Water Research 38 (2004) 2277–2284

ARTICLE IN PRESS

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doi:10.1016/j.w

Atrazine degradation in anaerobic environment by a mixedmicrobial consortium

Pranab Kumar Ghosha, Ligy Philipb,*aDepartment of Civil Engineering, Vellore Institute of Technology, Vellore, Pin-632 014, India

bDepartment of Civil Engineering, Indian Institute of Technology Madras, Chennai Pin-600 036, India

Received 1 May 2003; accepted 27 October 2003

Abstract

Atrazine degradation by anaerobic mixed culture microorganism in co-metabolic process and in absence of external

carbon and nitrogen source was studied at influent atrazine concentration range of 0.5–15mg/l. Wastewater of desired

characteristic was prepared by the addition of various constituents in distilled water spiked with atrazine. In co-

metabolic process, dextrose of various concentrations (150–2000mg/l) was supplied as external carbon source. The

reactors were operated in sequential batch mode in which 20% of treated effluent was replaced by the same amount of

fresh wastewater everyday, thus maintaining a hydraulic retention time (HRT) equal to 5 days. In co-metabolic process,

40–50% of influent atrazine degradation was observed. First-order atrazine degradation rate (expressed in day�1) was

better in co-metabolic process (5.5� 10�4) than in absence of external carbon source (2.5� 10�5) or carbon and

nitrogen source (1.67� 10�5). In presence of 2000mg/l of dextrose, atrazine degradation was between 8% and 15%

only. Maximum atrazine degradation was observed from wastewater containing 300mg/l of dextrose and 5mg/l of

atrazine. Influent atrazine concentration did not have much effect on the methanogenic bacteria which was clear from

methane gas production and specific methanogenic activity (SMA).

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Atrazine; Anaerobic; Biodegradation; Co-metabolism; Reaction rate; Sequential

1. Introduction

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-

triazine), probably the most commonly used herbicide in

the world [1], is quite persistent in neutral environment

and is toxic to various living organisms [2]. Many studies

were carried out so far on the biodegradation of atrazine

in wastewater and bioremediation of atrazine contami-

nated soil [3–6]. Most of these studies employed pure

culture microorganisms except a few by mixed bacterial

culture. Pure culture bacteria used atrazine as sole

source of carbon and/or nitrogen, but degradation was

partial and mineralization happened only in a few cases

[7,8]. Atrazine biodegradation rate and the degree of

ing author.

ess: ligy@civil.iitm.ernet.in (L. Philip).

e front matter r 2004 Elsevier Ltd. All rights reserve

atres.2003.10.059

degradation depended on the types of pure culture

bacteria, presence and absence of various external

carbon and nitrogen sources and their respective

concentrations, carbon/nitrogen ratio, pH and moisture

content [4–6]. The effects of above said factors on the

rate and degree of atrazine biodegradation are contra-

dictory. Furthermore, handling of pure culture bacteria

in actual field condition is a cumbersome job and

atrazine mineralization in situ condition was almost

non-existent [9]. Mandelbaum et al. [10] observed

mineralization of atrazine, only after mixing a number

of pure culture strains among two hundred isolated

strains.

Earlier studies showed that atrazine was better

degraded in anaerobic conditions than in aerobic one

[11]. Many chlorinated aliphatic and aromatic com-

pounds like Benzene Hexachloride (BHC) and DDT

d.

ARTICLE IN PRESSP.K. Ghosh, L. Philip / Water Research 38 (2004) 2277–22842278

[1,1-dichloro-2,2-bis(p-chlorophenyl ethylene], which

were earlier considered as recalcitrant in aerobic process,

were successfully degraded in anaerobic condition

[12,13]. Literature showed that the aromatic structure

of lignin, benzoate and other aromatic acids were

metabolized to gaseous end products in mixed cultures,

under methanogenic conditions [14]. However, degrada-

tion rate of such compounds by anaerobic mixed

microbial culture was slow, as the energy required for

bacterial growth was not adequate from such toxic

compounds. In such cases, addition of easily biodegrad-

able organic compounds stimulated the growth of

anaerobic bacteria, and thereby enhanced the degrada-

tion rate by utilizing recalcitrant compound as second-

ary substrate in co-metabolic process. Christiansen et al.

