7(4):639652
Asimiea OA, Sam-Wobo SO (2011) The impact of hydro-
ing agents for use in bioremediation of marine
carbon waste from brass oil terminal on the
environment. However, the high cost associated Phytoplankton and Periphyton communities of lower
with production and downstream processing hin- Brass River, Niger Delta, Nigeria. J Emerg Trends Eng
ders the commercial success of biosurfactants. Appl Sci 2:729733
Asselineau C, Asselineau J (1978) Trehalose containing
The use of cheap agro-based and industrial
glycolipids. Prog Chem Fat Lipids 16:5999
wastes as raw materials for production of biosur- Awashti N, Kumar A, Makkar R, Cameotra S (1999)
factant and the efcient isolation and purication Enhanced biodegradation of endosulfan, a chlorinated
process and optimization of growth conditions pesticide in presence of a biosurfactant. J Environ Sci
Health B 34:793803
for higher yield are the key steps in making their
Barkay T, Navon-Venezia S, Ron EZ, Rosenberg E (1999)
production more economically feasible. A better Enhancement of solubilization and biodegradation of
understanding on the biosurfactant production in polyaromatic hydrocarbons by the bioemulsier ala-
microorganisms and the mechanism involved in san. Appl Environ Microbiol 65:26972702
Baviere M, Degouy D, Lecourtier J (1994) Process for
the remediation process may pave the way for
washing solid particles comprising a sophoroside
enhanced clean up of the toxic wastewater pollut- solution. US Patent 5,326,407
ants and better remediation technology. These Bondarenko O, Rahman PKSM, Rahman TJ, Kahru A,
interesting surface-active molecules may serve as Ivask A (2010) Effects of rhamnolipids from
Pseudomonas aeruginosa DS10- 129 on luminescent
an efcient and environmental friendly way to
bacteria: toxicity and modulation of cadmium bio-
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Bordoloi NK, Konwar BK (2009) Bacterial biosurfactants
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Optimization of Cultural
Conditions for Marine Microbial 7
Biosurfactant Production: Future
Prospects from Untapped Marine

K.V. Deepika, Ganji Purnachandra Nagaraju,

and P.V. Bramhachari

Marine microbial biosurfactants are of great importance attributable to
their structural and functional diversity and industrial applications. Despite
huge number of reports describing biosurfactant (BS)/bioemulsier (BE)
applications and advantages, commercialization of these compounds
remains complicated and costly. This is mainly due to the usage of chemi-
cally synthesized media for growth of microorganism resulting in huge
monetary difference between the investment and achievable productivity
from the commercial point of view. Although numerous developments
have been taken place in the industrial biosurfactant sectors, unfortunately,
large-scale production remains economically challenging for many of
such molecules. Therefore, the knowledge on the genetic regulatory mech-
anisms and statistical and genetic algorithms would help to develop an
optimized biosurfactant production method with better product character-
istics and acquired capability of utilizing cheap agro-industrial wastes as
substrates. This review provides an overview of traditional biosurfactant
production methods, statistical optimization techniques, recent biotechno-
logical advances, the role and importance of molecular genetics, metage-
nomics, and gene regulation mechanisms involved in biosynthesis of
various marine microbial surfactants of commercial importance.

K.V. Deepika P.V. Bramhachari (*)

Department of Biotechnology and Botany, Krishna G.P. Nagaraju
University, Machilipatnam 521001, Department of Hematology and Medical Oncology,
Andhra Pradesh, India Winship Cancer Institute, Emory University,
e-mail: Atlanta, GA 30322, USA

Springer Science+Business Media Singapore 2017 105

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_7
106 K.V. Deepika et al.

7.1 Introduction The marine environment which occupies

nearly about three-fourth of the earths surface is
Marine microbes are well known for their numer- a robust reservoir of diverse microbes including
ous biomolecules of paramount signicance such biosurfactant producers. Previous reports on BSs
as antibiotics, enzymes, biopolymers, pigments of marine origin mainly focused on applications
and toxins. It has been reported that more than of biosurfactants in environmental remediation
10,000 metabolites with broad-spectrum isolated from oil-degrading microbes (Rosenberg
biological activities and incredible medicinal et al. 1979; Schulz et al. 1991; Yakimov et al.
properties were isolated from marine microbes 1998; Thavasi et al. 2011; Peng et al. 2008) and
until now (Kelecom 2002). Nevertheless, due to other possible applications such as medical and
the vastness of the marine biosphere, most of the industrial sectors have not been studied compre-
marine microbial worlds remain unexplored. hensively (Rodrigues et al. 2006b; Marchant and
Perhaps only <0.1 % of marine microbial world Banat 2012a, b). Recently, potential application/
has been explored so far (Ramaiah 2005). In the properties of marine BSs such as antimicrobial
midst of diverse marine bioactive compounds, (Mukherjee et al. 2009), biolm disruption
microbial biosurfactants/bioemulsiers (BSs/ (Kiran et al. 2010d) and nanoparticle synthesis
BEs) are of immense importance due to their (Kiran et al. 2010a) have gained much attention.
structural and functional diversity and wide range Microbial surface-active molecules have prospec-
of commercial and industrial applications (Banat tive applications in environmental, food, pharma-
1995a, b; Rodrigues et al. 2006a). Microbial BSs ceutical, petroleum recovery and health-care
are mainly amphiphilic surface-active agents industries (Rodrigues et al. 2006b; Mukherjee
synthesized by bacteria, fungi, yeast and et al. 2006; Banat et al. 2010). Although biosur-
Actinomycetes. They are categorized into glyco- factants exhibit such important advantages, they
lipids, glycolipoproteins, glycopeptides, lipopep- have not been yet employed extensively in indus-
tides, lipoproteins, fatty acids, phospholipids, try because of relatively high production costs.
neutral lipids, lipopolysaccharides (Banat et al. One promising approach for reducing costs is the
2010) and glycoglycerolipids (Wicke et al. 2000). utilization of alternative substrates such as agro-
The applications of BSs include detergency, industrial wastes (Mercade and Manresa 1994).
emulsication, foaming, dispersion, wetting, The main problem related to the use of alternative
penetrating, thickening, microbial growth substrates as culture medium is to nd a waste
enhancement, antimicrobial agents, metal with the proper balance of nutrients that allow
sequestering and enhanced oil recovering. All cell growth and product accumulation (Makkar
these remarkable aspects make BSs potential and Cameotra 1999). The establishment of waste-
candidates to substitute most of the chemical sur- based medium for biosurfactant production also
factants that are currently in use. Besides, BSs encounters another difculty, with regard to the
offer several advantages over chemically synthe- properties of nal product which are reliant on
sized surfactants, such as in situ production using the composition of the culture medium (Besson
economical substrates, lower toxicity, biodegrad- and Michel 1992).
ability and ecological compatibility (Marchant In recent years, the increasing demand for
and Banat 2012a, b). Strikingly, marine microbes natural polymers for pharmaceutical, food and
produce BSs with certain remarkable properties other industrial applications has led to a remark-
including tolerance and stability to pH (312), able concern in extracellular polymeric sub-
temperature (20100 C) and salinity (0.52.0 %) stances produced by marine bacteria (Nichols
(Nerurkar et al. 2009 ). The biosurfactant- et al. 2005; Poli et al. 2010). Bacteria-producing
producing microorganisms reported till date polymers with novel structures and innovative
are principally isolated from terrestrial sources. properties have been isolated in atypical environ-
Nevertheless, studies on BEs from marine ments, including extreme environments (Chi and
sources are very scanty. Fang 2005; Nichols et al. 2005). Therefore,
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 107

marine biosurfactants are one of the prospective in ANN models for training the target data set.
targets in sustainable production processes and Among them, the resilient back propagation
have been considerably increased in the last (RBP) rst-order optimization algorithm exhibits
years. The global market for industrial surfac- improved performance characteristics by recover-
tants is estimated to be more than US$27 billion ing the data over-tting problems during the train-
(Geys et al. 2014). Taking into consideration of ing, which signicantly reduces the time period
the growing demands of microbial surfactants for taken (Mastorocostas 2004; Sivapathasekaran
numerous industrial applications, it is essential to et al. 2010b). Although various statistical methods
improve the yield and productivity of biosurfac- have been benecially employed for process opti-
tant by fermentation (Das and Mukherjee 2007). mization in a bioreactor, there are hardly any
So far, the application of biotechnologically pro- reports on the use of sophisticated optimization
duced surfactants is mainly constrained to certain techniques like ANNGA for enhancing biosur-
specialized areas, as production processes, in factant production in a laboratory-scale fermenter.
general, yet cannot compete with synthetic and This review summarizes fundamental insights
chemical surfactants from an economical related to the factors inuencing the product
viewpoint. This is mainly due to relatively low yields and statistical optimization techniques
product yields, high-priced raw materials, and along with the molecular engineering aspects.
expensive downstream processing. Moreover, Also it emphasizes the modern approaches for
the choice of selected carbon source (sugar or optimized biosurfactants and future prospects of
nonsugar sources) in the growth medium repre- both naturally occurring and genetically modied
sents the rst step for the optimization of biosur- bacterial strains in bioremediation and industrial
factant production (Poli et al. 2010). Therefore, and therapeutic applications.
the optimized culture conditions can modify the
biosurfactant yield and the structure of polymers
by Quesada et al. (2004). 7.2 Factors Affecting
Among several approaches, statistical model- Biosurfactant Production
based optimization of media components that cru-
cially affect the product formation has been the Microbial productions of primary or secondary
most indispensable approach toward promoting metabolites are regulated by the genetic makeup
the production of biosurfactants. Some of the of the producer strain; however, nutritional and
widely used effective statistical techniques environmental factors play a crucial role on the
include response surface methodology and overall output of the metabolite. In addition, the
factorial design (Sen and Swaminathan 1997; knowledge about the regulation mechanisms of
Sivapathasekaran et al. 2010b). Alternatively, the chosen microorganism is highly imperative
mathematical tools such as articial neural net- for the production of microbial metabolites on a
work modeling and genetic algorithm optimiza- large scale. Generally, biosurfactant production
tion (ANNGA) are widely used to optimize can be induced by hydrocarbons or water-
complex nonlinear problems in the eld of bio- insoluble substrates. Several factors inuence
process technology (Pal et al. 2009). The combi- the production of microbial metabolites like
nation of ANN and GA is currently used as a the nature of carbon, nitrogen, and phosphate
powerful tool for product optimization, which is sources; metal ions and other additives used in
much more advanced to response surface method- the media formulation; culture conditions like
ologies (Pal et al. 2009; Sivapathasekaran et al. pH, temperature, agitation rate and oxygen avail-
2010a), particularly for complex processes, such ability; the nature of the selected microorganism;
as biological phenomenon. There are several and nally the adopted fermentation strategies
algorithms that are used for network architecture for large-scale production.
108 K.V. Deepika et al.

7.3 Nutritional Factors surfactant production. Pepi et al. (2005) reported

an Antarctic psychrotrophic Halomonas sp.
7.3.1 The Effect of Carbon Source ANT-3b producing emulsifying compounds when
grown on n-hexadecane, but not on mineral
Several studies revealed production of biosurfac- medium supplemented with D-fructose. On con-
tants by various microorganisms on carbohy- trary, Kokare et al. (2007) isolated a marine
drates, water-immiscible substrates and also Streptomyces sp. S1 showing signicant growth
hydrocarbons (Table 7.1). A signicant observa- on maltose yeast extract medium. Despite the
tion by Banat (1995b) revealed that modest various research reports on carbon substrates for
amount of biosurfactant yield was obtained when biosurfactant production, Raza et al. (2007) stated
cells were grown on readily available carbon that the type, quality and quantity of biosurfactant
sources; nevertheless, the biosurfactant produc- are affected and inuenced by the nature of the
tion was triggered when all the soluble carbon carbon substrate. Biosurfactant yield was limited
was consumed and water-immiscible hydrocar- with galactose or starch as carbon sources because
bon was available in the medium. Robert et al. of inhibition due to the decrease in pH is probably
(1989) reported production of rhamnolipid from caused by the production of secondary acid such
Pseudomonas aeruginosa using a variety of car- as uronic acid (Abouseoud et al. 2008). A marine
bon sources such as C11 and C12 alkanes, succi- Marinococcus sp. isolated from Alang coast,
nate, pyruvate, citrate, fructose, glycerol, olive Gujarat, was able to utilize crude oil along with
oil, glucose and mannitol. Some studies have evi- glucose to produce lipopeptide biosurfactants
denced the use of water-soluble compounds such (Sakalle and Rajkumar 2009). Das et al. (2009b)
as glucose, sucrose, and glycerol as source of car- investigated that glycerol was the best substrate
bon substrates for biosurfactant production for biosurfactant production by marine B. circu-
(Desai and Banat 1997). However, cost-effective lans. Malavenda et al. (2010) isolated an
substrates like molasses (Makkar and Cameotra Antarctic marine biosurfactant-producing bacteria
1999), peat hydrolysate (Sheppard and Mulligan Acinetobacter sp. 11/4, capable of utilizing soy-
1987) and potato processing efuents (Fox and bean oil as carbon source. Interestingly, Al-Nahas
Bala 2000) were suggested for biosurfactant pro- et al. (2011) exhibited highest biosurfactant yield
duction. An interesting approach was put forth of 9.8 g/L in Pseudoalteromonas sp. using glu-
from the study by Mata-Sandoval et al. (2001); cose as a carbon source. IbacacheQuiroga et al.
hydrophobic substrates like corn oil, lard (rich in (2013) isolated a marine biosurfactant-producing
unsaturated and saturated fat) and long-chain bacterial strain Cobetia sp. MM1IDA2H-1, grow-
alcohols signicantly maximized the biosurfac- ing in the presence of sulfur-containing heterocy-
tant production (100165 mg/g substrate), while clic aromatic hydrocarbon dibenzothiophene
hydrophilic substrates like glucose and succinate (DBT). However, in a recent report Singh et al.
acid were shown with poor yields (1236 mg/g (2013) demonstrated that for the production of
substrate). Experimental results showed that the biosurfactant, the preference carbon source
presence of either residual substrate or hydropho- mainly depends on the behavior of strain. More
bic by-products in the rhamnolipid crude mix- recently, Antoniou et al. (2015) reported few
tures can inuence their capabilities to solubilize marine hydrocarbon-degrading bacterial strains
hydrophobic compounds (Mata-Sandoval et al. Pseudomonas pachastrellae strain KMM330,
2001). Pagilla et al. (2002) used soluble acetate Marinomonas vaga strain 40, Thalassospira
and sparingly soluble hexadecane as carbon sub- lucentensis strain QMT2, Roseovarius
strate for Gordonia amarae growth and biosur- crassostreae, Alcanivorax borkumensis SK2,
factant production in large-scale batch reactors. Paracoccus marcusii and Sulfitobacter pontiacus
In another study, Ilori et al. (2005) identied ChLG-10, capable of producing biosurfactant
diesel and crude oil as best carbon sources for bio- using crude oil as a carbon source.
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 109

Table 7.1 Various carbon sources used for the production of biosurfactants by marine microorganisms
Microorganisms Biosurfactant Carbon source Yield Reference
Pseudomonas aeruginosa Glycolipid Glucose 1.5 g/L Guerra-Santos et al.
Pseudomonas aeruginosa Glycolipid Corn oil 46 g/L Linhardt et al.
Bacillus cereus lipopeptide Sucrose 1.6 g/L Cooper and
Goldenberg (1987)
Alcaligenes sp. PHY 9 L.86 Extracellular Tetradecane 1.1 mg/L Goutx et al. (1987)
Arthrobacter sp. EK 1 Trehalose tetraester n-alkanes 4.8 g/L Passeri et al. (1991)
Hahella chejuensis Exopolysaccharide Sucrose 9.23 g/L Ko et al. (2000)
Microbacterium sp. DSM Glycoglycerolipids Glucose 200 mg/L Wicke et al. (2000)
Yarrowia lipolytica, NCIM lipidcarbohydrate Alkanes or crude Zinjarde and Pant
3589 protein oil (2002)
Bacillus licheniformis strain Lipopeptide Glucose 165 mg/L Maugeri et al.
(B3-15) (2002)
Geobacillus sp. 4004 Exopolysaccharide Sucrose 90 mg/L Moriello et al.
Halomonas alkaliantarctica Exocellular Acetate 2.9 g/g Poli et al. (2004)
strain CRSS polysaccharides
Halomonas sp. ANT-3b Glycolipid n-Hexadecane Pepi et al. (2005)
Myroides sp. SM1 Ornithine lipids Weathered crude Maneerat et al.
oil (2006)
Pseudomonas aeruginosa Glycolipid Olive oil 6.58 g/L Thaniyavarn et al.
A41 (2006)
Yarrowia lipolytica Glycolipopeptide Glucose Amaral et al. (2006)
Corynebacterium kutscheri Glycolipopeptide Peanut oil cake 6 4 mg/mL Thavasi et al. (2007)
Rhodococcus sp. TW53 Lipopeptide n-Hexadecane 3.4 g/L Peng et al. (2008)
Pseudomonas aeruginosa Glycolipid Soybean oil 1.42 g/L Prieto et al. (2008)
Azotobacter chroococcum Glycolipid Peanut oil cake 4.6 mg/mL Thavasi et al. (2009)
Bacillus circulans Lipopeptide Glycerol 2.9 0.11 g/L Das et al. (2009b)
Bacillus velezensis strain Lipopeptide Starch Liu et al. (2010)
Alcanivorax dieselolei Proline lipid Diesel oil Qiao and Shao,
strain B-5T (2010)
Brevibacterium aureum Lipopeptide Molasses Kiran et al. (2010b)
Nocardiopsis lucentensis Glycolipid Kerosene Kiran et al. (2010c)
Bacillus licheniformis TR7 Lipopeptide Molasses 3.30 g/L Saimmai et al.
Bacillus subtilis SA9 Lipopeptide Molasses 3.78 g/L Saimmai et al.
110 K.V. Deepika et al.

Table 7.1 (continued)

Microorganisms Biosurfactant Carbon source Yield Reference
Pseudoalteromonas sp. AM Exopolysaccharide Glucose 10.51 g/L Al-Nahas et al.
Zunongwangia profunda Exopolysaccharide Lactose 8.90 g/L Liu et al. (2011)
Lactobacillus delbrueckii Glycolipid Peanut oil cake 5.3 g/L Thavasi et al. (2011)
Pseudomonas nitroreducens Lipopeptide Sodium benzoate 2.9 g/L de Sousa and Bhosle
TSB.MJ10 (2012)
Leucobacter komagatae 183 Lipopeptide Commercial 3.03 g/L Saimmai et al.
sugar (2012b)
Oleomonas sagaranensis Glycolipid Molasses 5.30 g/L Saimmai et al.
AT18 (2012a)
Nocardiopsis sp. B4 Glycolipid Olive oil Khopade et al.
Streptomyces VITSSB2 Exopolysaccharide Mannitol Shubhrasekhar et al.
decanal (2013)
Klebsiella sp. strain RJ-03 Glycopeptide Starch 10.1 0.11 g/L Jain et al. (2013a)
Klebsiella sp. strain RJ-03 Glycopeptide Corn powder 15.40 0.21 Jain et al. (2013b)
Pseudomonas aeruginosa Glycolipid Sucrose 1.35 mg/mL Gomathy and
Senthilkumar (2013)
Inquilinus limosus KB3 Lipopeptide Palm oil decanter 5.13 g/L Saimmai et al.
cake (2013b)
Selenomonas ruminantium Lipopeptide Molasses 5.02 g/L Saimmai et al.
CT2 (2013a)
Aeribacillus pallidus 418 Exopolysaccharide Maltose 0.13 g/L Radchenkova et al.
Pantoea strain BM39 Exopolysaccharide Glucose 21.30 g/L Silvi et al. (2013)
Streptomyces sp. MAB36 Glycolipid Fructose Manivasagan et al.

