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Assessment of pesticides removal using two-stage Integrated Aerobic

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DOI: 10.1016/j.biortech.2017.03.080

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Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Assessment of pesticides removal using two-stage Integrated Aerobic

Treatment Plant (IATP) by Bacillus sp. isolated from agricultural field
S.R. Geed, B.S. Shrirame, R.S. Singh, B.N. Rai ⇑
Department of Chemical Engineering and Technology, IIT (BHU), Varanasi 221 005, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Performance evaluation of IATP for

treatment of wastewater.

Isolation of Bacillus sp. species from
the pesticide contaminated site.

The results of GC–MS confirmed the
biodegradation of pesticides.

The kinetics parameters were
obtained using kinetic study.

a r t i c l e i n f o a b s t r a c t

Article history: The biodegradation of synthetic wastewater containing Atrazine, Malathion and Parathion was studied in
Received 29 January 2017 two stage Integrated Aerobic Treatment Plant using Bacillus sp. (consortia) isolated from agricultural
Received in revised form 10 March 2017 field. The influent stream containing these pesticides with initial COD of 1232 mg/L were fed to first reac-
Accepted 11 March 2017
tor and treated effluent of first reactor was fed to second reactor. The maximum removal of pesticides in
Available online xxxx
IATP was found to be greater than 90%. The various process parameters such as pH, DO, Redox potential
and BOD5/COD were monitored during the treatment. The degradation of pesticides and its metabolites in
Keywords:
the treated effluent were confirmed by GC–MS. Kinetic parameters such as first order rate constant (Kobs),
Aerobic treatment plant
Bacillus sp.
cell yield (YX/C) and decay coefficients (Kdp) were evaluated and found to be 0.00425 per hr, 0.696 mg of
Redox potential COD/mg MLSS and 0.0010 per hr respectively. This integrated process was found more effective than
GC–MS physico-chemical treatment of pesticides.
Kinetics Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Increasing population and hence increased demand for food
grains has compelled for extensive use of pesticides in recent years.
Approximately 200 times more use of pesticides is in use these
days as compared to the traditional pattern of agriculture practiced
in the India 5–6 decades back (Martin et al., 2009; Environment
⇑ Corresponding author.
E-mail address: bnrai.che@itbhu.ac.in (B.N. Rai).
Agency, 2014). This pesticides being water soluble ultimately con-
taminate water bodies nearby agricultural field (Environment
Agency, 2014). United Nations reported that among all the pesti-
cides used in agriculture, less than 1% reached to the crops and
the rest contaminates the soil, air and water (Martin et al., 2009).
Hedegaard and Albrechtsen (2014) reported that the presence of
pesticides in soil and surface water ultimately reaches to rivers
and lakes. It was found that the pesticides are bio-accumulated
in higher species like raptors through the food chain (Cooper
et al., 2016). According to geological survey of Denmark in 2011,