[15] had investigated the influence of a supplemental

carbon source on the anaerobic dechlorination of

pentachlorophenol (PCP). Enhanced degradation rate

of atrazine was observed in both aerobic [5] and

anaerobic conditions [4], when external carbon sources

were supplied to pure and mixed bacterial culture.

Struther et al. [5] used citrate and sucrose as external

carbon source whereas Chung et al. [4] used four

different external carbon sources viz., methanol, sodium

acetate, acetic acid and glucose.

The present study explored the potential of anaerobic

microbial consortia to degrade atrazine under various

operating conditions. The effect of atrazine on

the performance of the anaerobic biosystem was

also evaluated in terms of methane production,

specific methanogenic activity (SMA) and COD removal

efficiency.

2. Materials and methods

2.1. Chemicals

All the chemicals used in the present investigation

were of analytical reagent grade (purity>98%). Rallis

India Limited, Mumbai, India, supplied atrazine of

technical grade (purity=98%). Five primary metabo-

lites of atrazine namely hydroxyl atrazine [2-hydroxy-4-

ethylamino-6-isopropylamino-s-triazine] (HA) (pur-

ity=98%), deethyl atrazine [2-chloro-4-amino-6-isopro-

pylamino-s-triazine] (DEA) (purity=99%), deisopropyl

atrazine [2-chloro-4-ethylamino-6-amino-s-triazine]

(DIA) (purity=99%), deethyl deisopropyl atrazine [2-

chloro-4,6-diamino-s-triazine] (DEIA) (purity=99%)

and deethyl deisopropyl hydroxyl atrazine [2-hydroxy-

4,6-diamino-s-triazine] (DEIHA) (purity=99.5%)

were procured from SUPELCO (Sigma Aldrich

Foreign Holding Company, USA). The synthetic waste-

water contained, K2HPO4=150; KH2PO4=50;

MgCl2 � 6H2O=300; CaCl2 � 2H2O=50; NH4Cl=200;

FeCl2 � 4H2O=5; ZnCl2=0.5; NiCl2 � 6H2O=0.5;

CoCl2 � 6H2O=0.5; MnCl2 � 4H2O=0.5 and yeast ex-

tract=50mg/l of distilled water. Dextrose of 150, 300,

500, 1000 and 2000mg/l and atrazine of 1, 5, 10 and

15mg/l were added to water along with the above

micronutrients to prepare specific characteristic waste-

water according to the requirement.

Sludge from an anaerobic digester at Bara Sewage

Treatment Plant, Jamshedpur, India, was used as

inoculum for the present study. The sludge was fed with

wastewater, for a month, to make the system acclima-

tized with the changed environment and was used for

further experiments.

2.2. Analyses

Unless otherwise specified, analyses of various para-

meters were done as per the procedure suggested in

standard methods for the examination of water and

wastewater [16]. Volatile fatty acid (VFA) and alkalinity

were determined as suggested by DiLallo and Albertson

[17]. Activity of sludge was determined as per the

procedure suggested by Valke and Vestrate [18] and

expressed as specific acetoclastic methanogenic activity

(SMA).

Biogas produced in anaerobic process was collected

by liquid displacement method and methane content was

determined by passing the total gas collected through

32% (w/w) KOH solution. Volume change due to

temperature variations was incorporated in the mea-

sured volumes. Periodically the biogas was analyzed in

ORSAT apparatus.

2.3. Atrazine extraction and determination

Atrazine was measured in both UV-Visible spectro-

photometer (Model: UV-160A, Shimadzu, Japan) and

gas chromatograph (GC) (Model: GC 14A, Shimadzu,

Japan). In both the methods, atrazine was extracted

from wastewater by liquid–liquid extraction method.

Dichloromethane or ethyl acetate was used as extractant

in case of UV-Visible spectrophotometer and GC,

respectively.