7.3.2 The Effect of Nitrogen Source source is apparently slower, simulating a

nitrogen-limiting condition that is favorable to
Earlier reports suggest that the nature and con- rhamnolipid production. Many strains of P. aeru-
centration of the nitrogen source also affected the ginosa use nitrates, ammonia and amino acids as
biosurfactant production (Table 7.2). Nitrogen nitrogen sources (Mulligan and Gibbs 1989).
limitation conditions enhanced the production of Some sources report that limiting concentrations
rhamnolipid and sophorose lipid biosurfactants. of nitrogen up to 23 g/L maximize the produc-
According to Mulligan and Gibbs (1989), nitrates tion of rhamnolipid (Linhardt et al. 1989).
should be reduced to nitrite and then to ammonia Generally, the presence of an organic nitrogen
for utilizing them as nitrogen sources. On the source promotes both the specic growth rate
other hand, lipid formation is the rate-determining and biosurfactant production (Farres et al. 1997).
factor during the biosynthesis of rhamnolipid. Conversely, biosurfactant production was shown
Nitrogen limitation may promote lipid accumula- to be higher at lower nitrogen concentration
tion. Consequently, in comparison with ammo- (Gorret et al. 2001). The observations of Mata-
nia, the assimilation of nitrate as a nitrogen Sandoval et al. (2001) have shown that limiting
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 111

Table 7.2 Nitrogen sources used for the production of biosurfactants by marine microorganisms
Microorganisms Biosurfactant Nitrogen source Yield Reference
Alteromonas sp. strain 1644 Exopolysaccharide Ammonium chloride Samain et al.
Pseudomonas aeruginosa Glycolipid Sodium nitrate 1.42 g/L Prieto et al. (2008)
Aspergillus ustus MSF3 Glycolipoprotein Yeast extract Kiran et al. (2009)
Bacillus velezensis strain Lipopeptide Starch Liu et al. (2010)
Brevibacterium casei Glycolipid Peptone Kiran et al.
MSA19 (2010a)
Brevibacterium aureum Lipopeptide Acrylamide Kiran et al.
MSA13 (2010b)
Nocardiopsis lucentensis Glycolipid Beef extract Kiran et al.
MSA04 (2010c)
Pseudoalteromonas sp. AM Exopolysaccharide Meat extract 10.51 Al-Nahas et al.
g/L (2011)
Nocardiopsis sp. B4 Glycolipid Phenylalanine Khopade et al.
Oleomonas sagaranensis Glycolipid NaNO3 5.30 g/L Saimmai et al.
AT18 (2012a)
Leucobacter komagatae 183 Lipopeptide Monosodium 3.03 g/L Saimmai et al.
glutamate (2012b)
Selenomonas ruminantium Lipopeptide Monosodium 5.02 g/L Saimmai et al.
CT2 glutamate (2013a)
Bacillus amyloliquefaciens Lipopeptide peptone 6.76 g/L Dhasayan et al.
MB-101 (2014)
Nocardiopsis MSA13A Glycolipid Yeast extract Kiran et al. (2014)
Streptomyces sp. MAB36 Glycolipid Yeast extract Manivasagan et al.

concentrations of nutrients are not only important the use of ammonium salts in the form of ammo-
to maximize the rhamnolipids production but nium chloride favored the cell growth but did not
also the supplementation of nitrogen sources facilitate the enhanced biosurfactant production
through time intervals. It has been reported that and likewise caused a signicant decrease in pH
organic nitrogen source can promote enhanced (4.03) (Prieto et al. 2008).
cell growth, but proved to be an unfavorable Kiran and colleagues (2009) investigated that
nutritional factor for the production of glycolipid the carbon/nitrogen ratio is one of the most fun-
biosurfactant (Kim et al. 2006). Similarly Chen damental factors which induced the production
et al. (2007) found that nitrate-based compounds of secondary metabolites and demonstrated that
and inorganic nitrogen sources were recognized C/N ratio of 3:2 (glucose/yeast extract) facili-
as an excellent source of nitrogen for the rhamno- tated the highest biosurfactant production in
lipid production by Pseudomonas aeruginosa S2, marine Aspergillus ustus (MSF3). A similar study
giving a maximum rhamnolipid concentration by Gandhimathi et al. (2009) showed that pep-
of 2300 mg/mL. Few investigators reported tone supplementation enhanced the lipopeptide
nitrogen-limiting conditions (C/N ratio of 100) of biosurfactant yield in marine Actinomycetes
soybean oil and sodium nitrate as favorable to Nocardiopsis alba MSA10. However, Kiran
biosurfactant production by Pseudomonas aeru- et al. (2010c) observed that the maximum pro-
ginosa. The same group established the fact that duction of biosurfactant by Nocardiopsis lucen-
112 K.V. Deepika et al.

tensis MSA04 occurred at a C/N ratio of 0.5 oxyethylene sorbitan monopalmitate), Tween 80
(kerosene/beef extract) predicting that a higher (polyoxyethylene sorbitan monooleate), CHAPS
amount of nitrogen source was required by the (3-[(3-cholamidopropyl) dimethyl ammonio]-
strain compared to that of carbon source. 1-hydroxypropane-sulfonate), and Triton X 100
However, Al-Nahas et al. (2011) reported that (nonaethylene glycol octylphenol ether) may
inorganic nitrogen sources weakly supported ameliorate oxygen concentration in the growth
growth and polysaccharide production and media increasing the production of exopolysac-
resulted in a substantial decrease in biomass and charides (Morin 1998). Furthermore, Mata-
product yield; on the other hand, organic nitrogen Sandoval et al. (2001) revealed that best results of
sources favor both growth and polysaccharide rhamnolipid production were obtained under
production. Khopade et al. (2012a) reported yeast minimal nutrient conditions which help in direct-
extract as best nitrogen source for biosurfactant ing the cellular metabolism to the production of
production by marine Streptomyces species B3. rhamnolipids rather than to increase the cell
The maximum production of the biosurfactant by population. According to Lee et al. (2001) high
marine Nocardiopsis occurred at a C/N ratio of aeration rates generally enhanced production of
2:1 using olive oil and phenylalanine as carbon extracellular substances and increased the viscos-
source and nitrogen sources (Khopade et al. ity of the culture broth of Hahella chejuensis. Yeh
2012b). These reports evidently signify that C/N et al. (2006) showed that the supply of oxygen
ratio is certainly an essential parameter for the acts as a limiting factor for growth and lipopep-
production of biosurfactant. tide biosurfactant production. Biosurfactants
production from the Bacillus sp. was found to be
an aerobic process, requiring aeration for their
7.3.3 The Effect of Other Factors/ growth and metabolite formation (Lee and Kim
Sources 2004).
Agitation is another key factor for biosurfac-
Production, composition and nal yield of bio- tant production, which helps to distribute the
surfactants also depend on the fermenter design oxygen evenly in the medium and maintain the
and nutrient composition (Mulligan and Gibbs dissolved oxygen concentration at the desired
1993). Besides trace elements, amino acids, sup- level. Previous studies reveal that Bacillus sp.
plementation of nanoparticles, aeration, agitation grown in a highly agitated reactor environment
and rheology of growth media also inuence bio- for better production of metabolites (Joshi et al.
surfactant production as evidenced in several 2008). The use of Fe metal nanoparticles as nutri-
studies. Earlier studies by Zobell (1941) reported ent supplements came into light with the studies
the use of SrCl2 to facilitate the growth of marine of He et al. (2006) to enhance biosurfactant
strains which act as a growth stimulant. Morin production by marine Actinobacterium. This
(1998) reported that fermentation broth may approach would reduce the impact of nonmetallic
develop non-Newtonian characteristics acting ions of the metal salts in a fermentation process.
as a pseudoplastic uid due to the presence of In optimizing the medium, besides the carbon
extracellular substances and their metabolic and nitrogen sources, the role of trace metals has
products, as well as the lack of homogeneity in also been reported to be critical in enhancing
terms of mixing, mass and oxygen. Thus, the lipopeptide biosurfactant production (Wei et al.
rheological shifts during the fermentation 2007). Mukherjee et al. (2008) during statistical
process could be used as a parameter to monitor screening of nutritional parameters by a marine
the constancy and quality of extracellular bacterium found that optimal concentration of
substances and their production. Moreover, the MgSO4 at 0.3 g/L could yield more biosurfactant
utilization of detergents such as Tween 40 (poly- compared with when MgSO4 was used at high
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 113

concentration. Kiran et al. (2010b) reported a production from Bacillus subtilis ATCC 21
marine Brevibacterium casei MSA19 producing 332 in a bioreactor.
maximum biosurfactant in the presence of FeSO4 A deep-sea psychrotolerant bacterium
and asparagine in culture medium. Enhanced bio- Pseudoalteromonas sp. SM9913 produced high
surfactant production by marine Nocardiopsis amount of extracellular substances (5.25 g L1) in
MSA13A through the supplementation of FeCl3 the temperature range 3010 C (Qin et al. 2007).
followed by CuSO4 was evidenced by the reports Previously, Chi et al. (2007) detected the produc-
of Kiran et al. (2014). tion of 22.34 g/L of exopolysaccharide by marine
Cyanobacterium Cyanothece sp. 113 at 29 C,
aeration rate of 7.0 l/min and continuous illumi-
7.4 Environmental Factors nation with 4300 lx. Additionally Su and co-
workers (2007) achieved 18.4 g/L of
There have been a number of scientic reports exopolysaccharide produced by the same strain
describing individual effects of temperature, pH, Cyanothece sp. 113 at 29 C in an optimized
salinity, aeration and agitation on biosurfactant medium with 70.0 g/L of NaCl, 0.9 g/L of MgSO4
production. Desai and Banat (1997) also stated and continuous illumination at 86.0 E/
that environmental factors such as pH, tempera- M2/S. Some authors reported the maximum bio-
ture, agitation and oxygen availability and growth surfactant production from Bacillus subtilis 20B
conditions also affect biosurfactant production (Joshi et al. 2008) and Bacillus subtilis HOB2
by inuencing their cellular growth or activity. (Haddad and Wang Ji 2009) was achieved at a
Studies by Gugliandolo and Maugeri (1998) low temperature of 30 C. Chayabutra and Ju
examined the highest production of (2001) found that the rate of rhamnolipid produc-
exopolysaccharides by a thermophilic isolate tion by Pseudomonas aeruginosa ATCC 10145
Bacillus thermodenitrificans strain B3-72 at an signicantly increased when pH was in the range
optimal temperature of 65 C and pH 7.0 in aero- of 6.56.7. A tropical marine strain of Yarrowia
bic conditions. Yakimov et al. (1998) isolated six lipolytica, NCIM 3589 require an initial pH of
heterotrophic, biosurfactant-producing, n-alkane- 8.0 for extracellular emulsier production
degrading marine bacterial strains grew at an (Zinjarde and Pant 2002). Previous studies
optimal temperature between 25 and 30 C, at revealed that most lipopeptide biosurfactants are
NaCl concentrations ranging from 10 % to produced better in acidic conditions. For exam-
12.5 %. A study by Moriello et al. (2003) reported ple, the enhanced surfactin production by
a marine Geobacillus sp. 4004 produced 90 mg/L Bacillus subtilis ATCC 21 332 (Wei et al. 2004);
of extracellular substances at a temperature of maximum surfactin concentration from Bacillus
60 C at pH 7.0. In addition, Mancuso Nichols subtilis ATCC 21 332 at a pH value of 6.25 (Yeh
et al. (2004) depicted the yield of extracellular et al. 2006); lipopeptide production by Bacillus
substances by Pseudoalteromonas CAM025 circulans at a pH of 6.5 (Sivapathasekaran et al.
grown at 2 and at 10 C was 100 mg exopoly- 2010b). The optimized pH value clearly docu-
saccharides per gram dry weight of cells. This is mented that enhanced biosurfactant production
attributed to the fact that production at low tem- by marine strains required acidic conditions.
peratures may be a mechanism for adaptation to Salinity was found to be one of the critical param-
cold temperature for this strain. These results fur- eter in the production of biosurfactant. Marine
ther concluded that, the production of extracel- bacteria are generally lysed in hypotonic media
lular substances may be a successful evolutionary and particularly in distilled water (Bertrand and
strategy for bacteria inhabiting extreme sea ice Larsen 1989). Salt concentrations affect biosur-
environments. Yeh et al. (2006) reported an factant production depending on its effect on
optimum temperature of 30 C for surfactin cellular activity. Some biosurfactants however,
114 K.V. Deepika et al.

were not affected by salt concentrations up to 7.5 Statistical Optimization

10 % (w/v), although slight reductions in the crit- Techniques
ical micelle concentrations (CMCs) were for the Production
detected (Abu-Ruwaida et al. 1991). NaCl acti- of Biosurfactants
vated biosurfactant activity of many strains,
which were isolated from seawater or petroleum The PlackettBurman design is a widely used
reservoirs (Yakimov et al. 1995). statistical design technique for the screening of
Fernandez-Linares et al. (1996) investigated the medium components (Plackett and Burman
the effects of NaCl concentration on growth, 1944). RSM was used to optimize the medium
biodegradation and the different modes of trans- composition. Response surface methodology
fer of hydrocarbons to the cell surface. Because (RSM) is a collection of different statistical tech-
cytoplasmic bacterial membrane is permeable to niques, including designing experiments, build-
water but not to most other metabolites, hyper- ing models and evaluating the effects of factors
or hypoosmotic shock causes instantaneous to trigger desirable responses (Li et al. 2002). It
efux or inux of water, which is, respectively, has the intense ability to interpret the interactive
followed by a decrease or an increase of the effects among input variables, which are some of
cytoplasmic volume. A tropical marine strain of the attractive features of RSM (Montgomery
Yarrowia lipolytica, NCIM 3589, requires 1997; Al-Araji et al. 2007). The statistical
sodium chloride at a concentration of 23 % for approach could overcome the limitations of clas-
extracellular emulsier production (Zinjarde sical medium optimization (Lotfy et al. 2007).
and Pant 2002). In another study, B. subtilis Response surface methodology (RSM) designs,
BBK-1 produces three types of lipopeptides help designers to quantify the relationship
bacillomycin L, plipastatin and surfactin in the between one or more measured responses and the
presence of NaCl up to 8 % (Roongsawang et al. vital input factors, were successfully applied by
2002). Marine isolate showing an optimum many researchers to build reliable models and
activity in the salt supplemented medium was nd the optimal medium for a greater biosurfac-
reported by Kiran et al. (2009). Another marine tant yield (Rodrigues et al. 2006c; Kiran et al.
isolate, Pseudoalteromonas agarovorans, was 2010b; Sivapathasekaran et al. 2010b). Therefore,
shown to produce high amount of extracellular the statistical experimental designs are powerful
substances at an increased NaCl concentration tools for searching the key factors rapidly from a
of 30 g/L (Choi et al. 2009). Subsequently, multivariable system and minimizing the errors
Kiran and colleagues (2010b) reported a marine in determining the effect of parameters (El-Sersy
Brevibacterium casei MSA19 producing maxi- 2012).
mum biosurfactant in the presence of 2 % Mukherjee et al. (2008) determined critical
NaCl in culture medium. Al-Nahas et al. factors affecting the growth and biosurfactant
(2011) reported that a marine bacterium production by a marine bacterium using a
Pseudoalteromonas sp. AM grew only when PlackettBurman-based statistical screening pro-
NaCl concentration was increased from 10 to 30 cedure. Optimized medium demonstrated 84.7 %
g/L showing an increase in cell growth and pro- increase in biosurfactant yield over the unopti-
duction of extracellular substances. However, in mized medium. Statistical optimization of bio-
a recent report, NaCl concentration up to 8 % surfactant production from the sponge-associated
and pH 68 worked most excellently in case of marine fungus Aspergillus ustus MSF3 using
halophilic bacteria Halomonas sp. BS4 (Donio RSM was reported by Kiran et al. (2009). The
et al. 2013). optimized bioprocess conditions for the maximum
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 115

production were pH7.0, temperature 20 C, salt signicantly inuenced by the variables such as
concentration 3 %, glucose and yeast extract as glucose, yeast extract, ferrous nanoparticles (Fe
carbon source and nitrogen sources, respectively. NPs) and inoculum size either interactively or
Statistical approaches aid in the formulation of independently. In another study, Mabrouk et al.
production medium of biosurfactant and may be (2014) statistically optimized the glycolipopep-
crucial to enhance the quantity of the product tide production by soft coral-associated marine
(Kiran et al. 2010b). In another study by Kiran et. Bacillus sp. E34 using Plackett-Burman design
al (2010b), optimization and characterization of a (PBD) and recorded signicant increase (1.28-
new lipopeptide biosurfactant produced by fold) in the emulsication index. Interestingly,
marine Brevibacterium aureum MSA13 in solid- Dhasayan and colleagues (2014) attained
state culture were reported. Thus, it is most likely maximum biosurfactant production of 6.76 g/L
that the statistical optimization with pretreated by B. amyloliquefaciens MB-101 using response
molasses as substrate and olive oil, acrylamide, surface statistical optimization methods in
FeCl3 and inoculum size as critical control fac- glycerol-containing medium under submerged
tors resulted in threefold increased lipopeptide fermentation and nal yield was 3.48-fold
production over original conditions. An interest- higher than that of non-optimized normal media.
ing result came from a study by Sivapathasekaran Manivasagan et al. (2014) isolated potential
and colleagues (2010b) by achieving an average glycolipid biosurfactant producer Streptomyces
biosurfactant concentration of 3.01 0.02 g/L by sp. MAB36 from marine sediment samples.
Bacillus circulans through statistical modeling- Medium composition and culture conditions for
based response surface methodology (RSM) only the glycolipid biosurfactant production by
after identifying the critical components in modi- Streptomyces sp. MAB36 were optimized, using
ed marine medium using the OFAT technique. two statistical methods: PlackettBurman design
Kiran et al. (2010c) validated signicant interac- was applied to nd out the key ingredients and
tive inuence of secondary control factors such conditions for the best yield of glycolipid biosur-
as copper sulfate and inoculum size in glycolipid factant production and central composite design
biosurfactant production by marine Nocardiopsis was used to optimize the concentration of the
lucentensis MSA04 using RSM experiments. four signicant variables, starch, casein, crude
Enhanced biosurfactant production was achieved oil, and incubation time in terms of emulsica-
under solid-state culture (SSC) conditions using tion index (E24%).
kerosene as carbon source, beef extract as nitro-
gen source and wheat bran as substrate. The most
convincing evidence to maximize biosurfactant 7.6 Metabolic Engineering
production by Marine Bacillus subtilis N10 was for the Production
demonstrated by El-Sersy (2012) by using of Biosurfactants
Plackett-Burman experimental design (PBD),
evaluated in terms of emulsication index (E24) The continuous demand to reduce production
(80 %), i.e., 1.14-fold increase when compared to costs and to generate compounds with enhanced
its production in basal conditions. These statisti- properties for absolute applications has conse-
cal techniques are therefore used for identifying quently initiated biosurfactant engineering
important factors from among many potential projects based on a variety of biotechnological,
factors. In the analysis of these designs, usually molecular, or genetic approaches (Fig. 7.1).
only main effects are estimated. Direct and combinatorial strategies were previ-
Statistical optimization of biosurfactant pro- ously attempted to produce arrays of modied
duction by marine Nocardiopsis sp. MSA13A products in particular of lipopeptide derivatives.
under solid-state culture was carried out by Kiran Several studies were focused on the lipopeptide
et al. (2014). It was found that the production was engineering, dynamics and new structural fea-
116 K.V. Deepika et al.

Fig. 7.1 Potential targets for optimization of biosurfactant production

tures of lipopeptide synthetases. Microorganisms enzymes, or manipulation of precursor structures

produce a set of related lipopeptides (LPs), and are often employed (Baltz 2006).
the composition of their production prole can be
inuenced by abiotic and nutritional culture con-
ditions. Biosurfactant production can be modu- 7.7 Concept of Strain
lated by the fermentation conditions and by Engineering in Biosurfactant
feeding of specic precursors (Pryor et al. 2007). Production
Further options for the structural tailoring of bio-
surfactants are to manipulate precursor pathways Although enormous efforts have been made to
or modifying enzymes that act posttranslational investigate the potential candidates to replace P.
on the synthesized compounds, e.g., by attach- aeruginosa, none of them were successful due to
ment of specic residues. Mutation of external signicantly lower yields and nal concentra-
enzymes involved in selection and activation of tions. Hence, the marine biologists presume to
the fatty acid can result in the formation of LPs investigate wild-type P. aeruginosa strains for
with modied fatty acid chains (Powell et al. economic yields and impending objectives for
2007). Feeding modied precursors or manipula- genetic optimization. Further enhanced yields
tion of specic precursor pathways can thus achievable with these wild-type strains may
result in new products (Amir-Heidari et al. 2008). present a strong basis for further optimization by
The N-terminal fatty acid moiety determines to a strain engineering. Several approaches were put
large extent the biological properties of LPs. forth to optimize the rhamnolipid production in
Complex combinatorial platforms that comprise producer strains by genetic modifications
a set of different approaches like enzymatic (Fig. 7.1). Previous studies suggested few alter-
module swapping, complete protein subunit natives to be considered in strain engineering: the
exchanges, modication of accessory tailoring use of alternative nonpathogenic production
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 117

strains, broadened substrate spectrum, metabolic et al. (2011). On the contrary, a yield of 1 g/L of
spectrum and pathways of producer strains, by- mono-rhamnolipids was obtained by the growth
product formation and optimized carbon of the nonpathogenic Pseudomonas chlororaphis
yield, product formation and genetic regulatory on 2 % glucose (Gunther et al. 2005).
mechanisms and availability of precursor mole- However, traditional engineering by random
cules (Henkel et al. 2014). The approach of strain and targeted genetic alteration, process design
engineering mainly focused on the improvement and recombinant strategies in rhamnolipid bio-
of product yields which require a complex inter- synthesis did not succeed. For enhanced process
actions of genetics (e.g., deletions, insertions, development, there is an urgent need of in-depth
alterations in promoter strength), analytics (e.g., information about the rhamnolipid production
metabolome and transcriptome analysis), and regulation during bioreactor cultivation to design
systems biology (e.g., metabolic ux distribu- knowledge-based genetic and process engineer-
tions and modeling), to quantify the effects of ing strategies (Mller and Hausmann 2011). No
modication and identify prospective hurdles signicant differences in the quantity or compo-
(Mller and Hausmann 2011). A signicant con- sition of the rhamnolipid congeners could be pro-
tribution from Koch et al. (1991) described rham- duced by manipulating the growth conditions.
nosyltransferase 1 complex (rhlAB) as the key Sequences for the rhamnolipid genes indicated
enzyme responsible for rhamnolipid production low levels of strain variation and the majority of
and genetic modication of rhamnolipid produc- polymorphisms did lead to amino acid sequence
ing P. aeruginosa was performed by inserting the changes that had no evident phenotypic effect.
E. coli lacZY genes into its chromosome so as to The expression of the rhlB and rhlC rhamnosyl-
make it capable of growing and utilizing lactose transferase genes showed a xed sequential
from whey for enhanced biosurfactant produc- expression pattern during growth and no signi-
tion. The industrial exploitation of P. aeruginosa cant upregulation could be induced by varying
is limited due to its pathogenic nature and pro- producer strains or growth media. The same
duction of wide variety of virulence factors. To study demonstrated that rhamnolipids are highly
surmount this difculty, the rhlAB genes from P. conserved molecules and their gene expression
aeruginosa were cloned and expressed in heter- has a rather stringent control. This leaves little
ologous hosts (Ochsner and Reiser 1995). The opportunity to manipulate and greatly increase
recombinant strains P. fluorescens ATCC 15453 the yield of rhamnolipids from strains of P. aeru-
and P. putida KT2442 having the rhamnolipid ginosa for biotechnological applications
gene cluster demonstrated higher yields of 0.25 (Perfumo et al. 2013). Increase in yield of natural
and 0.6 g/L, correspondingly, when compared LPs can be obtained by general engineering
with the parent strain. A similar strategy was approaches of the producer strains. Modifying
adopted by Cha et al. (2008), where the opportu- the gene regulatory regions involved in biosur-
nistic pathogen P. aeruginosa EMS1 (yielded 5 factant expression by up-mutations may be con-
g/L rhamnolipid) was replaced by P. putida by sidered. Simplied strategy would be to replace
molecular cloning of the rhlAB rhamnosyltrans- weak endogenous promoters with strong promot-
ferase gene. Apparently, this engineered strain ers that can even be better controlled by stable
yielded high amount of rhamnolipids by utilizing inducers. The common strategy for molecular
glucose as a carbon source (Wittgens et al. 2011). engineering approaches is to modify the desired
However, P. aeruginosa PAO1 was not preferred parts of the cloned biosynthetic pathways by
as the appropriate model organism due to its viru- standard techniques in E. coli, to transfer the
lence characteristics; it exhibited promising modied genetic elements into the producer
results in rhamnolipid yields. Surprisingly, the strain and to obtain stably engineered producer
yield of 39 g/L after 90 h of cultivation with sun- strains by recombination (Koglin et al. 2010).
ower oil as substrate and under nitrogen- Several authors studied the molecular engineer-
limiting conditions has been reported by Muller ing aspects of production of surfactants (Baltz
118 K.V. Deepika et al.