http://dx.doi.org/10.1016/j.biortech.2017.03.080
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
2 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx
most of the groundwater was contaminated with pesticides and
their residual metabolites (GEUS, 2011). European Parliament
and Council (EPC, 2000) set 0.1 mg/L as the limit of the pesticides
concentration in the drinking water and ground water for single
pesticide and 0.5 mg/L for the total of all pesticides (EPC, 2000;
Hedegaard and Albrechtsen, 2014; Environment Agency, 2014).
Atrazine, Malathion and Parathion shows several health hazards
such as an endocrine disruptor, carcinogenic, neurotoxic and affect
the reproductive system in human and animal (Douglass et al.,
2014; Environment Agency, 2014; WHO, 2015; Geed et al., 2016).
Thus, the control of pesticides is one of the most desirable and chal-
lenging tasks for the environmentalists. The various physico-
chemical techniques such as Fenton Oxidation, Adsorption, Floccu-
lation etc have been developed for the removal of pesticides
(Klancar et al., 2016; Gozzi et al., 2016). But, they have their own
disadvantages such as higher processing and equipment cost, high
energy consumption, sludge generation and production of toxic
byproducts (Yadav et al., 2014; Geed et al., 2016). To overcome
these problems, several researchers have focused on biological
treatment for wide range of pesticides (Cesar and Ros, 2013;
Hedegaard and Albrechtsen, 2014;Cooper et al., 2016). As pesticides
are toxic and difficult to degrade compounds therefore only specific
species are able to degrade such compound with reasonable rate
and this is why the selection of suitable microbial consortia is very
crucial in effective rapid degradation of pesticides (Heitkamp et al.,
1990). In the recent years, mixed microbial consortia have been
found successful to treat mixtures of pesticides. Heitkamp et al.
(1990) and Bhadbhade et al. (2002) reported that by employing
pure culture, it is difficult to efficiently degrade the mixtures of pes-
ticides. They also reported that the metabolic cooperation is essen-
tial for maintaining microbial consortia of multiple species to treat
mixtures of pollutants biologically. The interaction of different
microbial species permits the efficient removal of organic contam-
inants (Heitkamp et al., 1990; Bhadbhade et al., 2002).
The proper growth of the microorganism help to build-up bio-
mass, minimize any toxic effect of substrate inhibition and
improve the operational stability of aerobic treatment plant
(Singh et al., 2006; Yadav et al., 2014; Srivastva et al., 2016). Most
of the studies available on bioremediation of pesticides are limited
to batch experiments, but only continuous systems have potential
to provide the solution to practical problems related to pesticides
contamination (Yadav et al., 2014; Cooper et al., 2016; Geed
et al., 2017). The kinetic study of biodegradation of pesticides is
limited in the literature which provides useful information about
the potential of microbial species, and kinetic constants are used
for design and upscaling of continuous systems (Beltran-Heredia
et al., 2000; Martin et al., 2008; Affam et al., 2014).
Above literatures clearly indicates that there are very limited stud-
ies available on biodegradation, kinetics of biodegradation, identifica-
tion of metabolites produced during biodegradation of pesticides in
the pilot plant scale bioreactors. Keeping above fact in mind, the pre-
sent study has been planned to investigate the biodegradation of
Atrazine, Malathion and Parathion in IATP. For the treatment of above
pesticides, the potential bacterial species were isolated from pesti-
cides contaminated site. The performance evaluation of two stage
aerobic treatment plants and effect of various parameters were stud-
ied during biodegradation of pesticides. The important kinetic param-
eters were evaluated such as first order biological rate kinetic
constant (Kobs), cell yield (YX/C) and decay coefficients (Kdp).
2. Materials and methods
2.1. Chemicals and mineral salt medium
Analytical grade Atrazine, Malathion and Parathion procured
from Sigma-Aldrich, India with more than 99.0% purity was used
Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
in the experiments. The mineral salt medium (MSM) containing
3 g of KH2PO4, 6 g Na2HPO4, 2 g NH4Cl and 0.5 g NaCl per liter of
solution is used as source micro-nutrient for the growth of bacte-
ria. A trace element solution containing 0.04 g MgSO47H2O,
0.025 g CaCl22H2O, 0.025 g FeSO47H2O, 0.04 g CuSO45H2O, 0.2 g
ZnSO47H2O and 0.4 g MnSO4H2O per liter of distilled water
(Bramhachari et al., 2016) was also prepared to serve as a source
of micro-nutrient. MSM and trace element solutions were steril-
ized by autoclaving for 15 min at 121 °C, and final pH was adjusted
to 7.0 ± 0.2 and then used for the isolation of strains.
2.2. Isolation, biochemical and molecular characterization of bacterial
species
Pesticides contaminated soil samples were collected from the
agricultural farm of Banaras Hindu University, Varanasi, India
(25° 190 N; 83° 30 E, 129 m above mean sea level). From decades
the sampling spot had been exposed for rigorous agricultural activ-
ity and therefore contaminated with a wide range of pesticides.
The enrichment of isolated bacterial sp. was performed in MSM
using pesticides as a sole carbon source.
Morphological characterization of the bacterial isolates were
carried out using laboratory microscope (Dewinter Tech, India)
and Scanning Electron Microscope (SEM QUANTA 200F Nether-
land). The sample containing bacterium was first filtered using
the strips of the cellulose filter paper (Ultipor N66, Pall Life
Sciences, India) of size (0.2 mm) for separation of bacterium from
sample. Further, the filter papers containing bacterium were dried
overnight at 30 °C in the oven (Model 159SS NSW, India) and
coated with gold and carbon particles for better resolution. The
SEM analysis was performed under low pressure to minimize any
harm to the bacteria. Biochemical test (Gram’s staining, Catalase,
Oxidase, Motility, Voges-Proskaur, Urease test, Fermentation,
Methyl red and Indole etc.) were done for the isolated microorgan-
ism using high media biochemical kit according to guidelines of
Bergey’s Manual of bacteriology (Srivastva et al., 2016; Geed
et al., 2017).
Bacterium genomic DNA was extracted using the standard pro-
tocols of Sambrook et al. (2001). Details are described in elsewhere
(Shukla et al., 2010). Genomic DNA was subjected to PCR amplifi-
cation of 16S rRNA gene with universal primers Bac8F (50 AGA
GTT TGA TCC TGG CTC AG-30 ) and 1492R (50 GGT TAC CTT GTT
ACG ACT T30 ) (Edwards et al., 1989). The details of the DNA ampli-
fication were given in literatures (Shukla et al., 2010; Yadav et al.,
2014).
Sequencing of the bacterial isolates was out sourced and done
by Triyat Scientific Hyderabad India. The sequences were aligned
manually with published sequences in NCBI database by using
CLUSTALW multiple sequence alignment programs. The evolution-
ary history was inferred using the Neighbor-Joining method. The
evolutionary distances were computed using the Maximum Com-
posite Likelihood method (Tamura et al., 2004) and are in the units
of the number of base substitutions per site. All positions contain-
ing gaps and missing data were eliminated. Evolutionary analyses
were conducted in MEGA6 (Tamura et al., 2013).
2.3. Experimental setup: Reactor startup and operations
The two-stage Integrated Aerobic Treatment Plant (IATP) pro-
cured from TAE/1000 Pignat, France (Fig. 1) with the total and
working volume of 45 and 30 L respectively. IATP was used for
the treatment of synthetic wastewater containing mixtures of
50 mg/L each of Atrazine, Malathion and Parathion was selected
after optimizing concentration in batch incubation study varying
concentration ranges from 25 to 100 mg/L each of pesticides. The
COD of selected synthetic wastewater was measured and found
 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx 3