Ten ml of sample was taken in a 50ml conical

separating funnel after centrifuging for 10min. Five ml

of dichloromethane was added to the solution and

shaken vigorously for 3min. Excess pressure due to

volatilization of dichloromethane was released by open-

ing the bottom outlet keeping upward. The solution was

again shaken for 2min and finally for 1min with

releasing pressure after each shake. The whole content

was allowed to stand quiet to separate the water-

dichloromethane layer. Dichloromethane being heavier

(sp. gr. 1.32) remained in the bottom of the separating

layer. It was allowed to pass through a filter paper

topped with a bed of 2 gm of anhydrous sodium sulfate

kept on glass wool. The filtrate was collected in a 25ml

ARTICLE IN PRESSP.K. Ghosh, L. Philip / Water Research 38 (2004) 2277–2284 2279

volumetric flask. Above procedure was repeated twice,

using 5ml of dichloromethane each time. The collected

volume was adjusted to 10ml using dry nitrogen gas

before measuring the absorbance in UV-Visible spectro-

photometer. The spectra were taken at a range of 220–

250 nm keeping dichloromethane as reference. Max-

imum absorbance was observed at 228.8 nm. Extraction

efficiency of atrazine from water by this method was

9570.52%. Detection limit of atrazine was 0.1mg/l and

up to a concentration of 7.5mg/l, absorbance was linear.

Same procedure was followed for the extraction of

atrazine by ethyl acetate for GC analysis. Atrazine was

extracted from 25ml of wastewater thrice using 5ml of

ethyl acetate each time. Ethyl acetate being lighter than

water (specific gravity=0.9) remained in upper part of

separating funnel. Lower part (i.e. wastewater) of the

content of separating funnel was collected in a 25ml

volumetric flask without filtration, whereas the upper

part (ethyl acetate containing atrazine) was allowed to

pass through a filter paper topped with a bed of 2 gm of

anhydrous sodium sulfate kept on glass wool. Waste-

water portion was again transferred to the same

separating funnel and the procedure was repeated twice

using 5ml of ethyl acetate each time. Final volume was

adjusted to 10ml using dry nitrogen gas before analysis

in GC. Extraction efficiency of atrazine by this process

was 9370.55%. Electron capture detector (ECD,

Radioisotope Nuclide 63Ni) and 1.5% OV-17 column

of 3m length and 18

00internal diameter using pure

nitrogen as carrier gas was used for atrazine determina-

tion in GC. Initial and final column temperature was

210�C, injector temperature was 225�C, and carrier gas

flow was maintained at the rate of 70ml/min. 15ml ofextracted sample was injected into injection port for

atrazine determination. Detection limit of atrazine was

0.5mg.Before each run, instruments were standardized with

anticipated atrazine concentration range. For the

standardization of instrument, atrazine was dissolved

in respective solvents viz., dichloromethane or ethyl

acetate.

To check the build-up of atrazine in anaerobic

biomass, 20–50ml of biomass (as mixed liquor sus-

pended solid, MLSS) sample was centrifuged twice at

4000 rpm for 5min after resuspension in physiological

saline water. Atrazine was extracted three times with

15ml of methanol by agitating for 2 h in a rotary shaker.

Methanol was evaporated using dry nitrogen gas and

residue was redissolved either in dichloromethane of

10ml or in ethyl acetate of 5ml, for the determination of

atrazine in UV-Visible spectrometer or GC, respectively.

2.4. Determination of atrazine metabolites in HPLC

Atrazine metabolites were analyzed in High Perfor-

mance Liquid Chromatograph (HPLC) using Dionex

AD20, UV/VIS detector at a wavelength of 222 nm,

using Dionex Summit P580, HPLC pump. Analysis was

carried out as per the method reported by Chung et al.

[4]. A 20ml sample was collected and centrifuged. Five

ml supernatant was passed through Sep-pak C-18

cartridge (Waterst Sep-Paks, Milford, Massachusetts,

USA). The cartridge was washed by passing 5ml of

deionized water through it. The analytes were eluted

with 2ml methanol. Extraction efficiency by this method

was 98.571%. The analytes were filtered through a

0.20 mm nylon syringe filter. Concentration of

atrazine and its dealkylated metabolites were determined

with a 5mm reversed phase C-18 column (Keystone

Scientific Inc.). The injection volume was 50ml,the column temperature was 40�C, the mobile phase

was programmed to a 5-min gradient, a 20–40%

acetonitrile gradient with water, the flow rate was

1ml/min.

Column used for DEA, DIA and DEIA was C18,

Keystone, 0.5u, 4.6� 150mm2, (col#32). Hydroxy ana-

logue of atrazine was extracted by the above said solid

phase extraction procedure using sep-pak C 18 car-

tridge. Heated extraction method adopted by Chung

et al. [4] was not followed as atrazine hydrolyzed

rapidly at a temperature more than 70�C and at high

and low pH [2]. The mobile phase used a 10-min

gradient, a 100–70% acetonitrile gradient with

water, at a flow rate of 1.5ml/min. Hydroxy analogues

of atrazine (HA and DEIHA) were determined using a

10 mm amino phase column (Bondapak, Waters

Association). Detection limit of metabolites were

10 mg/l. Normalizing peak area method was adopted

for the quantification of various metabolites of atrazine

in HPLC. Atrazine was chosen as the reference

compound.