2009; Alexander et al. 2010). However, few large sequence datasets such as genomes and
problems may be associated with the molecular metagenomes for the presence of gene clusters
engineering of biosurfactants; in view of that, associated with marine biosurfactant molecules
small change in biosurfactant chemical structure of interest. One such tool, antiSMASH (the
may lead to signicant problems for the micro- antibiotic and secondary metabolite analysis
bial physiology of producer strains. Even though shell), quickly identies and annotates second-
much knowledge has been gained on biosynthe- ary metabolite gene clusters from genomic
sis of LPs, only few molecules were familiar on sequence data (Medema et al. 2011) and has
their secretion mechanisms and metabolic path- been used to identify biosurfactant-related
ways inside the cell. Compounds with altered genes in the genome of Serratia marcescens
bioactivities could turn toxic to the producer or strain Db11 (Gerc et al.2014). A number of
they might impart negative side effects to other screening methods are available for screening
cellular processes. Export systems might not large-insert metagenomic clone libraries for
efciently recognize modied LP structures, or biosurfactant activities. Recent successes
they could become overloaded by increased include the heterologous production of biosur-
biosurfactant synthesis resulting in intracellular factants. Recently, few noteworthy contribu-
product accumulation. Manipulation of precursor tions were made in this area with more similar
or posttranslational modication pathways discoveries highlighting the wide scope of these
could affect other biosynthetic systems with advanced techniques in sustainable production
consequences that are difcult to envisage. of biosurfactants, cloning of srwW gene of S.
Co-engineering of associated pathways and marcescens into E. coli using a constitutively
enzymes might therefore be indispensable in expressed promoter (Thies et al. 2014), employ-
order to ascertain stable and efcient producer ing PCR, gene-walking and cloning to express
cell lines for modied or newly designed biosur- a sophorolipid-related glucosyltransferase gene
factants (Koglin et al. 2010). (gtf-1), from C. bombicola, in Saccharomyces
cerevisiae (Solaiman et al. 2014), expression of
partial biosurfactant production genes in S.
7.8 Metagenomics for Marine cerevisiae (Huang et al. 2014). Nonetheless, the
Biosurfactant Discovery complex regulatory mechanisms governing bio-
surfactant production may become main dif-
Metagenomics can lead to an invention of novel culty to metagenomic approach of marine
biosurfactants, tackling issues of low produc- biosurfactant synthesis. In view of the fact that
tion yields. Metagenomic analyses comprise production is interlinked to growth through
both sequence-based and function-based strate- quorum sensing mechanisms and stress or
gies. Sequence-based analyses have advanced nutrient depletion responses, strategies like
enormously over the last decade with the prog- addition of precursor molecules to growth
ress of next-generation sequencing (NGS) plat- media and optimization of bioprocess engineer-
forms and ensuing scalability, with signicant ing techniques are highly warranted in the
cost reductions. This has enabled the querying upcoming years. The use of metagenomic DNA
of enormous metagenomic sequence datasets to from suitable marine environments, proper
identify protein-coding sequences based on functional screening and suitable heterologous
homology to reference sequence data in curated host systems will collectively aid in the discov-
sequence databases (Kanehisa and Goto 2000). ery of novel molecules with remarkable surface-
Software tools are also now available to query active and emulsication properties.
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 119

7.9 Modern Approaches order to select the critical medium components

for Optimization for marine biosurfactant production. This model
of Biosurfactant Production revealed that production process optimization
was maximized by involving an articial neural
As the process economy plays an imperative role network and genetic algorithm (ANN-GA)-aided
in determining the absolute use of the product, it experimental modeling yielding 4.61 0.07 g/L
has become very crucial for researchers to after 22 h of fermentation. In one of the research
enhance the yield and biosurfactant productivity. report, free Fe2+ was reported to be sequestered
The utilization of cheaper substrates has emerged by the marine Bacillus megaterium lipopeptide as
as one of the attractive options, which further jus- it was produced. Therefore, Rangarajan et al.
ties the demand of biosurfactant for low-end (2012) adopted a time-dependent multi-dosing
applications such as enhanced oil recovery, waste Fe2+ feeding strategy for the enhancement in the
water treatment and bioremediation (Das and maximum lipopeptide product yield (YP/S) when
Mukherjee 2007; Mukherjee et al. 2006; Das compared with glucose mineral salt medium
et al. 2009a). Further, the adoption of various (GMSM) without Fe2+ supplementation.
process intensication strategies (Table 7.3) has In addition to that, a comparative performance
resulted in the improved biosurfactant produc- evaluation of batch and semicontinuous reactor
tion. Thus, several attempts were made to operations was demonstrated for the optimized
enhance the product yield and process productiv- production of a marine microbial surfactant
ity by modern techniques (Sivapathasekaran and using a LuedekingPiret model. Interestingly,
Sen 2013a). Interestingly, Abbasi and Ahmadian Sivapathasekaran and Sen (2013a) demonstrated
(2012) developed a Sugeno-type adaptive neural that unsteady state fed-batch (USFB-I) operation
fuzzy inference (ANFI) modeling technique in showed improvement in the biosurfactant

Table 7.3 Modern approaches for optimization of biosurfactant production in marine microorganisms
Microorganisms Biosurfactant Method Yield Reference
Bacillus circulans Lipopeptide CCD, ANN-GA 4.61 0.07 Sivapathasekaran and Sen
(DMS-2)MTCC 8281 g/L (2013b)
Bacillus circulans Lipopeptide Unsteady state 6.2 g/L Sivapathasekaran and Sen
(DMS-2)MTCC 8281 fed-batch strategy (2013a)
Bacillus megaterium Lipopeptide Bioprocess design 5.34 0.1 Rangarajan et al. (2015)
MTCC 8280 g/L
Bacillus megaterium Lipopeptide Intermittent Fe2+ dosing 4.2 0.15 (Rangarajan et al. 2012)
Bacillus subtilis RB14 Lipopeptide Heat-induced 4 g/L Rahman et al. (2006)
Bacillus subtilis ATCC Lipopeptide Cell immobilization and 320 mg/L Chtioui et al. (2010)
21332 lipopeptide recovery by
Bacillus subtilis ATCC Lipopeptide Bubbleless membrane 230 mg/L Coutte et al. (2010)
21332 bioreactor
Bacillus subtilis GB16 Lipopeptide Novel oxygenation Lee and Kim (2004)
Bacillus subtilis DSM Lipopeptide In situ removal by Glazyrina et al. (2008)
21393 automated surface
Bacillus subtilis ATCC Lipopeptide Non-foaming modied 787 mg/L Chtioui et al. (2014)
21332 rotating discs reactor
120 K.V. Deepika et al.

production by about 35 % over batch mode com- obligate hydrocarbonoclastic bacteria (OHCB) to
pared to USFB-II. Lee and Kim (2004) developed thrive, becoming the majority of the total microbial
a novel oxygenation method to achieve signi- population (Yakimov et al. 2007). The surface-
cant enhancement in the lipopeptide production active compounds produced by these organisms
by increasing the dissolved oxygen concentration mediate the dissolution of hydrocarbons by inter-
during the microbial production of lipopeptides acting with the insoluble compounds, reducing
by Bacillus sp. GB16. In another study, Rahman the interfacial tension, and making them avail-
et al. (2006) investigated the production of lipo- able to the microbes (Gnanamani et al. 2010).
peptides using induced germination of the spores The diverse assortment of hydrocarbons present
by heat activation and nutrient supplementation. in crude oil involves resource partitioning by
A new method to remove and separate biosurfac- microbial populations, and microbial modica-
tants from complex mixtures by compressing and tion of oil components and the surrounding
harvesting the liquid surface layer was developed environment will lead to temporal succession.
by Glazyrina et al. (2008). The exploitation of Several reports put forth the utilization of
bubbleless bioreactors with aeration by a hollow marine microbes as potential hydrocarbon and
ber membrane airliquid contactor was devel- toxic organic pollutant degraders. Alcanivorax
oped by Coutte et al. (2010) for the production of borkumensis is known for its ability to grow
lipopeptides by B. subtilis ATCC 21332. Chtioui metabolizing only alkanes and producing glyco-
et al. (2010) reported the enhanced synthesis of lipid biosurfactants (Yakimov et al. 1998).
both lipopeptides and especially of the fengycin Melcher et al. (2002) isolated phenanthrene or
by the immobilized aerobic cells of Bacillus sub- chrysene strains from San Diego Bay sediments
tilis ATCC 21332. Chtioui et al. (2014) illustrated representing the genera of Vibrio, Marinobacter,
the production of lipopeptides fengycin and sur- Cycloclasticus, Pseudoalteromonas,
factin in rotating disk bioreactor which could be Marinomonas and Halomonas. Coelho et al.
very useful for the production of other molecules (2003) investigated quinoline-degrading marine
using bioprocesses requiring bubbleless oxygen bacterium Pseudomonas sp. strain GU 104 iso-
supply. More recently, Rangarajan et al. (2015) lated from coastal waters of Goa, India. Another
elucidated the effect of limiting oxygen and interesting study by Thavasi et al. (2006)
nitrogen sources on lipopeptide selectivity described the hydrocarbon-degrading potential of
through a bioprocess design for selective a marine bacterium Azotobacter chroococcum
enhancement of fengycin production by marine isolated from Tuticorin harbor where degradation
Bacillus megaterium resulting in the enhanced of 58 % crude oil was evidenced in experimental
lipopeptide production. studies. In cold marine environments, the obli-
The bioremediation of coastal and marine gate alkane-degrading psychrophile, Oleispira, is
environments is signicantly beneted with the commonly associated with oil spills (Coulon
use of biosurfactants/bioemulsiers. Apparently, et al. 2007). Edlund and Jansson (2008) revealed
marine microbes present higher percentage of a remarkable diversity of putative PAH degraders
surfactant production compared to terrestrial belonging to the genera Exiguobacterium,
species (Maneerat 2000). Since marine source is Shewanella, Methylomonas, Pseudomonas,
the critical contaminated site, the microbial Bacteroides, as well as Deltaproteobacteria and
population of marine sources may have the inher- Gammaproteobacteria from marine sediments.
ent capacity to remediate the contaminants at the The dominant benzo[a]pyrene-degrading
fastest rate and have robustness in solubilizing bacteria from a marine enrichment were isolated
and degrading the PAHs (Plante et al. 2008). and faster degradation was seen when the three
Spills of oil and hydrocarbons in marine environ- strains (Ochrobactrum, Stenotrophomonas and
ments stimulate the indigenous community of Pseudomonas spp.) were combined than when
7 Optimization of Cultural Conditions for Marine Microbial Biosurfactant 121

tested individually (Luo et al. 2009). New genera 1. Limited searches have been made for new
of obligate alkane degraders, Oleibacter sp., producer organisms, since many strains are
were discovered by Teramoto et al. (2009). already known but not fully investigated with
Similarly, Kostka et al. (2011) isolated potential different functionalities applicable at low-
oil degraders that belong to Gammaproteobacteria, temperature environments.
including representatives of genera with known 2. Investigating microorganisms capable of pro-
oil degraders (Alcanivorax, Marinobacter, ducing biosurfactants as tools for bioremedia-
Pseudomonas and Acinetobacter), from Gulf of tion of marine environments.
Mexico beach sands impacted by the deepwater 3. Detailed investigations are warranted on the
horizon oil spill. Interestingly, Naether et al. structure and functionality of marine micro-
(2013) explained that the strategy of A. borku- bial biosurfactants to identify those with
mensis to get access to hydrocarbon involves unique characteristics.
both the solubilization of the substrate within 4. Studies on novel methodologies are essential
micelles and the subsequent direct uptake of the in biosurfactant production conditions for
nely dispersed droplets and also stated cistrans optimized yield, downstream processing and
isomerization of unsaturated fatty acids was raw material consumption.
proven as one adaptive mechanism of Alcanivorax 5. Investigations on assessment of the genetic
borkumensis SK2 to the toxic organic solvents and metabolic control of biosurfactant pro-
like chlorophenols and alkanols. Microbial duction by target marine microorganisms are
surfactant-mediated degradation of anthracene highly indispensable.
by marine Bacillus licheniformis MTCC 5514 6. Laboratory and eld investigations are perti-
was reported by Swaathy et al. (2014). nent for the potential use of marine microbial
Furthermore, a particular strain isolated from biosurfactant producers as an augmentation
sediments, Paracoccus marcusii, was proposed tool for bioremediation.
as an optimal choice for bioremediation purposes
as its biomass remains trapped in the hydrocar-
bon phase and thus remains undisturbed from 7.11 Concluding Remarks
potential dilution effects by sea currents
(Antoniou et al. 2015). Fundamental studies on marine microbial biosur-
factants are relatively few in number compared to
other polysaccharides. There are indeed
7.10 Future Prospects many gaps in our knowledge on basic questions
in Exploitation of Marine such as optimized production, strain engineer-
Biosurfactants ing, metagenomics, regulatory and metabolic
network of many biosurfactants biosynthesis.
Large numbers of microbial strains producing In-depth information about the biosurfactant pro-
biosurfactants have already been isolated from duction regulation during bioreactor cultivation
diverse environments, including marine environ- to design knowledge-based genetic and process
ments (Kiran et al. 2010a, b, c, d). A range of engineering strategies is also warranted. The data
major international companies is actively investi- currently available indicate that marine microbial
gating potential applications for the biosurfac- biosurfactants have considerable biotechnologi-
tants with interests in a wide range of market cal importance, but further studies are necessary
niches and potential applications. The following to evaluate the feasibility of their practical appli-
are priorities for further investigation of micro- cation. This may limit the practical application of
bial biosurfactants, principally glycolipids, from marine microbial biosurfactants in several
marine environments: prospective applications. Even though diverse
122 K.V. Deepika et al.

microbial biosurfactants have been studied in Al-Nahas MO, Darwish MM, Ali AE et al (2011)
Characterization of an exopolysaccharide-producing
the last years, only little information is available
marine bacterium, isolate Pseudoalteromonas sp.
regarding their hyperproduction and protable AM. Afr J Microbiol Res 5:38233831
yields. Improve the fermentation processes of Amaral PFF, Da Silva JM, Lehocky M et al (2006)
marine microbial biosurfactants, it is possible Production and characterization of a bioemulsier
from Yarrowia lipolytica. Process Biochem
to operate at different levels, by searching for
the ideal nutritional conditions resulting in the Amir-Heidari B, Thirlway J, Mickleeld J (2008)
enhanced yield of biosurfactants with more suit- Auxotrophic-precursor directed biosynthesis of nonri-
able biotechnological proprieties, as well as to bosomal lipopeptides with modied tryptophan resi-
dues. Org Biomol Chem 6:975978
operate at the genomic level by inserting the
Antoniou E, Fodelianakis S, Korkakaki E et al (2015)
genes that are directly involved in carbohydrate Biosurfactant production from marine hydrocarbon-
metabolism. Metagenomic approaches were one degrading consortia and pure bacterial strains using
of the fast-emerging trends in biosurfactant crude oil as carbon source. Front Microbiol 6:274
Baltz RH (2006) Molecular engineering approaches to
production. The above strategies, possibly when
peptide, polyketide and other antibiotics. Nat
applied together, can lead to an interdisciplinary Biotechnol 24:15331540
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system biological interactions and supported by lipopeptides in Streptomyces roseosporus. Methods
Enzymol 458:511531
statistical and genetic algorithm tools and further
Banat IM (1995a) Biosurfactants characterization and use
predictive studies for the sustainable production in pollution removal: state of the art. A review. Acta
of marine biosurfactants. Biotechnol 15:251267
Banat IM (1995b) Biosurfactants production and use in
microbial enhanced oil recovery and pollution reme-
Acknowledgments The authors gratefully acknowledge
diation: a review. Bioresour Technol 51:112
University Grants Commission, New Delhi, Govt. of
Banat IM, Franzetti A, Gandol I et al (2010) Microbial
India for nancial support as SRF under the Grant No:
biosurfactants production, applications and future
potential. Appl Microbiol Biotechnol 87:427444
Bertrand f-c, Larsen H (1989) In: Bianchi M et al (eds)
Micro- organisms dans les ecosystems oceaniques. La
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Denitrifying Bacteria in Marine 8
Environmental Sites

Trelita de Sousa

Denitrifying bacteria are ubiquitously distributed in marine ecosystems
and especially widespread in hydrocarbon-contaminated systems. Their
unique exible respiratory mechanism and ability to degrade a broad
range of aliphatic, aromatic, and polycyclic aromatic hydrocarbons
enable them to withstand varying oxygen uxes prevalent in oil-contam-
inated sites. This chapter emphasizes the signicance of denitrifying bac-
teria in hydrocarbon-contaminated environments. It elucidates the various
mechanisms adopted by denitrifying bacteria to degrade hydrocarbons
including aliphatic, aromatic, and polycyclic aromatic hydrocarbons and
adapt effectively to oxygen uxes prevalent in petroleum-contaminated
systems. In order to facilitate hydrocarbon degradation, denitrifying bac-
teria produce amphiphilic metabolites like biosurfactants and bioemulsi-
ers. Such surface-active compounds isolated from indigenous bacteria
have received increasing interest over the past few decades due to their
important applications in bioremediation projects designed to combat oil
spills and in handling, transportation, and recovery of oil. This chapter
focuses on the signicance of biosurfactant-producing denitrifying bacte-
ria in marine petroleum-contaminated sites for effective use in bioreme-
diation studies.

