48

CHAPTER 3
LITERATURE REVIEW

3.1

GENERAL
Bioremediation has been proven to be an effective, environmentally

friendly and less expensive treatment option for remediation of aquifers

contaminated with hydrocarbons (Shen and Wang, 1994; Jardine and Taylor,
1995; Ganguli and Tirupathi, 2002). Biotransformation is the process by which
a highly toxic compound is converted to less toxic/no toxic compound using
biological process. This process can be aerobic / anaerobic / anoxic or
combination of these three, based on the microorganisms. It has been reported
that several microorganisms, under various environmental conditions, can
decontaminate the dam sediments very effectively. This process depends on
carbon source, pH, temperature, dissolved oxygen, ORP, and presence of other
oxyanions and metal cations (Chen and Hao, 1998).
Bioremediation is the use of biological treatments, for the clean-up
of hazardous chemicals in the environment. At present, employing the,
biochemical abilities of microorganisms is the most popular strategy for the
biological treatment of contaminated soils, sediments and waters (Head, 1998).
Now a days, great emphasis is placed on environmental biotechnology and
attaining sustainable development: in particular, biological techniques can be
applied effectively in the remediation of sediments contaminated by organic
pollutants from a variety of sources.

49
3.2

BIOAUGMENTATION, BIOSTIMULATION AND

NATURAL ATTENUATION IN THE REMEDIATION OF
CONTAMINATED SOILS SEDIMENTS (BIOPILE STUDY)
The choice of bioremediation technologies used for site remediation

is often dictated by whether it is possible or practical to physically manipulate

the contaminated materials. If the contaminated materials can be removed and
treated ex situ in a cost-effective manner, then this is often the approach
selected because it offers the advantages of better process control, more rapid
rates of degradation, and greater minimization of risk. However, if the amount
of contaminated material is large or if the material is not easily accessed, then
treatment of the material in situ is often the only practical and cost-effective
alternative. This approach is also advantageous if there is no imminent risk to
human health or the environment and if the time required to reach closure is
not a critical factor.
Bioremediation

of

contaminated

soils/sediments

has

been

investigated since the late 1940s, but interest in the field did not become wide
spread until the Exxon Valdez oil spill in 1989 (Margesin and Schinner, 1997;
Jackson and Pardue, 1998). Consequently, there have been a large number of
studies conducted, and bioremediation has almost always been found to be an
effective treatment of hydrocarbon-contaminated sites (Huesemann and
Moore, 1993; Li et al., 1995; Zhou and Crawford, 1995).
Studies were conducted by Jee et al (1996) to evaluate the
bioremediation of C14 phenanthrene contaminated sediments under simulated
inplace slurry reactor conditions. This included experiments investigating the
rate of oxygen demand exerted by the sediments, the rate and extent of
phenanthrene release under slurry conditions, and the fate of phenanthrene in
an aerated slurry bioreactor.

Results of these studies provide useful

50
information into operation of slurry reaction systems and potential methods for
enhancing the performance.
Jussara and Fransisca (1999) has reported bioremediation of light
Arabian oil in sandy sediment by the mixed culture designated ND, obtained
from land farming, associated to indigenous microorganisms resulted in 42.9%
reduction of the heavy fraction of oil in 28 days when a phosphate and a
nitrate source were provided. Tests performed only in the presence of native
flora, have achieved 11.9% removal of these compounds. These results have
demonstrated the importance of utilizing cultures adapted to pollutants to
enhance efficiency and productivity of the bioremediation process.
In the field of biostimulation, nutrient supplementation for
hydrocarbon degradation has traditionally focused on addition of N and P,
either organically or inorganically. Because C is a major constituent of
petroleum fuels, its traditional role in bioremediation research has typically
been as an index to determine the amount of N and P that need to be added to
reach the optimal C:N:P ratio (Riser-Roberts, 1998). Moreover, the role of C
supplementation in hydrocarbon bioremediation in low organic matter or
otherwise C-poor soils has never been investigated. Various nutrient sources
such as, inorganic fertilizer, urea, sawdust, compost, manure, and biosolids
have been used in biostimulation (Rosenberg et al., 1992; Walworth and
Reynolds, 1995; Cho et al., 1997; Williams et al., 1999; Namkoong et al.,
2002).
Runes et al (2001) established the potential of pesticide
biodegradation in constructed wetland sediment. Under microcosm conditions,
bioaugmentation of sediment with small quantities of an atrazine spill-site soil
(1: 100 w/w) resulted in the mineralization of 25-30% of l4C ethyl atrazine (1-

51
10g g-1 sediment) as

14

CO2 under both unsaturated and water-saturated

conditions; atrazine and its common metabolites were almost undetectable

after 30 days incubation.
Frische and Hoper (2003) discussed in situ bioremediation as a
useful strategy for cleaning up contaminated soils. The subject of this study
was the significance of two bioassays for monitoring purposes. The work was
performed within the scope of a research project on the in situ bioremediation
of topsoil contaminated with 2, 4, 6-trinitrotoluene (TNT). To evaluate
changes within different experimental fields during a 17- month remediation
period, the results of soil microbial assays and luminescent bacteria assays
were compared with chemical monitoring data.
Ruberto et al (2003) performed microcosms systems (250 g soil in
flasks) in Jubany Station (King George Island, South Shetland Islands) to
analyze biodegradation of gas-oil in Antarctic soils under natural conditions.
Abiotic loss of hydrocarbons, biodegradation activity of indigenous microflora
and biostimulation with N and P were studied. In addition, biaugmentation
with a previously isolated psychrotolerant strain (B-2-2) was analyzed.
Hydrocarbon

concentration,

heterotrophic,

and

hydrocarbon-degrading

bacterial counts and predominant bacterial groups were evaluated during 51

days.
Ghazali et al (2004) investigated the bioremediation of hydrocarbon
in contaminated soils by mixed cultures of hydrocarbon-degrading bacteria.
The strains were selected based on the criteria that they were able to display
good growth in crude oil, individual hydrocarbon compounds or both. Their
ability to degrade hydrocarbon contamination in the environment was
investigated using soil samples that were contaminated with diesel, crude oil

52
or engine oil. Vezzulli et al., 2004 evaluated the potential of bioremediation
for mobilisation of carbon in organic-rich sediments. Both bioaugmentation
(bio-fixed microorganisms) and biostimulation (oxygen release compoundsORC) protocols had been tested and the response of the bacterial community
has been described to assess the baseline for bioremediation potential.
Sabate et al (2004) proposed a simple protocol for biotreatability
assays in two phases. In the first phase of the assays they examined the type
and metabolic activity of the indigenous microorganisms at the site, and the
presence of possible inhibitors. The biodegradability of contaminants in soil
slurries under optimal conditions was also tested. In the second phase several
parameters, such as the influence of nutrients and the addition of surfactant
and specialized inocula, were evaluated in microcosms with 2.5 kg soil.
Mrayyan & Battikhi (2005) described bioremediation as costeffective, environmentally friendly treatment for oily contaminated sites by the
use of microorganisms. In their study, laboratory experiments were conducted
to establish the performance of bacterial isolates in degradation of organic
compounds contained in oily sludge from the Jordanian Oil Refinery plant. As
a result of the laboratory screening, three natural bacterial consortia capable of
degrading total organic carbons (TOC) were prepared from isolates enriched
from the oil sludge. Experiments were conducted in Erlenmeyer flasks under
aerobic conditions, with TOC removal percentage varied from 0.3 to 28%
depending on consortia type and concentration.
Margesin et al (2005) evaluated soil biological activities as a
monitoring instrument for the decontamination process of a mineral oilcontaminated soil was made using measurements of microbial counts, soil
respiration, soil biomass and several enzyme activities. Biodegradation could

53
be monitored well by soil biological parameters. The residual hydrocarbon
content correlated positively with soil respiration, biomass-C (substrateinduced respiration), and with activities of soil of dehydrogenase, urease and
catalase. Soil lipase activity and the number of hydrocarbon utilizers
correlated negatively (P < 0:0001) with the remaining hydrocarbon content.
Similarly Morelli et al (2005) evaluated biological and chemical analyses of
the effectiveness of bioremediation of sludge from the petrochemical industry
in systems containing artificially contaminated soil. During bioremediation 8695% of the hydrocarbons were eliminated. Simultaneously, soil bacterial
populations and inhibition of seed germination by aqueous extracts increased
in all sludge-soil systems during the first 180 days of treatment. Furthermore,
residual levels of hydrocarbons and seed germination inhibitory effect
decreased sharply.
Maria et al (2005) reported qualitative and quantitative changes of
microbial communities in soil microcosms during bioremediation which were
determined throughout one year. Sarkar et al (2005) compared two methods of
biostimulation in a laboratory incubation study with monitored natural
attenuation (MNA) for total petroleum hydrocarbon (TPH) degradation in
diesel-contaminated Tarpley clay soil with low carbon content. Results suggest
that biosolids addition is a more effective soil amendment method for
biostimulation than the commonly practiced inorganic fertilizer application,
because of the abilities of biosolids to supplement carbon. Bento et al 2005,
studied bioremediation of diesel oil in soil by natural attenuation, or treated by
biostimulation or bioaugmentation. After 12 weeks of incubation, all three
treatments showed differing effects on the degradation of light (C12-C23) and
heavy (C23-C40) fractions of TPH in the soil samples.