2.5. Reactors employed for the study

The whole experiment was carried out in anaerobic

suspended growth process at controlled temperature of

3571�C. Four reactors namely, R-0, R-1, R-2 and R-3

were operated to assess the atrazine degradation

potential of anaerobic mixed microbial culture and its

effect on the system at different feeding and operating

conditions. Reactor R-0 and reactor R-1 were operated

in sequential batch mode with a hydraulic retention time

(HRT) of 5 days (described below). Reactor R-0 was

operated as a control reactor fed with the same

strength wastewater as that of reactor R-1 but without

atrazine. Reactor R-2 and R-3 were operated in batch

mode.

Reactor R-1 had a working volume of 4 l and a HRT

of 5 days. Microbial mass in the reactor was maintained

at 4000mg/l expressed as MLSS. The first cycle of

operation of reactor R-1 was carried out with waste-

water containing 150mg/l of dextrose and 0.5mg/l of

ARTICLE IN PRESSP.K. Ghosh, L. Philip / Water Research 38 (2004) 2277–22842280

atrazine along with other nutrients. Next day onwards,

20% of treated effluent was discarded from the reactors,

at an interval of 24 h. and replaced by equal volume of

fresh wastewater. Operation was continued for 5 days to

complete a cycle. Second cycle was started after

discarding the whole supernatant and replacing by equal

amount of same strength wastewater. The reactor was

operated for such 5 consecutive cycles with a particular

strength wastewater. Average value of five cycles of

operation and the standard deviation are given in

Table 1. Next experiment was conducted in a similar

way with the wastewater containing 150mg/l of dextrose

and 1mg/l of atrazine. Reactor R-1 was fed with various

strength wastewaters (containing dextrose of 150–

2000mg/l) spiked with different atrazine concentrations

(0.5–15mg/l).

Reactor R-2 was operated without external carbon

source but with nitrogen supplement as nitrate. Reactor

R-3 was operated without external carbon and nitrogen

source. Both the reactors R-2 and R-3 were operated in

batch mode keeping initial microbial concentration

around 4000mg/l. A control reactor was operated in

batch mode without any biomass to quantify the abiotic

losses of atrazine.

Table 1

Performancea of reactor R-0 and R-1 on COD removal

Initial atrazine in R-1 (mg/l) Influent dextrose concentration

150 (mg/l) 300 (mg/l)

0.5 86.573.1 88.673.5

1 86.173.2 88.373.3

2 85.673.5 88.172.4

5 86.273.1 87.972.6

10 85.673.2 87.172.55

15 8473.4 85.272.4

No atrazine (Reactor R-0) 88.272.2 90.173.8

aPerformance is being expressed as percentage COD reduction. D

operation. Standard deviation is also given along with the value.bENP=Experiment was not performed.

Table 2

Atrazine reduction in co-metabolic process from R-1

Initial atrazine in R-1 (mg/l) Influent dextrose concentration

150 (mg/l) 300 (mg/l

0.5 43.270.18a 4470.17

1 44.270.22 44.870.20

2 44.770.16 46.070.19

5 45.370.23 48.070.18

10 44.270.18 46.870.17

15 43.570.17 44.670.12

aAverage percentage atrazine removal (of initial atrazine concentrabENP=Experiment not performed.

3. Results and discussion

3.1. Atrazine degradation in co-metabolic process

Atrazine degradation potential of the mixed anaerobic

consortium was evaluated under various atrazine and

organic carbon concentrations and the results are given

in Tables 1 and 2. The biosystem could degrade around

43–45% of atrazine within 5 days from the wastewater

containing 150mg/l of dextrose, irrespective of the initial

atrazine concentrations. Though dextrose concentration

in the wastewater was varied from 300 to 1000mg/l, to

represent various strength wastewaters, there was no

significant change in atrazine removal efficiency. How-

ever, when the COD of wastewater was above 2000mg/l,

only 8–15% of atrazine removal was observed in the

system. There was no accumulation of atrazine in the

sludge and the loss of atrazine in the control reactor was

negligible. This shows that atrazine removal from the

system was due to biodegradation.

