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4 Inhibition of Reductive Dehalogenases (Enzyme Level)

4 Inhibition of Reductive Dehalogenases (Enzyme Level)

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K. Wei et al.



Such classical enzyme assays can be adapted to characterizing the dehalogenases in OHRB with careful consideration of their oxygen sensitivity and the

volatile nature of many organohalide substrates (Loffler et al. 1996; Rosner et al.

1997). Because reductive dehalogenases are not easily purified nor heterologously expressed, dehalogenase enzyme assays have often been applied to crude

cell extracts, typically prepared from mixed cultures. This approach works well

despite uncertainties in true active protein concentrations, provided that the same

batch of crude extract is used for an entire series of concentrations of substrate

and inhibitor. Enzyme assays for reductive dechlorination typically use titanium

citrate-reduced methyl viologen as the artificial electron donor under anaerobic

conditions and halogenated organic as the electron acceptor. As mentioned before,

the analytical requirements to determine velocity are also a little tricky owing to

the fact that the substrates are typically volatile and hydrophobic. These single

substrate dechlorination assays have been adapted to explore the inhibitory effects

of different halogenated compounds in mixed cultures (Chan et al. 2011; Grostern

et al. 2009). Briefly, to perform tests for inhibition, cells from active dechlorinating cultures are collected by centrifugation, disrupted by sonication in lysis buffer,

and the resulting CFE are dispensed into aliquots and stored frozen at −80 °C

until use. All manipulations are carried out without exposure to oxygen. Crude

protein extracts in assay buffer containing electron-accepting substrates, inhibitors,

and reduced methyl viologen as electron donor are incubated anaerobically for

1–3 h and then sampled to determine the extent and rate of dechlorination normalized to total protein concentration. The data obtained from each substrate/inhibitor

combination are then fit to enzyme kinetic models to quantify and categorize the

nature of the inhibition. What results are estimates of kinetic and model parameters, including the inhibition constant that defines the strength of the inhibition.

This approach is illustrated with examples below.

Chlorinated ethenes (e.g., PCE and TCE) are frequently found in groundwater

in combination with chlorinated ethanes (e.g., 1,1,1-TCA) and methanes (e.g., CF)

at industrial sites, and dechlorination rates are negatively impacted in these situations, thus inhibition by these co-contaminants has been the focus of several investigations. Inhibition was observed to act in both directions: chlorinated ethenes

inhibited the reductive dechlorination of chlorinated alkanes, and vice versa. These

examples are described below, in Sects. 13.4.1 and 13.4.2.



13.4.1 Inhibition of Chloroalkene Reductive Dehalogenases

by Chlorinated Alkanes

The inhibitory effects exerted by chlorinated alkanes on the reductive dechlorination of chlorinated ethenes were investigated in mixed cultures enriched on

PCE or TCE. Cell-free extract from three chloroethene-dechlorinating enrichment cultures, including KB-1 (Duhamel et al. 2002), OW (Daprato et al. 2007),

and Biodechlor Inoculum (BDI) (Ritalahti et al. 2006) were amended with



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TCE, cDCE, or VC and challenged with 1,1,1-TCA, 1,1-DCA or CF to determine the extent of the inhibitory effects (Chan et al. 2011). These three cultures contain Dehalococcoides and express the vinyl chloride reductase, VcrA,

but were enriched from geographically different contaminated sites using different electron donors to provide energy (hydrogen) and carbon (acetate) to the

dechlorinating organisms. All three of these mixed cultures include additional

dechlorinating genera beyond Dehalococcoides, such as Geobacter (KB-1 and

BDI) and Dehalobacter (OW). However, none of these enrichment cultures

dechlorinates the suspected inhibiting compounds, 1,1,1-TCA, or CF.