8.1 Introduction

Petroleum comprises natural gas, condensate,

and crude oil and is a complex mixture of various
aliphatic, aromatic, and polycyclic aromatic
hydrocarbons (Hassanshahian and Cappello
T. de Sousa (*) 2013; De Sousa 2015). There is no doubt that
Department of Microbiology, Goa University,
Taleigao Plateau, Goa 403206, India petroleum commands widespread anthropogenic
e-mail: usage and global economic importance

Springer Science+Business Media Singapore 2017 129

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_8
130 T. Sousa

(Hassanshahian and Cappello 2013). However, ability through dispersion, desorption, surface,
when ruthlessly released into the environment, and emulsication properties and enhancing the
this vital commodity becomes a serious threat to diffusion of the recalcitrant hydrocarbons into the
both aquatic and terrestrial ecosystems including bacterial cell (Ron and Rosenberg 2001;
deep-sea, coastal, and estuarine systems, due to Anyanwu et al. 2011; De Sousa and Bhosle
their deleterious effects to every marine organism 2012a).
from bacteria, planktons, algae, and fungi to cor- Because of their facultative ability to switch
als, sh, crustaceans, birds, plants, and animals between O2 and NO3 as their terminal electron
(Ingole and Sivadas 2007; De Sousa 2015). acceptor, denitrifying bacteria have been favored
An estimated 1.3 million tones of petroleum in bioremediation projects of hydrocarbons under
enters the marine environment each year mixed electron acceptor conditions (Wilson and
(McGenity et al. 2012). Cataclysmically devas- Bower 1997; Zumft 1997; Nestler et al. 2007;
tating oil spills like the Exxon Valdez oil spill at Bae et al. 2009), and the addition of nitrate to oil-
Prince William Sound, Alaska (1989), the contaminated sites has been successfully demon-
Prestige oil spill in Galicia (2002), and the strated to stimulate and enhance indigenous
Deepwater Horizon in the Gulf of Mexico (2010) bioremediation mechanisms (Wilson and Bouwer
only add to the already alarming petroleum bur- 1997; Atlas 2011; Van der Zaan et al. 2012; De
den of coastal and ocean ecosystems (Atlas 2011; Sousa 2015). Denitrifying bacteria have been
Jain et al. 2011; McGenity et al. 2012; De Sousa reported to utilize alkanes, toluene, xylene, phe-
2015). Bioremediation using indigenous micro- nols, cresols, phthalate, cyclohexanol, benzenes
organisms formulates an ideal strategy to over- (including halobenzenes), benzoate and other
come this urgent global issue (Atlas 2011; Jain aromatic acids, alcohols, aldehydes, and polycy-
et al. 2011). Petroleum compounds are after all clic aromatic hydrocarbons (Zumft 1997;
organic substrates, and being in constant contact Chayabutra and Ju 2000; Shinoda et al. 2004;
with these hazardous compounds, the autochtho- Nestler et al. 2007; Lu et al. 2011). Several new
nous microorganisms have developed well- isolates of denitriers capable of mineralizing a
advanced pathways to degrade and utilize them wide spectrum of aliphatic (Chayabutra and Ju
(Atlas 1981; Van Hamme et al. 2003; Chikere 2000), aromatic (Shinoda et al. 2004; Nestler
and Okpokwasil 2011). et al. 2007), and polycyclic aromatic (Lu et al.
Petroleum hydrocarbons especially the higher 2011) compounds have been isolated, and their
polycyclic aromatics are either partially or spar- number is steadily increasing. Research on the
ingly soluble in water impeding its bioavailabil- hydrocarbon-degrading denitrifying bacteria,
ity to the bacteria for effective breakdown (Yadav Thauera aromatica and Azoarcus evansii, has
and Hassanizadeh 2011). This may be resolved signicantly advanced the understanding of
by the production of biosurfactants which are anaerobic degradation of hydrocarbons (Zumft
potent surface-active agents produced on micro- 1997; Song and Ward 2005; Schmeling and
bial cell surfaces or excreted extracellularly Fuchs 2009).
(Nerurkar et al. 2009; Makkar et al. 2011; Reis The prevalence of denitrifying bacteria in
et al. 2011). They are amphiphilic in nature and petroleum-contaminated sites (Wilson and Bower
contain a combination of hydrophobic (unsatu- 1997) and their efcient potential to degrade a
rated or saturated fatty acids) and hydrophilic wide range of aliphatic, aromatic, and polycyclic
(amino acids or peptides; mono-, di-, or polysac- aromatic hydrocarbons favor their use in biore-
charides) moieties that reduce surface tension mediation of petroleum pollutants especially
and interfacial tension between individual mole- under prevailing oxygen uxes (Wilson and
cules at the surface and interface of two immis- Bower 1997; Zumft 1997; Nestler et al. 2007;
cible liquids (De Sousa 2015). These Bae et al. 2009; De Sousa and Bhosle 2012b; De
surface-active compounds expedite the degrada- Sousa 2015). Denitrifying bacteria are also
tion of hydrocarbons by increasing their bioavail- known to produce biosurfactants that assist the
8 Biosurfactant-Producing Denitrifying Bacteria in Marine Petroleum-Contaminated Environmental Sites 131

uptake of hydrocarbons thereby enabling an Denitrication differs from assimilatory

effective degradation process (Chayabutra and Ju nitrate reduction in being involved in energy con-
2000; De Sousa 2015). This chapter will focus on servation (bioenergetics) as opposed to cell bio-
biosurfactant production by denitrifying bacteria synthesis. However, the assimilatory and
in response to hydrocarbons. It will comprehen- dissimilatory reduction of nitrate may occur con-
sively discuss the signicance of denitrifying currently, in the sense that when a denitrifying
bacteria in petroleum-contaminated ecosystems, bacterium is able to assimilate nitrate, the reac-
the mechanisms adopted to degrade hydrocar- tion may proceed simultaneously with dissimila-
bons under both aerobic and anaerobic condi- tory nitrate respiration. The dissimilatory branch
tions, the production of surface-active agents by comprises denitrication and dissimilatory
denitrifying bacteria as a strategy to facilitate nitrate reduction to ammonium (DNRA) both
hydrocarbon degradation, and the various meth- commonly initiated by respiratory nitrate reduc-
ods of screening for biosurfactant-producing tion (De Sousa and Bhosle 2012b). Formerly it
denitrifying bacteria for effective use in oil reme- was thought that there is no known bacterium
diation and recovery. capable of both, denitrication and DNRA
(Zumft 1997). However, recent studies have pro-
vided evidence that some species of Paenibacillus,
8.2 Significance of Denitrifying which is a facultative anaerobic, endospore-
Bacteria in Hydrocarbon- forming bacterium, originally included within
Contaminated Environments the genus Bacillus and only in 1993 reclassied
as a separate genus (Ash et al. 1993), was capable
Denitrication is the sequential exergonic dis- of heterotrophic nitrication, DNRA, and denitri-
similatory reduction of NO3 and NO2 to the gas- cation (Rtting et al. 2011). Out of all the path-
eous nitrogen oxides, nitric oxide (NO) and N2O, ways of the intricate nitrogen cycle, denitrication
and ultimately to dinitrogen gas (N2) brought seems to be the most conducive to thrive in
about by aerobic to facultatively anaerobic het- petroleum-rich systems especially in the marine
erotrophs (Knowles 1982; Zumft 1997; De Sousa systems. Elevated levels of hydrocarbon in
and Bhosle 2012b). It has signicant global petroleum-contaminated sites correlate with
implications in the environment especially in the increased denitrication rates, rapid nitrate con-
biogeochemical cycling of nitrogen in coastal sumption, and net accumulation of dinitrogen
and estuarine systems (De Sousa and Bhosle (Scott et al. 2014).
2012b). The process acts as a natural sink for During denitrication, a nitrogen oxide,
nitrate removal in estuaries (Gruber and Galloway instead of oxygen (O2), serves as the terminal
2008), counteracts eutrophication in estuaries electron acceptor to generate an electrochemical
and coastal waterways (Schlesinger 2009), and gradient across the cytoplasmic membrane result-
helps maintain the marine productivity in the ing in the sequential transformation of NO3 to N2
ocean (Lam et al. 2009) with crucial implications through the concerted action of four sequential
to global warming and climate change (Zehr enzymes: nitrate reductase (NaR), nitrite reduc-
2009). This process has also found important tase (NiR), nitric oxide reductase (NOR), and
applications in efcient municipal/industrial nitrous oxide reductase (N2OR) to form the ulti-
wastewater treatment (Zhao et al. 2009) and mate product, dinitrogen (N2). The denitrifying
groundwater remediation (Hunter and Shaner genes (nar for nitrate respiration, nir for nitrite
2010). But more signicantly, the exible respi- respiration, nor for nitric oxide respiration, and
ration system of denitriers enables them to form nos for nitrous oxide respiration) are generally
ideal cleanup systems in the bioremediation of assembled in clusters, and their function and
hydrocarbon contamination (Wilson and Bouwer expression is regulated through a complex signal
1997; Cao et al. 2009). transduction system which is generally triggered
132 T. Sousa

by anoxia and the presence of a nitrogenous compounds like hydrocarbons. The abundant
oxide (Zumft 1997; Stouthamer et al. 1997; De availability of electron donors causes an excess
Sousa and Bhosle 2012b). of electrons, and these can be simultaneously
When bacteria shift to denitrication, the ini- shuttled to both O2 and NO3 (Wilson and
tial reactions in the aerobic electron transport Bouwer 1997; De Sousa and Bhosle 2012b). In
chain, involving the pyridine nucleotides, avins fact, research on degradation of hydrocarbons
and quinones, remain unchanged (Fig. 8.1, by denitrifying bacteria stems from the fact that
Wilson and Bouwer 1997; Arai 2011). However, dispersal of oil and fuel in the environment may
from cyt b onwards, separate and specic lead to the pollution of deep aquifers which are
enzymes, regulated by the redox conditions of frequently anaerobic (Wilson and Bouwer
ubiquinone, function to determine and control the 1997; Cao et al. 2009). Dynamic alterations in
electron transport to O2 or NO3 (Wilson and microbial communities in petroleum hydrocar-
Bouwer 1997; Zumft 1997; Arai 2011). Formerly, bon spills and the proliferation of heterotrophs
denitrication was thought to be a strictly anaero- in the presence of hydrocarbons as, invariably,
bic process mostly due to the inhibitory effect of the sole electron donor can, inevitably, result in
oxygen on NaR (Knowles 1982; Wilson and the rapid depletion of oxygen in these ecosys-
Bouwer 1997). However, a co-respiration of O2 tems through aerobic respiration (Scott et al.
and NO3 with both O2 and NO3 (or NO2) serv- 2014).
ing concomitantly as electron acceptors has been The availability of organic carbon, the ready
well established during, what is also called, aero- occurrence of nitrate, and the prevalence of
bic denitrication (Okada et al. 2005; De Sousa oxygen uxes in petroleum-contaminated sys-
and Bhosle 2012b). tems are seemingly tailor-made for the prolif-
Aerobic denitrication is especially eration of denitrifying bacteria (De Sousa and
prevalent in systems accumulating organic Bhosle 2012b; Scott et al. 2014) including

Fig. 8.1 A schematic

representation of the electron N2
transport chain in denitrifying
bacteria (Wilson and Bouwer
1997; Arai 2011) Cytochrome aa3 Nitrate oxide reductase


O2 Cytochrome c Nitrate reductase

Cytochrome o

Cytochrome b Nitrate reductase

Ubiquinone NO3

Regulatory dehydrogenase


Organic substrate
8 Biosurfactant-Producing Denitrifying Bacteria in Marine Petroleum-Contaminated Environmental Sites 133

Pseudomonas, Azoarcus, Thiosphaera, which then proceeds via the normal aromatic
Thauera, Rhodopseudomonas, Bradyrhizobium, breakdown pathways (Van Hamme et al. 2003;
Ochrobactrum, Paracoccus, Mesorhizobium, De Sousa 2015).
Ensifer, and Acidovorax (Chayabutra and Ju Although the importance of oxygen in hydro-
2000; Shinoda et al. 2004; Song and Ward carbon breakdown is very well apparent, it must
2005; Nestler et al. 2007; De Sousa and Bhosle be remembered that systems accumulating
2012b). petroleum hydrocarbons are also inevitably
exposed to varying oxygen uxes (Wilson and
Bower 1997). Hydrocarbon-polluted aquifers,
8.3 Mechanism of Hydrocarbon aquatic sediments, and submerged soils invari-
Degradation by Denitrifying ably become anoxic (Cao et al. 2009) mostly
Bacteria Under Aerobic because of the quick depletion of oxygen during
and Anaerobic Conditions respiration of easily utilizable substrates, low
solubility in water, and low rate of transportation
O2 is the most common electron acceptor for in soils and sediments (Bae et al. 2002).
microbial respiration (Cao et al. 2009). Therefore, Therefore, microbial processes like denitrica-
it is not surprising that the breakdown of petro- tion, iron (III) reduction, sulfate reduction, and
leum compounds, especially the aromatics, is methanogenesis, capable of utilizing alternative
limited by the supply of oxygen (Wilson and electron acceptors such as nitrate (NO3), iron
Bower 1997). Aerobic degradation of straight- (Fe3+), sulfate (SO42), and carbon dioxide (CO2),
chain aliphatic hydrocarbons (alkanes) requires a respectively, are of increasing interest for intrin-
membrane-bound monooxygenase in conjunc- sic and engineered bioremediation designs
tion with soluble rubredoxin and rubredoxin (Wilson and Bower 1997; Cao et al. 2009; Prez-
reductase to shunt electrons through NADH to Pantoja et al. 2010; Philipp and Schink 2011). In
the hydroxylase, in order to convert the alkane to terms of energy acquisition, NO3 is the pre-
alcohol, which is then oxidized to aldehyde and ferred electron acceptor because its yield is close
acid before proceeding to the -oxidation and tri- to that of O2 in comparison to Fe3+, SO42, and
carboxylic acid cycles (Van Hamme et al. 2003; CO2. Furthermore, it is highly water-soluble
Wentzel et al. 2007; De Sousa 2015). During (660 g/L), does not precipitate oxides, is inex-
aerobic biodegradation of aromatic compounds pensive (can be therefore used in in vitro stud-
(benzoate, benzene, toluene), oxygen not only ies), and is nontoxic to aquifer microorganisms
acts as an electron acceptor but is also involved as at concentrations below 500 mg/L (Wilson and
a highly reactive co-substrate in the initial Bower 1997). Van der Zaan et al. (2012) have
hydroxylation reactions catalyzed by mono- and also reported that the highest rates for anaerobic
dioxygenases. The monooxygenases transform degradation of benzene were obtained with NO3
the benzene ring into a few central intermediates as the electron acceptor in comparison to SO42,
such as catechol, protocatechuate, gentisate, and chlorate, and Fe3+.
hydroxyl benzoquinols. These are then further The anaerobic mechanism for alkane degrada-
cleaved by different dioxygenases to the tricar- tion is proposed to transpire either by alkane car-
boxylic acid intermediates via the ortho- or meta- boxylation leading to the formation of fatty acids,
pathways (Cao et al. 2009; Prez-Pantoja et al. as seen with sulfate reducers or by addition of
2010; De Sousa 2015). In the case of polycyclic alkane to the double bond of fumarate giving suc-
aromatic hydrocarbons like naphthalene, ace- cinate substituted with alkane-derived alkyl
naphthene, anthracene, uoranthene, pyrene, and chains as in the case of denitriers (Wentzel et al.
chrysene, molecular oxygen is introduced into 2007). In the anaerobic biodegradation of aro-
the aromatic nucleus by multicomponent non- matic compounds, the peripheral pathways cata-
heme iron oxygenase enzyme systems, like the lyzed by the oxygenases converge to benzoyl-CoA
naphthalene dioxygenase, forming a dihydrodiol (occasionally to resorcinol or phloroglucinol).
134 T. Sousa

Specic multicomponent energy-requiring 8.4 Biosurfactants Produced

reductases catalyze the de-aromatizing reactions by Denitrifying Bacteria
(Cao et al. 2009; Philipp and Schink 2011). in Response
Polycyclic aromatic hydrocarbons are also anaer- to Hydrocarbons
obically metabolized in a similar way via carbox-
ylation or by the addition to fumarate (Van Biosurfactant production is widespread in
Hamme et al. 2003). hydrocarbon-utilizing bacteria. These natural
Denitrifying bacteria can thus utilize hydro- products are a desirable alternative to syn-
carbons under both aerobic and anaerobic condi- thetic surfactants because of their selectivity,
tions by suitably modifying their pathways in biodegradability, low toxicity, and stability
adaptation to the availability of oxygen in the (Nerurkar et al. 2009; Vignesh et al. 2011; De
environment. These exible organisms possess Sousa and Bhosle 2012a) and are increasingly
enzymes that enable them to use aerobic degrada- in huge demand in hydrocarbon bioremedia-
tion pathways to metabolize hydrocarbons in the tion, microbial-enhanced oil recovery, agricul-
presence of molecular oxygen, and when oxygen ture, cosmetic, pharmaceutical, detergent,
levels diminish, the bacteria switch to nitrate res- food, textile, paper, and paint industries (Janek
piration (Wilson and Bouwer 1997; Prez- et al. 2010; Lima et al. 2011; Zheng et al.
Pantoja et al. 2010). 2012).
In the case of aliphatic hydrocarbons like Surface-active compounds are mainly catego-
hexadecane by Pseudomonas aeruginosa, the rized on the basis of their chemical composition,
initial oxygen-requiring transformation of the function, and point of origin (Vignesh et al.
alkane is taken care of using oxygen as the elec- 2011). Biosurfactants reduce surface tension at
tron acceptor. The oxygenated metabolites are the air-water interface and include low-molecular-
then further degraded under anaerobic conditions weight molecules including glycolipids, lipopep-
using nitrate as the electron acceptor (Chayabutra tides, lipopolysaccharides, polysaccharide-protein
and Ju 2000). Such sequential mechanisms are complexes, lipoprotein, phospholipids, fatty
also observed in the degradation of aromatic acids, lipids, polymeric biosurfactant, and par-
hydrocarbons like toluene by Thauera and ticulate biosurfactants (Makkar et al. 2011;
Azoarcus. The initial degradation of toluene Vignesh et al. 2011; De Sousa 2015).
occurred through a dioxygenase-mediated path- Bioemulsiers, although sometimes also referred
way in the presence of oxygen and proceeded to to as biosurfactants (Franzetti et al. 2012), on the
the benzyl-succinate pathway under denitrifying other hand reduce the interfacial tension between
conditions (Shinoda et al. 2004; Song and Ward immiscible liquids or at solid-liquid interface and
2005). also include mostly low-molecular-weight com-
Degradation of especially the aromatic com- pounds like lipopeptides and glycolipids (Vignesh
pounds like benzoate and toluene by denitrifying et al. 2011; De Sousa and Bhosle 2012a).
bacteria is enhanced by the presence of readily Extracellular polymeric substances or exopoly-
consumable carbon sources like succinate and saccharides (EPS) or bioemulsans are generally
acetate (Kariminiaae-Hamedaai et al. 2004; high-molecular-weight emulsiers which include
Martnez-Hernndez et al. 2009; De Sousa and polymers of polysaccharides, lipopolysaccha-
Bhosle 2012c). Chnier et al. (2003) also reported rides, proteins, or lipoproteins (Franzetti et al.
the enhancement of hexadecane mineralization 2012).
and denitrication with nutrient amendment Extracellular ionic surfactants like rhamnolip-
through the addition of glucose, ammonium ids or sophorolipids (produced by Pseudomonas
chloride, and phosphorous (K2HPO4). and Torulopsis spp., respectively), lipopeptides/
8 Biosurfactant-Producing Denitrifying Bacteria in Marine Petroleum-Contaminated Environmental Sites 135

lipoproteins, namely, surfactin or subtilisin (pro- Peng et al. 2008; Nerurkar et al. 2009; Janek et al.
duced by Bacillus subtilis), and rhodofactin (pro- 2010; Anyanwu et al. 2011; Franzetti et al. 2012),
duced by Rhodococcus sp.) act by emulsication. only a few have been isolated from denitrifying
Extracellular or cell-bound nonionic surfactants, bacteria in response to hydrocarbons such as the
capable of altering the structure of the cell wall, rhamnolipid biosurfactant from Pseudomonas
include lipopolysaccharides or liposan (produced aeruginosa (Chayabutra and Ju 2000), the lipo-
by Candida lipolytica, C. tropicalis, Rhodococcus peptide bioemulsier from Pseudomonas nitrore-
erythropolis, Mycobacterium sp., and ducens (De Sousa and Bhosle 2012a; De Sousa
Arthrobacter sp.) and emulsan (synthesized by 2015), and the exopolysaccharide produced by
Acinetobacter sp.). Several other effective bio- Halomonas ventosae (Martnez-Cnovas et al.
surfactants have also been reported which include 2004). A list of previously isolated biosurfactant/
xylolipid (produced by Lactococcus lactis), bioemulsier/EPS-producing marine/estuarine
mycolates and corynomycolates (produced by hydrocarbon-degrading denitrifying bacteria is
Corynebacteria, Mycobacteria, and Nocardia given in Table 8.1.
spp.), and ornithinlipids (produced by Besides enhancing growth on hydrophobic
Pseudomonas rubescens, Gluconobacter cer- substrates like hydrocarbons, biosurfactants/bio-
inus, and Thiobacillus ferrooxidans) (Peng et al. emulsiers assume different functions due to the
2008; Nerurkar et al. 2009; Saravanakumari and complexities in chemical structure and surface
Mani 2010; Vignesh et al. 2011). activity thereby providing varied advantages in
Despite their structural and functional differ- different ecological niches. They play an impor-
ences, all three types of surface-active agents tant role in regulating the attachment-detachment
(biosurfactants, bioemulsiers, and EPS) have of microorganisms to and from surfaces and are
found potential use in bioremediation designs and therefore also involved in bacterial pathogenesis,
have garnered an increasing interest over the past quorum sensing, biolm formation, and confer-
several decades in petroleum abatement ring antibacterial/antifungal properties to the pro-
(Chayabutra and Ju 2000; Martnez-Cnovas ducing microorganisms (Ron and Rosenberg
et al. 2004; De Sousa and Bhosle 2012a). Although 2001).
numerous surface-active compounds have been The production of surfactants/emulsiers by
isolated from various marine systems especially bacteria can be suitably optimized for potential
those contaminated with petroleum hydrocarbons application in bioremediation of hydrocarbons
(Batista et al. 2006; Maneerat and Phetrong 2007; and heavy metals and in enhanced oil recovery