54
Shuchi et al (2006) tested the ability of three bacterial strains,
Bacillus sp. SV9, Acinetobacter s., SV4 and Pseudomonas sp. SV17 from
contaminated soil in Ankleshwar, India to degrade the complex mixture of
petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes),
sediments, heavy metals and water known as oily sludge. Results showed that
Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30C
whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and
35%.
Julia et al (2007) studied the impact of the herbicide concentration
(0.10-10000 g kg-1) and addition of organic and inorganic nutrients on 2,4-D
and 2,4,5-T mineralization in an aquifer and soil samples in laboratory
experiments. 2,4-D was most rapidly mineralized followed by 2,4,5-T. The
mineralization rate of higher herbicide concentrations was significantly
stimulated by the amendment of inorganic nutrients.
Gideon and Dike (2008) revealed the applicability and the
effectiveness of natural attenuation and forced aeration in the remediation of
oil-impacted sediment in a typical Niger Delta setting through microcosm
studies. The initial hydrocarbon concentration was 90,212 mg/kg of sediment.
After 60 days, the natural attenuation processes accounted for 31.9% of the
total hydrocarbon removed while 13% was attributable to forced aeration,
bringing the cumulative hydrocarbon removed to 44.9%. Liu et al (2008)
studied

the

feasibility

of

applying

nutrient

enhancement

(NE),

bioaugmentation (BA) and biostimulation (BS), on diesel contaminated soil

through soil column experiments. The diesel-degradation efficiency was 67%,
80%, 45%, and 24% for NE, BA and BS respectively.

55
Rodrigo et al (2008) evaluated the capacity of a defined microbial
consortium isolated from a PAHs contaminated landfarm site to degrade and
mineralize different concentrations (0, 250, 500 and 1000 mg kg-1) of
anthracene, phenanthrene and pyrene in soil. This consortium mineralized
78%, on average, of the different concentrations of the 3 PAHs in soil after 70
days. Contrarily, the autochthonous soil microbial population showed no
substantial mineralization of the PAHs. Bacterial and fungal isolates from the
consortium, when inoculated separately to the soil, were less effective in
anthracene mineralization compared to the consortium. This signifies
synergistic promotion of PAHs mineralization by mixtures of the mono culture
isolates (the microbial consortium).
Lopez et al (2008) studied the effect of a combined biostimulationbioaugmentation treatment applied to a silty-loam soil polluted with 60,600
mg kg-1 of a complex mixture of total petroleum hydrocarbons (TPH), which
comprises 40% aliphatic hydrocarbons (AH) and 21% polycyclic aromatic
hydrocarbons (PAH). The native fungi were able to grow in a complex solid
mixture of hydrocarbons of high molecular weight, after previous
acclimatization in liquid culture. Bioaugmentation was able to remove 36%,
30% and 17% more TPH, AH and, PAH respectively in comparison with
biostimulation.
3.3

COMPOSTING TECHNOLOGIES FOR THE TREATMENT

OF HAZARDOUS WASTES
The composting process has always occurred in nature. One of the

first organized composting operations top be reported upon in the literature

was carried out in India in the early 1930s under the direction of Howard and
associated. The process was named for the location in India where it was
developed. In its simplest form, the process involves excavating a trench in the

56
ground 2 to 3 ft deep in which successive layers of putrescible materials such
as solid waste, night soil, animal manure, earth, and straw are placed. The
procedure was to turn the material only twice during the composting process,
which lasted six months or longer. Thus the composting of yard wastes,
municipal wastewater sludges, and municipal solid wastes are long
established.
However, composting of soils and sediments contaminated with
hazardous materials is still an emerging ex situ biotreatment technology.
Composting has been demonstrated to be effective in biodegrading polycyclic
aromatic hydrocarbons (PAHs) (Canet et al., 2001; Potter et al., 1999),
chlorophenols (Laine et al., 1997), polychlorinated biphenyls (Block, 1998),
explosives (Gray, 1999) and petroleum hydrocarbons (Namkoong et al., 2002)
at the laboratory and/or field-scales.
3.3.1

Composting of Explosives
Composting of explosives has been investigated by several research

groups. In one of the first studies to address this issue Kaplan and Kaplan,
(1982) described the biotransformation of

14

C-labelled 2,4,6-trinitrotoluene,

(TNT) by thermophilic microorganisms in a composting environment. In this

study no TNT mineralization was detected although the formation of

14

C-

labelled reduction products including various aminonitrotoluenes was

described. Composting was used to treat soils and sediment contaminated with
explosive compounds. Louisiana Army Ammunition Plant (LAAP) lagoon
sediments contaminated with 2,4,6-trinitrotoluene (TNT), 1,3,5-hexahydro1,3,5-trinitrohydrazine

(RDX),

1,3,5,7-hexahydro-1,3,5,7-tetranitrotriazine

(HMX), and n-2,4,6-tetranitro-N-methylaniline (Tetryl) were composted with

horse manure, alfalfa, and horse feed in the form of ventilated static compost
piles (Usathama, 1988; Garg et al., 1991). The total explosive compounds

57
decreased with half-lives of 27.36 and 16.6 d for mesophilic and thermophilic
conditions, respectively.
Williams et a1 (1992) investigated the feasibility of using
composting to bioremediate sediments contaminated with 2,4,6-trinitrotoluene
(TNT) and soils contaminated with nitrocellulose under thermophilic and
mesophilic conditions. Under thermophilic conditions extractable TNT and
nitrocellulose residues were reduced from 11,840 to 3 mg kg-1 and 13,090 to
16 mg kg-1 respectively. Under mesophilic conditions, solvent extractable TNT
residues were reduced from 11,190 mg kg-1 to 50 mg kg1. Breitung et al (1996)
further investigated the issue of TNT-contaminated soil bioremediation using
two different composting regimes. This

study aimed to elucidate

biodegradation and immobilization processes that led to the detoxificadon of

the contaminated soils. The first composting system was aerated from the start
of the incubation while the second was aerated for 95 days after a 65-day
anaerobic phase. In the solely aerated system there was a rapid decline in
extractable TNT (approx. 92% reduction); however TNT was still detectable
after 28 days. In the anaerobic/aerobic system TNT was almost completely
transformed to amino dinitrotoluenes in the anaerobic phase and completely
removed from the extractable phase following aeration.
Bruns-Nagel et al (1998) composted TNT-contaminated soil with
chopped sugar beet and straw anaerobically for 19 days then aerobically for a
further 58 days. Under the initial anoxic conditions approximately 90% of the
TNT was transformed to mono-and diaminonitrotoluene intermediates which
were further transformed under aerobic conditions to putatively less toxic
acetylated and formulated metabolites. The authors concluded that they had
developed an, efficient system for bioremediating TNT-contaminated soils.
The studies indicated the formation of TNT transformation products.

58
Williams and Keehan (1993) reviewed and reported research on destruction of
explosives (trinitrotoluene, nitrocellulose, and others) during composting of
soils and organic matter. They concluded that explosives are reduced to low
concentrations during composting.
Stryner et al (2002) studied composting in loam soil from
Pennsylvania incubated with 5 g kg-I of 15N-Iabeled 2,4,6-trinitrotoluene
(TNT) and 200 Ci kg-1 of

14

C-TNT for 3 days and then amended with

compost at a 1:2 soil to compost ratio. At the end of the aerobic phase, most of
the radioactivity was associated with organic matter; only 8.7% could be
extracted with water and methanol, but no TNT was present in the extracts as
determined by high-performance liquid chromatography. The NMR spectra
indicated that nitro groups of TNT had been reduced to amino groups that
were subsequently involved in the formation of covalent bonds with soil
organic matter.
3.3.2

Composting Poly Aromatic Hydrocarbons

Valo and Salkinoja-Salonen, (1986) carried out a field-scale

composting study in 50m3 windrows (using bulking agent, organic matter

source). The soil concentrations of chlorophenols were reduced from 212 to
30 mg kg-1 in the first summer (4 months) and further reduced to 15 mgkg-1 in
the second summer. All the chlorophenol congeners were degraded. These
microbes originated from the bulking agent, i.e., bark chips and straw
compost. The addition of ripe or mature compost to soil polluted with PAHs
can include the removal of these hydrocarbons from the soil. In an early study
Martens (1982) looked at the changes in concentration of four to six-ring
PAHs in two types of compost; fresh composts and mature composts, which
had been allowed to ripen for 3-12 months in stacks. The author found that
there were lower concentrations of four- six-ring PAHs in the mature compost