An example of the data that is obtained from kinetic assays is shown in Fig. 13.3

depicting (A) the effect of 1,1,1-TCA on VC dechlorination in whole cell suspension (CS) assays, as well as (B) the effect of TCE on CF dechlorination in cell-free

extract assays. The inhibition constant, Ki, can be thought of as that concentration of

inhibitor that results in a 50 % decrease in dechlorination rate relative to the uninhibited case: the lower the Ki, the stronger the influence of the inhibitor. In a study

comparing the effect of 1,1,1-TCA on VC dechlorination in three distinct mixed

dechlorinating cultures, a very similar response was observed despite different rates

of dechlorination. The key findings from this study were that 1,1,1-TCA strongly

inhibited VC dechlorination in cell-free extract assays, suggesting that 1,1,1-TCA

specifically interacts with the VC reductase associated with VC-to-ethene reductive dechlorination, i.e., the protein VcrA common to the three cultures (Table 13.1).

An inhibition constant, Ki, of around 2.0 µM (270 µg/L) 1,1,1-TCA was calculated

from these experimental data (Table 13.1). The reductive dehalogenases involved

in cDCE and TCE dechlorination were also inhibited by 1,1,1-TCA, but to a lesser

extent. In sharp contrast, 1,1-DCA had no pronounced inhibitory effects (i.e. large

Ki) on any chlorinated ethene reductive dehalogenases (Table 13.1), indicating that

removal of 1,1,1-TCA via reductive dechlorination to 1,1-DCA is a viable strategy to relieve inhibition (Grostern et al. 2009). Interestingly, 1,1,1-TCA was less

inhibitory to the TCE reductive dehalogenases in consortia BDI and OW suggesting that the reductive dehalogenases in the TCE to cDCE dechlorinating microbes

(Geobacter and Dehalobacter) present in these cultures may be slightly less inhibited by 1,1,1-TCA than Dehalococcoides—which was actually confirmed using a

Geobacter enrichment from KB-1 (Chan et al. 2011).



13.4.2 Inhibition of Chloroalkane Reductive Dehalogenases

by Chlorinated Alkenes

Inhibition was also found to occur in the reverse direction, where chlorinated

ethenes inhibit the reductive dechlorination of chlorinated alkanes. These experiments were conducted using the Dehalobacter-containing enrichment culture

referred to as ACT-3 (Fig. 13.2) that dechlorinates 1,1,1-TCA, 1,1-DCA, and

CF, but none of the chlorinated ethenes (Grostern et al. 2009). Cell-free extracts

of ACT-3 were amended with the substrates 1,1,1-TCA, 1,1-DCA or CF, and



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K. Wei et al.



Fig. 13.3  Michaelis Menten kinetics for two distinct enrichment cultures in the presence of

inhibitors. Panel A Kinetics of vinyl chloride (VC) dechlorination in cell suspensions of Dehalococcoides-containing culture KB-1 in the presence of increasing concentrations of the inhibitor

1,1,1-TCA. Panel B Kinetics of chloroform (CF) dechlorination in cell free extracts of Dehalobacter-containing culture ACT-3 in the presence of increasing concentrations of the inhibitor

TCE. Symbols are experimental data and solid lines represent the best fit to each data set based

on nonlinear regression to a competitive model (panel A) or uncompetitive model inhibition

model (panel B). Adapted from Chan et al. (2011) and Wei (2012)



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Table 13.1  Ranked inhibition constants for chloroethene dechlorination inhibited by chlorinated

ethanes and methanes



Culture

Substrate Inhibitor Preparation

Cell Suspensions (Competitive Model):

OW

VC

1,1,1-TCA CS

BDI

VC

1,1,1-TCA CS

a

KB-1

VC

1,1,1-TCA CS

Cell Suspensions (Non-Competitive Model):

KB-1

TCE

CF

CS

KB-1

VC

CF

CS

Cell-Free Extracts (Non-Competitive Model):

KB-1

TCE

1,1,1-TCA CFE

KB-1

TCE

CF

CFE

KB-1

VC

1,1,1-TCA CFE

OW

VC

1,1,1-TCA CFE

BDI

VC

1,1,1-TCA CFE

KB-1/Dhc

TCE

1,1,1-TCA CFE

KB-1

VC

CF

CFE

KB-1/Geo

TCE

1,1,1-TCA CFE

BDI

cDCE

1,1,1-TCA CFE

KB-1

cDCE

CF

CFE

KB-1

cDCE

1,1,1-TCA CFE

OW

TCE

1,1,1-TCA CFE

BDI

TCE

1,1,1-TCA CFE

OW

cDCE

1,1,1-TCA CFE

OW

VC

1,1-DCA

CFE

BDI

cDCE

1,1-DCA

CFE

OW

cDCE

1,1-DCA

CFE

BDI

VC

1,1-DCA

CFE

KB-1

VC

1,1-DCA

CFE

KB-1

cDCE

1,1-DCA

CFE

KB-1

TCE

1,1-DCA

CFE



Ki ± 95%CI

µM



µg/L (ppb)



