An anaerobic bioreactor allows the efficient

degradation of HCH isomers in soil slurry

J.C. Quinteroa,

M.T. Moreirab,

J.M. Lemaa,

G. Feijooa,

Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, E-15782

Santiago de Compostela, Spain

Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, E-15782

Santiago de Compostela, Spain

http://dx.doi.org/10.1016/j.chemosphere.2005.08.043, How to Cite or Link Using DOI

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Abstract
The insecticide gamma-hexachlorocyclohexane ( -HCH or lindane), which has been
extensively used for agricultural and medical purposes, presents high persistence and toxicity to
the environment and low solubility. This study intends to assess the efficiency of an anaerobic
reactor to degrade HCH isomers contained in soil slurry cultures. This study was developed in
two phases: experiments in flasks to optimize the process parameters, and assessment of the
slurry process in the anaerobic slurry reactor operated for an approximate period of a year. The
influence of different environmental conditions was evaluated: the HCH concentration (25
100 mg HCH kg1), the type of substrate (volatile fatty acids or starch), the sludge concentration
(28 g VSS l1) and the replacement of spiked soil to simulate a fed-batch operation (1050%).
The best results were obtained when the reactor was operated with a sludge concentration of
8 g VSS l1, starch concentration of 2 g COD l1 and soil replacements of 1020%. Under these
conditions, - and -HCH were completely degraded after 10 d while nearly 90% - and HCH were removed only after 50 d. According to the obtained results related to the total
degradation of the HCH isomers and the degradation rates, especially high for - and -HCH,
the anaerobic slurry reactor appears to be a good alternative for the degradation of the HCH
isomers present in polluted soil.

Keywords

Hexachlorocyclohexane;

HCH;

Anaerobic degradation;

Slurry soil remediation treatment

1. Introduction
Lindane (-HCH) is an organochloride insecticide that has been used worldwide for agriculture
and public health (Breivik et al., 1999 and Li, 1999). During its production, 85% of the product

containing other isomers, mainly -, - and -HCH, was dumped as waste, causing serious
soil pollution (Braun et al., 1991).
Although biologically mediated degradation of HCH isomers has been demonstrated under
aerobic and anaerobic conditions (Deo et al., 1994 and Singh et al., 2000), the high adsorption
of the pollutant to the soil and its restricted availability for the biological action of endogenous or
exogenous microorganisms limits the extent and rate of degradation (Bollag et al.,
1992 and Harms and Bosma, 1997). One possibility to favor the mobility of the pollutants is their
transfer from the soil to the liquid phase. The slurry-phase system is a bioremediation technique
where the contaminated soil is combined with water and other additives in a stirred tank
bioreactor (Alexander, 1994). This system minimizes the formation of aggregates and allows a
faster equilibrium between the solid and liquid phases by means of the suspension and vigorous
agitation of the soil in a liquid medium. Both effects favor mass transfer and thus, enhance the
biodegradation rate of the pollutants (Rogers et al., 1993). Although there are several papers
reporting the anaerobic degradation of HCH isomers in slurry systems in laboratory flask scale,
there is lack of studies at a higher scale (Bachmann et al., 1988 and Van Eekert et al., 1998).
This study intends to assess the efficiency of an anaerobic reactor to degrade HCH isomers
contained in soil slurry cultures. However, the proposal of a soil detoxification system for its
viable application at a more realistic scale requires a further knowledge of the operational
parameters involved in the process. Therefore, this study was developed in two phases:
experiments in flasks to optimize the process parameters, and assessment of the slurry process
in the anaerobic slurry reactor operated for an approximate period of a year.
Different environmental conditions were evaluated in the flask experiments: the toxicity of HCH
isomers on the methanogenic activity, the type of the substrate (volatile fatty acids or starch) as
an adequate electron donor, the sludge concentration (28 g VSS l1) and the concentration of
the pollutant. Once the most adequate experimental conditions were established from the batch
assays, the slurry process was performed in the anaerobic slurry reactor, which operated with
the replacement of spiked soil to simulate a fed-batch operation strategy (1050%) (Eweis et al.,
1998). In this bioreactor configuration, the spiked soil was suspended in a stirred reactor vessel
and mixed with water, nutrients and anaerobic sludge to a concentration that was determined by
the pollutant concentration in the soil, the rate of biodegradation, and the physical nature of the
soil.