59
over those found in fresh compost. Additionally, when these composts were
incubated with

14

C-labelled anthracene, benz[a] anthracene, benzo[a]pyrene

and dibenz[a,h] anthracene it was found that there were significantly higher
levels of mineralization to 14CO2 in the mature composts. Maximal values for
the four 14C-labelled PAHs in fresh and mature composts were 19 and 62% for
anthracene, 8 and 58% for benz[a]anthracenej 0.5 and 19% for benzo[a]pyrene
and 1.4 and 21% for dibenz[a,h] anthracene, respectively over a l0-week
incubation period.
Adenuga et al (1992) showed that pyrene could be degraded in the
composting of soil/sludge mixtures although the rate and extent were not
mentioned in this study. This study showed that although the benzo[a]pyrene
appeared to be removed, there was no appreciable difference between the
uninoculated and inoculated systems with 65.6% and 62.8% removal
respectively after 95 days. Creosote is a complex mixture of PAHs (~85%),
phenolic compounds (~10%), and N-S- and O-heterocycles (~5%). Of the 150200 compounds present in creosote, only a few are present in concentrations of
1% (Mueller et al., 1989). Straw compost produced in the mushroom industry
has been investigated as an inoculum in the bioremediation of chlorophenol
contaminated soil.

Valo and Salkinoja-Salonen (1986) carried windrow

composting of soil contaminated with 212 mg/kg chlorophenol form wood

preserving facilities and allowed for degradation of the pollutant to levels of
about 30 mg/kg in four months.
The composting of wood containing PAHs was studied under
laboratory conditions by Loser et al (1997). Wood was artificially
contaminated with PAHs (phenanthrene and pyrene) or was obtained from
preservative treated wood (such as chipped railroad ties). The wood was
soaked with liquid hog manure before composting. After 61 days of

60
composting in dewar vessels, PAH concentrations in the artificially
contaminated wood were reduced from 1,000 mg/kg of each PAH to 26 mg
phenanthrene/kg and to 83 mg pyrene/kg. For the preservative treated wood
the degradation of PAHs was somewhat slower than with the artificially
contaminated wood; this result attributed to the presence of more complex,
condensed types of PAHs. Adding liquid manure to the compost mix
accelerated temperature rises in the vessels and evolution of carbon dioxide
relative to wood receiving no supplement or to wood receiving a source of
nutrients.
Degradation of coal tar polycyclic aromatic hydrocarbons (PAHs)
was investigated by Wischmann and Steinhart, (1997) in three soil systems
which had been artificially contaminated. Studies showed that in unamended
soil only aromatics with upto three fused benzene rings were considerably
degraded during the first 15 weeks whereas soil supplementation with compost
helped to enhance elimination of all compounds. Composting strategies have
been used to treat aromatic compounds like benzene, toluene, ethyl benzene
and o, m and p-xylenes which are commonly found in petroleum contaminated
sites and are of major concern because of their toxicity and carcinogenicity.
Mattean and Ramsay (1997) first reported the feasibility of using thermophilic
and mesophilic phases during the composting of leaves and alfalfa to
biodegrade toluene. Under thermophilic conditions toluene was degraded at a
rate of 110 g m-3h-1 whereas under mesophilic conditions the aromatic
compounds were removed at the reduced rate of 98 gm-3h-1. Additionally
benzene was degraded under mesophilic conditions at a similar rate to that of
toluene.
Sukesan and Watwood (1997) investigated the fate of gaseous
trichloroethylene (TCE) in two different composts, namely leaf compost and

61
wood chip-bark compost, as well as in hydrocarbon-induced and uninduced
composts. The authors found that TCE removal was greater in the leaf
compost than in the wood chip-bark compost with 95 and 15% TCE removed,
respectively. They also found that composts, which had been induced using
methane or propane, were stimulated equally into removing TCE from the gas
phase. Further improvement in TCE removal was achieved with the
introduction of granular activated carbon. They also went on to characterize
the growth of TCE-utilizing microflora within the composts showing that
enrichment with methane or propane resulted in increase in methanotrophic
and propanotrophic populations, respectively.
BTEX compounds are toxic products of the petroleum industry
(Semple et al., 2001). The degradation of benzene was assessed in spent
mushroom compost after a 3-month enrichment period in the presence of a
variety of BTEX compounds (Semple et al., 2001). It was found that as the
incubation temperature was raised from 18C to 37C to 50C, there was a
commensurate increase in the mineralization of [UL-14C) benzene over 14
days. Similarly Joyce et al (1998) investigated the fate of a mixture of three
and

four-ring

PAHs

(fluorine,

anthracene,

phenanthrene,

pyrene,

benz[a]anthracene) under composting conditions with solid municipal waste

monitored over a 60-day period (30 days of active composting followed by 30
days of compost curing). The fate of the PAHs was also monitored in HgCl2
treated systems to compare the impact of biotic and abiotic processes. The
results of this study showed that the loss processes occurred during the active
phase of composting (first 30 days). Anthracene, phenanthrene and pyrene
were removed effectively during the composting process by a combination of
biotic and abiotic mechanisms principally dominated by the biotic processes.
Fluorine proved to be too volatile and so most of the compound (approx. 75%)
was removed abiotically in the gas phase. Additionally benz[a]anthracene

62
proved to be resistant to biodegradation throughout the composting incubation
with between approximately 40-50% being lost abiotically.
The fate of 16 polycyclic aromatic hydrocarbons (PAHs), targeted
by the USEPA agency has been investigated by Amir et al (2005) during
composting of lagooning sludge. Composting showed efficient decrease of the
content and the bioavailability of each PAH. Biodegradation and sorption were
suggested as the main mechanisms contributing to this decrease. During the
stabilization phase of composting, extensive microbial degradation of PAHs,
mainly those with a low number of aromatic rings were achieved following
development of intense thermophilic communities. However, partial sorption
of PAH to non-accessible sites temporarily limits the mobility mainly of PAHs
with a high number of aromatic rings plus acenaphthene and acenaphthylene,
and allows them to escape microbial attack. During the maturation phase, the
development of a mesophilic population could play an important role in the
degradation of the remaining PAH. During this phase of composting, PAH
sequestration and binding of their oxidative metabolites within new-formed
humic substances might also explain PAH decrease at the end of composting.
The tendency of change of content or bioavailability of various PAH
compounds during composting are found to be strongly related to the number
of their aromatic rings, their molecular weight and structure.
Blanca et al (2007) studied the biodegradation of 16 USEPA listed
polycyclic aromatic hydrocarbons ( PAHs) with accompanying humification
and microbial community structure changes during simulated in-vessel
composting of an aged coal-tar-contaminated soil amended with green waste
over 56 days. This study proved that using thermophilic temperatures (70C)
towards the end of in-vessel, composting processes (TP2) resulted in greater
PAHs removal than using other variable temperature profiles (TP1, TP3), as

63
long as the increase was stepwise via an intermediate temperature (55C).
Phospholipid fatty acid (PLFA) signatures indicated that use of thermophilic
temperatures towards the end of the in-vessel composting bioremediation
(TP2) resulted in a higher fungal to bacterial PLFA ratio and a lower Grampositive to Gram:-negative (G(+)/G(-)) bacterial ratio. Fluorescence excitationemission matrix (EEM) showed the presence of peaks typical of humic-like,
(Ex/Em wavelength pair 340/460 nm) and fulvic-acid-like substances,
indicating mineralization and/ or maturation of the compost. Varying the
temperature during in-vessel composting to comply with regulatory
requirements for pathogen control, promoted contaminant biodegradation
microbial activity and compost maturation.
Blanca et al (2008) investigated the microbial community structural
changes of an aged-coal-tar soil contaminated with polycyclic aromatic
hydrocarbons (PAHs) during simulated bioremediation at the laboratory scale
using an in-vessel composting approach. Relative changes in microbial
populations were investigated by following the dynamics of phospholipid
fatty acid (PLFA) signatures during in-vessel composting over 98 days. The
results of this investigation indicated that fungal to bacterial PLFA ratios were
significantly influenced by temperature (p<0.05) and Gram-positive to Gramnegative bacterial ratios were significantly influenced by temperature
(p<0.001) during in-vessel composting. Additionally, the Gram-positive to
Gram-negative bacterial ratios were correlated to the extent of PAH losses
(p<0.005) at 70 0C.
3.3.3

