Chemosphere 54 (2004) 1573–1580

www.elsevier.com/locate/chemosphere
Short Communication
Acclimation of anaerobic sludge degrading chlorophenols
and the biodegradation kinetics during acclimation period
a,* b
Fen-xia Ye , Dong-sheng Shen
a
Department of Chemical Engineering, Ningbo College, Cuibai Road, Ningbo 315016, PR China
b
Department of Environmental Engineering, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, PR China
Received 8 October 2001; received in revised form 6 August 2003; accepted 21 August 2003

Abstract

The acclimation of sludge from Hangzhou citrate factory and Hangzhou municipal wastewater treatment plant for
degradation dechlorination of chlorophenols (CPs) compounds, and its biodegradation kinetics were studied in batch
process with or without addition of sucrose. Three monochlorophenols (2-CP; 3-CP; 4-CP) and pentachlorophenol
(PCP) were concurrently fed to different bioreactors. The parameters that were monitored included biogas production,
biogas composition and chemical oxygen demand (COD). The results showed that acclimation with chlorophenol can
increase the degradation activity of anaerobic sludge and degradation rate of chlorophenolic compounds, and reduce
the lag time. Degradation dechlorination activity of the acclimated sludge strongly depended on sludge source, mi-
croorganism population and chlorophenol congener. 2-CP was more easily acclimated than 3-CP and 4-CP. Among the
four tested compounds, 4-CP was the most difficult to be acclimated. The observed degradation rate with presence of
sucrose was higher than that with absence of sucrose, suggesting that addition of the external carbon source can
stimulate the formation of acclimated sludge which could effectively degrade chlorophenols. Kinetic equations of
biodegradation of chlorophenols were also presented in this paper.
Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic sludge; Acclimation; Chlorophenols; Dechlorination; Kinetics