2. Materials and methods

2.1. Chemicals
-, - and -HCH (9899% purity) were purchased from Riedelde HanFluka and -HCH
isomer (99% purity) was purchased from SigmaAldrich. Stock solutions of HCH isomers at
concentrations of 2 g l 1 were prepared in acetone were added to clean soil previous to the
experiments. Chlorobenzene (CB), dichlorobenzene isomers (1,2-DCB, 1,3-DCB, 1,4-DCB) and
trichlorobenzene isomers (1,2,3-TCB, 1,2,4-TCB, 1,3,5-TCB) (9999.9% purity) were purchased
from Riedelde HanFluka. Other chemicals such as hexane and acetone were of analytical
grade from Panreac commercial source.

2.2. Anaerobic sludge

The granular sludge used in this study comes from an up-flow anaerobic sludge blanket reactor
treating a synthetic wastewater from the winery industry. The chemical composition of the
effluent is 10 g COD l1, 175 mg NH4Cl l1, 25 mg KHPO4 l1, 50 meq NaHCO3 l1, and pH 7. The
reactor had been operated for at least 2 years with an organic loading rate of 5 kg COD m3 l1,
previous to sludge sampling. The granular sludge had excellent sedimentation characteristics,
with an average diameter of 1.5 mm and a volatile suspended solids (VSS) concentration of
60 g l1 and a specific methanogenic activity of
. The sludge was
stored at 4 C and washed thoroughly with distilled water to remove residual soluble substrate
before being used in the experiments.

2.3. Soil characterization

A sandy slime soil collected from a forest site was used for the experiments (Table 1). For
experiments, soil was sieved to select a particle size smaller than 0.5 mm in order to provide
high superficial area for interaction between pollutants and microorganisms. The soil initial
microorganism content was of 5.1 105 CFU g1 dry soil.
Table 1. Physico-chemical characterization of the soil
Structure

Sandy slime soil

pH

4.04

Carbon concentration

7.85%

Nitrogen concentration

0.55%

Moisture content

18%

Organic matter

9.8%

Ash content

90.2%

CEC (cmolc kg1)a

4.98

Granulometry of soil (mm)

Higher than 2.0

34.8%

Between 1.0 and 2.0

20.4%

Between 0.5 and 1.0

23.6%

Lower than 0.5

21.2%

a
CEC: cationic exchange capacity, defined as the amount of exchangeable cations that can be adsorbed onto the soil at pH 7.

Table options

2.4. Methanogenic toxicity assays

The batch anaerobic assays were performed in 125 ml serum bottles as described by Soto et al.
(1993). Each bottle contained 2 g VSS l1 anaerobic sludge, NH4Cl (170 mg l1), NaHCO3 (2 g l1),
CaCl2 2H2O (8 mg l1), KH2PO4 (37 mg l1), MgSO4 4H2O (9 mg l1), 1 ml l1 of a trace element
solution, 2 g COD l1 as volatile fatty acids (VFA) with a ratio 4:1:1 of acetic:propionic:butyric and
polluted soil (100 g l1).
Several assays at different HCH isomer concentrations (0, 100, 250 and 500 mg isomer HCH kg
soil) were carried out with spiked soil (100 g l 1). In addition, assays with a mixture with
identical concentrations of -, -, - and -HCH were also performed to reach final
1

concentrations of HCH: 0, 200, 400, 500, 600, 800 and 1000 mg total HCH kg1 soil.
Na2S 9H2O was added to a final concentration of 100 mg l1 and liquid and headspace flasks
were flushed with nitrogen gas (containing 20% CO 2) to secure anaerobic conditions. Finally,
the flasks were connected to the biogas measuring device and placed in a thermostatic bath at

30 C. Methane production was periodically measured by volumetric displacement of an

alkaline solution (NaOH 25 g l1) to retain the produced CO2.