High degradation rate of atrazine at comparatively

high atrazine concentration may be due to the effect of

concentration gradient. At high concentration gradient,

the pollutant has a higher chance to be exposed to the

in R-0 and R-1

500 (mg/l) 1000 (mg/l) 2000 (mg/l)

ENPb 85.174.2 ENPb

85.374.9 84.574.4 82.173.5

ENPb 84.473.8 ENPb

85.573.7 8473.2 82.972.55

85.573.5 84.673.1 81.973.56

83.573.2 82.473.2 82.372

87.073.9 8675.5 83.473.7

ata given in the table is average value obtained in 5 cycles of

in R-1

) 500 (mg/l) 1000 (mg/l) 2000 (mg/l)

ENPb 43.870.24 ENP

43.970.15 40.070.21 8.670.58

ENP 41.870.19 ENP

44.070.18 43.370.15 11.5470.54

44.270.23 43.870.21 12.7470.33

43.970.29 44.270.25 14.4770.38

tion) with standard deviation of 5 sets of experimental data.

ARTICLE IN PRESS

Fig. 1. Atrazine degradation rate profile for various initial

atrazine and dextrose concentrations.

P.K. Ghosh, L. Philip / Water Research 38 (2004) 2277–2284 2281

cell and/or penetrates through the cell, which is essential

for biodegradation/biotransformation. As the concen-

tration of dextrose increased, atrazine degradation rate

also improved to a certain extent, and then gradually

reduced. In co-metabolic processes, by utilizing primary

carbon source, microbes produce enzymes which are

responsible for the degradation of secondary substrates

(toxic compounds). At low primary substrate concen-

tration, bacterial growth usually gets affected by

substrate limitation. At very high dextrose concentra-

tion, the competition among dextrose and atrazine

might have prevented atrazine degradation. This might

also be due to the inhibition of the secretion of atrazine

degrading inducible enzyme, at high carbon source, as

the microbial consortia will always prefer easily degrad-

able non-toxic substrates [19,20].

3.2. Rate of atrazine degradation

Most of the biological reactors have been designed as

per first-order reaction kinetics. In the present investiga-

tion, almost all cases, atrazine reduction profile fitted

well with first-order reaction kinetics.

Fig. 1 shows the variation of atrazine degradation rate

constant ‘K1’ [day�1] in reactor R-1, with respect to

initial atrazine concentration and strength of waste-

water. The biomass concentration was kept as a

constant i.e., 2000mg/l. For low strength wastewater

(with initial dextrose concentration of 150 and 300mg/l)

value of K1 was increasing with the increase of initial

atrazine concentration from 0.5 to 5.0mg/l and then

decreased slowly up to 15mg/l. Degradation rate was

slightly more in wastewater containing 300mg/l of

dextrose irrespective of the atrazine concentrations.

At a dextrose concentration of 500mg/l, value

of K1 remained almost same for all atrazine

concentration tried though the value was slightly less

than the values obtained for a dextrose concentration of

300mg/l.

Increase in the value of K1 (first-order rate constant,

[day�1]) with the increase in initial atrazine concentra-

tion from 0.5 to 5.0mg/l in low strength wastewater

might be due to the effects of concentration gradient of

atrazine. Slight decrease at 10 to 15mg/l of atrazine

addition might be due to the inhibition on microorgan-

ism. At 1000 and 2000mg/l of dextrose addition,

inhibition on atrazine degradation was not observed

but the degradation rate increased slowly due to the

increase in pollutant concentration gradient. Low value

of K1 at 2000mg/l of dextrose addition might be due to

the masking effects of atrazine by high concentration of

dextrose. Almost same K1 value irrespective of the

atrazine concentration in case of 500mg/l of dextrose

concentration might be due to the balance of the effects

of high gradient of atrazine concentration and masking

of atrazine toxicity on atrazine degrading microorgan-

isms. But, a combination of 5mg/l of atrazine and

300mg/l of dextrose has given maximum atrazine

degradation rate. This may be due to the proper

balancing of primary carbon source and secondary

carbon source for the co-metabolic system.