1. Introduction Chlorophenols (CPs) were extensively used as wood

Xenobiotic compounds are man-made chemicals
characterized by structures or substituents that make
them resistant to biodegradation and hence persistent in
the environment. The large production at industrial scale
and the intensive use of synthetic chemical products have
provoked the dispersion of noxious compounds in the
environment. The chlorinated compounds, and more
concretely the chloroaromatic compounds are notewor-
thy for their toxicity and recalcitrant nature in the envi-
ronment.
*
Corresponding author. Tel./fax: +86-574-87080072.
E-mail address: yefenxia@hotmail.com (F.-x. Ye).
0045-6535/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2003.08.019
preservers, pesticides, herbicides, biocides and dyes.
Chlorophenols are persistent, toxic and health risk re-
lated (Yee and Wood, 1997; Vallecillo et al., 1999;
Manimekalai and Swaminathan, 2000), so that they
have been characterized as first priority pollutants by
both the EU (European Union) and US EPA (United
State Environmental Protection Agency) (Ruzgas et al.,
1995). Because of their ecological risk, these compounds
become the target of investigations focusing on their
possible biodegradation (Schollhorn et al., 1994; Suidan
et al., 1996; Khodadoust et al., 1997; Wilson et al.,
1997). Due to its toxicity, biological treatment of
the industrial wastes containing high concentration of
chlorophenols is difficult.
The biodegradation of chlorophenols has been
studied in both anaerobic (Boyd et al., 1983; Boyd and
1574 F.-x. Ye, D.-s. Shen / Chemosphere 54 (2004) 1573–1580
Shelton, 1984; Christiansen et al., 1995) and aerobic
systems (Puhakka and Jarvinen, 1992; Armenante et al.,
1999). Microbial biodegradation is one of the most im-
portant factors affecting the concentration of chloro-
phenols in the environment. In quantitative models,
this is usually described by a rate constant. However,
rate constants do not accurately describe the acclimation
period prior to the onset of detectable degradation after
a community is exposed to a new chemical. The degra-
dation capacities of anaerobic sludge can be enhanced
by the acclimation of the inoculum (Buitr
on and Cap-
deville, 1995; Buitr
on et al., 1998). Different phenomena
have been described to explain the acclimation phase.
Wiggings et al. (1987) suggested that there is a selection
and a multiplication of specialized microorganisms
during this phase. In aerobic microbial communities, the
acclimation periods typically range from several hours
to several days (Wiggings et al., 1987; Timothy et al.,
1989), but for anaerobic communities, these periods
typically vary from two weeks to six months or longer
(Horowitz et al., 1983). Because of its length and broad
range, the acclimation period is probably more impor-
tant in predicting chemical exposure concentration in
habitats than the shorter biodegradation period. In
particular, it would seem difficult to ignore the accli-
mation period in predicting models of environmental
transport and fate, especially when anaerobic microbial
communities play important roles in this process.
Chlorophenols can easily migrate within different
aqueous environments and contaminate groundwater
(Smith and Novak, 1987) because of their relatively high
solubility in water. Monochlorophenols can be formed
during wastewater chlorination, and as a result of the
breakdown of pesticides and chlorinated aromatic
compounds (Pritchard et al., 1987). Mono- and, to a
lesser extent, dichlorophenols can be metabolized by
aerobic microorganisms (Fava et al., 1995; Kobaloo
et al., 1995), but aerobic attack becomes less effective to
more highly chlorinated compounds. On the other hand,
reductive dechlorination of chlorophenols has been
shown to occur under anaerobic conditions (Togna
et al., 1995; Buitr
on and Gonz
aes, 1996). The rate of
dechlorination under such conditions is actually greatest
for more heavily chlorinated compounds, but progres-
sively slower as the chlorophenol molecules become
more dechlorinated. In those cases in which anaerobic
dechlorination of more highly chlorinated chlorophe-
nols was shown to be followed by anaerobic mineral-
ization of the resulting monochlorophenols, the rate of
mineralization was reported to be significantly slower
than that of the initial dechlorination steps (Kohring
et al., 1989; Zhang and Wiegel, 1990). Therefore, the
accumulation of monochlorophenols occurs easily in the
environment, especially in the anaerobic environment.
From the above discussion, it follows that a possible
strategy to degrade absolutely more highly chlorinated
phenols is to acclimate anaerobic sludge which can
biodegrade chlorophenols, especially some monochloro-
phenols which are the intermediates of highly chlori-
nated phenols, since the detoxification of a chlorophenol
containing waste requires total mineralization of the
compounds.
Therefore, the objectives of this investigation are
to (i) study the acclimation of anaerobic sludge for
biodegrading the less highly chlorinated compounds to
completely degrade chlorophenols, (ii) investigate the
biodegradation potential of some chlorophenolic com-
pounds [pentachlorophenol (PCP); 2-monochlorophenol
(2-CP); 3-monochlorophenol (3-CP), and 4-chlorophe-
nol (3-CP)] in anaerobic sludge, and (iii) determine the
effect of chemical structure, sludge source and external
carbon source on the length of the acclimation period.
2. Materials and methods
2.1. Chemicals
All chlorophenolic compounds [PCP (98.5%), 2,4-
TCP (96.0%), 3,5-TCP (96.0%), 2-CP (96.0%), 4-CP
(96.0%)] used in this study were purchased from
ShangHai Reagent factory, Shanghai, China, except 3-
CP, which was obtained from Fluka AG. Chem. Fabrik
CH-9470 Bucks. The CPs were used without further
purification. Stock solutions (5000 mg/l) of four CPs
were prepared in 0.05 mol/l NaOH and used to make
additions to the anaerobic sludge in bioreactors.
2.2. Source of anaerobic sludge
The initial anaerobic enrichment cultures used were
the anaerobic digested sludge of Hangzhou municipal
wastewater treatment plant, and anaerobic digested
sludge of Hangzhou citric acid factory in Hangzhou.
The former sludge contained a TS and VS contents of
10.2% and 8.4%, respectively, while TS and VS of the
latter sludge were 9.6% and 7.6%, respectively. The
specific methane production rates were 0.53 g CH4 -
COD/(g VSS d) and 1.12 CH4 -COD/(g VSS d), respec-
tively.
2.3. Acclimation procedures
About 1500 ml anaerobic sludge was incubated in an
anaerobic batch bioreactor (2.5 l) and fed with the
synthetic wastewater (500 ml) containing a different CPs
congener. The chemical oxygen demand (COD) of the
synthetic wastewater was 5000 mg/l. The bioreactors
were located in a temperature controlled room at 28 °C.
The course of acclimation was arranged in Table 1.
 F.-x. Ye, D.-s. Shen / Chemosphere 54 (2004) 1573–1580 1575