2.5. HCH anaerobic biodegradation in slurry soil flasks

Biodegradation studies in slurry soil were performed in 500 ml flasks in triplicate. Each flask
containing 40 g dry soil spiked with the four HCH isomers (in an individual concentration of each
isomer of 100 mg HCH kg 1soil), Na2S 9H2O (0.1 g l 1), NaHCO3(2 g l 1), NH4Cl (170 mg l 1),
CaCl2 2H2O (8 mg l1), KH2PO4(37 mg l1), MgSO4 4H2O (9 mg l1), 1 ml l1 of a trace element
solution according to Soto et al. (1993), and water for a volume of 400 ml, was incubated at
30 C, pH 7.0 and 180 rpm for up to 13 weeks in an orbital shaker.
The effects of biomass concentrations and type of substrate were studied in the anaerobic
slurry batch assays, according to the following conditions: 2, 4 and 8 g VSS l1 with 2 g COD l
1

as VFA for the biomass assays and 2 g COD l 1 as VFA or starch as energy sources with

2 g VSS l1 of anaerobic sludge in the substrate assays.

2.6. Degradation of HCH isomers in the soil slurry reactor

A 5-l stirred tank reactor equipped with a single turbine propeller, pH and temperature sensors
was used in the anaerobic biodegradation studies. The biogas outlet was cooled down to get
volatile compounds condensed and returned to minimize losses. The slurry reactor was
operated at pH 7, 30 C and 350 rpm.
The anaerobic reactor was operated for a year under different environmental conditions (Table
2). The environmental conditions of the start-up period (stage A1: days 0 51) were the
following: 400 g of spiked soil with a concentration of 100 mg kg1 of each HCH isomer (-, -,
- and -HCH); 2.0 g VSS l 1 of granular sludge; 0.1 g l 1 Na2S 9H2O; 2.0 g l 1 NaHCO3;
2.0 g COD l 1 as VFA (acetic:propionic:butyric in a ratio 4:1:1), 170 mg l 1 NH4Cl, 8 mg l
1
CaCl2 2H2O, 37 mg l 1 KH2PO4, 9 mg l 1 MgSO4 4H2O, 1 ml l 1 of a trace element solution
according to Soto et al. (1993), and water to reach a final volume of 4 l. At day 51 (stage A2),
the slurry reactor was reinoculated with new granular sludge to reach a final concentration of
8 g VSS l1 and a potato starch solution was fed every three days to reach a concentration of
2 g COD l 1. At day 69 (stage A3), a pulse addition of HCH to reach a concentration of
100 mg kg 1 of each HCH isomer was injected into the reactor. From day 89, different
replacements of soil were performed under the specific conditions presented in Table 2.
Table 2. Operational conditions of the soil slurry reactor
Variable

Stage
A1

A2a

A3

B1

B2

C1

C2

Day

051

51
69

69
89

89
104

104
156

156
217

217
280

280
350

350
377

HCH concentration in
spiked soil (mg kg1)

1000

1000

1000

1000

1000

500

500

100

100

Replacement (%)

NRb

NR

NR

10

10

20

20

50

20

HCH concentration in the

reactor (mg kg1)c

100

100a

100

100

100

100

100

50

25

CODd (g l1) addition every

3d

NS

2.0

2.0

2.3

0.75

1.5

1.5

3.4

0.75

a
Reinoculation of the biomass at day 51 to reach a final concentration of 8 g VSS l1.

b
Operation with no soil replacement.

c
HCH concentration corresponds to each isomer. The concentration of the total HCH mixture is fourfold.

d
Addition of starch solution, except in stage A1 where VFA was added only at day 0.