0.2 ± 0.1

0.4 ± 0.1

0.8 ± 0.2



33 ± 11

58 ± 13

100 ± 27



6.8 ± 0.3

59 ± 4.9



800 ± 38

7000 ± 585



1.5 ± 0.6

2.0 ± 0.1

2.0 ± 0.3

2.0 ± 0.4

2.1 ± 0.4

2.2 ± 0.6

4.2 ± 0.2

5.1 ± 2

5.5 ± 0.8

11 ± 0.9

19 ± 4

40 ± 9

43 ± 17

86 ± 17

104 ± 24

110 ± 18

130 ± 57

162 ± 39

224 ± 111

830 ± 280

No inhibition



210 ± 74

240 ± 13

270 ± 40

270 ± 50

280 ± 50

300 ± 75

500 ± 26

690 ± 260

730 ± 110

1300 ± 110

2,500 ± 600

5,300 ± 1,200

5,800 ± 2,300

11,500± 2,300

10,300± 2,400

11,000± 1,800

13,000± 5,600

16,000± 3,700

29,700±11,000

82,000±28,000

No inhibition



Data compiled from Chan et al. (2011) for TCA and DCA and Wei (2012) for CF. The noncompetitive model was typically the best fit for all cell-free extract experiments

CFE Cell-free extract

CS Whole (resting) cell suspension

KB-1/Dhc (Highly enriched Dehalococcoides subculture of KB-1)

KB-1/Geo (Geobacter enrichment from KB-1 dechlorinating PCE to cDCE only)

Shaded rows at the bottom of the table highlight high inhibition constants with 1,1-DCA, and

thus little to no inhibition by this compound

aData set plotted in Fig. 13.3 panel A



challenged with individual chlorinated ethenes to determine the extent to which

TCE, cDCE or VC inhibited reductive dechlorination (Grostern et al. 2009). In

­particular, VC was found to profoundly inhibit chloroform reductive dechlorination. The inhibition constant Ki was estimated to be as low as ~0.6 µM (40 µg/L)



K. Wei et al.



296



Table 13.2  Kinetic parameters (Vmax, Km and Ki) for 1,1,1-TCA, 1,1-DCA and CF reductive

dechlorination in cell-free extracts and resting whole cell suspensions of a Dehalobacter enrichment culture in the presence of chlorinated ethenes

Substrate



Inhibitor



Preparation



1,1,1-TCA

1,1,1-TCA

1,1,1-TCA

1,1-DCA

1,1-DCA

1,1-DCA

CF

CF

CF

CF

1,1,1-TCA

1,1,1-TCA

1,1,1-TCA

1,1-DCA

1,1-DCA

1,1-DCA



TCE

cDCE

VC

TCE

cDCE

VC

TCE

cDCE

VC

VC

TCE

cDCE

VC

TCE

cDCE

VC



CFE

CFE

CFE

CFE

CFE

CFE

CFEa

CFE

CFE

CS

CS

CS

CS

CS

CS

CS



Vmax (nmol/

min/mg)

102 ± 7

86 ± 11

73 ± 8

63 ± 6

44 ± 5

53 ± 3

19 ± 0.63

23 ± 1.0

24 ± 1.0

9.4 ± 0.12

4.5 ± 0.3

5.0 ± 0.3

3.0 ± 0.3

2.2 ± 0.2

2.2 ± 0.1

5.6 ± 0.4



Km

(μM)

42 ± 6

34 ± 10

33 ± 8

46 ± 64

289 ± 68

396 ± 42

22 ± 2.4

17 ± 3.4

23 ± 4.0

1.5 ± 0.11

14 ± 3.0

11 ± 3.0

13 ± 4.0

87 ± 30

147 ± 15

192 ± 26



Ki

(μM)