Table 8.1 Biosurfactant/bioemulsier/exopolysaccharide-producing hydrocarbon-degrading denitrifying bacteria iso-

lated from marine petroleum-contaminated sites
Denitrifying isolate Type of surface-active compound Reference
Pseudomonas aeruginosa ATCC 10145 Rhamnolipid biosurfactant Chayabutra and Ju (2000)
Pseudomonas nitroreducens TSB.MJ10 Lipopeptide bioemulsier De Sousa and Bhosle (2012a)
Pseudomonas sp. Biosurfactant Grishchenkov et al. (2000)
Brevibacillus sp. Biosurfactant Grishchenkov et al. (2000)
Halomonas ventosae Exopolysaccharide Martnez-Cnovas et al. (2004)
Dietzia maris Wax ester-like biosurfactant Nakano et al. (2011)
Pseudomonas nitroreducens Rhamnolipid biosurfactant Onwosi and Odibo (2011)
136 T. Sousa

processes (Anyanwu et al. 2011; Lima et al. 8.5.1 Cell Hydrophobicity by the BATH
2011; Franzetti et al. 2012; Zheng et al. 2012). (Bacterial Adherence
The amount of microbial surfactants produced to Hydrocarbons) Assay
depend primarily on the producer organism
and on factors like carbon and nitrogen sources, This is one of the most popular and simplest meth-
trace elements, temperature, pH, and aeration ods used to screen for cell hydrophobicity, and not
conditions (Santos et al. 2002; Onwosi and many changes have been made to the original
Odibo 2011; Vignesh et al. 2011). It has been method proposed by Rosenberg (1984). The opti-
suggested that the production of these surface- cal density of the cells suspended in phosphate
active agents is concurrent with the onset of buffer before and after mixing with a small volume
the stationary phase induced by molecular sig- of hexadecane at high speed is measured. A
nals involved in quorum sensing (Ron and decrease in absorbance of the aqueous layer after
Rosenberg 2001; Reis et al. 2011). However, an incubation time of 30 min on mixing with hexa-
certain studies have also reported their synthe- decane reects the hydrophobicity of the cells.
sis during the exponential growth phase
(Batista et al. 2006). Their potential is largely
dependent on their chemical nature and their 8.5.2 Emulsification Activity
activity may be enhanced by simple media
modication (Mutalik et al. 2008; Nerurkar The cell-free culture broth is used for screening
et al. 2009). for emulsication activity. In the method described
by Maneerat and Phetrong (2007), equal quanti-
ties of cell-free supernatant are mixed with hexa-
8.5 Screening Methods decane or xylene at high speed, and the resulting
of Denitrifying Bacteria emulsion (De Sousa 2015) is allowed to stabilize
for Biosurfactants for 24 h and the emulsication index (E24), dened
as the per cent height of the emulsion layer divided
Because of their wide range of applications, var- by the total height of the mixture, is calculated.
ious methods and techniques are designed to
maximize the isolation of biosurfactants.
Biosurfactant production generally depends on 8.5.3 Quantitative Oil Displacement
the fermentation conditions, environmental fac- Assay
tors, and nutrient availability (Santos et al. 2002;
Gakpe et al. 2007; Onwosi and Odibo 2011; In this simple method, cell-free culture broth is
Vignesh et al. 2011). The denitrifying isolates carefully placed on the center of an oil lm
are cultivated in a suitable medium with the formed by pouring a mixture of parafn oil and
hydrocarbon of choice prior to the screening an oil-soluble dye (oil red O) over water in a
methods. Care must always be taken to incorpo- clean petri plate. An appearance of a clear halo
rate a nitrate source in the cultivation medium (De Sousa 2015) within 30 s is positive for sur-
when culturing denitrifying bacteria (De Sousa face activity (Morikawa et al. 1993).
and Bhosle 2012b). KNO3 has been observed to
be the best source for their cultivation (De Sousa
et al. 2013). Succinate also enhances denitrica- 8.5.4 Qualitative Drop-Collapsing
tion activity (De Sousa and Bhosle 2012c). Activity
Denitrifying bacteria can be screened for biosur-
factants by the following methods routinely used A small amount of cell-free culture broth is
for biosurfactants. placed onto the surface of an oil droplet on a
8 Biosurfactant-Producing Denitrifying Bacteria in Marine Petroleum-Contaminated Environmental Sites 137

clean grease-free slide, and its shape is observed 8.5.6 Type of Emulsion
for a minute. Flat drops (De Sousa 2015) indicate
a positive result, while round drops are negative Biosurfactants and bioemulsiers are character-
for surface activity (Youssef et al. 2004). ized on their ability to produce stable emulsions
which allow the microorganisms to adhere
strongly to the hydrocarbons thus facilitating the
8.5.5 Surface Tension Measurement degradation process (Nerurkar et al. 2009). They
and Estimation of Critical are distinguished on their hydrophile-lipophile
Micelle Concentration (CMC) balance (HLB): those that possess a low HLB are
strongly lipophilic and therefore generally used
The surface tension of the cell-free culture broth as water-in-oil emulsiers, while those that have
collected at different time intervals is determined a high HLB are strongly hydrophilic and appro-
by the ring method using a du Nouy tensiometer priately used as oil-in-water emulsiers (Nerurkar
(Onwosi and Odibo 2011). The concentration at et al. 2009). A drop of oil red O in parafn oil
which micelles begin to form is represented as and/or crystal violet solution is added to the cell-
the CMC which is determined by plotting the sur- free culture broth. These solutions are then mixed
face tension as a function of the biosurfactant with equal amounts of hexadecane at high speed
concentration. The surface tension can also be for 2 min and allowed to settle thereafter, for an
measured by the drop-weight and drop-count hour. If the emulsion is formed in the aqueous
methods using a Traubes stalagmometer phase, then it is an oil-in-water type of emulsion,
(Fig. 8.2) (De Sousa and Bhosle 2012a). whereas if the emulsion is formed in the organic
phase, it is a water-in-oil type of an emulsion
(Fig. 8.3).

8.6 Characteristics
of Biosurfactants
for Effective Use
in Petroleum Bioremediation

Because of their hydrophobicity, bacteria gener-

ally adhere to hydrocarbons. However, only the
hydrocarbon molecules that are dissolved in the
aqueous phase are accessible to microorganisms
for biotransformation. Thus, the rate of dissolu-
tion of such hydrocarbons is critical for its bio-
availability. The amphipathic nature of
biosurfactants aids in the dispersion of these par-
ticles via dissolution in the aqueous phase thereby
rendering them available for uptake by the cell
(Ron and Rosenberg 2001; Anyanwu et al. 2011;
De Sousa and Bhosle 2012a).
Biosurfactants possess several advantages
over chemical surfactants including biodegrad-
ability, low toxicity, biocompatibility, digestibil-
ity, acceptable economics (cheaper raw materials
Fig. 8.2 Traubes stalagmometer used to measure surface and lower production costs), better environmen-
tension by the drop-weight and drop-count methods tal control, and specicity (Gakpe et al. 2007).
138 T. Sousa

Fig. 8.3 Water-in-oil type of

emulsion exhibited by
bioemulsiers produced by a
denitrifying strain
Pseudomonas nitroreducens
TSB.MJ10 (De Sousa and
Bhosle 2012a; De Sousa
2015). In the absence of the
biosurfactant (control tubes),
oil-soluble oil red O goes into
the organic phase, while the
water-soluble crystal violet
goes into the aqueous phase.
On mixing the hydrocarbon
with the biosurfactant, both oil
red O and crystal violet go into
the emulsion layer indicating a
water-in-oil type of emulsion
as opposed to bringing the
dyes into the aqueous layer
seen in oil-in-water emulsions

Fig. 8.4 Emulsication of various hydrocarbons by a lipopeptide bioemulsier produced by a denitrifying strain
Pseudomonas nitroreducens TSB.MJ10 (De Sousa and Bhosle 2012a; De Sousa 2015)

However, the most important factor governing for effective use under extreme environmental
the suitability of biosurfactants in bioremediation conditions (De Sousa and Bhosle 2012a).
designs is their stability (Janek et al. 2010; Lima Ideal biosurfactants form stable emulsions
et al. 2011; Vignesh et al. 2011; De Sousa and with a broad range of aliphatic, aromatic, polycy-
Bhosle 2012a). The stability of biosurfactants is clic aromatic, and petroleum hydrocarbons (Fig.
tested against a wide range of pH, temperature, 8.4). Stable emulsication of petroleum and
and salt concentrations. It is desirable that the crude oil facilitates their transportation and man-
biosurfactants demonstrate a stable E24 under agement (Lima et al. 2011). Viscosity reductions
extreme pH, temperature, and salt concentrations of petroleum uids by biosurfactants also help
8 Biosurfactant-Producing Denitrifying Bacteria in Marine Petroleum-Contaminated Environmental Sites 139

overcome the difculties encountered during denitrifying bacteria. This chapter provides sim-
handling, recovery, and transportation of oil, ple methods for effective screening of
which signicantly contribute to their biosurfactant-producing denitrifying bacteria and
cost-effectiveness (Anyanwu et al. 2011; Lima encourages the isolation of more surface-active
et al. 2011; De Sousa and Bhosle 2012a; Franzetti agents including biosurfactants, bioemulsiers,
et al. 2012). and exopolysaccharides for effective use in petro-
Reduction of surface tension by biosurfactants leum bioremediation.
denes their efciency for use in bioremediation
studies (Batista et al. 2006; Gakpe et al. 2007;
Lima et al. 2011). Lipopeptides show potent sur- References
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biosurfactant production by Serratia marcescens
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Chnier MR, Beaumier D, Roy R, Driscoll BT, Lawrence
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Environ Microbiol 69:51705177
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Chikere CB, Okpokwasil GC (2011) Monitoring of
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Interaction of Haloarchaea
with Metals 9
Sanika Shivdas Naik and Irene Furtado

Haloarchaea are predominant microora of hypersaline econiches such as
solar saltern, salt lakes, and salt deposits and so on. Urbanization and
industrialization including mining, agriculture, and waste disposal in
coastal countries result in the discharge of efuents containing toxic metal
ions into rivers, estuaries, and marine econiches. Saltpans located along
the estuary often serve as a sink of these metal toxicants. Moreover, solar
salterns are sites where microorganisms thrive and where haloarchaea are
predominant indicating their survival in metal-toxicated environment to be
the result of resistance mechanism specialized to overcome the stress. This
chapter reviews and focuses on the various resistance strategies adopted
by Archaea especially haloarchaea to survive the metal-contaminated

9.1 Introduction and application of agrochemicals in agricultural

elds which add a considerable quantity of heavy
Metal pollution is ceaselessly on rise as a conse- metals in the environment (Kumar et al. 2011).
quence of anthropogenic activities, industrializa- The concentration of very common heavy metals
tion, urbanization, and natural sources (Hu et al. such as Cd, Cr, Cu, Ni, Pb, and Zn in marine sedi-
2014). Metal pollutants ultimately nd its way ments is reported to range from 0.09 to 88.6 g
into soils and water bodies including freshwater g1 (Zhuang and Gao 2014). These metal pollut-
as well as marine seas (Zhuang and Gao 2014) ants are accumulated in living organisms includ-
from industrial operations such as mining, smelt- ing microorganisms, animals, and humans having
ing of ore, manufacturing of iron and steel prod- its toxic effect on the whole food chain and are
ucts, manufacturing of alkaline storage batteries, thus responsible for causing metabolic and physi-
ological disorder (Matyar et al. 2010).
Metal binding capabilities of microorganisms
S.S. Naik (*) I. Furtado have been explored by various researchers across
Microbiology Department, Goa University, the world which is proven to be an economical as
Taleigao Plateau, Goa 403206, India well as eco-friendly solution to remediate the

Springer Science+Business Media Singapore 2017 143

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_9
144 S.S. Naik and I. Furtado

metal-contaminated sites (Ahmed and Malik Other metals, including Tc7+, Cr6+, Co3+, Mn4+
2012). (Kashe and Lovley 2000), and Au3+ (Kashe
Archaea belong to one of the three domains of et al. 2001), are also reduced by P. islandicum in
life and are inhabited by diverse environments the presence of hydrogen as the electron donor.
such as shallow or deep-sea anaerobic sediments Pyrococcus furiosus and the archaeal strain 234
including free-living and endosymbiotic are also able to reduce gold, as well as Fe3+, in the
methanogens, hot springs, or deep-sea hydrother- presence of H2 (Kashe et al. 2001). Metal
mal vents and highly saline landlocked seas com- transformations leading to the formation of
prising methanogens, sulfate reducers, and insoluble precipitates may also result from
extreme halophiles (Silveira et al. 2013). metabolic processes. For example, anaerobic
However, Archaea are also reported and explored oxidation of methane (AOM) can be carried out
in non-extreme environments, including marine, using different electron acceptors, including the
freshwater, and mangrove ecosystem belonging metals manganese or iron (Beal et al. 2009).
to Group I Archaea and Groups II and III However, under such conditions, AOM proceeds
Euryarchaeota (Silveira et al. 2013; Delong at signicantly slower rates than those observed
2007). Also, the discovery of numerous archaeal for sulfate-dependent AOM (Beal et al. 2009).
species from metal-rich environment that con- Haloarchaea belonging to the domain Archaea
tributes to the highly oxidative environment such comprise 36 genera and 129 species identied
as mining sites, salterns, and metal-contaminated (Oren 2012). The members of this family are
soils has enhanced the interest in studying metal predominant microora of hypersaline
resistance in these microbes (Maezato and Blum environments such as solar salterns, soda lakes,
2012). and salt deposits (Tabak et al. 2005). These
environments serve as avenues for transformation
of native microora into potential metal-resistant
9.2 Transformations of Metals strains.
by Archaea

Archaebacteria are reported to impact the 9.3 Metal Resistance

mobility of metals in the environment by mode of in Haloarchaea
oxidation or reduction of elements with the aim
of energy conservation or detoxication or 9.3.1 Intracellular Proteins
indirectly by altering the pH or redox conditions
of their environment, which in turn affects metal Haloarchaea are reported to have
precipitation or solubilization (Bini 2010). -glutamylcysteine (-GC) that is analogous to
Archaeal species are also able to transform metals glutathione (GSH) and involved in maintaining a
into their insoluble forms in biomineralization reducing environment within the cell that aids in
processes that lead to the formation of mineral overcoming oxidative and disulde stress. The
deposits of the corresponding metal ores. In resistance to toxic metal ions is attributed to the
hyperthermophilic habitats, the presence of thiol group of cysteine in g -GC that can chelate
magnetite and uraninite has been proposed to be the metal ions. Further, in case of Halobacterium
due to the activity of P. islandicum. Cell cultures salinarum, a multimeric nonheme ferritin DpsA-
of P. islandicum are capable of Fe3+ oxide and U4+ like protein is found to sequester iron in response
mineral reduction, leading to the formation of to the oxidative stress exerted by excess iron
ultrane magnetite and UO2, respectively (Reindel et al. 2005). This protein was
(Kashe et al. 2008a, b). downregulated under iron-decient conditions. It
exhibits the features of nonheme bacterial
9 Interaction of Haloarchaea with Metals 145

ferritins that are expressed to sequester the excess of these metals may be responsible for substituting
iron. Their expression is repressed under with Ca2+. This implicates that the tolerance to
conditions of iron starvation. Kaur et al. (2006) these metal ions may be mediated in the course of
have also proposed that the chelation of Fe2+ by binding with EPS. Furthermore, in the presence
the ferritin DpsA is a mechanism for detoxifying of certain other metals lacking this electronic
Fe2+ in Halobacterium sp. strain NRC-1. conguration, such as Mg2+ and Sr2+ (alkali earth
Transcription of DpsA is upregulated by Fe2+, metals), Mo2+, Cd2+and Sn2+, and Hg2+and Pb2+
Co2+, and Zn2+ and downregulated by Mn2+and Fe belonging to fth and sixth periods, respectively,
deciency conditions; the transcription regulators no conglomeration was observed. Bolhuis et al.
for this mechanism have not been identied. (2006) have proposed a cbp, encoding the cell
Furthermore, among haloarchaea, Halococcus surface calcium-binding acidic-repeat protein
saccharolyticus, Halogeometricum sp., that is involved in Ca2+-dependent aggregation in
Halorubrum saccharovorum, and Haloterrigena Haloquadratum walsbyi, Haloarcula
turkmenica are reported to produce carboxylate- marismortui, Haloterrigena turkmenica, and
type siderophores (Dave et al. 2006). Halobacterium sp. strain NRC-1, although its
Overexpression of siderophores in haloarchaea role in this process remains to be demonstrated.
increases chelation in case of iron deciency. On Iron uptake studied in Halobacterium salinarum
the other hand, repression of the siderophores in by Hubmacher et al. (2007) revealed the role of
the presence of excess iron may avoid uptake xenosiderophores (triacetylfusarinin) for iron
(Malki et al. 2009; Dave et al. 2006). uptake that is an energy-dependent process,
either dependent on the respiratory chain or on
the existence of the membrane potential. Iron
9.3.2 Metabolites transport across the cell membrane was proposed
to include a reductive step that is located at the
Haloarchaea are able to synthesize cell surface or existence of a ligand exchange
exopolysaccharide (EPS) as a protective mechanism at the cell wall or at the membrane.
mechanism for survival under stressed conditions
such as nutrient depletion, variation in
temperature, and the presence of toxic chemical 9.3.3 Efflux Pumps
compounds (Poli et al. 2011). Further, Kawakami
et al. (2007) have reported a Ca2+-dependent As reviewed by Srivastava and Kowshik (2013),
aggregation system in Halobacterium salinarum efux pumps (Fig. 9.1) are one of the most
CCM 2090 wherein the divalent ion binds to common mechanisms of resistance to inorganic
certain aggregation factors present on the cell ions in microbes including Archaea.
surface which stimulates ionic cross-linking
between the EPS molecules resulting in
conglomeration of the haloarchaeal cells. 9.3.4 P1B-Type ATPases
Moreover, certain receptor proteins are also
demonstrated to be present on the cell surface The P1B-type ATPases are a large family of
that interact with Ca2+ to form cell aggregates. As integral membrane proteins driven by ATP
explained by Kawakami et al. (2007), Ca2+ being hydrolysis (Fagan and Saier 1994). Metal ions
the twentieth element found in the fourth row of such as Mg2+, Ca2+, Cu2+, Ag 2+, Zn 2+, and Cd2+
the periodic table could be replaced by other act as substrates to these ATPases (Saier 1994).
transition metal ions such as Mn2+, Cr2+, Fe2+, These transporters serve the purpose of uptake of
Co2+, Ni2+, Cu2+, and Zn2+, belonging to the same essential elements and efux of toxic elements,
row. It is proposed that during aggregate thus conferring resistance to the expelled metal
formation, the distinctive electronic conguration ion (Snavely et al. 1989; Nies 2003). In a system
146 S.S. Naik and I. Furtado

Fig. 9.1 Various metal transporters reported in Halobacterium sp. strain NRC-1

level analysis of Halobacterium sp. strain NRC-1 cellular demands (Tottey et al. 2001; Solioz and
demonstrated by Kaur et al. (2006), the Stoyanov 2003). In Halobacterium sp. strain
functionality and role of such transporters in NRC-1, the cpx gene that encodes CPx P1B-type
metal resistance exhibited upregulation of yvgX, ATPases was found to be downregulated by Fe2+,
a P1B-typeATPase, in response to Cu2+and Cu2+, and Ni2+ to prevent the upsurge of toxic
Zn2+metal stress. In bacteria, the yvgX family is metal within the cell (Kaur et al. 2006).
known to encode two kinds of CopA proteins,
CopA1 and CopA2 (Klein and Lewinson 2011).
CopA1 is essential for copper inux and 9.3.5 Cation Diffusion Facilitators
tolerance, while CopA2 is involved in the inux/ (CDF) Metal Transporters
efux of Cu and its transport to Cu-containing
enzyme cytochrome oxidase c (Klein and The CDF family of transport proteins is
Lewinson 2011; Raimunda et al. 2011). Archaea ubiquitously present in all three domains of life
are reported to prefer CopA2 over CopA1 (Paulsen and Saier, Jr. 1997). CDF functions as
(Coombs and Barkay 2005). The yvgX of homo-dimeric antiporters, transporting metal
Halobacterium sp. strain NRC-1 was found to be ions against concentration gradients using H+/K+
more specic for Cu2+ efux family as the yvgX to create electrochemical gradient. Based upon
strain was susceptible to Cu2+ and not to Zn2+or their substrate specicity, CDFs have been
Co2+and therefore belongs to the CopA2 family classied as Zn2+-CDF, Fe/Zn-CDF, and Mn-CDF
of proteins and is found in Haloarcula (Montanini et al. 2007). They usually possess six
marismortui, Haloarcula hispanica, and transmembrane domains (TMDs) with a
Haloquadratum walsbyi (Orell et al. 2012). In cytoplasmic N- and C-terminal and a histidine
uptake of copper, Cu-CPx-type ATPases have loop of variable length between TMD IV and V
also been shown to be involved to satisfy the (Haney et al. 2005; Anton et al. 1999). The
9 Interaction of Haloarchaea with Metals 147

amphipathic domains TMD I, II, V, and VI are 9.3.7 Metal Resistance Operons
involved in metal transfer and are the most
conserved, while the hydrophobic TMD III and Haloarchaea are reported to possess large
IV are critical for zinc specicity and mutations plasmids known as minichromosomes or
wherein these domains alter substrate specicity megaplasmids. These minichromosomes possess
(Montanini et al. 2007). The genome of genes for antibiotic resistance or metal resistance
Halobacterium sp. strain NRC-1 has disclosed a that may be indispensable for survival of
putative CDF Cd 2+transporter ZntX, which haloarchaea (DasSarma et al. 2009). The model
confers resistance against Ni2+, Cu2+, and Zn2+ organism Halobacterium sp. strain NRC-1
besides Cd2+ (Kaur et al. 2006). The role of this harbors pNRC100 (191 kb), one of the two
transporter in metal resistance has been conrmed megaplasmids that possess arsADRC gene
by upregulation of ZntA in response to heavy cluster, which confers resistance to arsenate
metals (Cu and/or Zn) and poor growth of zntA (As5+) and arsenite (As3+)/antimonite (Sb3+)
strain in the presence of Ni2+, Cu2+, Zn2+, and Cd2+ (Kaur et al. 2006). As5+can be taken up by the
(Kaur et al. 2006). In Haloarcula hispanica and cells through phosphate transporters (pit/pst) and
Haloarcula marismortui, ZntA transporter for As3+ by aquaglyceroporins (glycerophorin
Zn2+transport has also been elucidated. For membrane transport proteins) (Mukhopadhyay
inorganic metal ion transport, a putative CDF et al. 2002) or hexose transporters (Suzuki and
family protein has also been found on the Matsushita 1968) followed by its conversion to
chromosome of Natrialba magadii (Orell et al. As3+by arsenate reductase encoded by arsC. arsA
2012). codes for P1B-type ATPase transporters that help
in extrusion of As3+/Sb3+ from the cell. arsR and
arsD encode trans-acting repressors of the
9.3.6 ATP-Binding Cassette (ABC) operon. ArsR and ArsD bind to As3+/Sb3+ resulting
Transporters in expression of the arsA and arsC. Arsenate
reductase encoded by arsC is expressed weakly
ABC transporters are ubiquitously present in all in Halobacterium sp. strain NRC-1, and therefore
three domains of life from bacteria to eukaryotes deletion of arsC and arsADRC was found to be
and Archaea. They play a crucial role in substrate ineffective in conferring arsenate sensitivity. The
uptake, export, and osmoregulation (Albers et al. operon arsADRC was found to be inducible by
2004). ABC transporters for sugar and arsenite and antimonite. Halobacterium sp. strain
polypeptide have been found in Haloferax NRC-1 also harbors both arsA in ars operon on
volcanii (Hartman et al. 2010), Haloarcula the megaplasmid pNRC100 and arsB on the main
marismortui (Baliga et al. 2004), Halobacterium chromosome. However, arsB was found to play
sp. NRC- 1 (Kaur et al. 2006), Natronomonas no signicant role in arsenic resistance in this
pharaonis (Falb et al. 2005), and Haloquadratum organism. Thus, it has been proposed that
walsbyi (Bolhuis et al. 2006). Many of the ABC Halobacterium sp. strain NRC-1 harbors a novel
transporters are metal ion transporters in transporter unrelated to arsB although with a
Halobacterium sp. such as cbiNOQ for similar function (Wang et al. 2004). Another type
Co2+transport (Roth et al. 1993), hemUV for iron of arsenite resistance operon is found in
uptake (Faraldo-Gomez and Sansom 2003; Halobacterium sp. strain NRC-1, arsR2M, which
Schneider and Paoli 2005), nosFY for copper is present upstream of arsADRC on pNRC100,
(Zumft et al. 1990), and zurMA for zinc transport. where arsR2 is constitutively expressed while
148 S.S. Naik and I. Furtado