Table options

2.7. Extraction and GCMS analysis

The procedure for the extraction of HCH residues in the soil slurry reactor was performed as
follows: (i) 200 ml of the sampling port were purged and returned back into the reactor; (ii)
aliquots of 50 ml of the soil slurry reactor were transferred to Erlenmeyer flasks with magnetic
stirring; (iii) aliquots of 3 ml of this homogenized medium were immediately transferred to a
15 ml volumetric tubes and mixed with 6 ml of 1:1 hexaneacetone solution. The remaining
volume of the sample: 44 ml is returned to the reactor; (iv) the tubes were hermetically sealed
and shaken for 10 min in a vortex in order to attain the transport of HCH and their metabolites
from water or soil to the organic phase, and centrifuged (2500 rpm for 10 min) for separation of
the organic and aqueous phases; (v) an aliquot of the organic phase was taken for GCMS
analysis. The average extraction efficiencies (%) were 82 4, 96 3, 84 5 and 90 5 for
-, -, - and -HCH, respectively.
Residual HCH and intermediate metabolites were analyzed in a Varian-Saturn GC/MS (CP
3900), equipped with a split splitless injection port (CP-8400) and connected to a mass
spectrometer with ions trap (Varian-Saturn 2100). The capillary column used was a CP-Sil 8 CB
Low Bleed/MS fused silica WCOT (30 m length and 0.25 mm ID). The oven temperature was
programmed as follows: hold time at 60 C, 2 min; ramp rate at 20 C min1 to 180 C; ramp
rate at 5 C min1 to 200 C and finally, a ramp rate at 10 C min1 to 300 C. The injection
volume was 1 l via a split-less injection at 280 C. Helium was used as a carrier at a flow
rate of 1.0 ml min1. Under these conditions, the retention time of HCH isomers and intermediate
metabolites detected were: -HCH (11.27 min), -HCH (11.83 min), -HCH (12.04 min), HCH

(12.69 min),

pentachlorocyclohexane

isomers PCCH

(8.56

and

9.56 min),

tetrachlorocyclohexene TCCH (8.50 min), 1,2,3-trichlorobenzene 1,2,3-TCB (6.80 min), 1,3dichlorobenzene1,3-DCB (5.33 min), chlorobenzeneCB (3.25 min). HCH concentration was
quantified with external standard calibration, which was linear in the range of 0.055.0 mg l1.
Biodegradation expressed as percentage was calculated on the basis of the difference between
remaining HCH in controls and treated samples. The recovery of the HCH isomers for the
control assay was higher than 80%.

3. Results and discussion

This study intends to assess the efficiency of an anaerobic reactor to degrade HCH isomers
contained in soil slurry cultures. To perform the effective degradation of HCH isomers the
knowledge of the operational parameters involved in the process is necessary. Therefore,
different environmental conditions were evaluated in the flask experiments: the toxicity of HCH
isomers on the methanogenic activity, the type of the substrate (volatile fatty acids or starch) as

an adequate election donor, the sludge concentration (28 g VSS l1) and the concentration of
the pollutant.

3.1. Anaerobic toxicity of the HCH isomers

The potential toxicity of the HCH isomers on the methanogenic bacteria was evaluated in batch
assays (Fig. 1). At 2 g VSS l1 the addition of HCH at a concentration of 250 mg HCH kg1 soil
had a variable negative effect on the methanogenic activity relative to controls dependent on the
isomer considered: 95%, 80%, 18% and 11% of methanogenic activity reduction for -, -, and -HCH, respectively. A higher concentration of 500 mg of HCH kg1 soil resulted in a total
inhibition effect of the methanogenic activity except for -HCH. The highest toxicity of the HCH isomer is in agreement with other works. Farghaly et al. (1998) have described a
significant effect on the soil microbial activity at a concentration of 10 mg -HCH kg 1 soil
andErguder et al. (2003) have reported an unfavorable effect on the biogas production of
methanogenic bacteria from a concentration of 10 mg -HCH l1.