Composting of Petroleum Hydrocarbons

Beaudin et al (1996) studied the composting of hydrocarbon-

contaminated soil with alfalfa and maple leaves. For soils contaminated with
mineral oils and grease (40% aliphatic, 32% polar, 28% aromatic

64
hydrocarbons) at 17,000 mg total hydrocarbons per kilogram, 60% of the
aliphatic, 54% of the aromatic, and 83% of the polar compounds had degraded
in 180 days of composting. This result compared with only 50% degradation
of mineral oils and grease in land. After 287 days of composting, 73% of all
mineral oils and grease had degraded.
Milne et al (1998) studied considered composting with a variety of
bulking agents as a method for dealing with this recalcitrant mixture. Three
bulking agents were used in this study namely chopped barley straw, heattreated peat moss and Solv-II (a preparation of peat moss enriched with
nutrients and oil-degrading microbes). The author found that both the Solv-II
and the peat moss were equally good at activating biodegradative activity in
the composting processes. However over a composting period of 800 h, there
was a reduction of approximately 25% in total petroleum hydrocarbons
(TPHs) in the composting systems containing the barley and the peat moss.
But in the composting systems containing the Solv-II bulking agent there was
a 55% reduction in TPHs along with high CO2 production suggesting high
microbial respiratory activity. This study suggested that composting coupled
with bioaugmentation, was a successful approach to take in the remediation of
oily residues.
Gestel et al (2003) worked on the soil spiked with diesel oil mixed
with biowaste (vegetable, fruit and garden waste) at a 1:10 ratio (fresh weight)
and composted in a monitored composting bin system for 12 weeks. Pure
biowaste was composted in parallel. In order to discern the temperature effect
from the additional biowaste effect on diesel degradation, one recipient with
contaminated soil was hold at room temperature, while another was kept at the
actual composting temperature. Measurements of composting parameters
together with enumerations and identifications of microorganisms demonstrate

65
that the addition of the contaminated soil had a minor impact on the
composting process. The first-order rate constant of diesel degradation in the
biowaste mixture was four times higher than in the soil at room temperature,
and 1.2 times higher than in the soil at composting temperature.
Zytner et al (2006) evaluated various factors affecting the efficiency
of the composting process, including environmental conditions, microbial
population present and composition of the hydrocarbon, impact of mixture
composition on individual compound and overall degradation. Biodegradation
experiments were conducted in sealed, 1-litre bioreactor / respirometer vessels
containing soil spiked with hydrocarbon compounds in isolation and in
mixtures. The influence of bacteria and fungi on the degradation process was
also monitored. The degradation behavior of the various compounds was
monitored using the fraction of contaminant remaining and first-order
degradation coefficients based on hydrocarbon loss.
3.3.4

Composting of Municipal Solid Wastes

The decomposition of seven different organic waste mixtures

prepared with sewage sludge, animal manures, city refuse and industrial and
plant residues, during their aerobic incubation with soil was studied by Bernat
et al (1998). The waste mixtures were composted by the Rutgers static pile
system, and four samples of each mixture were collected at various
composting stages: the initial mixture, and samples taken during the
thermophilic phase, at the end of the active phase and after maturation. These
samples were added to a calcareous silt loam soil at a rate of 200 mg per 10 g
soil, and the CO2C evolution was determined during 70 days of aerobic
incubation at 28oC. Carbon mineralization decreased as the composting time
lengthened. The lowest values of C mineralization were found for the mature
samples, and only compost, which had not attained an advanced degree of

66
maturation, gave results higher than 25% of TOC. Carbon mineralization
followed a combined first- and zero-order kinetic model in most of the
samples, suggesting that the organic C of the composting wastes was made up
of two organic pools of differing degrees of stability. However, the differences
in the slow C mineralization pool at the end of the active phase and after
maturation were very small, indicating that the organic matter at both stages
was of a similar microbial stability. Comparing the C mineralization, which
takes place in soil and during composting, it can be concluded that composting
is the best way of obtaining maximum C stabilization, which is an important
factor in soil conservation and reclamation.
Sanchez Monedero et al (1999) dealt with the relationships between
the degradation and humification processes which the organic matter
underwent during the composting of six different organic-waste mixtures. Four
of them were prepared by the Rutgers forced ventilation composting system
and the other two by the mobile (turn over) pile system. The main components
were sewage sludge, sorghum bagasse and municipal solid waste. Different
degradation rates were observed for the three main components (cellulose,
hemicellulose and lignin) of the organic matter during composting. In the case
of the first two components, the degree of degradation ranged from 70 to 85%
during the whole process, depending on the starting mixture, whereas only
3050% of the initial concentration of lignin was degraded in the mixtures
prepared with municipal solid wastes (MSW) and lignocellulosic materials.
Water-soluble carbohydrate and phenol degradation were studied because they
have been proposed as precursors of the humification processes. In the
experiment described, they had different degradation rates during composting
depending on the starting mixture and the composting system used. The watersoluble carbohydrate was the most intensely degraded fraction in the piles
prepared with urban refuse although no appreciable degradation was measured

67
in the other three mixtures, whereas there was an appreciable reduction in the
water-soluble phenol fraction of all six mixtures during composting, values of
less than 0.1 % being reached in the mature composts. Humification processes
were studied by quantification of the extractable humic-like substances and
the generally accepted (EXC/TOC), humic acid carbon to total organic carbon
(HAC/TOC), humic acid carbon to extractable carbon (HAC/EXC) and the
humic acid carbon to fulvic acid carbon (HAC/FAC) and by determining the
cation exchange capacity of the mixture during composting. All the indices
increased during composting and followed a similar trend. The humic-like acid
fraction was mainly responsible for these changes, showing that the
composting involves a process of humification. The cation exchange capacity
to total organic carbon ratio showed itself to be a useful humification index
during composting since this index clearly increased more than the others.
A batch composting study was performed by Fang & Wong (1999)
to evaluate the feasibility of co-composting sewage sludge with lime, aiming
at reducing the availability of heavy metals in the sludge compost. Sewage
sludge with sawdust as bulking agent was amended with lime at 0, 0.63, 1.0,
and 1.63% w/w, and composted for 100 days in laboratory batch reactors. The
changes in temperature, pH, electrical conductivity (EC), and extractable
heavy metal contents were measured while compost maturity was determined
by C/N organic and cress seed germination during the composting period.
Liming raised pH of compost effectively at the initial stage of composting and
caused a decrease in EC through precipitation of soluble ions. Lime
amendment also significantly reduced water-soluble and Diethylene triamine
pentaacetic acid (DTPA)-extractable metal contents. The maximum reductions
were 60 and 40% for Cu, 80 and 40% for Mn, 55 and 10% for Zn, and 20 and
25% for Ni at the end of the composting period for the lime-amended sludge
as compared to the control. In spite of the inhibitory effect of lime amendment

68
on the decomposition activity of sewage sludge, all treatments reached
maturation after 63 days of composting as indicated by the results of C/N
organic ratio and cress seed germination test results. A lime amendment of
41.0% is recommended to co-compost with sewage sludge.
3.3.5

Composting of Contaminated Soils / Sediments

Namkoong et al (2002) investigated on the appropriate mix ratio of

organic amendments for enhancing diesel oil degradation during contaminated

soil composting. Sewage sludge or compost was added as an amendment for
supplementing organic matter for composting of contaminated soil. The ratios
of contaminated soil to organic amendments were 1:0.1, 1:0.3, 1:0.5, and 1:1
as wet weight basis. Target contaminant of this research was diesel oil, which
was spiked at 10,000 mg/kg sample on a dry weight basis. The degradation of
diesel oil was significantly enhanced by the addition of these organic
amendments relative to straight soil. Degradation rates of total petroleum
hydrocarbons (TPH) and n-alkanes were the greatest at the ratio of 1:0.5 of
contaminated soil to organic amendments on wet weight basis. Preferential
degradation of n-alkanes over TPH was observed regardless of the kind and
the amount of organic amendments. The first order degradation constant of nalkanes was about twice TPH degradation constant. Normal alkanes could be
divided in two groups (C1O-C15 versus C16-C20) based on the first order
kinetic constant. Volatilization loss of TPH was only about 2% of initial TPH.
Normal alkanes lost by volatilization were mainly by the compounds of C10 to
C16. High correlations (r=0.80-0.86) were found among TPH degradation rate,
amount of CO2 evolved, and dehydrogenase activity.
The feasibility of fungi bioaugmentation in composting of a flare pit
soil was studied in lab-scale composter by Baheri and Meysami (2002). The
preliminary screening tests, using a range of bulking agents and white rot

69
fungi strains were conducted to determine best strain and bulking agent for the
main experiments. The initial total petroleum hydrocarbon (TPH) of the flare
pit soil was found to be 16%. The effect of moisture and bulking agent content
and the fungi application on biodegradation of hydrocarbons were then
evaluated based on a fractional factorial design over a 3-months period.
Analysis of the TPH content of the soil after 98 days (using gravimetric
method) showed an average of 29% reduction in most jars. Furthermore, gas
chromatograph (GC) analysis of the oil extract from the samples showed 7099% reduction in the peak area of the selected hydrocarbons. However
statistical analysis of the results did not show any significant effect due to the
fungi application or the change in the moisture content (30-50% range). The
results showed that the change in the bulking agent content was marginally
significant for the hydrocarbon loss.
Ma et al (2003) Microbial activity of an anthracene-spiked soil
mixed with kitchen waste during laboratory composting at 56-59oC was
studied using an in-vessel technology. The effect of old compost containing
acclimated microorganisms on the composting efficiency was also
investigated. Microbial succession, microbial enzyme activity, microbial
diversity and anthracene removal rate were analyzed during 42 days of
composting. The results demonstrated that inoculating with old compost
increased the amounts of thermophilic microorganisms, but did not
significantly increase anthracene removal. A microbial succession from
mesophilic bacteria to thermophilic bacteria and thermophilic actinomycetes
was observed during composting. Polyphenol oxidize activity decreased while
catalase activity varied irregularly.