Table 1
The arrangement of anaerobic sludge acclimated to chlorophenols
Reactor Substrate Volume (ml) of addition of chlorophenols
Day 1 Day 14 Day 21 Day 42 Day 65 Day 100 Day 116
1 PCP 4 5 10 10 10 10 10
2 4-CP 4 5 10 10 10 0 0
3 3-CP 4 5 10 10 10 0 0
4 2-CP 4 5 10 10 10 10 10
5 PCP 4 5 10 10 10 10 10
6 4-CP 4 5 10 10 10 0 0
7 3-CP 4 5 10 10 10 0 0
8 2-CP 4 5 10 10 10 10 10
9 PCP 4 5 10 10 10 10 10
10 4-CP 4 5 10 10 10 0 0
11 3-CP 4 5 10 10 10 0 0
12 2-CP 4 5 10 10 10 10 10
13 PCP 4 5 10 10 10 10 10
14 4-CP 4 5 10 10 10 0 0
15 3-CP 4 5 10 10 10 0 0
16 2-CP 4 5 10 10 10 0 0
The anaerobic sludge of 1–8 bioreactors was from Hangzhou municipal wastewater treatment plant; the anaerobic sludge of 9–12
bioreactors was from Hangzhou citrate factory.
Addition of 120 g/l sucrose (20 ml) weekly in 5–8 and 13–16 bioreactors; addition of distilled water (20 ml) weekly as the control in 1–4
and 9–12 bioreactors.

2.4. Analytical methods
Wastewater was taken from bioreactors weekly for
the purpose of monitoring the variation of residual CPs
concentrations, COD and pH values. All experiments
were performed in triplicate.
For the determination of residual phenolic com-
pounds, wastewater samples were extracted with aceto-
nitrile, centrifuged for 20 min at 3000 rpm, and filtered
through 0.45 lm filters. Extracts were analyzed via high
performance liquid chromatography (HPLC) (Waters
510). The HPLC was equipped with a C-18 reverse-col-
umn and a variable wavelength UV absorbance detector
operated at 280 nm. The mobile phase used a 60:38:2
mixtures of methanol, water, and acetic acid. The de-
tection limit of phenolic compounds was set to 0.01 mg/l.
Methane was quantified by a gas chromatograph
equipped with a packed column and a flame ionization
detector. Column, injector, and detector temperatures
were maintained at 55, 90, and 90 °C, respectively. Ni-
trogen was employed as a carrier at a flow rate of 20 ml/
min.
3. Results and discussions
3.1. Lag phase of anaerobic sludge degrading chlorophe-
nols
The anaerobic biodegradation of four CP isomers by
two kinds of anaerobic sludge were investigated. De-
gradation activity appeared after a lag period of two
weeks. When being added sucrose, regardless of anaer-
obic sludge source, the lag times of PCP, 2-CP and 3-CP
were all less than seven days, and lag time of 4-CP was
7–14 days. In the case of absence of sucrose, for the
sludge from the citric acid factory, the lag times were
(days): 7–14, 14, 7, 14 for 2-CP, 3-CP, PCP and 4-CP,
respectively. But for sludge from municipal wastewater
plant, the lag times were 7, <7, 7, 14 days, respectively.
The lag period of 4-CP was longest (14 days), and PCP
and 2-CP were most easily to be acclimated. Relatively
rapid degradation occurred after two months of incu-
bation (Figs. 1 and 2). The results suggested that addi-
tion of sucrose can enhance the acclimation of sludge to
chlorophenols, which was more obvious in the case of 3-
CP and 2-CP, and ortho-position and PCP were more
easily acclimated than para-position and meta-position
monochlorophenol. The results also implied that an-
aerobic microorganisms had potential of degradation of
chlorophenols. Induction of this potential might be ob-
tained in a short time, but it will take a long time to
enrich sufficient amount of biomass with degradation
activity. The results were consistent with the previous
study by Droste et al. who found that ortho-chlorine
atoms were most readily removed from CPs. CPs con-
taining chlorine atoms in the para-position were the
most toxic agents (Droste et al., 1998). Takeuchi et al.
also thought that the chlorine atom at the ortho-position
was the most easily dechlorinated, whereas dechlorina-
tion rate at the para-position was lowest (Takeuchi et al.,
2000).
1576 F.-x. Ye, D.-s. Shen / Chemosphere 54 (2004) 1573–1580