As3+/Sb3+ induces the expression of arsM (Wang 9.3.8 Scope of Haloarchaea: Metal
et al. 2004). The arsR2 is analogous to arsR and Interaction Studies
arsM encodes a putative As3+-methyltransferase
that is reported to be analogous to mammalian Although haloarchaea is reported to interact with
methyltransferases and S-adenosyl methionine- metal ions also as a bioadsorbent of Mn2+, Pb2+,
dependent methyltransferases of Cr2+, Ni2+, Zn2+, As2+, Cd2+, Al2+, and Hg2+ (Naik
Magnetospirillum magnetotacticum. and Furtado 2014; Popescu and Dumitru 2009;
ArsM is involved in converting As3+ to meth- Williams et al. 2012), it is also explored for
ylated species like dimethylarsinate (DMA), tri- synthesis of metal nanoparticles extracellularly
methylarsine oxide (TMAO), or trimethylarsine (Patil et al. 2014) and intracellularly (Srivastava
(TMA) gas (Cullen and Bentley 2005). arsM et al. 2013). Haloarchaeal members are bestowed
shows an increased sensitivity to arsenite but with unique cell envelope structure consisting of
fails to do so toward arsenate or antimonite S-layers comprising protein or often glycoprotein
(Wang et al. 2004). Thus, arsM confers two pos- subunits that form a self-assembled two-
sible mechanisms of As3+resistance. First, a con- dimensional array enclosing the whole cell
centration gradient is generated that results in surface and glycerol ether core lipids in their
the movement of methylated arsenite (negatively membranes (Asker et al. 2002). Three haloar-
charged/uncharged) out of the cell. Second, As3+ chaeal S-layer glycoproteins have been studied in
is eliminated by the formation of volatile tri- detail, i.e., the S-layer glycoproteins of
methylarsine out of the cell (Yuan et al. 2008). In Halobacterium salinarum (Lechner and Sumper
Halobacterium sp. strain NRC-1, the arsenite 1987), Haloferax volcanii (Sumper et al. 1990),
resistance is brought about by arsM gene present and Haloarcula japonica (Wakai et al. 1997).
as a part of the arsR2M operon (Wang et al. Recently, S-layers of Bacillus sphaericus JG-A12
2004). The mer operon confers resistance to were used as a template to fabricate well-
mercury in Archaea and bacteria that is involved separated Pd, Pt, and Au nanoparticles that are
in detection, regulation, transport, and reduction ordered in regular arrays (Pollman and Matys
of Hg2+ (Osborn et al. 1997). The merRHAI 2007; Merroun et al. 2007). Consequently, halo-
operon of thermoacidophilic archaeon Sulfolobus archaeal S-layers can be explored as a prospec-
solfataricus is the best studied mercury resis- tive cell surface structure for nanoparticle studies
tance operon in Archaea. The MerR regulator of nanotechnological signicance.
maintains the operon under control, which then
represses the operon in the absence of Hg2+ and
thus in its presence augments the transcription. 9.3.9 Future Aspects
Hg2+is bound by a TRASH (trafcking, resis-
tance, and sensing of heavy metals) domain Haloarchaea being predominant in solar salterns
MerH, a metallochaperone, while the reduction located along the estuary which often serve as a
and detoxication to volatile Hg (0) are carried sink of metal toxicants (Chapman and Wang
out by MerA, a mercuric reductase (Schelert 2001; Litcheld and Gillevet 2002) are sparsely
et al. 2006). Some mer operons carry additional explored for their metal resistance mechanisms.
mer genes, notably merB, an organomercurial Development of genetic tools and understanding
lyase that cleaves the C-Hg bonds of organomer- the molecular mechanism underlying the metal
curials, and the released Hg2+ is reduced to Hg resistance pattern in haloarchaea would assist in
(0) by MerA. merA and merB genes are also enlightening the knowledge of metal physiology
found in Halobacterium sp. strain NRC-1 and in haloarchaea that would make haloarchaea a
Haloterrigena, respectively, (Osborn et al. potential candidate for remediation of metal-
1997). polluted hypersaline environment (Table 9.1).
9 Interaction of Haloarchaea with Metals 149

Table 9.1 Heavy metal resistance of haloarchaea isolated from various econiche
Haloarchaeal sp. Isolation site Metal ion(s) MIC (mM) References
Halobacterium sp. Solar salterns, Zn2+, Cu2+, Ag+, 0.5, 12.5, 0.5, Nieto et al.
Spain Hg2+, As3+, Cd2+ 0.01, 20, 2.5 (1987)
Halocula sp. Solar salterns, Zn2+, Cu2+, Ag+, 0.05, 2.5, 0.05, Nieto et al.
Spain Hg2+, As3+, Cd2+ 0.01, 10, 0.05 (1987)
Halobacterium sp. Estuarine Li+, As5+, As3+, Mn2+, Ni2+, 200, 60, 3, 50, 5, Khandavilli
salterns, Goa, Cu2+, Zn2+/Cd2+/Fe2+/Fe3+ 2.5, 2 et al. (1999)
Halobacterium sp. Salt lake Fe2+, Mn2+, Co2+ 7.5, 2, 0.6, 0.05, Kaur et al.
NRC-1 Zn2+, Ni2+, Cu2+ 2, 1.2 (2006)
Haloferax mediterranei Solar saltern Zn2+, Ni2+, Pb2+, Cr6+ 1, 2.5, 2.5, 5.0 Popescu and
Dumitru (2009)
Haloferax sp. TL 5 Telega salt lake, Zn2+, Ni2+, Pb2+, Cr6+ 0.5, 2.5, 2.5, 5.0 Popescu and
Romania Dumitru (2009)
Halobacterium Solar salterns Zn2+, As3+, Cd2+ 0.01, 0.001, Williams et al.
saccharovorum, H. Tamil Nadu, 0.001 (2012)
salinarum, India
Haloarcula sp. IRU1 Hypersaline As5+ 0.65 Taran et al.
Urmia lake, Iran (2013)
Haloferax volcanii Estuarine Zn2+ 1, 0.5, 0.5, 1, Salgaonkar
BBK2, Halorubrum strain salterns, Goa, respectively. et al. (2015)
BS17, Haloarcula India
japonica BS2,
Halococcus strain BK6

Bolhuis H, Palm P, Wende A et al (2006) The genome of

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J Biochem 192(3):591599
Manganese-Tolerant Bacteria
from the Estuarine Environment 10
and Their Importance
in Bioremediation
of Contaminated Estuarine Sites

Flory Pereira

Eco-sensitive environs such as coastal and marine ecosystems of the world
are constantly facing the risk of destruction. Estuaries, mangroves and
solar salterns being fragile econiches are very susceptible to disturbances,
both natural and anthropogenic. Metal mining is currently one of the most
polluting anthropogenic activities, projected to have a major impact on the
ecosystem. Estuaries provide a major pathway for transferring pollutants
such as metals into the oceans and back again from the oceans to the rivers
which ow into the adjoining areas such as salt marshes, mangroves and
salterns. The sediments of these econiches serve as an ecological sink,
where the metals concentrate to several orders of magnitude above the
normal background levels. Consequently, microorganisms inhabiting
these sites are adapted to both the estuarine and their own unique environ-
ment. Several groups of multimetal-tolerant bacteria have been isolated
from such environments. Manganese (Mn), though an indispensible metal
for biological function, could result in toxicity at elevated concentrations.
Consumption of solar salt contaminated with metals like manganese could
be a major route for human exposure. The mitigation of manganese from
such contaminated sites by Mn-tolerant bacteria provides a safe and
environment-friendly alternate technology for the future. Manganese-
tolerant bacteria are capable of scavenging not only Mn but also many
other metal contaminants, viz. Co, Ni, Zn, Cu, Pb, Cd and Hg. Cumulative
strategies by which these estuarine bacteria resist high concentrations of
manganese include extracellular sequestration, biosorption, precipitation,
oxidation and regulation of stress proteins. This article seeks to give an
insight into some of the molecular mechanisms adopted by halotolerant

F. Pereira (*)
Department of Microbiology, PESs Shri Ravi
Sitaram Naik College of Arts and Science,
Farmagudi, Ponda, Goa, India

Springer Science+Business Media Singapore 2017 153

M.M. Naik, S.K. Dubey (eds.), Marine Pollution and Microbial Remediation,
DOI 10.1007/978-981-10-1044-6_10
154 F. Pereira

bacteria from the estuarine environment for tolerating manganese, as they

could be a key to effective minimization and mitigation of mining impacts
in contaminated estuarine sites.

10.1 Introduction pogenic sources into ve main groups: (1) metals

released from natural atmospheric deposits such
Due to the toxic and persistent character of met- as copper, chromium, cadmium, uranium, lead,
als, contamination with metals in coastal and arsenic and mercury; (2) metals from mining and
marine environments is one of the critical issues smelting of ores such as iron, manganese, lead,
(Varol and Sen 2012; Zhan et al. 2010) for both cadmium, mercury and arsenic (Zouboulis et al.
the naturally stressed marine ecosystem and 2004); (3) metals used in industries such as
humans who are dependent on marine resources cobalt, nickel, zinc, copper, chromium, cadmium,
for food, recreation and industry (Naser 2013). arsenic and mercury; (4) metals in agriculture
Estuaries form a zone where a transition from a (zinc, copper, cadmium, selenium, uranium, lead
river environment to a maritime environment and arsenic); and (5) metals contained in dis-
takes place (Yang et al. 2014); therefore, they are posed wastes such as copper, zinc, chromium,
governed both by riverine and marine inuences. cadmium, lead, mercury and arsenic. When the
Since they receive large volumes of nutrient threshold levels are exceeded, all metals can be
inputs, both from freshwater runoffs and pollut- toxic to marine and estuarine organisms, as well
ants entering seawater, they represent some of the as to their consumers at higher trophic levels
most biologically productive ecosystems on the including man. This critical limit depends on the
planet (Merrield et al. 2011; Edgar et al. 2000). availability of the metal to the target species,
They also provide invaluable ecosystem services which is further governed by environmental
upon which all organisms depend (Daily et al. factors.
1997). Metals gain entry into coastal and marine Until a few years back, not much attention
environments via varied sources and activities, was paid to manganese globally, perhaps because
including anthropogenic disturbances and coastal it was commonly perceived to be a trace element
development. High concentrations of metals are that is essential but comparatively non-toxic
introduced into the environment during metal (Kehres and Maguire 2003). However, manga-
mining activities and during the processing of nese, being a constituent of multiple enzymes as
metals for industrial use. Other factors that con- well as functioning as an activator of many other
tribute to metal pollution in the estuarine envi- enzymes, has a vital role in a number of physio-
ronment are the disposal of wastes and efuents logical processes (Nielsen 1999). In vertebrates,
containing metals from various industries such as it serves as a cofactor for arginase in urea forma-
the agriculture and petroleum industry (Oyetibo tion (Sekowska et al. 2000); in gluconeogenesis,
et al. 2010). Bonnevie et al. (1994) and Al-Masri it serves as a cofactor for pyruvate carboxylase. It
et al. (2002) have reported local sources speci- also promotes activities of other enzymes such as
cally known to be the leading causes of metal transferases, for example, glycosyltransferase in
pollution in sediments, viz. metal-based indus- cartilage formation (Keen and Zidenberg-Cherr
tries (e.g. Cd, Cr and Zn from electroplating; Ni, 1996), glutamine synthetase and aminopeptidase
Pb and Cu discharge from smelters; Pb and As P (Yocum and Pecoraro 1999) in protein metabo-
from petroleum reneries; paint and dye formu- lism (Abell et al. 1995) and farnesyl pyrophos-
lators containing metals such as Cu, Zn, Cd, Cr, phate synthetase in fat metabolism (Institute of
Se, Pb and Hg), as well as efuents from plants Medicine 2001). Manganese enzymes are partic-
manufacturing chemicals. On similar lines, Ross ularly essential in detoxication of superoxide
(1994) divided metal contamination from anthro- radicals by superoxide dismutase (Leach and
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 155

Harris 1997). Besides, Mn also has diverse func- gicide products, alloys of iron and steel as well as
tions within the bacterial cells. Manganese is a livestock feeding supplements (HSDB 2001;
cofactor for enzymes such as PEP synthase (Chao ATSDR 2000; IPCS 1999). Manganese, due to its
et al. 1993), 6-phospho--glucosidase (Thompson versatility, nds a variety of applications such as
et al. 1999), 3-phosphoglycerate mutase (Chander in the removal of green colour caused by the
et al. 1998) and pyruvate carboxylase presence of iron contaminants in glass, as an
(Mukhopadhyay et al. 1998) involved in carbo- additive in various products such as dry cell bat-
hydrate metabolism. teries for the prevention of hydrogen formation
The importance of manganese in living sys- and in black paints as a drying agent. The use of
tems is undeniable. In Cyanobacteria, oxygenic manganese containing potassium permanganate
photosynthesis occurs in the reaction centre com- as a bleaching agent and as an oxidant for clean-
plex of photosystem II, which requires a tetra-Mn ing and disinfection purposes is already well
cluster (Morgan et al. 1998). The importance of known (ATSDR 2000; HSDB 2001). In some
Mn in bacterial signal transduction has been doc- locations, manganese greensands have been used
umented by Missiakas and Raina (1997). In for the treatment of drinking water (ATSDR
nucleic acid synthesis, endonuclease IV (Hoseld 2000). Methylcyclopentadienyl manganese tri-
et al. 1999) and ribonuclease HII (Ohtani et al. carbonyl (MMT), an organic manganese com-
2000) are Mn-dependent enzymes. All living pound, nds extensive application in the United
cells are protected from reactive oxygen species States of America (USA) in unleaded petrol as an
(ROS) because of its detoxication by the octane-enhancing agent. Similar use of MMT has
enzymes mangani-catalase (Whittaker et al. also been reported from Canada, South America,
1999) and superoxide dismutase (Fridovich Europe and Asia (Lynam et al. 1999). All these
1995), both of which require Mn2+. Mn2+- products would nally form primary sources of
containing enzymes such as ortho-phosphatases contamination. In addition, sewage sludge and
are known to be involved in regulating vegetative municipal wastewater discharges also contain
growth and cell density in bacteria during station- manganese. Consequently, considering the vast
ary phase, fruiting body development, assimila- applications of manganese, it is being investi-
tion of carbon and nitrogen, response to stress, gated by scientists as a possible emerging con-
cell segregation (Shi 2004), formation of bacte- taminant, especially in the marine environment.
rial spores and cell wall stabilization (Doyle Whether manganese is toxic or not would
1989). In addition, non-enzymatic Mn2+ is critical depend on the permissible/safe levels in our
for appropriate functioning of diverse products of waters and the dietary intake from all sources that
bacterial origin, such as antibiotics (Archibald has the ability to produce harmful effects.
1986) and exopolysaccharides (Appanna 1988), According to estimates by the World Health
and for oxidation of poly--hydroxy butyrate in Organization (WHO), the daily dietary consump-
Sphaerotilus discophorus as reported by Stokes tion of manganese in adults is between 0.7 and
and Powers (1967). Kirchner and Grabowski 10.9 mg, with the intake being higher for vegetar-
(1972) determined that even in the environment, ians consuming large proportions of manganese-
manganese plays the indirect yet signicant role rich nuts, legumes and grains in their diet as
of controlling the availability of nutrients in compared to the non-vegetarians (WHO 1973).
freshwater, by complexing it with iron. WHO has set the acceptable limit of manganese
However, excessive levels of essential ele- in drinking water as 0.4 mg/l. Based on a review
ments too can be toxic at elevated levels (Gadd by Greger (1999), the Food and Nutrition Board
1992). During the past century, industrialization of the Institute of Medicine (IOM 2002) sets the
has been occurring at a very fast pace prompting tolerable upper intake level of manganese for
large-scale production of compounds containing adults at 11 mg/day and the adequate intake lev-
manganese such as metallurgic and chemical els at 2.3 mg/day for men and 1.8 mg/day for
products, varnishes, ceramics, fertilizers and fun- women. However, based on epidemiological
156 F. Pereira

studies on the effects of manganese exposure via 10.2 Manganese Biogeochemistry

drinking water on the central nervous system of
humans, it has been estimated that 0.8 mg/kg/day In order to study manganese as a pollutant, it is
was the lowest observed adverse effect level imperative to comprehend the behaviour of man-
(LOAEL). More recent epidemiological and ganese in the environment. In nature, manganese
experimental studies however conrm that an is present in various oxidation states, from +2 to
undeniable link exists between manganese expo- +7; but the three main oxidation states between
sure and detrimental changes in the central ner- which it cycles is Mn2+ (which exists as the highly
vous system. According to Adekunle et al. soluble Mn(H2O)62+), Mn3+ and Mn4+ (which pre-
(2014), manganese is a toxic essential trace ele- cipitate typically as sparingly soluble oxyhydrox-
ment, meaning that though it is necessary for the ides) (Hansel and Francis 2006). Since Mn2+ is
survival of humans, it is also toxic when present unstable, it tends to oxidize and precipitate or
at high concentrations in the human body. dissociate to either Mn3+ or Mn4+ (Tekerlekopoulou
Manganese toxicity occurs due to exposure to and Vayenas 2008). Cycling between these forms
high levels of manganese, causing manganese is typically due to abiotic or biotic oxidation and
madness or Parkinson-like diseases (Gerber reduction reactions (Schamphelaire et al. 2008).
et al. 2002; Erikson and Aschner 2003; Perl and The Mn2+ form is the only stable form in soil
Olanow 2007), neurological changes (Santamaria solution, while Mn3+ and Mn4+ are stable in the
and Sulsky 2010) and abnormalities of the solid soil phase (McBride 1994). How manga-
immune (Vartanian et al. 1999) and reproductive nese is transported and partitioned in water
(Kim et al. 2012) systems. In mammals, large depends upon its solubility, which in turn is gov-
amounts of manganese have been reported to be erned by other factors such as the oxidation-
toxic to the embryo and foetus and are also reduction potential, pH and the associated anions.
known to affect fertility. On the other hand, de- If neutral conditions exist, then the Eh (redox
ciency of manganese has also been reported to be potential) inuences manganese mobility more
teratogenic and affect fertility (Gerber et al. than the pH. Under conditions where there is a
2002). In general, carcinogenicity of compounds deciency of oxygen, manganese occurs as the
containing manganese follows the order MnCl2 > highly soluble Mn2+, but under oxygen-rich con-
KMnO4 > MnSO4. The toxicity though largely ditions, it is found as the oxidized, highly insolu-
depends on the route of administration and the ble, stable MnO2 form (Calvert and Pedersen
valence state, with Mn(II) being more toxic than 1996). The concentration of manganese could
Mn(III). Further, manganese toxicity also thus be much higher in anaerobic groundwaters
depends on the associated anion; for example, the compared to aerobic surface waters (Marbaniang
chloride ion is less toxic than the citrate ion 2012), because manganese would be released
(Smith 1972). In many cases, metal toxicity is a from its minerals and reduced to Mn2+ which is
direct outcome of biomagnication. Although its more soluble form, when the conditions are
biomagnication of manganese in food chains is anaerobic. As the depth increases, anaerobic con-
insignicant (ATSDR 2000), bacterial mobiliza- ditions in the soil would increase, thereby
tion and immobilization of manganese from sedi- increasing the reduction potential. Under such
ments are noteworthy, especially in waters like circumstances, oxygen is the rst to be used up,
that of solar salterns, where consumption of and then nitrate and manganese are used as elec-
heavy metal-contaminated solar salt could be a tron acceptors. As the reducing conditions pro-
major route for human exposure, with potential gressively increase, iron gets reduced, followed
long-term implications on human health. by sulphate. Therefore, even though manganese
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 157