Microbial diversity increased drastically

when temperature elevated from 35 to 56oC, but decreased when temperature

maintained at 56-59C.

70
Faundez (2007) evaluated the feasibility of aerated in-vessel
composting at a laboratory scale as a bioremediation technology to clean-up
contaminated desert mining soils (fuel concentration> 50,000 mg kg-I) and
sawdust (fuel concentration> 225,000 mgkg-1) in the Atacama Region. The
results of this research indicate that bioremediation of an aged contaminated
mixture of desert mining soil and sawdust with fuel oil is feasible. This study
recommends a soil : sawdust ratio of 1:3 and a correct nutrient balance in order
to achieve a maximum overall hydrocarbon removal of fuel, oil in the
weathered and aged contaminated wastes.
3.3.6

Composting of other Organic Wastes

Paredes et al (2002) experimented on the use of maize straw (MS)

or cotton waste (CW) as bulking agents in the composting of olive mill

wastewater (OMW) sludge by studying the organic matter (OM)
mineralization and humification processes during composting and the
characteristics of the end products. Both composts were prepared in a pilotplant using the Rutgers static-pile system. The use of CW instead of MS to
compost OMW sludge extended both the thermophilic and bio-oxidative
phases of the process, with higher degradation of polymers (mainly lignin and
cellulose), a greater formation of nitrates, higher total nitrogen losses and a
lower biological nitrogen fixation. The CW produced compost with a more
stabilized OM and more highly polymerised humic-like substances. In the pile
with CW and OMW sludge, OM losses followed a first-order kinetic equation,
due to OM degradation being greater at the beginning of the composting and
remaining almost constant until the end of the process. However, in the pile
with MS and OMW sludge this parameter followed a zero-order kinetic
equation, since OM degraded throughout the process. The germination index
indicated the reduction of phytotoxicity during composting.

71
The effect of C source on N losses by volatilization during
composting was measured by Barrington et al (2002) using four bulking
agents, each at three humidity levels and composted in duplicate under passive
and active aeration. The bulking agents were pine shavings alone and
corrected with soybean, chopped grass hay alone and corrected with urea, long
(unchopped) wheat straw and chopped oat straw.
Dyaz et al (2002) investigated two composts which were obtained
by co-composting of a concentrated depotassified beet vinasse and cotton gin
waste using two different aeration systems: static aerated pile (forced aeration
provided by a blower whom operated in the positive mode) and windrow
(turned pile). The composting mixtures were cotton gin trash (55%) and
vinasse (45%) (dry weight). In static pile, the total amount of vinasse was
added at the beginning of the process whereas; in windrow two additions of
vinasse were performed. Differences in temperature changes between both
composting systems were found: a faster increase of temperature in the
windrow (54C at 7 days) than in the static pile (45C at 21 days) was
observed. Probably in the static pile system, the compaction of the substrates
made difficult the correct distribution of the air inside the pile. Moreover, after
the second addition of vinasse a new thermophilic phase was started in
windrow. The different aeration systems and the way of addition of vinasse
could cause differences in organic matter (OM) degradation and in weight
(22.6% for the static pile and 26.7% for the windrow) and gas losses during
the process. Nevertheless, the composts obtained by the two systems had a
high fertilizer value (25.1 g kg-1 N, 3.2 g kg-1 P2O5, 21.4 g kg-1 K2O, C/N 8) for
compost obtained in static pile and (16.2 g kg-1 N, 3.4 g kg-1 P2O5; 16.1 g kg-1
K2O, C/N 12) for compost obtained in the windrow). A high degree of stability
was reached in the final composts. Composting of vinasse with cotton gin

72
waste serves two objectives, disposal of wastes and recycling of waste
components.
Pelaez et al (2003) reported the quantitative determination of
enzyme activities can be used to evaluate the dynamics of the composting
process. An enzyme activity assay in solid phase was developed in which the
enzyme reaction is performed by mixing the compost sample and the
corresponding substrate with a water content equivalent to the water retention
capacity (WRC) of the compost sample. As opposed to common activity
assays that extract and quantify soluble enzymes, this solid phase method
evaluates total enzyme activities under conditions closer to those found in the
actual composting process. Preliminary results on enzyme activities of a
compost of poultry manure and sawdust include amylase, invertase, cellulase,
phosphatase, protease, and dehydrogenase activities. With the exception of
proteases, all increased from day 9 to 43 of the composting process.
Domeizel et al (2003) studied the humification of organic matter
during composting of sewage sludge and green waste by the quantification and
monitoring of the evolution of humic substances. The final objective was to
evaluate the state of humification based on a spectrum of total humic,
substances using the method of UV spectral deconvolution. Compared to the
humic acid to fulvic acid ratio, the new index obtained by UV spectral
deconvolution showed more representative results: the humic acid to fulvic
acid ratio for nonmature compost was abnormally high, suggesting that the
compost was mature, whereas the UV index proposed showed that the
compost was really young and not yet humified.
Contreras-Ramos et al (2004) reported that wastewater from the
leather industry in Leon (Guanajuato, Mexico) is discharged into the Turbio

73
river without treatment. Tannery effluent was composted with cow manure
electrolytic conductivity (EC) of 28.1 ms cm-l, cation exchange capacity of
17.7 meq 100 g-l, an absorbance at 645 nm of 0.0175, a respiration rate of
0.062 mg CO2-C kg-1 compost-C day-1, pH 8.5 and C:N ratio 7:1 with a
germination index for cress (Lepidium sativum) of 48% after 90 days. Less
than 10 faecal coliforms and no Salmonella sp. Shigella sp. or eggs of
helminthes were detected in the compost while total coliforms decreased by
log10 of 2. Total concentrations of lead (Pb) were 8.9 mg kg-1 dry compost,
chromium (Cr) 77 mg kg-1 dry compost, cadmium (Cd) 0.4 mgkg-1 dry
compost, copper (Cu) 10.3 mgkg-1 dry compost and sodium (Na) 14,377 mg
kg-1 dry compost. The compost characteristics indicated that it was mature, but
the germination index for cress of less than 50% suggested possible remaining
phytotoxic compounds. However, the large salt concentrations (especially Na)
might have inhibited cress development and thus reduced the germination
index. The large salt concentration might thus limit the use of this kind of
compost. The compost can not be used for farming due to large salt content. It
can be disposed in secured landfill.
Enzymatic activity (EA) was explored by Claudio Mondini et al
(2004) as a possible tool for composting characterization. Three composts
(yard wastes, cotton wastes and a mixture of the two) were sampled during
different phases of the process and divided in two fractions. The first was
immediately analysed for microbial biomass C (BC) and EAs (b- glucosidase,
arylsulphatase, acid and alkaline phosphatase). Therefore, the development of
a stable enzyme activity in air-dried compost could represent a simple measure
of compost stabilization in routine analysis of composting process.
Elemental, functional and spectroscopic analyses (FTIR, 13CNMR) were performed by Baddi et al (2004) to study fulvic acids of