40 80
A B

3-CP Conc. (mg/l)

2-CP Conc. (mg/l)
30 60

20 40

10 20

0 0
0 20 40 60 80 100 0 20 40 60 80 100

70 50
C
60 D
4-CP Conc.(mg/l)

40

PCP Conc. (mg/l)

50
40 30
30 20
20
10
10
0 0
0 20 40 60 80 100 0 20 40 60 80 100
incubation time (d) incubation time (d)

Fig. 1. Chlorophenols degradation during the acclimation period of anaerobic sludge from citrate factory ( : without sucrose; N:

without sucrose).

50 80
A B
2-CP Conc. (mg/l)

3-CP Conc. (mg/l)

40
60
30
40
20

10 20

0 0
0 20 40 60 80 100 0 20 40 60 80 100

80 60
C D
50
4-CP Conc. (mg/l)

PCP Conc. (mg/l)

60
40
40 30
20
20
10

0 0
0 20 40 60 80 100 0 20 40 60 80 100
incubation time (d) incubation time (d)

Fig. 2. Chlorophenols degradation during the acclimation period of anaerobic sludge from municipal wastewater treatment plant


( : without sucrose; N: without sucrose).

3.2. Effect of chemical structure weeks to six months and were not correlated with
complete mineralization of the substrates in this habitat.
Acclimation periods prior to significant substrate It has been shown previously that the toxicity of chloro-
depletion and product formation ranged from three phenol increased with the number of substituted chlo-
 F.-x. Ye, D.-s. Shen / Chemosphere 54 (2004) 1573–1580 1577

rine atoms, except 2,4-DCP which was more toxic than
2,4,6-TCP (Vallecillo et al., 1999). Among the four tes-
ted chlorophenols, the toxicity of PCP was largest, but
the acclimation effects of PCP and 2-CP were the best.
The degradation rate of these two chlorophenolic com-
pounds significantly increased over the time course. 3-
CP and 4-CP were difficult to be acclimated, resulting in
the accumulation of 3-CP and 4-CP in the sludge, which
in turn influence the degradation and acclimation of
3-CP and 4-CP (Figs. 1–3). It was indicated that in
acclimation phase, the chemical structure of target
compounds was a decisive factor. The more highly
chlorinated, the more easily reductive dechlorinated,
resulting in more rapid of degradation rate. The sub-
stituted ortho-position on the aromatic ring was the
preferred attack position. Meta- and para-positions were
less reactive and resulted in a lower degradation rate
than the ortho-position.
3.3. Effect of external carbon source
Figs. 1–3 show that, addition of sucrose can enhance
the acclimation effect of anaerobic sludge. The external
carbon source not only reduced the lag period, but also
increased the degradation rate of acclimated sludge. The
results was agreed with the observation of Vallecillo
et al. who investigated the anaerobic biodegradation of
2,4,6-trichlorophenol and found that the addition of the
external carbon source (acetic acid) stimulate the de-
chlorination (Vallecillo et al., 1999). The addition of an
external carbon source was needed to assure the biode-
gradation of chlorophenols (Chang et al., 1999; Gavala
and Lyberatos, 2001).
3.4. Effect of anaerobic sludge source
Lately, there are some reports in the literature which
have shown significant differences in the response of
different seed cultures, with respect to their ability to
degrade compounds and their acclimation rates (Razo-
Flores et al., 1996). These observations emphasize the
importance of the seed sludge source in treating aro-
matic compounds. Consequently, anaerobic sludges
from two different sources were tested to evaluate their
biodegradation capacities with respect to acclimation
period, the degradation rate and the degradation po-
tential. The microorganism species in the anaerobic
sludge obtained from wastewater treatment station of
Hangzhou citrate factory was few because the compo-
sition of wastewater treated was simple and steady.
Comparatively, the sludge from Hangzhou municipal
wastewater plant where various wastewater were trea-
ted, probably had been exposed to some recalcitrant
pollutants, so abundant microorganism population had
been developed in this sludge. As a result, the lag period
of the former sludge was longer than the latter during
the acclimation period, such as the 3-CP and 2-CP, the
lag periods from citrate factory were 14 days and 7–14