comprises a negligible chemical constituent of saltern well within permissible limits. However,
fresh and marine water, the conversion of soluble the impact of environmental factors cannot be
Mn2+ to insoluble Mn3+ and Mn4+ oxides and oxy- ignored. Experiments conducted by Spratt and
hydroxides is environmentally signicant. This is Hodson (1994) showed that manganese oxidation
supported by the fact that after oxygen, the oxides rate in sediments from a salt marsh was linked to
and oxyhydroxides of manganese are the most salinity and decreased with an increase in hyper-
potent oxidizing agents in the environment and saline conditions. However, in a similar study,
therefore may have an important role to play in Atkinson et al. (2007) showed that rather than
aquatic geochemical cycles (Sunda and Huntsman changes in dissolved oxygen concentration
1990; Laha and Luthy 1990). In addition, oxides (38 mg l1) or salinity (1545 %), a change in
and oxyhydroxides have a remarkable charge dis- pH of the overlying water from 5.5 to 8.0, and
tribution and surface area, which makes them disturbance of the sediment by physical mixing,
among the most important and strongest sorbents resulted in greater metal release and sequestra-
and scavengers of many metals (Tebo et al. 2004). tion rates. Balzer (1982) reported that below
They thus form potentially rich reservoirs of 16 % saturation, i.e. under hypoxic conditions,
adsorbed metals (Shi et al. 1999; Bratina et al. the concentration of dissolved manganese
1998). increased to levels approaching 1500 g/l, which
The estuarine environment is known to be a is above that normally found in seawater.
highly dynamic environment, subject to uctua- Huntsman and Sunda (1980) detected an increase
tions in various parameters. Manganese like all in rate of oxidation of manganese when a cata-
other metals is more soluble in freshwater com- lytic surface like manganese dioxide was used or
pared to saline water. Pereira et al. (2013) when the pH was increased. Manganese oxides
reported that metal cycling and solubility in salt- have been reported to be precipitated extremely
ern water are affected by several parameters such rapidly following oxidation, in a stream where
as salinity, pH, Eh and dissolved oxygen. there was acid mine drainage carrying manganese-
According to them, the mean value of manganese rich inows (Scott et al. 2002). Olaniran et al.
recorded in the overlying water of the Ribandar (2013) conducted experiments on the solubility
Saltern in Goa, India, was 0.6 ppm during the of cadmium, wherein they observed that when
salt-making season, whereas for the non-salt- the pH was increased from 6 to 7 in 1.3 mM
making season, it was lower at 0.4 0.3 ppm. phosphate, its solubility decreased 8.8-fold. They
Similarly, in the saltern sediment, manganese demonstrated that metal solubility and bioavail-
concentration varied by 34 % with a change in ability could decrease by several orders, even
salinity. As the salinity increased, the metal- with a small pH change. Similar observations
borne suspended particulate material quickly were made by Pereira et al. (2013), who found
settled down due to increased coagulation and that the level of dissolved manganese in the salt-
occulation, thereby sedimenting the metal. ern decreased approximately twofold, when the
When the salinity or pH of the overlying water pH was increased from 7.4 to 8.4. Recent studies
decreased, the metals which had sedimented by Hallberg and Johnson (2005) concluded that
were released back into suspension, thereby mak- very little oxidation of Mn2+ can be observed
ing the metals bioavailable to the microorgan- below pH 8 and that on many occasions it pro-
isms. Saltern sediments have been shown to be ceeds even more slowly in the presence of iron.
dramatically inuenced by cyclic changes in Nairn and Hedin (1993) demonstrated that Fe2+
salinity and pH of the overlying water, thereby impedes biological oxidation of Mn2+ at ferrous
making metals more bioavailable and subse- iron concentrations exceeding 1 mg/l, thus pre-
quently resulting in a larger metal-tolerant bacte- venting its removal from aerobic wetlands. In a
rial population. These metal-tolerant bacteria, study on the Ribandar Salterns of Goa, India,
originally of estuarine origin, play an important receiving its input from the adjacent Mandovi
role in maintaining the metal concentration in the Estuary, Pereira et al. (2013) observed that the
158 F. Pereira

salterns receive a fair share of contaminants, 10.3 Manganese as a Pollutant

especially ferrous iron, with concentrations in in the Marine Environment
excess of 1 mg/l. In the presence of such high
levels of Fe2+, biological oxidation of Mn2+ is The measure of manganese naturally occurring in
expected to be hampered. Thermodynamically, at groundwater is between 1 and 10 ppm, whereas
neutral pH, the spontaneous oxidation of Mn2+ to the levels are as high as 1001000 ppm in river
Mn4+ should occur when the waters are well aer- water (Nealson 1983). Barceloux (1999) detected
ated. However, because Mn2+ requires higher concentrations in freshwater typically ranging
activation energy compared to ferrous iron, the from 1 to 200 g/l, contrary to the large amount
oxidation of Mn2+ would be greatly slowed down (0.2 mg kg1) found in seawater by Bowen (1979)
in non-acidic waters, making it far more stable and Ehrlich (2002). According to ATSDR (2000),
than ferrous iron. Additionally, iron can form US EPA (1984) and Zeri et al. (2000), average
insoluble sulphides, but the same does not hap- manganese concentration in seawater is about 2
pen with manganese; consequently, iron can be g/l (Barceloux 1999), with the ambient concen-
removed continuously from the environment, but trations ranging from 0.4 to 10 g/l. In anaerobic
manganese almost never precipitates as the sul- regions of open seawater, Lewis and Landing
phide forms. Hence, it is generally precipitated as (1991, 1992) have reported concentrations as
rhodochrosite (MnCO3), and its removal by high as 500 g/l. In the ocean, Glasby (2006)
sulphate-reducing bacteria is uncommon. There obtained levels of dissolved manganese (Mn2+)
have been solitary reports of -MnS (rambergite) up to 0.23 nmol kg1. Smith et al. (1987) sam-
nanoparticles being produced in the deep anoxic pled 286 rivers and streams from the United
marine basins of the Baltic Sea, by the bacterium States and detected an average dissolved manga-
Shewanella oneidensis MR-1 due to dissimila- nese concentration of 24 g/l. Howe et al. (2004)
tory metal reduction. The biogenic manganese found manganese ranging from 0.03 to 4.0 g/l in
sulphide produced was attributed to the simulta- the north-east Atlantic Ocean, the North Sea, the
neous reduction of MnO2 and thiosulphate English Channel and the Indian Ocean. Similar
together with the oxidation of H2 (Lee et al. levels of manganese, i.e. 0.225.5 g/l, were
2011). However, to date, no such reports of MnS reported by Alessio and Lucchini (1996) off the
production are available from the estuarine envi- coast of the United Kingdom, from the coastal
ronments. Given the extreme conditions preva- waters of North Sea and the Irish Sea. In the
lent in salterns, the removal of manganese from north-western cities of Madagascar, Ravelonabdra
saltern waters with concentrations exceeding et al. (2010) determined levels of manganese at
1.06 % in the sediment and 0.64 mg L1 in water, Nosy Be to be as high as 36.02119.1 g/g of
respectively, and with a relatively high pollution sediment. Evaluating the contamination of the
load index >1 poses quite a challenge. However, Aden Port sediments in Yemen, Nasir et al.
the abundance of manganese-tolerant bacteria (2006) reported levels of manganese ranging
proves benecial here (Pereira et al. 2013). Many from 138.23 to 658.87 g/g. In acidic waters
microorganisms capable of catalysing Mn2+ oxi- where the pH is less than 2.5 due to drainage
dation have been identied, and a few of them are from acid mines, Filipek et al. (1987) recorded
even known to utilize this reaction as a source of dissolved manganese concentrations as high as
energy (Lovley and Phillips 1988; Nealson and 4400 g/l. According to Reimer (1999), dis-
Myers 1992; Ehrlich 1996; Tebo et al. 2005). The solved manganese concentrations in natural
activation energy barrier for oxidation of manga- waters free from anthropogenic pollution range
nese being overcome biologically, the adsorption from 10 to 10,000 g/l, with the concentration
of Mn2+ onto MnO2 and its subsequent precipita- generally not exceeding 200 g/l (Pearson and
tion as Mn+4 become much more rapid. Greenway 2005). Howe et al. (2004) also
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 159

documented the levels of manganese from vari- Estuary of Goa. This estuary receives a consider-
ous sites in a report called the Concise able supply of inorganic nitrogenous nutrients
International Chemical Assessment Document from a variety of sources such as mining rejects
63. According to the report, lakes receiving inputs (De Souza 1999), land runoff (Sardessai and
from urban areas, industries as well as windborne Sundar 2007) and sewage efuents (Ansari et al.
dust from old mine dumps contained manganese 1986). Alagarsamy (2006) reported manganese
ranging up to 13,400 mg/kg dry weight; sedi- ranging from below detection limits to 1.61 %
ments from the river contained concentrations as from the surface sediments of the same estuary.
high as 4106700 mg/kg dry weight, whereas Interestingly, the Ribandar Saltern, which lies
sediments from the intertidal mudats had 100 along the Mandovi Estuary and evaporates the
1000 mg/kg. Similarly, concentrations of manga- water it receives, contained an average manga-
nese as high as 35508960 mg/kg dry weight nese concentration of 0.72 0.16 % in the sedi-
have been reported from the Baltic Sea surface ment during the non-salt-making season, which
sediments and were attributed to the load of fer- showed an increase by 42 % in the salt-making
romanganese in the river. season (Pereira et al. 2013).
India has manganese ore mines at Karnataka, Heavy metal contamination in aquatic marine
Odisha, Madhya Pradesh, Maharashtra and Goa. ecosystems is found to be localized near highly
In Karnataka, in the central estuarine region rep- urbanized, industrialized areas along the coast.
resented by Gurpur and Swarna Estuary, manga- These contaminants being associated with very
nese concentrations as high as 90 5 % and ne particles often remain in suspension for an
77 10 %, respectively, have been reported from innite period of time. Eventually, they get
the mudat sedimentary environment (Fernandes deposited in the sediments, where they may be
and Nayak 2015). In the proximity of the mining concentrated tenfold to 100-fold higher than in
regions of Jharkhand, Orissa and West Bengal, solution. Therefore, sediments behave as sinks
manganese levels detected were as high as 0.024 for all pollutants, from where they are slowly
0.165 % in the surface sediments of the Dhamra released into the surrounding long after the pri-
Estuary (Barik and Panda 2014). High levels of mary source has disappeared, thereby emerging
manganese (12.59 mg/kg) were obtained in the as an important secondary source of contamina-
sediment of Siddheshwar Dam constructed on tion (Temara et al. 1998; Pereira et al. 2013).
Purna River in the Hingoli district of Maharashtra Mangroves and estuaries serve as nurseries
state (Parveen and Bhosle 2013). The highest and and breeding grounds for a number of commer-
lowest levels of manganese recorded in the waters cially important marine organisms such as shell-
of Manjara Dam of Maharashtra were 0.1866 sh, sh, shrimps and crabs. Exposure of human
mg/L and 0.057 mg/L, respectively (Mane et al. beings to toxic heavy metals occurs when they
2013). In Madhya Pradesh, water samples of consume seafoods contaminated with metal.
Birsinghpur town and its neighbouring areas in Metal accumulation by an organism from its sur-
the District of Satna were found to be contami- rounding water indirectly reects the extent of
nated with manganese ranging from 0.125 to pollution in the estuarine water. The extent of
0.292 mg/l in all surface waters and 0.0120.248 heavy metal bioaccumulated by an organism
mg/l in 30 % of groundwater samples (Shrivastava depends on the rate of uptake of metal by the
and Mishra 2011), which exceeded the limit of organism and its physiology. In the case of sh,
0.1 mg/l recommended by the WHO. Wasim the major factors that determine the rate of accu-
et al. (2010) detected manganese concentrations mulation are age and size of the sh, time taken
as high as 0.0221.78 mg/l in the surface water of to complete its life cycle, its feeding habits and
Ganga River around Kolkata in West Bengal, ecological needs as well as other environmental
whereas Attri and Kerkar (2011) reported an parameters such as salinity, pH, hardness and
average manganese concentration of temperature of the water (Canli and Atli 2003).
0.19 0.002 %, in the sediment of the Mandovi Even at lower trophic levels, aquatic biota can
160 F. Pereira

bioconcentrate manganese from water to a large Kasimpur in Aligarh receives wastewater con-
extent. Howe et al. (2004) have estimated taining pollutants, namely, y ash and heavy met-
bioconcentration factors (BCFs) for various als, from the Harduaganj Thermal Power Station
marine species such as sh (35930), mussels (HTPS). In a study on sh exposed to efuents
(800830), phytoplankton (25006300), marine from this power plant, Javed and Usmani (2013)
and freshwater plants (200020,000) and mac- noticed bioaccumulation of heavy metals such as
roalgae (3005500). Zingde et al. (1976) detected Mn in body organs such as the gills, liver and kid-
metals such as manganese, zinc, copper and arse- ney and in muscle tissue and integument of the
nic in the ora and fauna from the coastal waters sh Mastacembelus armatus, which exceeded
and estuaries of Goa and attributed it to the fer- severalfold the maximum permissible limit of
romanganese ore-bearing landmass and the min- Mn (1.0 ppm) set by the FAO (1983). The liver
ing operations. Among seaweeds, they identied seemed to have the maximum afnity for bioac-
Sargassum tenerrimum as the most efcient cumulation of Mn (49.96 mg Kg1.dry weight),
accumulator of trace metals, followed by Padina whereas muscle tissue had the least afnity
tetrastromatica. Among crustaceans, highest (9.03 mg Kg1.dry weight). Similar studies for
metal values were found in crabs, while prawns, comparison of heavy metal content were con-
mussels and bivalves showed intermediate levels. ducted by Gawade et al. (2013) on grey mullet
Oysters showed least preference for arsenic and sh (Liza aurata) and oysters (Crassostrea
manganese. A study by George and Kureishy madrasensis) from Gosthani Estuary in
(1979) on bioaccumulation factor in zooplankton Bheemunipatnam which lies on the east coast of
from a limited area of the Bay of Bengal showed India and clam (Polymesoda erosa) samples col-
that their bioaccumulation factor was highest for lected from the Mandovi Estuary which lies on
Fe (15,638), followed by Co (1955), Zn (1042), the west coast of India. In the Gosthani Estuary,
Ni (442), Mn (358) and Cu (228), respectively. average concentrations of Fe were 754.3 350.2
KesavaRao and Indusekhar (1989) reported the g/g in sh gills, 415.1 134.7 g/g in oyster and
trend in concentration factor of the elements as 60.5 40.59 g/g in muscle tissue of sh, respec-
Mn > Zn > Cu > Co > Ni in seawater and sea- tively, whereas Mn was 42.2 16.7 g/g in tissue,
weeds (green, brown and red) from Saurashtra 380.9 176.1 g/g in gills and 48.4 20.7 g/g in
coast. A study on trace metals bioaccumulation oyster, respectively. In the Mandovi Estuary,
conducted by Alam et al. (2010) on four soft- clam showed average accumulation of
bottom polychaetes from the mangrove wetlands 711.7 206.3 g/g Fe and 53.9 27.6 g/g Mn.
of the Indian Sundarban showed Mn enrichment High Fe (79.5 %) and Mn (9.4 %) concentrations
as high as 730 mg/kg body weight. In Uppanar in clams collected from Mandovi Estuary have
Estuary which lies on the southeast coast of India, been attributed to Fe and Mn ore mining. The
metals such as Mn, Pb, Cd and Cr were beyond metal accumulated in an organism from its food
the permissible limits set by the World Health source thus gets concentrated many times higher
Organization. As expected therefore, Pravinkumar at higher tropic levels as biomagnication occurs.
et al. (2014) found metals accumulated to differ- In the estuarine environment, heterotrophic
ent levels in the liver, muscle and gill of nsh marine bacteria play a signicant role in food
and tissues and the gill of shellsh collected from web dynamics, metal speciation and bioavailabil-
both the Uppanar Estuary and the Vellar Estuary ity, as well as in the biogeochemical cycling of
from the same vicinity. Tissue samples of nsh the metals such as Mn, by accumulating them to
and shellsh showed high levels of metal accu- varying degrees and releasing them back into the
mulation, i.e. Mg (9.7431.55), Mn (0.0911.12), water column in dissolved form, depending on
Fe (1.429.85), Al (1.263.95), Zn (0.312.31), environmental changes, thus either making them
Cr (0.252.91), B (0.170.51), Pb (0.140.50), more bioavailable than their inorganic forms or
Cd (0.100.72), Cu (0.050.62), Ni (0.020.30) by immobilizing them and mitigating toxic levels
and Co (00.18) g g1 dry weight, respectively. (Pereira et al. 2013).
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 161

10.4 Manganese-Tolerant oxidizers with activities exceeding 50 mM MnO2

Bacteria were isolated by Yang et al. (2013) from among
the phyla Proteobacteria (Escherichia,
Extensive studies have been carried out on the Cupriavidus, Ralstonia, Variovorax),
use of microorganisms for bioremediation of Actinobacteria (Agromyces, Cellulomonas,
manganese by sequestration, precipitation or Microbacterium) and Firmicutes (Bacillus).
alteration of the oxidation state. Some of the Pereira et al. (2012) isolated halotolerant
most remarkable manganese-tolerant bacteria are Chromohalobacter beijerinckii which was toler-
Bacillus, Leptothrix discophora, Pseudomonas ant to manganese at concentrations as high as 10
putida and Pedomicrobium (Geszvain et al. 2013; mM at a salinity of 20 % (w/v), from saltern sedi-
Ridge et al. 2007; Saratovsky et al. 2006; ments of Ribandar in Goa, India. This saltern is
Villalobos et al. 2003; Brouwers et al. 2000); fed by waters from the Mandovi Estuary which is
among fungi Acremonium, Cephalosporium sp., a waterway for shipping ferromanganese ores.
Phanerochaete, Paraconiothyrium, Phoma, They obtained Mn-tolerant bacteria with counts
Periconia sp., Coniothyrium and Sporothrix sp. as high as 105106 CFU g1 from the sediment
are well known (Sasaki et al. 2006; Saratovsky and 104 CFU ml1 in the overlying water of the
et al. 2009; Timonin et al. 1972), whereas algae Ribandar Saltern, respectively (Pereira et al.
like Cladophora and Gloeothece magna 2012). Of the total viable population tested on
(Mohamed 2001; Duggan et al. 1992) play an nutrient media, they found that 82 % isolates
important role in bioremediation of metal in the were resistant to manganese, iron and lead at a
environment. Because of their ability to catalyse salinity of 1.5 % (w/v) and 74 % were resistant to
the oxidation or reduction of manganese (Cerrato manganese at 20 % salinity. At a lower salinity of
et al. 2010; Gounot 1994), manganese-oxidizing 1.5 % (w/v), multimetal tolerance to a minimum
bacteria which are ubiquitous and found in just of ve metals was observed in 20 % of Fe and Mn
about any habitat exposed to high levels of man- isolates, 18 % of Co isolates and 11 % Ni isolates,
ganese are ideal candidates for manganese with some showing tolerance even to seven met-
cycling, especially in freshwaters, marine waters, als. Interestingly, at a higher salinity of 20 %
sediments and submarine basalt surfaces, hydro- (w/v), a higher percentage of bacteria showed
thermal vents, ferromanganese nodules and cave multimetal tolerance, i.e. 28 % Mn isolates, 18 %
deposits (Hansel and Francis 2006). Previous Pb isolates, 14 % Ni isolates and 10 % Fe and Cd
studies conducted by Tebo et al. (2004), on isolates.
Mn(II)-oxidizing bacteria in freshwater and
marine habitats, showed that the majority of these
bacteria belonged to either alpha- beta- and 10.5 Mechanisms of Manganese
gamma-Proteobacteria, Gram-positive Tolerance in Bacteria
Firmicutes having low GC content or Gram-
positive Actinobacteria with high GC content. The importance of metal ions such as manganese
Similar studies by Sullivan and Koppi (1993) on as vital cofactors for metalloproteins and enzymes
alsol soil having large Mn deposits yielded in many biological reactions is well known. Trace
Citrobacter freundii and Pseudomonas sp. capa- metals are required for such metabolic processes
ble of Mn(II) oxidation. Fe-Mn nodules and the such as photosynthesis, carbon xation, nutrient
soils surrounding these nodules were also a rich assimilation and respiration (Morel et al. 1991),
source of phylogenetically diverse bacteria (He in redox processes as a source of electrons in
et al. 2008). Hitherto unrecognized, Roseobacter- energy production and in anaerobic respiration as
like bacteria were shown to play a role in the oxi- electron acceptors (Doelman et al. 1994) and for
dation of Mn(II), especially in the coastal structural stability (Ji and Silver 1995; Poole and
environments (Hansel and Francis 2006). Mn(II) Gadd 1989; Hughes and Poole 1989). The role of
162 F. Pereira

Mn in protecting cells against reactive oxygen ions directly bind to negatively charged func-
species by serving as a cofactor for enzymes that tional pyruvyl, carboxyl, phosphoryl, hydroxyl,
remove harmful free radicals produced during succinyl and uronyl groups on the exopolymer.
metabolism is well studied (Tseng et al. 2001; Manganese entrapped in the EPS can be oxidized
Niven et al. 1999). Manganese forms non- directly by enzymatic reactions involving extra-
proteinaceous manganese antioxidants which can cellular polysaccharides (EPS) (Tebo et al. 1997;
combat oxidative damage without actually being Beveridge 2005; Ghiorse and Hirsch 1979). The
affected by Fenton chemistry. However, to sur- exopolymer matrix which surrounds the cell is
vive in any environment, it is critical for all the most common site for Mn(II) oxidation in
microorganisms to maintain homeostasis, i.e. -Proteobacteria, -Proteobacteria and low-GC
they need to keep a balance between the amount Gram-positive bacteria. However, the location
of metal actually available to them from their may vary. For example, both strains MnB1 and
immediate surroundings and that which is taken GB1 of Pseudomonas putida oxidized Mn(II)
up, in accordance with physiological needs, enzymatically on the outer membrane glycocalyx
because any imbalance would have serious con- (de Vrind et al. 1986; Caspi et al. 1998; Brouwers
sequences. It is necessary to regulate the metal et al. 1999), whereas strain SG-1 of Bacillus sp.
acquisition system based on the conditions pre- was found to deposit it on the exosporium (van
vailing in the surrounding. During metal starva- Waasbergen et al. 1996; Francis and Tebo 2002).
tion, the activity of the transporters and the The sheath of the mesophilic bacterium
expression of the relevant genes need to be Leptothrix discophora SS-1 was shown to be
upregulated, but when there is a surplus of met- encrusted with manganese oxides due to the sorp-
als, efux pumps need to be activated (Wakeman tion of these metal ions. This enzymatic oxida-
and Skaar 2012). Bacteria strictly maintain metal tion of Mn(II) was possible due to a protein
homeostasis by employing numerous transcrip- which is normally present on its extracellular
tional and biochemical regulators for the uptake sheath (Adams and Ghiorse 1986, 1987;
and export of metals, thus enabling them to adjust Brouwers et al. 2000; Corstjens et al. 1997).
to changing environmental conditions. A combi- Deposition of MnO on EPS was also reported by
nation of two or three fundamental mechanisms Ghiorse and Hirsch (1979) and Sly et al. (1990)
is employed for resistance and homeostasis of in Pedomicrobium spp. In Leptothrix discophora,
many metals, which are categorized as (1) use of isolated from Pinal Creek, near Globe, Arizona,
permeability barriers such as extracellular poly- the outer surface of the cell wall and holdfasts of
saccharides (EPS) and cell walls; (2) decreased the bacteria were found to be the main sites for
uptake or enhanced efux; (3) intracellular accu- concentration of Mn (Robbins and Corley 2005).
mulation; (4) extracellular precipitation as oxa- Studies by Gutierrez et al. (2012) revealed that
lates, phosphates, sulphides, oxides and EPS of Halomonas sp. TG39 showed similar
carbonates; and (5) enzymatic detoxication. binding capacity for Fe, Mn, Ca, Mg, Si and Al.
Interestingly, even though Halomonas sp. from
Carlsberg Ridge could only oxidize and precipi-
10.5.1 Use of Permeability Barriers tate Mn2+ extracellularly (Fernandes et al. 2005),
such as Cell Walls the same isolate in the absence of Mn2+ but in the
and Extracellular presence of other metals such as Ni and Co could
Polysaccharides (EPS) assimilate metals both intra- and extracellularly
(Antony et al. 2010; Sujith et al. 2010). In another
Gadd (1990) reported that tolerance to heavy study on R. etli M4 isolated from an environment
metals could be an intrinsic property of the contaminated with MnSO4-rich acid mine drain-
microorganism, for example, production of extra- age (AMD), Foster et al. (2000) demonstrated the
cellular mucilage or polysaccharide or an imper- remarkable ability of the EPS produced by this
meable cell wall. The positively charged metal organism to bind Mn(II) ions in the form of
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 163