to be 1232 mg/L. The synthetic wastewater was stored in a plastic Table 1

tank of 200 L capacity. The initial characteristics of wastewater Physico-chemical characteristics of the wastewater.

were shown in Table 1. The first stage in the Integrated Aerobic Parameters Wastewater sample
Treatment Plant was stirred aerobic bioreactor (Reactor-1) as Before treatment After treatment
shown in Fig. 1. The stirring was done by motor-1 (IKA, Eurostar
Nitrate (ppm) 1.227 –
40) with Rushton turbine rotating continuously at 160 rpm, and Sulfate (ppm) 4.265 –
the air was supplied using an air compressor (KNF, Labport). The Phosphate (ppm) 4.073 –
timer was set at an interval of 30 min/h to maintain proper aera- Fluoride (ppm) 0.890 0.270
tion in the reactor. The parameters like pH (HD 2305.0; Delta Chloride (ppm) 51.906 0.461
Bromide (ppm) 0.295 –
OHM; Italy), Redox potential (HD 2305.0; Delta OHM; Italy) and
Calcium (ppm) 111.811 10.211
DO (HD 2109.1; Delta OHM; Italy) were measured using respective Magnesium (ppm) 40.364 2.571
probes was provided by Pignat, France. Initially the isolated bacte- Sodium (ppm) 65.706 6.461
rial species was cultured in Nutrient broth (100 mL) in screw cap Potassium (ppm) 18.492 0.267
Lithium (ppm) 0.089 –
bottles. The well grown bacterial biomass was co-cultured with
TDS (ppm) 224.7 391.6
glucose (100 g/L), MSM and trace element solution in the
Reactor-1 and kept for 240 h (or 10 days) for proper growth of
the bacterial species. After 240 h, the synthetic wastewater with CODinitialCODfinal
COD of 1232 mg/L was fed into Reactor-1. Every day during the % COD Removal ¼  100 ð1Þ
CODinitial
treatment process, 10 L fresh wastewater (periodic feeding; 2 h
feeding and 10 h treatment in each the day) was fed to the
Reactor-1 for the treatment and proper performance of the reactor.
The treated effluent from Reactor-1 was transferred to the Reactor- 2.4. Effluent analysis
2 (second stage; settling tank) which was equipped with stirring
motor-2. In Reactor-2, the remaining parental pesticides and Chemical oxygen demand (COD) and Biological oxygen demand
metabolites were again treated with some part of effluent recycled (BOD5) of initially prepared synthetic wastewater and treated sam-
from Reactor-1. The recycle pump (KNF, Liquiport) was operated at ples were performed according to the standard methods (APHA
constant recycle flow rate of 6 L/h. The samples were taken twice et al., 2005) in the laboratory. The physico-chemical characteristics
at an interval of 12 h from the sampling valve of Reactor-1 and of wastewater were analyzed by Ion Chromatography (Dionex ICS
Reactor-2 for analysis. The aerobic plant was operated for 30 days 3000).
(720 h) and parameters such as pH, Redox, DO, COD and COD/BOD5 The residual concentration of mixture of pesticides and their
were regularly monitored. The ANOVA analysis was performed for metabolites were analyzed by HPLC (ELICO, India) equipped with
the significant effect of pH, DO, Redox and BOD5/COD ratio on COD C-18 column, UV detector (HD469) and pump (HD464) at wave-
removal. The COD removal was calculated using Eq. (1) length of 210 nm using mobile phase (acetonitrile:water; 45:55)

Fig. 1. Schematics of experimental setup two-stage Integrated Aerobic Treatment Plant.

Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
4 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx
for 15 min with a constant flow of 1.0 mL/min (Douglass et al.,
2014).
For GC–MS analysis samples were prepared by extracting resid-
ual metabolites of pesticides with the addition of an equal volume
(5 mL) of chloroform and degraded sample. Then prepared samples
were shaken vigorously to separate the organic layer. The
extracted samples were analyzed using GC–MS-QP2010 Ultra (Shi-
madzu, Japan). The column flow was maintained at 1.21 mL/min
with a split ratio of 10. The initial oven temperature was set at
50 °C for 2 min and then increased at 5 °C/min to 250 °C, where
it was maintained for 2 min. Then it again increased at the rate
of 15 °C/min to 280 °C, where it was maintained for 14 min. Injec-
tor and oven temperature was 260 and 280 °C respectively. The MS
measurements were done at MS ion source temperature of 230 °C,
interface temperature of 270 °C, total time 20 min and solvent
delay 3 min; chromatographic data were collected and recorded
by GC–MS (Geed et al., 2017).
2.5. Kinetic study
Bailey and Ollis (1986) reported numerous models related to
growth and substrate utilization rate with the available substrate
concentration. After referring several literatures, the Monod model
was found wide applicability to mathematically represent the
growth of microorganism in such systems (Monod, 1949). The
model equations are as follows
1 dx lmax  C
l¼ ¼ ð2Þ
X dt Ks þ C
By simplifying Eq. (2) get
dx XC
¼ lmax
dt Ks þ C
where m specific growth rate, per hr; mmax maximum specific growth
rate, per hr; C substrate concentration (mg/L) and Ks half saturation
rate constant (mg/L). In biological processes, rg = (dx/dt) the growth
rate can be defined as (4)
rg ¼ l  X
where rg is the rate of bacterial growth, mg/L/h, and X microorgan-
ism dry weight cell mass (mg/L).
Monod model can be modified as shown in equation, the sub-
strate concentration (Ks  C) (Beltran-Heredia et al., 2000)
dx
¼ K obs  C
dt
where Kobs biological first order rate kinetic constant (per day)
As Co > 0, the differential equation is solved and gets the follow-
ing equation
C0
Ln ¼ K obs  t
Ct
where Ct and Co (COD mg/L) are the residual concentration of sub-
strate at treatment time t (day; after cultivation time) and the initial
concentration respectively; Kobs (per day) the rate constant.
A plot of Ln(Ct/Co) vs. treatment time t (day; after cultivation
time) as shown in Fig. 5a and the value of constant Kobs and corre-
lation coefficient R2 were calculated.
Cell yield coefficient (Yx/c) and biomass decay coefficient (Kdp)
are very important parameters related to the biomass concentra-
tion evolution over the entire period of the aerobic treatment
plant. The Yx/c represents the mg of biomass produced per mg of
substrate utilized, and Kdp indicates the importance of endogenous
metabolism. The Yx/c and Kdp were calculated using specific growth
rate equation (Beltran-Heredia et al., 2000) as given below
Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
l ¼ Y XC  q  K dp ð7Þ
where m is specific growth rate (per hr); q is the specific substrate
utilization rate; Yx/c is the ratio of the mass of cell formed to the
mass of substrate utilized; and Kdp is kinetic constant for biomass
death phase (per hr) (Affam et al., 2014). m and q was calculated
using Eqs. (8) and (9).
1 DX
l¼ ð8Þ
X Dt
1 DC
q¼ ð9Þ
x Dt
3. Results and discussion
3.1. Bacterial identification: morphological, biochemical and molecular
characterization
The bacterial isolates were found to be rod-shaped and motile
in nature (laboratory microscope and SEM images). Biochemical
characterizations of microorganisms provide the metabolic activi-
ties regarding their growth requirements and enzymatic activities
using chemicals, media, and nutrients to understand observable
changes. The isolate was shown positive results for Gram’s stain-
ing, Catalase, Oxidase, Motility, Voges-Proskaur, Urease, and Fer-
mentation while Methyl red and Indole production were
indicated negative results.
The molecular characterization of bacterial nucleotide
sequences was outsourced by Tri Scientific, Hyderabad India.
Nucleotide sequences of 16S-rRNA determined for bacterium sp.
were deposited in GenBank database under the accession number
ð3Þ KX158859, KX158860 and KX158862. Nucleotide sequence analy-
sis based on 16S-rRNA fragment and phylogenetic investigation
using BLAST software identified as Bacillus sp. S1, Bacillus sp. S2
and Bacillus sp. S4 as the most abundant species. Convectional bac-
teriological tests confirmed the identity of the isolates. The phylo-
genetic tree has been drawn in Fig. 2 with the help of nucleotides
sequence of the microorganisms. The various researchers used
ð4Þ
Bacillus species for the biodegradation of different pesticides
(Singh et al., 2009; Tang and You, 2012).
3.2. Startup and performance of IATP
In this study, typical experiments were conducted with isolated
ð5Þ potential microbes to examine the performance of aerobic plant
(Fig. 1) for 30 days under varying process and operating parame-
ters. The isolated bacterial consortia were allowed to grow in the
Reactor-1 (before supply of wastewater) by feeding with 100 g/L
of glucose as a carbon source along with MSM and trace element
solution for the period of 240 h (10 days; start up time). According
ð6Þ to Tang and You (2012) Bacillus sp. could degrade triazophos from
sewage sludge wastewater through co-metabolism when fed with
nutrients like peptone, yeast extract, and glucose. During start-up
stage, the complete degradation of glucose was achieved in the
Reactor-1 with stable biomass content and almost no fluctuation
in pH (neutral).
After the startup stage, the synthetic wastewater (Table 1) with
COD 1232 mg/L was fed into the Reactor-1 with flow rate 2.5 L/h.
The COD (mg/L) of the effluent from Reactor-1 and Reactor-2 were
measured at a regular interval of 12 h. Fig. 4 shows the variation of
pH, DO, Redox and BOD5/COD (process parameters) along with %
COD removal with time in Reactor-1. The overall increasing trend
of % COD removal with time was observed which show that the
COD in the Reactor-1 was decreasing with time. However, some-
times % COD removal decreased which might be due to periodic
 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx 5