Ki

(µg/L)

42 ± 6 5500

126 ± 38 12,000

35 ± 8 1600

No inhibition

No inhibition

No inhibition

40 ± 3.1 5300

6.7 ± 0.70

650

0.56 ± 0.052

35

8.4 ± 1.1

530

242 ± 124 32,000

872 ± 498 85,000

228 ± 167 14,000

No inhibition

189 ± 27 18,000

83 ± 19 5200



Data compiled from Grostern et al. (2009) for substrates 1,1,1-TCA and 1,1-DCA and Wei

(2012) for CF

The best fit for all data was to an uncompetitive model

Error values represent 95 % confidence intervals

CFE Cell-free extract

CS Whole cell suspension

aData set plotted in Fig. 13.3 panel B



in cell-free extracts (Table 13.2). The strong inhibition of CF dechlorination by

VC is consistent with the problematic persistence of CF at contaminated sites.

Moreover, although the same enzyme catalyzes both 1,1,1-TCA and CF dechlorination, 1,1,1-TCA dechlorination was significantly less inhibited by VC and cDCE

(Table 13.2) than CF. These observations are consistent with unusual isotope

fractionation behavior with these same reactions (Chan et al. 2012) and suggests

a rather strong interaction between 1,1,1-TCA and the enzyme. In contrast, none

of the chlorinated ethenes inhibited 1,1-DCA dechlorination in cell-free extract

assays. This is consistent with the proven existence of two distinct Dehalobacter

strains in the ACT-3 culture, one that dechlorinates 1,1,1-TCA and the other 1,1DCA. One strain produces a reductive dehalogenase (CfrA) that dechlorinates

1,1,1-TCA and CF, but not 1,1-DCA, while the other strain produces a reductive

dehalogenase (DcrA) that dechlorinates 1,1-DCA but not 1,1,1-TCA nor CF (Tang

and Edwards 2013). These enzymes are very similar at the sequence-level, and

thus kinetic observations point to ways that one can begin to investigate mechanisms of substrate interactions and protein structure in more detail.



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13.5 Inhibition in Whole Cell Assays

Once inhibition is quantified in cell-free extracts, the next step is to extend the

analysis in order to determine if the inhibiting substance might have an effect on

critical cell components other than reductive dehalogenases. For this, whole CS

assays can be used. These assays are similar to CFE assays except that concentrated, intact cells are used instead of disrupted cells. Hydrogen or another physiological electron donor is provided instead of reduced methyl viologen and the

assay is run in growth medium, not lysis buffer. The reaction is initiated with

addition of the electron acceptor and is terminated 1–3 h later, before any significant cell growth has occurred, hence these assays are sometimes called “resting

cell assays”. Cell growth can be assumed to be negligible over a period of 3 h or

less because OHRB tend to have relatively long doubling times on the order of

0.8–3 days (Grostern et al. 2010; Loffler et al. 2013; He et al. 2007). This assay

will detect any inhibition on dehalogenases as in CFE assays, but inhibition will

be modulated by the presence of other cellular components, such as intact cytoplasmic and outer membranes, that might alter the rate-determining step. Since

these assays require a functional electron transport chain, inhibition of hydrogenases may also be detected in CS assays. One of the advantages of whole CS

assays is that the kinetic constants are more relevant for use in models of microbial growth and dechlorination since the rate constants can be normalized to the

number of dechlorinating organisms present, and the inhibition and half-saturation

constants are completely transferrable to models involving growing cells. The

next two sections (13.5.1 and 13.5.2) illustrate some of the features of whole cell

assays.



13.5.1 Cross-Inhibitory Effects Examined in Whole Cell

Suspension Assays

The cross-inhibitory effects of chlorinated ethenes and alkanes described in

Sects. 13.4.1 and 13.4.2 were also investigated resting CS assays (Grostern et al.

2009; Chan et al. 2011; Wei 2012). The key findings in CS assays as well as the

comparison with the results obtained from cell-free extract assays are summarized

in this section.