3.3. Gas production

Performance of anaerobic reactors R-0 and R-1 were

monitored in terms of total and methane gas production

and the results are presented in Table 3. Though

oxidation reduction potential (ORP) measurements were

not carried out, methane gas production was treated as

an indicator of anaerobic environment, as methanogens

are strict anaerobes.

The result showed that up to an atrazine concentra-

tion of 10mg/l there was no significant reduction in total

and/or methane gas production. The average methane

content in the biogas was 79% almost in all the cases.

At an initial atrazine concentration of 15mg/l, small

reduction was observed in gas production, which

may be due to the inhibitory effect of atrazine

on the mixed microbial culture used in the present

investigation.

As the organic loading rate increased (due to increase

in influent COD), the gas production also increased in

all the cases irrespective of atrazine concentrations.

However, for the same initial organic loading rate, the

gas production, both total and methane, decreased with

increase in atrazine concentration (Table 3). The

decrease was more significant in case of 15mg/l

of atrazine concentration. As the organic loading

ARTICLE IN PRESS

Table 4

Activity of sludgea of reactor R-0 (without atrazine) and R-1 at

various atrazine concentration

Dextrose (mg/l) Atrazine concentration

0.0 (mg/l)b 0.5(mg/l) 5.0(mg/l) 15.0(mg/l)

150 0.3318 0.3267 0.3166 0.2608

300 0.3495 0.3419 0.3267 0.2760

500 0.3596 0.3545 0.3469 0.2912

1000 0.3672 0.3647 0.3596 0.3242

2000 0.3697 0.3697 0.3545 0.3495

aActivity of sludge expressed as Specific Acetoclastic

Methanogenic Activity (SMA) (Unit=g of CH4-COD/

gMLVSS-day).bColumn shows the SMA of the sludge of the reactor R-0

that was operated without atrazine.

Table 3

Total and methane gas production in reactors R-0 and R-1

Initial

atrazine

(mg/l)

Influent dextrose concentration in wastewater of reactor R-1

150mg/l 300mg/l 500 (mg/l) 1000 (mg/l) 2000 (mg/l)

Total

biogasaCH4 gas

a Total

biogas

CH4 gas Total

biogas

CH4 gas Total

biogas

CH4 gas Total

biogas

CH4 gas

0.5 20079b 16174 239710 18875 ENPc ENP 283712 19375 ENP ENP

1 19678 15575 240712 19074 274713 20276 281713 19378 370715 26078

2 197710 15475 24177 18873 ENP ENP 282712 19274 ENP ENP

5 20379 16373 246711 19275 270713 20076 283713 19375 368713 25977

10 194711 15376 237714 18775 267711 19875 280715 19175 367718 25678

15 18378 14974 22879 17773 251710 18676 278710 18476 366715 25579

Reactor

R-0

209712 17075 250710 19575 279710 20677 325715 23878 370713 26377

aAverage total and methane gas production (ml/g COD reduction at 35�C and atmospheric pressure).bResults shown represent the average of 5 sets of results with standard deviation.cENP=Experiment Not Performed.

P.K. Ghosh, L. Philip / Water Research 38 (2004) 2277–22842282

increased, the methane content of the total biogas was

decreased.

The total gas production per gram of COD reduction

was less for low strength wastewater and increased with

high strength wastewater. This might be due to solubility

of CO2 and CH4 in aqueous phase. Carbon dioxide has a

solubility of 665ml/l whereas methane has only a

solubility of 27.6ml/l of water at 1 atmospheric pressure

and at a temperature of 30�C [21]. As the working

volume of the reactor was constant, the amount of CO2

and CH4 in aqueous phase would be same in all the

cases. For high strength wastewater, the total gas

production was higher, whereas the liquid volume

remained same. Hence, the amount of gas being

solubilized also remained same. Because of this, more

CO2 was available in gaseous phase for high strength

wastewater, which might have lowered the percentage

methane content of the gas.

3.4. Sludge activity

SMA of the sludge from reactors R-0 and R-1 is given

in Table 4. The results showed that SMA of the reactor

sludge R-1, treating low strength wastewater (dextrose

content of 150–300mg/l) containing atrazine up to 5mg/

l was almost same as that of SMA of reactor sludge R-0,

while treating same strength wastewater. A little

reduction of SMA was observed when atrazine concen-

tration in the feed was 15mg/l. But the negative effect of

atrazine on SMA reduced as the dextrose concentration

in the wastewater increased. The values obtained were in

the range of 0.26–0.35 g of CH4-COD/gMLVSS-day for

a dextrose concentration of 150–2000mg/l.