0.3
(
degradation rates (mg g-1.d-1

A B

0.2

0.1

0
1 2 3 4 1 2 3 4
degradation rates (mg g -1.d -1)

0.4
C D

0.3

0.2

0.1

0
1 2 3 4 1 2 3 4

Fig. 3. Effect of external carbon source on chlorophenols degradation rate of anaerobic sludge from municipal wastewater treatment
plant (A and B) and citrate factory (C and D) during acclimation period (j: with sucrose; : without sucrose; 1: PCP; 2: 4-CP; 3: 3-CP;
4: 2-CP; A and C: during 42–65 days; B and D: during 65–100 days).
 1578
Table 2
Kinetic equations of biodegradation of chlorophenols during acclimation period of anaerobic sludge originated from Hangzhou citrate factory and Hangzhou municipal wastewater
treatment plant

F.-x. Ye, D.-s. Shen / Chemosphere 54 (2004) 1573–1580

Sludge Chlorophenols Sucrose Acclimation period
source Day 21–42 Day 42–65 Day 65–100
Equation r Equation r Equation r
S1 4-CP W – – – – C ¼ 243:40  43:33 ln t )0.9850
3-CP W – – ln C ¼ 3:98  0:01t )0.9792 ln C ¼ 4:14  0:005t )0.9668
2-CP W C ¼ 16:51  4:86 ln t )0.9908 C ¼ 15:00  4:41 ln t )0.9971 C ¼ 15:29  4:66 ln t )0.9961
PCP W ln C ¼ 3:71  0:07t )0.9088 C ¼ 17:33  6:20 ln t )0.9937 C ¼ 13:65  4:44 ln t )0.9740
4-CP N C ¼ 48:36  2:10 ln t )0.9954 – – C ¼ 57:77  1:32 ln t )0.9966
3-CP N – – ln C ¼ 3:99  0:01t )0.9414 – –
2-CP N C ¼ 21:94  3:87 ln t )0.9511 C ¼ 71:91  4:22 ln t )0.9948 C ¼ 19:93  5:29 ln t )0.9961
PCP N – – ln C ¼ 3:91  0:03t )0.9087 C ¼ 29:68  7:57 ln t )0.9736