Whole CS assays were used to determine the inhibitory effects of chlorinated

ethanes on TCE sequential reductive dechlorination. The smallest most potent

inhibition constants, ranging from 0.3 or 0.8 µM (40–100 µg/L) were measured

for 1,1,1-TCA inhibiting VC dechlorination in these assays. As the inhibition

constants were similar between CS assays and cell-free extract (CFE) assays, the

data suggest that 1,1,1-TCA acts directly on the reductive dehalogenase enzyme

system and does not exert a general toxic effect of Dehalococcoides cells (Chan

et al. 2011). When all experiments and associated uncertainties were considered,



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K. Wei et al.



the inhibition constant for 1,1,1-TCA on VC ranged between 30–270 μg/L. This

range represents the threshold where VC dechlorination rates will be significantly

impacted relative to conditions without 1,1,1-TCA, providing direct guidance to

site managers (Chan et al. 2011).

Whole CS assays were also used to examine the inhibitory effects of chlorinated alkenes on the reductive dechlorination of chlorinated ethanes by a

Dehalobacter-containing culture, ACT-3. A comparison of the cell-free extract

and whole CS data revealed that inhibition was less pronounced in whole CSs

compared to cell-free extracts for both CF and 1,1,1-TCA, as shown by higher Ki

values by an order of magnitude in whole CSs (Table 13.2). For example, the Ki

measured for VC inhibition of CF dechlorination was ~8 µM (500 µg/L) versus

0.6 µM (40 µg/L) in cell-free extracts. These findings suggest that when the reductive dehalogenase activity is assayed in intact cells of Dehalobacter, the membrane offers some form of protection against inhibition. In these experiments, the

rate of the dehalogenation reaction on a total protein basis (V0) was much faster

in cell-free extracts (where methyl viologen is electron donor) than in CS assays

(Table 13.2), suggesting that electron transport from H2 and not dechlorination

is rate-limiting in whole cell assays. This observation has also been reported by

others (Nijenhuis and Zinder 2005), and can certainly confound interpretation of

results. In whole cell assay, VC was much less inhibitory to 1,1-DCA dechlorination compared to 1,1,1-TCA or CF, with a Ki ~ 80 µM or 8 mg/L (Table 13.2).

Although relatively high, a Ki of 8 mg/L is nonetheless a concentration relevant to

some DNAPL sites and certainly relevant to enrichment cultures where inhibition

was indeed observed (Grostern and Edwards 2006a). As illustrated, these whole

cell inhibition constants are a useful guide to determine when inhibition needs to

be considered and when it is appropriate to omit.



13.5.2 Inhibition Model Type and Fit and Underlying

Mechanisms

The kinetic model that best fit the data presented in the examples (Tables 13.1 and

13.2) was not the same. In experiments with chlorinated ethene-dechlorinating cultures (Dehalococcoides-dominated), the noncompetitive model fit best, except for

in assays for 1,1,1-TCA inhibition with whole cell where the competitive model

fit better (Table 13.1). Perhaps different rate-limiting steps or interactions were

interrogated in cell free versus whole cell assays. The ability to truly discriminate

between different models is a function of the number of experiments conducted

and reproducibility, and thus one should be cautious in over-interpreting the data.

Nevertheless, noncompetitive inhibition assumes that the inhibitor binds somewhere other than at the active site, which is a likely interaction in the light of the

recent 3D structure of a reductive dehalogenase, suggesting a substrate-tailored

opening into a cavity containing the active site (see Chap. 20). It may be that 1,1,1TCA and CF, being nonplanar, occlude this opening in chlorethene reductases.



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In all of the experiments conducted with the Dehalobacter-containing ACT-3

culture, the best fit of the data was to the uncompetitive model, which assumes

that the inhibitor binds only to the enzyme–substrate complex (Table 13.2). The

differences in inhibitory mechanism suggested by model fit may simply reflect

experimental error and not reflect a governing underlying principle. But perhaps

they may point to real mechanistic differences and avenues for future research.

The diversity of reductive dehalogenase sequences and associated proteins provides ample possibility for differences in protein interactions. It is clear that each

enzyme will have different kinetic constants and different interactions with potential inhibitors. A better understanding of the active enzymes in a given system, and

their structures, will ultimately improve our fundamental understanding of inhibition and how to represent these phenomena in models.