74
composted olive mill wastes plus cereal straw, in order to follow the maturity
of the final product during composting. The extracted fulvic acids were
characterized by high nitrogen, acidic functional group and phenolic hydroxyl
contents that might have resulted from the high degree of humification and the
synthesis of more condensed humic complexes. This was confirmed by
decrease of alcoholic and aliphatic structures and an increase of aromatic
structures, as shown by the FTIR and 13C-NMR analyses. The results showed
that stability of the final product was reached after 12 months of composting
and that fulvic acid levels could constitute an additional tool to assess final
product maturity and its agronomic value.
Huang et al (2004) evaluated the effect of C/N on the composting
process of pig manure with the purpose of reducing the amount of sawdust
normally used as co-composting materials. Two aerobic static piles were
prepared consisting of pig manure mixed with sawdust at an initial C/N of 30
(pile A) and 15 (pile B), respectively. Pile B containing larger amount of pig
manure showed a slower rise in temperature, lower maximum temperature,
and shorter thermophilic phase than pile A. It also resulted in higher pH and
electrical conductivity (EC) values, and even higher contents of soluble NH4-N
and volatile solids throughout the composting period. Chemical and biological
parameters including dissolved organic carbon (DOC) (4932 mg kg-1), soluble
NH4-N (371 mgkg-1), C/N solid (18.3), C/Naqueous (5.8) and seed germination
index (GI) (66.5%) indicated that pile A achieved maturity after 49 days of
composting. After 63 days of composting, pile B contained 5352 and 912
mgkg-1 of DOC and soluble NH4-N content, respectively, which was much
higher than the criterion of 5% and 400 mgkg-1, indicating its immature nature.
Pile B showed a relatively low GI value of 46%, which may be due to its high
indigenous EC value as a result of larger amount of pig manure. Therefore, cocomposting of pig manure with sawdust at low initial C/N would require a

75
composting longer than 63 days, and the high salinity due to the large amount
of pig manure would pose a potential inhibition on plant growth.
Sundberg et al (2004) reported that during composting of household
waste, the acidity of the material affects the process during the initial phase of
rising temperature. In this study, the effects of temperature (36- 46oC) and pH
(4.6-9.2) on the respiration rate during the early phase of composting were
investigated in two different composts. A respiration method where small
compost samples were incubated at constant temperature was used. The
respiration rate was strongly reduced at 46C and pH below 6, compared to
composts with-a higher pH or lower temperature. The combination of high
temperature and low pH is a possible adverse factor in large-scale composting
of food waste.
Goyal et al (2005) determined changes in organic C, total N, C:N
ratio, activities of cellulase, xylanase and protease, and microbial population
during composting of different organic wastes such as mixture of sugarcane
trash and cattle dung, press mud, poultry waste and water hyacinth biomass.
The study shows that no single parameter can be taken as an index of compost
maturity. However, C:N ratio and CO2 evolved from finished compost can be
taken as the most reliable indices of compost maturity.
Compost studies were conducted by Meunchang et al (2005) to
determine how rapidly this material can be converted to a stable product that
may be useful in crop production and to characterize the N transformations.
Two kinds of sugar mill by-products were composted filter cake and filter cake
mixed with bagasse, at a 2:1 ratio to reduce the C:N ratio in an attempt to
reduce nitrogen loss during composting. Materials were mixed manually at 35 day intervals during the composting process. Both composts were analyzed

76
at least weekly to measure temperature, pH, NH4, NO-3, total N content, C
loss, and germination index. For both mixtures, the thermophilic stage lasted
15-20 days and was higher than ambient for nearly 80 days. The degradation
of organic matter (OM) was rapid in both mixtures to approximately 40 days,
after which it began to stabilize. Both mixtures achieved maturity at
approximately 90 days as indicated by a stable C/N, low NH4/NO-3 lack of
heat production and a germination index higher than 80%. Mixing filter cake
with bagasse helped conserve nitrogen during composting. Because nitrogen
was in excess, approximately 12-15% was lost from the composts. Mixing
more bagasse with the filter cake may result in further reduction in nitrogen
losses. Both composts have potential for use in crop production.
A respirometric method was set up by Tremier et al (2005) to study
kinetics of biological reactions involved in the treatment of organic wastessludge mixed with pine barks-by composting. Oxygen consumption rates of
this type of mixture were monitored during 10-20 days, using a 10 1
respirometric cell kept at constant temperature and moisture. Oxygen
consumption kinetics was modeled and organic matter composition was
characterized as biomass, easily-biodegradable, slowly-biodegradable and
non-biodegradable organic matter. The influence of temperature on kinetics
was tested. Results show that this respirometric method is a useful tool for the
characterization of solid organic matter biodegradability and for the modeling
of the biological kinetics of the composting process.
Sellanli et al (2008) studied co-composting of two olive mill wastes,
exhausted olive cake (EOC) and paste of olive mill wastewater naturally
dehydrated (POMW) with 25% sesame bark (SB). The humification process
was evidenced by quantifying the humic substances and humification indices:
(i) the ratio of humic acid (HA) carbon to fulvic acid (FA) carbon (CHA/CFA),

77
(ii) the ratio of water soluble organic carbon (Cw) to total organic nitrogen
(Cw/Norg), (iii) the ratio of humic acid carbon to total organic carbon CHA/Corg
and by determining the absorbance ratios: E2/E4, E2/E6 and E4/E6. The results
showed that the time required to reach maturity was dependant on the
chemical properties of the initial raw materials used. The compost including
EOC had more nitrogen and synthesised more polymerised HA, the POMW
compost also had acceptable degrees of stability and maturity at the end of the
process. Maturation was confirmed by a decline in Cw below 1.7, an increase
in nitrogen in HA, in CHA/ CFA and an elimination of phytotoxicity.
Yue et al (2008) investigated use of a settlement measurement
device to track the settlement of different layers in composting piles and time
courses of settlement velocity, bulk density distribution, and effective bulk
density during the composting sewage sludge composted with different
bulking agents. The physical compressibilities of composting mixtures are
night-soil sludge and garden refuse (1:1) > sewage sludge and cornstalks (5:4)
> night-soil sludge and garden refuse (2:3) > sewage sludge and bark (1:1).
Adding the proportion of bulking agent in the proper extent effectively
retarded compo sting settlement.
Jeyasingh et al (2010) carried out bioremediation studies for the
treatment of Cr (VI) bearing sludge using indigenous microorganisms
isolated from a chromium contaminated site. Effects of moisture content,
initial substrate and biomass concentrations on the bioremediation process
were studied by conducting batch and continuous experiments. The
leachability of total chromium and Cr(VI) from remediated soil was evaluated
and compared with that of untreated soil. Experimental data was used to
determine biokinetic parameters and validate a mathematical model. Single
objective and multi-objective management models were developed by

78
embedding the mathematical model describing the process in a simulation
optimization framework. Single objective management models considered
either cost minimization or minimization of time for treatment. Genetic
Algorithm, available in MATLAB tool box was used for solving the
optimization problems. Applicability of proposed management models was
demonstrated for the remediation of Cr(VI) bearing sludge in Ranipet,
Tamilnadu, India. Multi-objective management model was used to derive the
Pareto-optimal front, which describes the trade off between the cost of
treatment and the time taken for treatment.
Gopalakrishnan et al (2010) had made a comparative study on
biosorption of Zn (II), Cu(II) and Cr(VI) from textile dye effluent using
sawdust and neem leaves powder. They reported that adsorbent dosage, pH
and contact time were taken as parameters for biosorption study. Removal of
heavy metals ions from the textile dye effluent increases with increase in
adsorbent dosage. The influence of pH and contact time was maximum for
removal of heavy metal ions. The presence of the decreased heavy metal
toxicity in the treated textile dye effluent was evaluated through the
percentage of seed germination of Vigna mungo L. On comparison, sawdust
was found to be good adsorbent compared to neem leaves powder.
Robert et al (1993) reported the in-situ method for decreasing heavy
metal leaching from soil or waste. A method of treating solid waste in soil or
solid disposed waste containing unacceptable levels of leachable metals, such
as lead, cadmium, arsenic, zinc, copper and chromium, includes mixing the
solid waste or soil in place with a phosphate source or a carbonate source or
ferrous sulfate. After the solid waste and the additive are mixed, if needed an
additional pH controlling agent is mixed into the soil or waste and additive.
After the solid waste and additive and pH controlling agent are mixed under

79
conditions which support reaction between the additive, pH controlling agent
and metals. The metals will be converted to non-leachable forms which are
relatively stable under normal environmental conditions. The treatment
additives can be introduced and contacted with the soil or waste in any of the
following techniques: spreading the additives on top of the soil or waste and
mixing with a mechanical device, such as a rotary tiller, adding the treatment
chemical through an infiltration gallery as a solution or slurry, injecting a
soluble additive through injection nozzles or injection wells, and adding a
treatment additive through a hollow-shaft auger and in-place mechanical
mixing. It is a method for treating disposed solid waste or soils containing
unacceptable levels of chromium. The chromium will be converted to nonleachable forms which are stable under normal environmental conditions.
3.4