Fig. 1. Methanogenic activity of anaerobic sludge (2 g VSS l 1) with individual and mixture of HCH
isomers. Symbols: () HCH mixture; () -HCH; () -HCH; () -HCH; () -HCH; () HCH
mixture with an anaerobic sludge concentration of 8 g VSS l1. The activity value for controls at 100%
were

.
Figure options

The influence of the HCH isomers on the methanogenic activity was dependent on both
chemical and sludge concentration. Therefore, different anaerobic sludge concentrations (2 and
8 g VSS l1) were considered (Fig. 1). For a bacterial culture concentration of 2 g VSS l1, the
addition of a mixture of HCH isomers (100 mg for each HCH isomer kg1, that is to say, 400 mg
total HCH kg 1 soil) decreased methanogenic activity relative to the control by 50%. If these
results are compared with the individual effect of each isomer: 10% inhibition for 100 mg kg1 for
-, - and -HCH and 20% inhibition for 100 mg kg 1 -HCH, there was an evident
accumulative toxic effect. At a higher sludge concentration of 8 g VSS l1, the detrimental effect
on the methanogenic activity remarkably decreased. For a concentration of 400 mg HCH kg
1

soil, the degradation decreased by 10% and only total inhibition was observed for a

concentration of 1000 mg HCH kg1 soil.

3.2. HCH biodegradation vs. biomass concentration

Biodegradation assays of the different HCH isomers were performed in slurry phase for different
concentrations of the anaerobic sludge: 2, 4 and 8 g VSS l1, VFA of 2 g COD l1 added as a
substrate and HCH concentrations of 100 mg kg 1 soil. The sludge concentration had a
significant effect on the degradation extent of the HCH isomers, except for -HCH, which was
totally degraded regardless of the biomass concentration (Fig. 2). The degradation percentage
for -HCH was increased twofold when the biomass concentration was changed from 2 to
4 g VSS l 1, however, no further rise was observed in the experiments with 8 g VSS l 1. In
contrast, for -HCH the degradation was directly proportional to the biomass concentration; the
best results (60% degradation) corresponded to the assays with 8 g VSS l1. Buser and Muller
(1995) reported a degradation percentage of -HCH threefold when using a sludge
concentration of 25 g dry matter l1, which is a value 2.5 higher than the one here considered.
Finally, for -HCH there was only 36% degradation for assays performed with 8 g VSS l1. The
positive effect of the sludge concentration on the degradation was also observed for other
organochloride compounds; as an example, the chlorophenol degradation was increased 2.5
times when the anaerobic sludge concentration was augmented from 10 to 20 g l1 (Ribarova et
al., 2002).

Fig. 2. Biodegradation of HCH isomers with different concentrations of anaerobic sludge biomass after
30 d. Symbols: 2 g VSS l 1 (white bars); 4 g VSS l 1 (dotted bars); 8 g VSS l 1 (grey bars). Standard
deviations were obtained from experiments in triplicate.
Figure options

3.3. HCH biodegradation vs. substrate

Anaerobic sludge is a mixture of different types of microorganisms that are grouped in different
trophic categories. Each group can degrade a certain substrate type and the substances
generated as residue are used as nutrients for another group in the consortium (Verstraete et
al., 1996). Therefore, to perform the degradation of specific pollutants with anaerobic sludge is
necessary to provide the adequate substrates according to the trophic groups contributing to the
degradation. To evaluate this effect, the HCH biodegradation assays were performed with VFA
(a ratio 4:1:1 of acetic:propionic:butyric), substrate for acetogenic and methanogenic bacteria,
or starch, complex substrate for hydrolytic, acidogenic, acetogenic and methanogenic bacteria.
The degradations of - and -HCH at 100 mg HCH isomer kg1 soil were not influenced by the
type of the substrate used at 2 g COD l 1 (Fig. 3). These results could be explained by the
capability of acetogenic and methanogenic bacteria to degrade these isomers. Several reports
have evidenced the capability of these bacteria to carry out both the dehalogenation of
haloalkanes (Holliger and Schraa, 1994) and that of -HCH (Fischer and Pecher, 1993). In this
work, the degradation of - and -HCH was higher when starch was used as electron donor