S2 4-CP W – – C ¼ 40:20  0:78 ln t )0.9569 ln C ¼ 4:14  0:01t )0.9912

3-CP W – – – – C ¼ 59:96  1:78 ln t )0.9524
2-CP W C ¼ 25:60  2:90 ln t )0.9979 C ¼ 29:59  5:58 ln t )0.9940 C ¼ 28:81  3:10 ln t )0.8861
PCP W – – C ¼ 24:12  5:53 ln t )0.9519 C ¼ 18:49  3:77 ln t )0.9266
4-CP N – – – – ln C ¼ 4:19  0:005 ln t )0.9625
3-CP N – – C ¼ 50:21  1:20 ln t )0.8357 ln C ¼ 4:33  0:06t )0.9344
2-CP N C ¼ 25:07  3:24 ln t )0.9978 C ¼ 29:03  5:12 ln t )0.9722 C ¼ 31:41  1:87 ln t )0.8440
PCP N – – C ¼ 21:08  2:65 ln t )0.9654 ln C ¼ 4:18  0:04 ln t )0.9942
W: with sucrose; N: without sucrose; r: correlation coefficient;  : 5% significance level;  : 1% significance level; S1: sludge from Hangzhou citrate factory; S2: sludge from Hangzhou
municipal wastewater treatment plant.
 F.-x. Ye, D.-s. Shen / Chemosphere 54 (2004) 1573–1580
days, while in the case of sludge from wastewater
treatment plant, the lag periods were seven days and less
than seven days. However, the former sludge after ac-
climated can more rapidly adapted to various chloro-
phenols than the latter, and had higher degradation
rate (Figs. 1–3). As the two kinds of sludge were accli-
mated under the same condition, the microorganism
population originally in the sludge was the key factor.
Microscope observation found that bacillus was pre-
dominant in the sludge from citrate factory before ac-
climated, while coccus was major microorganism in the
sludge from municipal wastewater plant. After accli-
mation, no significant change on the microorganism
population in the two kinds of sludge was observed.
Similar microscope result was obtained in the later re-
actor operation period. In addition, all the anaerobic
microorganisms isolated by some investigators degrad-
ing PCP were bacillus, which implied that bacillus was
much more adapted to degrade the chlorophenols.
3.5. Kinetics of biodegradation of chlorophenols during
acclimation period
During the acclimation of anaerobic sludge, the re-
lationship between residual chlorophenols concentration
and incubation time gradually changed, and then
reached steady. Before acclimation was achieved, the
change of residual chlorophenol over time was compli-
cated. Once the sludge was completely acclimated to the
tested chlorophenols, when being added the compound
again, the configuration of degradation curve was sim-
ilar, independency of sludge source and compounds. In
general, the residual chlorophenols concentration was
highly correlated inversely with the logarithm of the
incubation time. During the acclimation period with
sucrose as external carbon source, this correlation was
more evident than without sucrose (Table 2). The data
from Table 2 showed that, the degradation rate of
chlorophenols in the acclimated sludge originated from
citrate factory was higher than the acclimated sludge
originated from municipal wastewater treatment plant;
the correlation between residual chlorophenol concen-
tration and time was better, and degradation was much
steadier than the latter.
4. Conclusion
The extensive temporal data in this study provide
documentation of the earlier indications that lengthy
acclimation periods are typical before apparent degra-
dation in anaerobic sludge. This acclimation phase was
depended on the chemical structure of tested com-
pounds, anaerobic sludge source and external carbon
source. The obtained results showed that the addition of
external carbon source can enhance the dechlorination
1579
process and ortho-position chlorine atom was more
easily to be acclimated than meta-position and para-
position chlorine atoms. The relatively short acclimation
period of pentachlorophenol (PCP) is more intriguing,
since this compound was more toxic, implying that
chemical structure, not the toxicity, play key role in the
acclimation period. Our results inspired that, because of
its long lag time, the acclimation period cannot be ig-
nored in understanding and predicting the transport and
fates of these chlorophenols and probably other pollu-
tants which may reside in environments, especially in
anaerobic environments. Pure evaluation of total bio-
mass cannot correctly estimate the biodegradation rate.
If acclimation effectiveness and acclimation period were
not appropriately taken into account, direct short-term
evaluations of anaerobic biodegradation potential can
lead to misunderstanding as to whether these chemicals
can be metabolized.
Acknowledgements
This work was supported by grants from the China
National Natural Science Funds Council.
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