MnSO4. Pulsawat et al. (2003) showed that the the cell envelope, which relies on the ability of
anion bound to Mn can signicantly affect both manganese-tolerant bacteria to sense Mn2+ and
the metal adsorption capacity and binding maintain homeostasis. Prokaryotes can accumu-
strength of the biosorbent. They reported a late Mn in high concentrations in their cytoplasm,
decrease in the specic adsorption capacity implying that the uptake of Mn must be carried
(qmax) of manganese to EPS, for example, when out by some energized pumping mechanism
sulphate was the corresponding anion, it showed (Frausto da Silva and Williams 1991). Such Mn2+
a maximum binding of 62 mg g1, followed by uptake systems are seen in many Gram-positive
nitrate (53 mg g1) and chloride (21 mg g1). bacteria like B. subtilis, L. plantarum and
Markus et al. (2002) have reported Mn adsorp- Staphylococcus aureus (Silver and Lusk 1987). It
tion of 43 mg g1 by dead biomass of Lecanora has been hypothesized that in some bacteria like
conizaeoides with Cl as the counter ion. In con- Deinococcus radiodurans, the hyperaccumula-
trast, Mn adsorption by Sargassaceae sp. was tion of low molecular weight Mn(II) complexes
approximately 100-fold lesser than the EPS from is a mechanism to protect these organisms from
R. etli M4 when the anions were sulphate (da oxidative stress and radiation damage to DNA
Costa et al. 1996). In the case of Rhizobium due to reactive oxygen species.
radiobacter F2, Wang et al. (2010) determined As per literature available, three kinds of Mn2+
maximum biosorption capacities of Mn(II) by uptake systems have been studied (Fig. 10.1).
extracellular polymeric substance (EPS) to be One is a P-type ATPase (MntA) with a high speci-
28.86 1.28 mg/g under optimum conditions. city for Mn2+ as seen in L. plantarum (Hao et al.
EPS, bacterial cell surfaces and manganese 1999). The second involves members of Nramp
oxides are all capable of absorbing heavy metals family, which is a natural resistance-associated
to different degrees and therefore profoundly macrophage protein (Nramp). Nramp (MntH) is a
inuence the fate and removal of heavy metals proton-dependent divalent cation transporter
from marine environments. However, reports of seen in several species of bacteria such as
manganese-resistant bacteria removing Mn from Escherichia coli, Salmonella and Brucella abor-
estuaries, mangroves or salterns by binding to tus. And the third conforms to the ABC trans-
EPS are limited. porter which belongs to the ATP-binding cassette
(ABC) superfamily described by Bartsevich and
Pakrasi (1995) who discovered the mntCAB
10.5.2 Decreased Uptake operon in a cyanobacterium Synechocystis sp.
and Enhanced Efux PCC 6803, which has a high requirement for
Mn2+ during photosynthesis. ABC permeases
The intracellular levels of Mn2+ are tightly con- directly utilize the hydrolysis of ATP to pump
trolled by regulating its import and export across manganese into the cytoplasm. These transport-

Mn Mn
H+ MntE Mn

Mn Mn

Fig. 10.1 Uptake and efux pumps for manganese resistance in estuarine bacteria
164 F. Pereira

ers comprise three proteins. One is a cation- also take place via manganese transporters.
binding protein (MntC) which is extracellular, the Transport of other divalent metals by manganese
second is MntA which is an ATP-binding protein transporters is possible when Mn2+ oxidation
in the cytoplasm, and the third is an intrinsic causes upregulation of the manganese transport-
transmembrane protein (MntB) that mediates ers. Such organisms wherein the export systems
transport. Substrates such as Mn2+ are exclusively can be regulated are ideal candidates for biore-
transported by inducible uptake systems like mediation of contaminated sites. Regulated sys-
ABC transporters in Streptococcus gordonii tems for export of Mn2+ may exist in bacteria, but
(Kolenbrander et al. 1998). In B. subtilis, a so far none have been reported from marine envi-
bifunctional metallorepressor protein (MntR) ronments or from mangroves.
acts on mntH such that the expression of Nramp
transporter is repressed when Mn2+ abounds in
the surrounding, but when Mn2+ is low it activates 10.5.3 Intracellular Accumulation
mntABCD transcription which encodes for the
ABC transporter (Que and Helmann 2000). In Intracellular traps can be biosynthesized as a
contrast to uptake systems, very few bacterial temporary measure for removal of metal ions.
Mn2+ efux systems are known. The only identi- Tolerance to heavy metal ions could be due to the
ed Mn2+ efux system is MntE in Streptococcus existence of metal-binding proteins such as
pneumoniae (Jakubovics and Valentine 2009) metallothionein that are capable of sequestering
and in Deinococcus radiodurans (Sun et al. metal and detoxifying it or exporting it out of the
2010), which is a new member of the cation dif- cells. These metal-binding ligand molecules
fusion facilitator (CDF) family. In addition to enable the organisms to survive even in an envi-
MntE, the P-type ATPase CtpC, from M. tubercu- ronment which has toxic levels of metals.
losis and M. smegmatis, and YebN (renamed Metallothioneins have the potential to sequester
mntP), a new system for export of Mn2+ in Hg, Cd, Pb, Zn, Ag and also Ni but with a lower
Xanthomonas oryzae pv. oryzae (Xoo), appear to afnity. There have been reports of metallothio-
facilitate manganese efux. MntP was found to neins in Cyanobacteria and several other phyla
be positively regulated by MntR, a transcription (Kagi and Nordberg 1979; Hamer 1986; Shaw
regulator dependent on Mn2+ (Li et al. 2011). Any et al. 1992). The role of metallothioneins in limit-
deletion in manganese transporting ATPases ing metal availability and thereby conferring pro-
(MntP) and cation-phosphate transporter (CtpC) tection against the harmful effects of toxic levels
genes resulted in manganese sensitivity due to of metal is documented in Anabaena PCC 7120,
accumulation of high levels of manganese inside Synechococcus PCC 7942, Pseudomonas aerugi-
the bacterial cell, implying that there may be a nosa and Pseudomonas putida (Blindauer et al.
threshold level of tolerance to Mn2+ inside bacte- 2002), but no reports of Mn sequestering are doc-
rial cells. Mn2+ functions as an antioxidant below umented, especially from marine environments.
this critical level, but when the threshold level is Cyanobacterial metallothionein gene SmtA from
crossed, it becomes toxic and induces excessive Synechococcus PCC 7942 was found to be hyper-
ROS production while decreasing the capacity to sensitive to Zn and Cd, but had no afnity for
scavenge ROS. Manganese therefore plays a sig- other metals (Turner et al. 1993). However, this
nicant role in bacterial physiology. In order to was not the case with E. coli, where smtA, besides
survive, the bacterial cells should be able to having high afnity for Zn and Cd, could also
respond to uctuations in external Mn2+. Here the bind Hg and Cu (Shi et al. 1992). Similar dis-
coordination between expression and activity of placement of Zn from the protein could also be
the transporters becomes crucial for the normal possible by Mn ions, though no such reports are
functioning of the cells. Many divalent transport- documented. The presence of pseudo-thioneins
ers can have broad substrate specicity. Kehres has also been reported by Higham et al. (1984) in
et al. (2000) showed that transport of other ions Cd-adapted Pseudomonas putida.
such as Fe2+, Ni2+, Cu2+, Co2+, Zn2+ and Cd2+ could
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 165

According to Cavat et al. (2003), the existence mechanism employed for its removal is by oxi-
of metallothionein gene in the bacteria may be an dation of Mn2+ to MnO2. A crust of Mn oxide
indication that the organism needs to sequester minerals was observed on the cell surface of
the metals which are present in its environment, in Pseudomonas putida GB-1 due to extracellular
order to prevent toxicity. In fact, high levels of Mn oxidation (Tebo et al. 1997). Here, the Mn
intracellular accumulation of Mn have been oxides not only protect the organism from exter-
shown to enable Lactobacillus plantarum, which nal reactive oxygen species (ROS) but may
lacks a superoxide dismutase, to survive under serve as a mechanism for reducing the toxicity
aerobic conditions (Archibald and Fridovich of Mn(II) and other toxic metals (Tebo et al.
1981). Similar studies by Daly et al. (2004) 2005). Mn(II) oxidation was visually apparent
showed that Deinococcus radiodurans could sur- in Roseobacter-like bacteria, as distinct brown
vive oxidative stress and high levels of ionizing oxide particles (<2 mm) after an incubation
radiation because it maintains low levels of iron period of 23 days (Hansel and Francis 2006).
(Fe) and high levels of Mn within the cell. Medicis Besides oxides, oxalates, phosphates, sulphides
et al. (1986) found that intracellular content of and carbonates also effectively cause extracel-
manganese in halophilic bacteria was higher, lular precipitation of manganese. Organic acids
implying that manganese could play a role in like malonate may be released into the sur-
haloadaptation. In many microbes, proteins like rounding medium, thereby binding manganese
phytochelatins (PCs) and metal-binding peptides and mitigating toxic levels from the
like the cysteine (Cys)-rich glutathione (GSH) environment.
confer resistance to metals by chelating the metals Precipitation of oxidized Mn was evident in
within the cell. In addition to peptides, intracellu- algae and Cyanobacteria, when the pH was
lar phosphates can bind manganese, forming greater than 8 during photosynthesis
phosphate-Mn complexes which are potent anti- (Richardson et al. 1988). Brachybacterium sp.
oxidants that have shown the capacity to react strain Mn32A, a manganese-oxidizing bacte-
efciently with superoxide, without displaying rium from deep-sea sediments, showed an
any pro-oxidant side effects shown by other exceptionally high ability to remove Mn(II) by
redox-active metals (Jensen and Jensen 2014). Mn oxidation, with resistance to Mn(II) as high
Hence, in a contaminated environment, metal as 55 mM (Fig. 10.2). Experimentally, the
accumulation, especially by phosphates within removal of Mn(II) by strain Mn32 occurred in
the cell, would be particularly signicant due to two steps. First, the soluble Mn(II) was oxi-
its unique application for remediation. High Mn dized to insoluble biogenic oxides of Mn such
concentrations may actually be benecial to the as manganite (MnOOH) and birnessite (MnO2
organism because it would result in a shift from a group); further, these biogenic Mn oxides
Fe-based metabolism to a Mn-based metabolism. served as adsorbants for more Mn(II) from the
When Fe is replaced by Mn, enzymes having Fe culture media (Wang et al. 2009). It was seen
cofactors exposed to the solvent are protected that the Mn oxides generated by strain Mn32
from oxidative stress because now Mn is bound to had better capacity to adsorb Zn(II) and Ni(II),
the cellular protein (Sobota and Imlay 2011). This i.e. it was two to three times higher than fresh,
is particularly signicant in an iron- and manga- synthetic commercial MnO2.
nese-rich environment such as an estuary contam-
inated with ferromanganese ores.
10.5.5 Enzymatic Detoxication

10.5.4 Extracellular Precipitation Heterotrophic bacteria can bring about Mn oxi-

dation rapidly using indirect mechanisms such as
The most efcient metal chelators are manga- by modifying the pH or redox conditions of their
nese oxide and organic acids. In manganese- surrounding or by releasing a chemical such as
contaminated environments, the most common superoxide which is an oxidant for Mn (Learman
166 F. Pereira

Fig. 10.2 SEM images of (a) Brachybacterium sp. strain surface of strain Mn32 when grown in liquid A medium
Mn32 cells grown in liquid A medium without MnCl2 for with 0.2 M MnCl2 for 9 days, (c) commercial MnO2, (d)
9 days, (b) encrustation of biogenic Mn oxides on the cell synthetic fresh MnO2 (Courtesy of Wang et al. 2009)

et al. 2011), as well as by direct mechanisms such was the direct catalyst of Mn(II) oxidation in
as the production of polysaccharides or by enzy- Leptothrix discophora and was also responsible
matic activity. When the pH of the environment for the oxidation of Mn by spores of the marine
ranges between 6 and 9, the natural indirect oxi- Bacillus species whose cells became encrusted
dation of Mn(II) to insoluble Mn(III, IV) oxides with amorphous Mn oxides, thereby efciently
is very slow. However, if the concentration of removing Mn(II) from solution (Rosson and
oxygen is about 35 mg L1 and the redox poten- Nealson 1982; van Waasbergen et al. 1996;
tial is above +200 mV (Schweisfurth et al. 1978), Francis and Tebo 2002; Dick et al. 2008). Francis
there is an increase in the rate of manganese oxi- and Tebo (1999) showed that the spores of
dation by four to ve orders, if Mn(II)-oxidizing Mn(II)-oxidizing marine Bacillus sp. strain SG-1
bacteria are present (Nealson et al. 1988; Tebo could be effectively used for bioremediation of
1991; Wehrli et al. 1995; Tebo et al. 1997). Mn due to their unique capacity to bind metal and
Reports show that bacteria can bring about catal- precipitate it oxidatively, besides their inherently
ysis of Mn to Mn oxides in seawater, by enzymes tough physical nature. Geszvain et al. (2013)
known as multicopper oxidases (MCOs). MCO identied the mnxG gene of Pseudomonas putida
genes are essential for Mn oxidation and observed GB1 by its similarity to the mnxG gene of
in all Mn(II)-oxidizing bacteria studied to date Bacillus sp. SG-1. In 1999, Larsen et al. observed
(Tebo et al. 2005). Corstjens et al. (1997) demon- a copper-dependent enzyme in Pedomicrobium
strated that the mnxG gene encoding for MCO sp. strain ACM 3067 which was similar to the
10 Manganese-Tolerant Bacteria from the Estuarine Environment and Their 167

multicopper oxidase which catalysed Mn(II) oxi- Because minerals of Mn such as Mn oxides
dation. Hansel and Francis (2006) isolated a are highly charged, they can adsorb and concen-
diverse group of Mn(II)-oxidizing Roseobacter- trate many other metals. The geochemical cycling
like species able to oxidize soluble Mn(II) rap- of Mn oxides can therefore inuence trace metal
idly to its insoluble Mn (III, IV) oxides, from the distribution (Huang 1991). Thus, manganese-
coastal estuary of Elkhorn Slough on the border tolerant bacteria capable of oxidizing manganese
of Monterey Bay in California. In the coastal are important in the mitigation of not only man-
waters, these bacteria were found not only to ganese but also other metals in contaminated
actively participate in manganese (Mn) oxidation estuarine environments. As observed by Barkay
and cycling but also to provide a nonconventional (1987) and Nies (2003), microorganisms from
method for oxidation of Mn(II) in the photic zone contaminated estuarine sites such as solar salt-
via a photo-oxidation pathway. A protein homol- erns may employ various mechanisms, such as a
ogous to multicopper oxidases encoded by shift towards a more resistant population, or the
CumA, which could actively mineralize Mn increased expression of stress-related genes, or
oxide from Mn(II) and deposit it on the outer the inter- and intraspecies transfer of metal-
membranes in Pseudomonas putida, was reported resistant genes. Haritha et al. (2009) have sug-
by Brouwers et al. (1999). Another mechanism gested the there is a strong possibility that
for metal resistance in microorganisms exposed transposition of metal resistance traits from plas-
to toxic metals is by the upregulation of genes mid to chromosome may take place during acute
involved in metal homeostasis and the redox con- metal stress, thereby disseminating the property
trol of defence. Analysis by SDS-PAGE to deter- of metal resistance.
mine which proteins are directly responsible for
catalysing the mechanism by which
Chromohalobacter beijerinckii from saltern sedi- 10.6 Ecological Signicance
ments of Ribandar, Goa, could oxidize Mn(II) of Manganese-Tolerant
biochemically revealed a protein band of 50 kDa Estuarine Bacteria
which was upregulated on addition of 10, 100 as Bioremediators
and 1000 M of Mn2+, while there was a down-
regulation of the 53 kDa fraction (Pereira et al. Induced tolerance in bacteria continuously
2012). Mn(II)-oxidizing proteins of varying sizes exposed to a particular pollutant, such as manga-
were reported from diverse Bacillus species and nese, for a long time provides a mode for indi-
were attributed to variations in gene size, or the rectly mitigating metal concentrations in the
additional proteins required for activity, or post- ecosystem (Blanck et al 1988; Hopkin 1993;
translational modications (e.g. proteolysis or Chapman et al. 1998) and maintaining it within
glycosylation) (Francis and Tebo 2002). Boogerd limits, thus preventing it from adversely affecting
and de Vrind (1987) and Adams and Ghiorse living organisms. Due to their exceptional prop-
(1987) consistently recovered a 110 kDa protein erties such as their capability to grow even at a
by SDS-PAGE, which was capable of oxidizing high anion and cation concentrations, halophilic
Mn(II) in L. discophora SS-1 gels, yet the mofA and halotolerant microorganisms are ideal candi-
gene for multicopper oxidase is predicted to be a dates for bioremediation processes (Ventosa et al.
174 kDa protein (Corstjens et al. 1997). Mn(II)- 1998). Hence, not only do they have a require-
oxidizing activity in P. putida GB-1 could be rec- ment for these elements, but they are also
ognized due to the formation of complexes of naturally tolerant to elements that may be toxic to
high molecular mass of about 250 and 180 kDa in their non-extremophilic counterparts (Amoozegar
native gels (Nieto et al. 1989). This suggests that et al. 2005). Mondal et al. (2008) used species
several proteins, including the multicopper oxi- from the Ralstonia family that were phylogeneti-
dase cumA, are required for activity. cally related to Cupriavidus metallidurans for
168 F. Pereira

Table 10.1 Manganese tolerance mechanisms employed by marine bacteria

Type of resistance
Marine sites Name of bacteria mechanism References
Tamar Estuary, Halomonas strains Manganese oxidation Vojak et al. (1985)
Mandovi and Zuari V. harveyi EPS Bramhachari and Dubey
River, Goa (2006)
Elkhorn Slough Roseobacter-like species Mn oxidation Hansel and Francis (2006)
Estuary, California
Carlsberg Ridge Halomonas species Mn oxidation and Fernandes et al. (2005)
extracellular precipitation
Pinal Creek, Arizona Leptothrix discophora EPS Robbins and Corley (2005)

bioremediation studies and recorded removal of property for remediation of toxic metals.
65.2 % Fe, 72.7 % Mn, 98.6 % Cu, 8 % As and Indigenous metal-tolerant microorganisms, with
99.3 % Zn, respectively, from water contami- the ability to naturally remove, attenuate or detox-
nated with metal. Hallberg and Johnson (2005) ify toxic substances from estuarine environments
used a simple xed bed bioreactor packed with such as salterns and mangroves contaminated
microorganisms capable of oxidation and pre- with ores from mines, pesticides and industrial
cipitation of Mn(II) as a highly effective method efuents, need to be explored and harnessed.
for the removal of metals from a synthetically
prepared metal-rich wastewater. Industries fre- Acknowledgement I would like to express my gratitude
quently make use of salts and then release efu- to my Ph.D. guide and mentor Dr. Savita Kerkar for
extending her constant support and guidance. I am also
ents containing brine into the environment.
grateful to H. O. D. Department of Biotechnology, Goa
Manganese-tolerant bacteria displaying multi- University; to the ex-director of the National Centre for
metal tolerance and adapted to live in hypersaline Antarctic and Ocean Research (NCAOR), Goa, Shri Rasik
habitats would prove useful as biological detoxi- Ravindra; and to Dr. K. P. Krishnan, scientist at NCAOR
for permitting use of the lab facilities and assistance. I
cants in saline aquatic polluted environments or
would like to thank Dr. Thamban Meloth (programme
even as bioindicators of pollution. Table 10.1 director, laboratories at NCAOR) for his constant support.
gives a summary of manganese tolerance mecha- I would also like to place on record the assistance given by
nisms reported in bacteria from different marine Mr. Rupesh Kumar Sinha in the eldwork.

10.7 Conclusion
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