substrate (Fig. 3). These results are in agreement with those obtained by other research
groups. Van Eekert et al. (1998) studied the degradation of -HCH using methanogenic
granular sludge with three different substrates: VFA, methanol and sucrose, finding better
degradation results in the experiments with sucrose. Moreover, Middeldorp et al.
(1996) observed that the degradation of -HCH was not related to the action of methanogenic
bacteria, since the use of a specific inhibitor: bromoethanol sulfonate, had no significant effect
on its degradation.

Fig. 3. Degradation of HCH isomers with different substrates (2 g COD l 1) for 30 d. Symbols: VFA
(white bar); starch (grey bar). Standard deviations were obtained from experiments in triplicate.
Figure options

There are, at least, two possible mechanisms, which can explain the biodegradation results of
- and -HCH: (i) cometabolism and (ii) the action of halorespiratory bacteria. An improved
activity of all the bacterial groups of the anaerobic consortia can favor the degradation by
cometabolism with starch as primary substrate, in comparison with the exclusive action of
acidogenic and methanogenic bacteria. Although the reductive dehalogenation can result as a
consequence of the cometabolic process, the dehalogenation as a respiration process by
means of halorespiratory bacteria can also play an important role, as was observed in the
biodegradation of chlorophenols by Desulfitobacterium species and tetrachloroethene
by Dehalospirillumspecies (Holliger and Schraa, 1994). In this mechanism, hydrogen is involved
as electron donor and the organohalogens are electron acceptors (Fetzner, 1998). The
hydrogen concentration is a key factor responsible of the dehalogenation mechanism since the
action of the halorespiratory bacteria are enhanced at hydrogen partial pressures between 10
4.3

and 10 5.2 Pa (Fennell et al., 1997). In anaerobic assays, the rate of hydrogen production

decreases according to the substrate used: sucrose > glucose > starch > cellulose (Logan et al.,
2002). Therefore, we supposed that the use of starch as a substrate would be beneficial for the
action of halorespiratory bacteria. In this sense, complex electron donors as tetrabutoxysilane
(TBOS) for reductive dehalogenation of trichloroethylene has been used (Yu and Semprini,
2002). This substrate is lowly hydrolyzed and fermented to butyrate and/or acetate until product
hydrogen.

3.4. HCH degradation in the soil slurry bioreactor

The degradation profiles of the HCH isomers during the different operational phases defined
in Table 2 of the slurry bioreactor are shown in Fig. 4. During the starting period (stage A), the
slurry reactor was operated with no soil replacement. In stage A1 corresponding to the reactor
start-up, there was only degradation of the - and -HCH isomers. This fact can be explained

by the low sludge concentration (2 g VSS l1) as well as the high concentration of the pollutant in
the soil (100 mg HCH kg1 soil), which caused the inhibition of the anaerobic activity. At day 51
(the beginning of stage A2), a reinoculation with anaerobic sludge to reach a concentration of
8 g VSS l 1 was performed in combination with the addition of starch as substrate electron
donor. During this period, there was a complete degradation of the most recalcitrant isomers:
- and -HCH. At day 69, the four isomers were freshly added to the reactor to reach a
concentration of 100 mg kg1. Total degradations of - and -HCH were obtained after 3 d,
whereas - and -HCH required a more prolonged period to attain degradations of 90%: days
71 and 86, respectively.