BIODEGRADATION OF HAZARDOUS ORGANICS

Generally the chemical industrial activities consists of various

process and operations. This will generate complex nature of effluent. The
multi-substrate nature of an industrial effluent is characterized by the presence
of a variety of compounds in varying concentrations (Godbole and
Chakrabarti, 1991). Persistent organic pollutants are among the most
dangerous chemicals released into environment by human. These include
polycyclic aromatic hydrocarbons (PAHs), nitro phenols, and chlorophenols
and they are found in wide variety of natural environments. Some of these
chemicals are considered priority pollutants due to their potential for toxicity,
mutagenicity and carcinogenicity (Keith and Telliard, 1979).
Haggblom and Young (1990) demonstrated that five chlorophenols
(2-1 chlorophonol, 3-chlorphenol, 4-chlorophenol, 2, 4-dichlorophenol and
2,6-dichlorophenol) can be degraded under sulfate reducing conditions.
Anaerobic degradation of chlorinated aromatic compounds such as

80
chlorobenzenes, chlorophenols and PCBs has been found to occur in a wide
variety of environments. Sylvewstre et al (1985) found mineralization of 4chlorobiphenol to be dependent on the interaction of two bacterial populations.
The first of these population transformed 4-chlorooiphenyl into 4chlorobenzoate, while the second mineralized the 4-chlorobenzoate to carbon
dioxide. Nelson et al (1986, 1987, 1988) have isolated an ammonia oxidizing
bacterium, Nitrosomonas euroaea, which can degrade chlorinated aliphatic
compounds. Microbial degradation of chlorinated acetophenones by mixed
culture which mineralizes 4- chloroacetophenone via the Baeyer- Villigeroxidation. Haggblom (1992) had reported that mono and dichlorophendls are
degraded via hydroxylation to the corresponding chlorocatechol, which are
then degraded via ortho-cleavage in higher chlorinated phenols as
hydroxylation which occurs in para position.
Microorganisms are largely responsible for the degradation of toxic
chemicals present in the environment (Alexander, 1995). The mechanisms of
enzymatic attack on halogenated compounds have been studied extensively
(Fetzner, 1994; Janssen et al., 1994). Despite the deactivating effects of
chlorine substitution a few aerobic microorganisms can degrade chlorinated
phenols. Mono and Dichlorophenols and their metabolites like chlorocatechols
are cometabolically formed by yeasts and molds.
Sadat et al (1995) studied the degradation of 3-Nitrotoluene
incubated with the resting cells of Pseudomonas putida OU83. Most of the 3nitrotoluene (70%) was metabolized via reduction of the nitro group to form 3aminotoluene (3-AT). A minor portion (30%) was degraded through a novel
pathway involving oxidation of 3-NT to form 3-nitrophenol through a series of
intermediary metabolites: 3-nitrobenzyl alcohol, 3-nitrobenzaldehyde and 3nitrobenzoic acid. Degradation of 3-nitrophenol occurred with the formation of

81
a transient intermediary metabolite, hydroxynitroquinone, which was further
degraded with the near stoichiometric release of nitrite into the medium. 3Nitrotoluene- induced cells showed increased oxygen consumption with 3nitrotoluene, 3-nitrobenzaldehyde, 3-nitrobenzoate, and 3-nitrophenol as
substrates in comparison to uninduced cells. Cell extracts prepared from strain
OU83

contained

benzylalcohol

dehydrogenase

and

benzaldehyde

dehydrogenase activities. The experimental evidence suggests a novel pathway

for the degradation of 3-NT in which C-l elimination is catalyzed by a
cofactor-independent deformylase rather than a decarboxylase or dioxygenase.
Annachhatre and Gheewala (1996) has reported that partial removal
of chlorophenols between 40-60% is usually observed in aerobic and
anaerobic processes. Removal can be enhanced by a combination of aerobic
and anaerobic operations. They also reported that aerobic degradation of
chlorophenols proceeds through the formation of catechols. Lee & Lee (1996)
showed that a newly isolated Arthrohacter ureqfacienir strain CPR706 could
degrade 4-chlorophenol via a new pathway in which the chloro substituent was
eliminated in the first step and hydroquinone was produced as a transient
intermediate. Strain CPR706 exhibited much higher substrate tolerance and
degradation rate than other strains that degraded 4-chlorophenol by the
hydroxylation at the second carbon position to form chlorocatechol. Strain
CPR706 could also degrade other para-substituted phenols (4-nitro-, 4-bromo-.
4-iodo-, and 4-fluoro-phenol) via the hydroquinone pathway.
Zdrahal (1998) identified 4-Nitrocatechol as a product of
transformation of 4-nitrophenol by bacterial strain Corynebacterium sp.8 using
direct acetylation of biodegradation samples by acetic anhydride followed by
GC-MS analysis. The identity of 4-nitrocatechol in the form of diacetate was
confirmed

by

electron-impact

spectra

and

spectra

recorded

under

82
chemicalionization conditions (positive and negative modes). Negative-ion
chemical ionization was used for quantification of 4-nitrocatechol in
biodegradation samples in a concentration range of 1-25 mg l-1.
Chauhan et al (1999) reported that pure cultures of bacteria isolated
with the ability to degrade many of the halogenated aromatic hydrocarbons
degradation is frequently the result of a consortium of microorganisms.
Degradation of 2,4,5-trichlorophenol and 2,3,5,6- tetrachlorophenol was
studied by using a two stage approach that utilized efficient pulse electric
discharge followed by biological degradation with consortium from acclimated
return activated sludge.
Alan

Farrell

and

Brid

Quilty

(2001)

demonstrated

that

Pseudomonas testosterone CPW301 degraded phenol and 4-chlorophenol

simultaneously, but degradation rates of these compounds were affected by 4chlorophenol. Aleksieva et al (2002) studied Trichosporon cutaneum R57
which was able to grow and utilize some phenol derivatives: resorcinol, 2,6dinitrophenol, 3-nitrophenol, 4-nitrophenol and m-cresol as sole carbon and
energy source. The results obtained showed the ability of the strain to develop
by degrading these compounds except 4-nitrophenol. Resorcinol and 2,6dinitrophenol were utilized in 27 h in yeast nitrogen base (YNB) medium
while 3-nitrophenol and m-cresol were only partly degraded in this period of
time. The growth of T.cutaneum using YNB containing one of the phenol
derivatives as the sole carbon and energy source was compared with the
growth of cells in YNB with the same concentration of the phenol derivative
plus 0.3 g l-1 phenol. Both compounds were simultaneously utilized by the
mechanism of cometabolic degradation. HPLC analysis of the centrifuged
culture medium showed an increased degradation rate of resorcinol.
Resorcinol and phenol were completely degraded in 18 h. No significant

83
change of the degradation rate for 2,6-dinitrophenol and 3-nitrophenol was
observed. The combination of m-cresol and phenol exhibited inhibiting effects
with negligible degradation of phenol and m-cresol in 27 h. The
biodegradation of the phenolic derivatives by cells of T.cutaneum R57
immobilized onto polyamide and polyacrylonitrile granules and fibers were
also studied.
Zouri et al (2002) investigated 4-Chlorophenol (4-CP) degradation
by suspended and immobilized Phanerochaete chrysosporium conducted in
static and agitated cultures. The best results were achieved when experiment
was carried out in a rotating biological contactor instead of an Erlenmeyer
flask, for both batch degradation and repeater batch degradation. The relative
contribution of lignin peroxidase (LiP) versus manganese peroxidase (MnP) to
the 4-CP degradation by P. chrysosporium was investigated. 4-CP degradation
slightly increased and a high level of MnP (38 nKat ml-1) was produced when
P. chrysosporium was grown at high MnII concentration. High LiP production
in the medium had no significant effect on 4-CP degradation. 4-CP
degradation occurred when P. chrysosporium was grown in a medium that
repressed LiP and MnP production. This result indicates that LiP and MnP are
not directly involved in 4-CP degradation by P. chrysosporium. Hela Zouari et
al (2002) reported 4-Chlorophenol (4-CP) degradation investigated by
suspended and immobilized Phanerochaete chrysosporium which was
conducted in static and agitated cultures
Ladislao & Galil (2003) investigated bioremediation of consecutive
spills of phenol, 2-chlorophenol (2-MCP), 2,4,6-trichlorophenol (2,4,6-TCP)
and pentachlorophenol as single pollutants in eight pilot plant scale sand
columns system (100 cm 1, 6 cm ID), simulating the conditions which could
be created in the saturated zone of a pristine aquifer following an accidental