Fig. 2. Phylogenic tree of isolated bacterial species 16S rRNA gene fragment with the sequences of closed affiliations by neighbor joining methods using Mega 6.0.

Fig. 3. Overall performance two stage Integrated Aerobic Treatment Plant during the treatment of synthetic wastewater.

feeding (2 h feeding and 10 h treatment in each the day) of (Celis et al., 2008; Li et al., 2014). Li et al. (2014) who reported
wastewater into the Reator-1. Another reason might be unavail- decline in % COD removal in case of wastewater containing pesti-
ability of sufficient biomass to maintain the increasing % COD cides because of limited bioavailability of pesticides for growth
removal which was supported by observation of some researchers and maintenance of microbial species. The reason behind decrease

Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
6 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx

Fig. 4. Monitoring the parameters such as pH, DO, Redox and BOD5/COD with the operation of aerobic treatment plant (Reactor-1).

in COD concentration was the demand for organic pollutant for the
growth and metabolism of the bacterial species.
The % COD removal was observed to be irregular on 336th,
420th, 492th and 612th hr of operation. To overcome this irregu-
larities of COD removal, pH and air flow rate were adjusted in
Reactor-1 to maintained the biomass concentration for the better
performance of the aerobic treatment plant. In the Reactor-1, the
% COD removal of 87.5% (COD of 154 mg/L) was achieved over
the 30th day of the operation as shown in Fig. 3 and Table 2 with
a constant retention time (RT) of 72 h (3 days). According to Cesar
and Ros (2013) RT has significant influence on degradation pro-
cesses but in the present study RT was kept constant. Li et al.
(2014) who reported effectively reduction of sludge and removal
of organic pollutant with fixed RT (1.3 days) was performed in pilot
scale continuous aerobic-anaerobic coupled (CAAC) process.
The variation of process parameters namely pH, DO, Redox and
BOD5/COD were monitored along with % COD and observed within
ranges pH (6.16–8.11), DO (2.14–5.52 mg/L) and Redox (74 to
232.5 mV).
pH is an important parameter in the biodegradation of organics
waste as reported by Russell and Dombrowski (1980) who
observed the decrease in biomass yield with pH lower than 7.0.
Cycon et al. (2009) reported the maximum bacterial growth at
pH 7.0 ± 1 for biodegradation of diazion in soil by Pseudomonas
species. In this study initially, the wastewater with pH 7.0 was
fed into Reactor-1 and pH was regularly monitored at an interval
of 12 h. The pH was fluctuated with the operation in the Reactor-
1 within the approximate range of 7.0 ± 1 as shown in Fig. 4. The
similar variation in the pH were found in the various literature
(Cycon et al., 2009; Yadav et al., 2014). No significant correlation
was between pH and % COD removal.
The variation of DO with time was monitored during the oper-
ation and shown in Fig. 4. The initial DO in the Reactor-1 was
5.52 mg/L, and then it show decreasing trend with some fluctua-
tion, and at the end of the operation (30th day) the DO was found
2.14 mg/L. The decrease in DO with time was supported by the
increase in% COD removal (Fig 4) because more oxygen will be con-
sumed at higher rate biodegradation corresponding to higher %
COD removal. The COD value of the treated wastewater in
Reactor-1 was decreased to 154 mg/L from the initial value
1232 mg/L at the end of the operation (720 h). The result was sim-
ilar result reported by Lim and Vadivelu (2014). Maintaining high
DO in the reactor by intermittent aeration process may result in
better removal pollutants (Hanhan et al., 2011; Lim and
Vadivelu, 2014).
Fig. 4 shows the variation of redox potential on the degradation
of pesticides. The positive value of redox potential indicates the
microbial breakdown of pesticides (Pett-Ridge and Firestone,
2005). In the initial phase of operation, the redox potential fluctu-
ated between negative and positive values and also support low %
COD removal. After 300 h of operation, the redox potential was
always found positive and fluctuated (6.3–232.5 mV) as shown in

Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx 7

Fig. 5. Kinetic study for (a) calculation of first order biological rate constant (Kobs) and (b) cell yield (YX/C) and decay coefficients (Kdp).

Fig. 4 and during this period % COD removal was also increased
continuously and attained 87.5% at the end of the operation.
Fulazzaky et al. (2015) who found that the redox value varies in
between 44 and 135 mV for the treatment of municipal wastewa-
ter in the alternating aerobic-anoxic system. Rabotnova and
Schwartz (1962) reported that aerobic microorganisms such as
Actinomyces sp. or Azotobacter sp. required higher redox potential
of positive range. According to Pett-Ridge and Firestone (2005) fre-
quent fluctuation of redox potential may be a strong selective force
on the phylogenetic bacterial communities and may promote
metabolic redox tolerance mechanisms. Bellucci et al. (2011) have
studied laboratory scale AAA reactor operated under oxygen-
limiting conditions at sludge retention time 15 day, resulted
average DO 0.63 mg/L, even if the complete nitrification can occur
at low DO level with intermittent aeration.
Annabi et al. (2016) have been recognized the BOD5/COD ratio
as the indicator of biodegradability of the solution. As degradation
proceeds in the bioreactors the BOD5/COD ratio should increased
which is an indication of successful biodegradation taking place
in the reactor (Kumar et al., 2010).
The lower value of BOD5/COD indicates that higher organic
loading. Initially, BOD5/COD ratio in the Reactor 1 was found to
be 0.0212 and it fluctuated as degradation proceeds (Fig. 4) with
increasing trend and attained maximum value of 0.1376 at the
end of operation. The BOD5/COD ratio in this study was improved
from 0.0212 to 0.1376 which is indication of efficient bacterial

Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
8 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx

Table 2 found for Parathion, Malathion and Atrazine at 1.86, 2.40 and
Characteristics and performance of Integrated Aerobic Treatment Plant. 2.65 min residence time (Supplementary Fig. S1). The similar anal-
Performance Parameters Aerobic treatment plant ysis was done for treated effluent samples. The Parathion
Reactor-1 Reactor-2 (1.85 min), Malathion (2.33 min) and Atrazine (2.59 min) and
along with other peaks which may be metabolites produced during
Reactor Volume (L) 30 30
Feed flow rate (L/h) 1.5 –
biodegradation were found at different residence time (Supple-
Recycle pumping rate (L/h) – 6 mentary Fig. S2) which was indicated the formation of
Speed of agitator (RPM) 160 1 intermediates.
Retention time (h) 72 – Effluent samples from Reactor-2 were analyzed using GC–MS to
Total pollutant treated (L) 200 200
identify the presence metabolites and confirmation of biodegrada-
COD (mg/L) 154–1232 101–1011
BOD (mg/L) 10.6–28.2 – tion of wastewater. The GC–MS analysis showed the parental com-
DO (mg/L) 2.14–5.52 – pound as well as intermediates (Supplementary Fig. S3; Table S1).
BOD/COD ratio 0.010485–0.137662 – The peaks Atrazine, Malathion and Parathion were found at resi-
pH 6.16–8.11 –
dence time of 28.87 min 32.99 min and 33.61 min respectively.
Redox potential (74) to 232.5 –
Temperature (°C) Room temperature Room temperature
Total three metabolites one of each of parental compounds Atra-
Operation time (day) 30 30 zine, Malathion and Parathion were identified as Biuret, Succinic
Removal Efficiency (RE)% 87.5 91.80 acid monomethylester and Dithiodiphosphoric acid tetraethyl
% Transmission – 46–73 ester at residence times of 11.17 min, 13.55 min and 26.82 min
respectively. The results (Table S1) clearly indicate that the
metabolites of the insecticides are less toxic which is desirable in
species able to degrade the mixture of pesticides. Affam et al. the bioremediation experiments.
(2014) reported that the BOD5/COD ratio while tratment of The similar results of GC–MS for Atrazine, Malathion and Para-
wastewater containing pesticides in SBR was increased from 0.02 thion biodegradation were reported in the literatures (Zhao and
to 0.31. According to Kumar et al. (2010) the BOD5/COD ratio Hwang, 2009; Fang et al., 2015; Geed et al., 2017). Biodegradation
was found in the range of 0.65–0.67 during treatment of pulp mill of pesticides is the series of enzyme catalyzed reactions during
industrial wastewaters. which both stable as well as unstable intermediates are formed
From the ANOVA analysis it was seen that pH, DO, Redox and depending upon the type of enzyme released by microorganism
BOD5/COD ratio had a significant effect (P < 0.05) on the treatment to metabolize the pesticides (Zhao and Hwang, 2009). Fang et al.
of pesticides. The average value and SD were found to be at pH 7.44 (2015) have proposed Atrazine degradation pathway with com-
and 0.46, DO 3.44 and 1.03, Redox 102.73 and 74.78 and BOD5/COD plete information about intermidiates formed during the degrada-
0.045 and 0.030 respectively. These results are similar with the tion. In the degradation pathway the enzyme atrazine chloro
reported values (p < 0.05) from various literature for the Chloropy- hydrolase played important role for the formation Hydroxya-
rifos and Malathion (Yadav et al., 2014; Geed et al., 2017). trazine. Further it was biodegraded to Cyanuric acid and then
The treated effluent from Reactor-1 (after 36 h of treatment) Biuret by several enzymatic activities of bacterial species. The
was transferred to the second stage (Reactor-2) for the treatment identified metabolites Biuret was less harmful than Atrazine
of untreated parental pesticides and intermediates and the COD (Fang et al., 2015; Douglass et al., 2014).
value of effluent were regularly monitored. The part of treated Malathion biodegradation details were given in the Geed et al.
wastewater from second reactor was recycle (6 L/h) to first reactor (2017). The identified metabolites Succinic acid monomethyl ester
continuously. The slow agitation and availability of substrate lead was formed during metabolism of Malathion. The enzyme succinic
to increase the biomass into Reactor-2 which was resulted in bet- acid monomethyl esterase is responsible for the breakdown of
ter biodegradation (Fig. 3 and Table 2) and thus overall perfor- Malathion the into Succinic acid monomethyl ester and further it
mance of aerobic treatment plant was improved. The maximum was degrade to Succincte with series of enzymatic activities
% COD removal was achieved to 91.80% (Fig. 3) in Reactor-2 at (WHO, 2015; Geed et al., 2017). Similar bacterial degradation
the end of operation (720 h) with RT 6 h. Fulazzaky et al. (2015) was occur for Parathion and intermediate was identified as
who reported that the performance AAA system increased approx- Dithiodiphosphoric acid tetraethyl ester which was further decom-
imately 53.4% by increasing the anoxic time (RT; Reactor-2) from 3 posed to ethythiophosphate and then to phosphate by several
to 6 h. In Reactor-2, the % COD removal was improved to 91.80 enzymatic metabolic reaction (Zhao and Hwang, 2009).
from 87.5 (Reactor-1) this may because of the gradual growth of Although few researchers have been noted that hydrolysis of
flocculent sludge. Almost constant and stable % COD removal was the ester bond is a common degradation step for organophospho-
observed from 640 h to 720 h which show the saturation of bacte- rus compounds, it is unlikely to be an important mechanism since
rial degradation capability in the Reactor 2. Similar trend in the the P–S bonds were intact (Zhao and Hwang, 2009). Pesticide
reactor were also observed by Liu et al. (2015) for treatment of degradation can occur by oxidase or hydrolase enzyme or mixed
slaughterhouse wastewater. Lim and Vadivelu (2014) reported that function of oxidase as well as a hydrolase (Geed et al., 2017;
90% COD removal of palm oil mill effluent removed successfully in Yadav et al., 2014).
SBR. The % COD removal in this work was higher than previous
works (Kumar et al., 2010; Othman et al., 2013; Zhang et al., 3.4. Biodegradation kinetics and evaluation of death phase
2011) in which performance aerobic reactors were evaluated for
the treatment of livestock wastewater and digested piggery For the design and upscaling of wastewater treatment plant, the
wastewater. substrates utilization kinetics is useful (Affam et al., 2014). In this
study Monod growth kinetic was found to fit with experimental
data well and kinetic parameters were estimated by Eq. (5). The
3.3. Intermediates/metabolites confirmation: GC–MS biological first-order kinetic rate constant (Kobs) was evaluated
as shown in Fig. 5a and found to be 0.00425 per hr (0.102 per
HPLC analysis was performed to find out the treated concentra- day). Affam et al. (2014) reported the value of Kobs as 0.01332
tion of mixtures of pesticides. The control sample of mixtures of per hr for the treatment of wastewater containing pesticides in
pesticides without treatment was run separately, and peaks were sequencing batch reactor. The kinetic parameter provides detail

Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
 S.R. Geed et al. / Bioresource Technology xxx (2017) xxx–xxx
about cell history, the organism’s intrinsic characteristics and the
existing environmental conditions (Beltran-Heredia et al., 2000).
Cell yield coefficient (Yx/c) and biomass decay coefficient (Kdp)
are very important kinetic parameters related to the evaluation
of biomass concentration over the entire period of the aerobic
treatment plant (Beltran-Heredia et al., 2000; Martin et al.,
2008). The Yx/c represents gram of biomass produced per gram of
substrate utilized and Kdp indicates the importance of endogenous
metabolism into cell culture (Beltran-Heredia et al., 2000). As the
plant operated the biomass concentration may be decreased due
to toxic effect of pollutant or metabolites formed during operation.
These parameters help to determine presence of biomass concen-
tration in the reactor during operation. For the growth cycle of bac-
terial species, the death phase has been taken into consideration,
when the reduction in the bacterial cell number was take place.
As Bailey and Ollis (1986) pointed out, the relative studies have
been performed on this phase, because many industrial microbio-
logical processes are terminated before the death phase begins. The
value of Yx/c and Kdp were obtained from a plot of m vs. q shown in
Fig. 5b. The values of Yx/c and Kdp were 0.696 mg COD/mg MLSS
(MLSS-Mixed liquor suspended solids) and 0.0010 per hr, respec-
tively. Similar results were obtained Affam et al. (2014) and
Martin et al. (2008) reported that Yx/c 0.5301 and 0.5 and Kdp
0.0072 and 0.004 per hr respectively for the biodegradation of pes-
ticides in wastewater. Beltran-Heredia et al. (2000) reported Yx/c
0.227 and Kdp 0.0069 per hr for the biodegradation of black olive
wastewater which was confirmed the biomass concentration. Both
exponential growth and death phase have been considered for the
kinetic study for evaluating the first order rate constant Kobs, Yx/c
and Kdp which is helpful for the metabolism of bacterial species
used in wastewater tratment process.
4. Conclusion
This study successfully demonstrated the treatment of syn-
thetic wastewater containing mixture of Atrazine, Malathion and
Parathion in two-stage IATP. The highly efficient Bacillus species
were successfully isolated from contaminated site and more than
90% removal was obtained. The GC–MS analysis results confirm
the presence of Biuret, Succinic acid monomethyl ester and
Dithiodiphosphoric acid tetraethyl ester which are common
metabolites reported during degradation of Atrazine, Malathion
and Parathion. Kinetic parameters such as first order biological rate
kinetic constant Kobs (0.00425 per hr), cell yield YX/C (0.696 mg of
COD/mg MLSS) and decay coefficients Kdp (0.0010 per hr)were
evaluated in order to determine the biomass concentration over
the period of operation.
Acknowledgements
The author (SRG) gratefully acknowledges Ministry of Human
Resource Development, India (MHRD, India) for providing financial
support and the Indian Institute of Technology (BHU) for providing
a lab for conducting research work. Author (SRG) also thankful to
AIRF LAB JNU, Delhi for providing GC–MS analysis facility.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.03.
080.
Please cite this article in press as: Geed, S.R., et al. Assessment of pesticides removal using two-stage Integrated Aerobic Treatment Plant (IATP) by Bacillus
sp. isolated from agricultural field. Bioresour. Technol. (2017), http://dx.doi.org/10.1016/j.biortech.2017.03.080
9
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