Fig. 4. Profiles of degradation of HCH isomers in slurry sequential batch reactor. Start phase (A); soil
replacement of 10% (B); soil replacement of 20% (C); soil replacement of 50% (D); soil replacement of
20% (E). Symbols: () -HCH; () -HCH () -HCH; () -HCH.
Figure options

From day 89, the reactor was operated with sequential replacements of spiked soil. Between
days 89 and 156, two consecutive replacements of 10% were performed (period B, Fig. 4).
Under these conditions, degradation higher than 90% for - and -HCH was obtained after
50 d, while the degradation periods for - and -HCH were shorter than 5 d. These results
were comparably worse than those obtained in phase A3 since significant decreases in the
degradation rates of -HCH and -HCH were observed (Table 3): from 15.9 to 5.05.1 mg kg

d1 and from 20.2 to 6.48.1 mg kg1 d1, respectively. This sharp decrease in the degradation

rate could be due to mass transfer limitations by the different addition procedures of HCH, as in
A3 the pollutant was added to the slurry suspension while in B the pollutant was previously
sorbed onto the soil and then added to the reactor. Limitations of mass transfer are marked
when ageing effect occurs and can affect the analysis of the results, but here is no case
because the age soil is the same for all experiments.
Table 3. HCH degradation rate for the different operational periods in the slurry reactor
HCH isomer

HCH degradation rate (mg kg1 d1)

A1

A2

A3

B1

B2

C1

C2

-HCH

3.10

76.6

81.7

36.6

23.8

17.1

5.1

1.4

-HCH

0.0

13.4

15.9

5.1

5.0

5.1

3.7

0.7

0.8

-HCH

7.0

172.4

270.2

96.1

51.1

33.8

23.6

6.7

-HCH

0.0

11.2

20.0

8.1

6.4

5.7

4.3

0.5

0.9
Table options

The replacement percentage was increased to 20% in phase C, between days 156 and 266.
During this period the bioreactor maintained stable conditions and high degradation rates.
However, this tendency changed in phase D, where the replacement was changed to 50% while
the spiked soil had 50 mg HCH kg1, an initial lower concentration of the pollutant (Table 2).
Under these conditions, the degradation rates decreased to 70%, 81%, 30% and 89% for -,
-, - and -HCH, respectively (Table 3). This decrease can be explained by the lower
biomass concentration in the reactor after the replacement. Moreover, lower concentrations of
the pollutant reduced the degradation rate of the process according to a first order kinetics. This
fact would be corroborated during phase E.

3.5. Metabolites production during HCH degradation

During the different degradation phases in the slurry reactor, traces of diverse intermediate and
end-products compounds were detected, such as pentachlorocyclohexane isomers (PCCH),
tetrachlorocyclohexene (TCCH), 1,2,3-trichlorobenzene (1,2,3-TCB), 1,3-dichlorobenzene (1,3DCB), chlorobenzene CB (Fig. 5). These low concentrations of the compounds and the
prolonged operational period of the reactor indicate that intermediate compounds were not
accumulated and they proceed to their further degradation to CB, the end-product in the
degradation mechanism (Haggblom et al., 2000).

Fig. 5. CG-MS detection of HCH metabolites from sequential anaerobic sludge slurry reactor at days 0
and 4 in D replacement: -HCH (11.27 min), -HCH (11.83 min), -HCH (12.04 min), -HCH
(12.69 min), pentachlorocyclohexane isomersPCCH (8.56 and 9.56 min), tetrachlorocyclohexene
TCCH (8.50 min), 1,2,3-trichlorobenzene 1,2,3-TCB (6.80 min), 1,3-dichlorobenzene 1,3-DCB
(5.33 min), chlorobenzeneCB (3.25 min).
Figure options

Although HCH toxicity may affect significantly the anaerobic microbial activity, the increase in
the biomass concentration solved this drawback by decreasing the specific HCH concentration.
Complex substrates as starch can increase HCH biodegradation due its slow liberation of
hydrogen that can be inhibitory at high concentrations for the activity of halorespiratory bacteria.
According to the obtained results related to the total degradation of the HCH isomers and the
degradation rates, specially high for - and -HCH, the anaerobic slurry reactor appears to be
a good alternative for the degradation of the HCH isomers present in polluted soil.