84
spill. Degradation curves demonstrated first-order kinetics. Biodegradation
rates (k1) were influenced by consecutive exposures. Calculated rate constants
for biodegradation for sole substrate experiments were in the range of 0.060.15 day-1, 0.21-1.20 day-1, 0.04-2.28 day-1 and 0.01-0.03 day-1 for phenol, 2MCP, 2,4,6-TCP and PCP respectively.
Gallego et al (2003) studied degradation and detoxification of a
mixture of persistent compounds (2-chlorophenol, phenol and m-cresol) by
using pure and mixed indigenous cultures in aerobic reactors. Biodegradation
assays were performed in batch and continuous flow reactors. Biodegradation
was evaluated by determining total phenols, ultraviolet spectrophotometry and
chemical oxygen demand (COD). Microbial growth was measured by the plate
count method. Scanning electronic microscopy was employed to observe the
microbial community in the reactor. Individual compounds were degraded by
pure bacteria cultures within 27 h. The mixture of 2-clorophenol (100 mg 1 -1),
phenol (50 mg 1-1) and m-cresol (50 mg 1-1) was degraded by mixed bacteria
cultures under batch conditions within 36 h: 99.8% of total phenols and 92.5%
of COD were removed; under continuous flow conditions 99.8% of total
phenols and 94.9% of COD were removed. Mineralization of phenolic
compounds was assessed by gas chromatography performed at the end of the
batch assays and in the effluent of the continuous-flow reactor.
Chu-Fang Yang et al (2005) had studied 10 indigenous PCPdegrading bacterial strains isolated from a PCP-degrading mixed culture, and
the potential of both the pure and mixed cultures for PCP degradation
compared. Kulkarni & Chowdary (2005) found a strain of Pseudomonas
putida capable of metabolizing p-nitrophenol (PNP) as a sole source of carbon,
nitrogen and energy. To explore the applicability of this strain for
bioremediation for controlling environmental PNP pollution, its degradation

85
potential at 300 and 500 ppm was examined in a medium devoid of carbon and
nitrogen source (minimal medium). At A600, 0.5 OD inoculum, the strain
metabolized 300 and 500 ppm within 36 and 72 h, respectively. The
degradation was accompanied by release of stoichiometric amount of nitrite.
Effect of glucose and nitrogen on PNP degradation under similar conditions
revealed that (i) glucose (0.4.g/l) at 20 and 50 ppm PNP did not accelerate the
rate of PNP degradation, while glucose (0.4 g/l) at 300 ppm PNP inhibited its
degradation, (ii) nitrogen supplement viz. sodium nitrate and ammonium
sulphate (0.04 and 0.4 g/l) in minimal medium with PNP (300 ppm) showed
no effect on PNP degradation, while glutamate alone (0.04 and 0.4 g/l) showed
mere rise in biomass (from 0.5 to 1.6 OD units), and (iii) acidic pH (4.0-6.5)
did not support PNP degradation, while alkaline pH (7.5-9.5) significantly
enhanced the rate of PNP degradation. The complete degradation of PNP at
high concentration (300 ppm) was confirmed by HPTLC analysis.
Gemini et al (2005) isolated an indigenous bacterial strain capable
of utilizing p-nitrophenol (PNP) as the sole carbon, nitrogen and energy from
river sediment in Buenos Aires (Argentina). The 168 rDNA sequence showed
a close relationship to Rhodococcus wratislaviensis. This indigenous bacterial
strain degrades 0.36 and 0.72mM PNP in 34 and 56 h respectively and releases
the nitro group from the compound as nitrite. Aerobic biodegradation assays
were performed using a 2L microfermentor at 28C with agitation (200 rpm).
Biodegradation was evaluated by spectrophotometry, gas chromatography and
microbial growth. As the nitrite released during PNP degradation is also an
environmental toxic agent it was removed by adding a denitrification step in
an anoxic process. Detoxification was evaluated by using a Daphnia magna
toxicity test.

86
Baggi et al (2007) investigated the degradative properties towards
chlorobenzoates by a 2-chlorobenzoate-degrading mixed culture (2MC).
Although 2MC did not grow on 2,3,5-; 2,3,6- and 2,4,6-trichlorobenzoates, it
was

able

to

completely

oxidise

2,3,5-

trichlorobenzoate

and

2,5-

dichlorobenzoate. This was degraded through the intermediate formation of 4chlorocatechol and 3-cis,cis-chloromuconate, suggesting a dioxygenation in
1,2 position. 4-Chlorobenzoate; 3,4- and 2,6-dichlorobenzoate; 2,4,6- and
2,3,6-trichlorobenzoate did not undergo any co-metabolic transformation, the
only 2,4-dichlorobenzoate was scarcely transformed into a dichlorinated
phenol as end-product. On the basis of its degradative properties, 2MC could
be a good candidate for use in the removal of ortho- and/or metachlorobenzoates that are the major end-products of PCB co-metabolism.
Alcocer et al (2007) studied biodegradation of mixed chlorophenols
by a microbial consortium in steady state continuous culture. Removal
efficiencies, cell growth yields, volumetric and specific biodegradation rates of
phenol and chlorophenols were compared to those obtained with pure strains
in the same culture conditions. Using phenol as the primary carbon and energy
source, the microbial consortium cometabolically degraded mono-, di- and trichlorophenols with overall removal efficiencies ranging from 95 to 99.8% and
COD removal efficiencies from 85 to 97.8%. In all cultures, the microbial
consortium showed better overall removal efficiencies than single strains.
Binary mixtures of chlorophenols produced a remarkably increase in specific
biodegradation rates of substrates qc, beyond those obtained when the
consortium grew on single compounds. The results suggest that chlorophenols
collapsed cell's proton motive force; thus, energy spilling, reflected by the qc
increase, could be the cell's response to compensate the short-circuit in
membrane electrochemical proton gradient.

87
Fang Yang and Lee (2007) isolated pure phenol-degrading strains
from enriched mixed cultures and monitored the variations of species during
the enrichment period. Two strains were isolated from the acclimated mixed
culture. They were identified as Pseudomonas resinovorans strain P-1 and
Brevibacillus sp. strain P-6. DGGE indicated that strain P.resinovorans
appeared at the beginning arid maintained well during the enrichment period.
The second strain Brevibacillus sp. did not appear in the initial stage but
showed up after 2 weeks of enrichment. The optimum growth temperatures for
P.resinovorans and Brevibacillus sp were 31 and 39oC, respectively. P.
resinovorans could degrade phenol completely within 57.5 h, when the initial
phenol concentration was lower than 600 mg 1-1. If the initial phenol
concentration was lower than 200 mg 1-1, Brevibacillus sp. could remove
phenol completely within 93.1 h. It was obvious that the phenol-degrading
ability of P.resinovorans was much better than that of Brevibacillus sp. The
metabolic pathway for P. resinovorans phenol degradation was assigned to the
meta-cleavage activity of catechol 2,3-dioxygenase.
Saravanan et al (2008) developed and studied an internal loop airlift
reactor (ILALR) for biodegradation of phenol/ m-cresol as single and dual
substrate systems under batch and fed batch operation using an indigenous
mixed microbial strain, predominantly Pseudomonas sp. The results showed
that the culture could degrade phenol/ m-cresol completely at a maximum
concentration of 600 mg l-1 and 400 mg l-1, respectively. Batch ILALR study
has revealed that phenol has been preferentially degraded by the microbial
culture rather than m-cresol probably owing to the toxic effect of the later.
Wei et al (2008) screened high strength phenol degrading bacteria
isolated from nodules of eight wild legumes species in the mining tailing
region of Shaanxi province, northwest of China, and cultivated them in a basal

88
salt (BS) medium supplemented with different phenol concentrations as a sole
carbon source. The phenol degradation was around.99.5 and 78.3%, with an
initial concentration of 900 and 1000 mg/l phenol in 62 and 66 hours
respectively. Kinetic studies indicated that the strain had a high KS (743.1 M)
and an extremely high KS (10,469 M) in Haldane's model. The phylogenetic
analysis based on 16S rRNA gene sequences showed that CCNWTB701
belonged to the Rhizobium genus, and it was closely related to Rhizobium
mongolense and Rhizobium gallicum.
Santos et al (2008) studied the degradation of phenol (2-30 mM) by
free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13
isolated from stainless steel effluents in batch cultures with saline solution not
supplemented with nutrients or yeast extract. Results suggested that inoculants
based on immobilized cells of A.pullulans. FEI3 have potential application in
the biodegradation of phenol and possibly in the degradation of other related
aromatic compounds.
3.5

SUMMARY
Several ex-situ treatment options are available for treatment of

contaminated dam sediments. Among various technologies, bio-remediation

appears to be more effective, economically viable, and environmentally
friendly treatment option for decontamination of dam sediments. In the past
three decades, many experimental studies have been undertaken to understand
biotransformation of sediments by different microbes, under different
environmental

conditions.

In

this

study

on

bioremediation

of

the

Orathupalayam dam sediments, two types of approaches were carried out (i).
bioremediation by microcosm, (ii) bioremediation by composting.