For a good anaerobic sludge, the SMA value should

be in the range of 0.08–0.3 g CH4-COD/g VSS-day. The

SMA value of the sludge was within this range in all the

cases, irrespective of the dextrose and atrazine concen-

tration. SMA slightly increased with increase in influent

COD. This may be due to the masking of the toxic

effects of atrazine by the high concentration of organic

matter. There was a significant reduction in methano-

genic activity when the atrazine concentration was

increased to 15mg/l. This shows that a high atrazine

concentration has an adverse effect on the anaerobic

microbial consortium. Even at this atrazine concentra-

tion, for high strength and very high strength waste-

waters, the inhibition on SMA was insignificant. This

also reinforces the concept of masking effect of organic

matter on atrazine toxicity.

ARTICLE IN PRESS

0

2

4

6

8

10

12

14

16

18

0 20 40 60 80 100 120 140 160

Time, (day)

Atr

azin

e re

mai

ning

(m

g/l)

1 mg/l 1 mg/l (control)5 mg/l 5 mg/l (control)10 mg/l 10 mg/l (control)15 mg/l 15 mg/l (control)

Fig. 3. Kinetics of atrazine degradation in reactor R-3.

P.K. Ghosh, L. Philip / Water Research 38 (2004) 2277–2284 2283

3.5. Degradation of atrazine as sole carbon source/carbon

and nitrogen source

Atrazine degradation studies were carried out by

providing atrazine as sole carbon source (Reactor R-2)

and as sole carbon and nitrogen source (Reactor R-3)

and the results are given in Figs 2 and 3. Although there

was significant atrazine reduction in R-2 (50–60% after

35–100 days) and R-3 (30–45% after 150 days) there was

also a reduction in microbial mass (1–2%) in both the

reactors. Atrazine loss from the ‘‘control reactor’’ was

insignificant and no build up of atrazine in sludge

sample was observed.

Atrazine degradation was more in co-metabolic

process compared to other two cases viz., using atrazine

as sole (a) carbon source, and (b) carbon and nitrogen

source. Moreover, except in co-metabolic processes, the

cell mass in the reactor gradually decreased with respect

to time. This showed that the anaerobic consortia used

in the present investigation might not be comfortable in

using atrazine as the sole carbon and energy (reactor

R-2) as well as nitrogen source (reactor R-3). In absence

of primary substrate, microbial cell mass (cell decay)

might have served as primary carbon source [22]. Again,

for the same percentage of atrazine reduction, time taken

by the microorganism was the maximum in reactor R-3

among all the three reactors (R-1, R-2 and R-3).

The degradation rate of atrazine was far better in case

of co-metabolic process (reactor R-1) than in absence of

external carbon source (reactors R-2 and R-3). Stimula-

tion of the growth of atrazine degraders in presence of

external carbon source (dextrose) in reactor R-1 might

have been the reason for better atrazine reduction [9]. In

addition, it was observed that atrazine degradation rate

Fig. 2. Kinetics of atrazine degradation in reactor R-2.

in R-2 was faster than that in R-3. This might be due to

the difficulty of anaerobic mixed culture microorganisms

in breaking the aromatic ring of the atrazine molecule.

Atrazine degradation pathway also showed that nitro-

gen could be available to the microorganism only after

the ring cleavage. Better degradation of atrazine in R-2

might be due to the availability of nitrogen source.

The major metabolites observed in the effluent of all

the reactors (R-1, R-2 and R-3) were DIA and/or DEA

but only in small amounts (0.1–0.6%). No other primary

metabolite (dealkylated and hydroxy analogue) was

observed in any of the samples of reactor R-1, R-2 and

R-3. Reduction in atrazine concentration observed

might be due to mineralization of atrazine.

4. Conclusion

Atrazine degradation by anaerobic mixed culture

microorganisms was better in co-metabolic process than

in absence of external carbon and nitrogen source. There

was no significant inhibition effect on mixed anaerobic

microbial consortia even at an atrazine concentration of

15mg/l. However, high organic content of the waste-

water reduced the atrazine degradation rate. Atrazine

degradation was dependent on the strength of wastewater

and the amount of atrazine in the influent. Atrazine

concentration in the wastewater did not affect the gas

production or methanogenic activity significantly.

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