13.6 Inhibition in Actively Growing Microbial Cultures

and Communities

Ultimately, the need is to quantify and predict the effects of inhibitors during

active culture growth as it occurs in laboratory batch and continuous experiments,

packed flowing columns, and especially in engineered or natural systems in the

field (Schaefer et al. 2008). The approach outlined herein of starting from CFE

and resting CS assays to deconvolute simultaneous effects can substantially narrow down the experimental conditions and concentration ranges that then need to

be tested in more complex realistic situations. The examples provided illustrating the specific effects between chlorinated ethanes, methanes, and ethenes have

revealed interesting patterns of cross-inhibition relevant to both the scientific

understanding of the underlying microorganisms and their respective reductive

dehalogenases, as well as to the application of dechlorinating cultures for bioaugmentation. There are certainly other experimental approaches to control variables

and extract meaningful kinetic data from mixed cultures, such as the batch multiequilibration method demonstrated by (Yu et al. 2005), which also minimizes

growth. As it has become easier to also monitor microbial populations and proteins

in cultures and field samples, these approaches will be informed with the knowledge of the actual concentration of OHRB and the identity of the specific proteins

expressed resulting in more accurate, specific, and transferrable kinetic parameters

and half-saturation and inhibition constants for use in models and hypothesis testing. Researchers should be encouraged to collect such information for their systems. The various models described in Sect. 13.2.1 already are extensively used

because they provide useful frameworks to integrate complex simultaneous phenomena and to improve conceptual understanding of site data and remediation

performance. Understanding the molecular basis for inhibition is improving significantly with new developments in microbial community analysis and protein

characterization, and this knowledge will certainly translate to improved predictive

ability and utility of modeling efforts.



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K. Wei et al.



In this chapter, we have so far only surveyed data for chlorinated ethenes, chloroform, and two chlorinated ethanes. Needless to say, many more interactions are

possible among the myriad of chemicals that are present in the environment and at

contaminated sites. Many investigations have revealed other complex patterns of

inhibition that are often difficult to understand. In a study with chlorinated ethanes

with halogens on vicinal carbons, such as 1,1,2-TCA and 1,2-DCA that tend to be

dechlorinated via dihaloelimination (see Fig. 13.1), 1,2-DCA was not dechlorinated by Dehalogenimonas until 1,1,2-TCA reached low concentrations (Dillehay

et al. 2014). Lai and Becker (2013) used a dual Monod model to predict population abundance and survival of two PCE-dechlorinating genera by incorporating

PCE and TCE inhibition on VC dechlorination by Dehalococcoides, and of VC

inhibition of PCE and TCE dechlorination by Dehalobacter in the coculture.

Considering more complex molecules beyond chlorinated aliphatics, such as

chlorinated and brominated aromatic compounds (He et al. 2006), polychlorinated biphenyls (PCBs) (Demirtepe et al. 2015), halogenated pesticides, dioxins

(Häggblom and Bossert 2003), and many more described in this book, the possible combinations of substrates and inhibitors become truly daunting. Complex patterns of dechlorination have been observed with chlorinated benzenes and toluenes

(Nelson et al. 2014) and PCBs (Bedard 2008; Demirtepe et al. 2015), underlying

multiple strains and species that carry out distinct reactions. Nevertheless, these

mixed microbial populations can and eventually dechlorinate these compounds.

Knowledge of these interactions can help to promote more productive pathways.

A key factor in the transformation of some of the more chlorinated hydrophobic

compounds is that their concentrations are typically limited by the low aqueous

solubility of the parent compound and thus may not reach critical thresholds for

inhibition, meaning that slow but steady dechlorination can persist. So while on

the one hand low aqueous concentrations means slower dehalogenation, it may

also permit a greater diversity of reactions and ultimately more complete dehalogenation. In these slower systems with lower concentrations of substrates, competition for nutrients beyond the halogenated electron acceptor plays a major role, as

introduced in the next section.



13.7 Cross-Feeding and Competition in Anaerobic

Dehalogenating Microbial Communities

In the context of microbial ecology, intraspecies and interspecies competition

exists within anaerobic dehalogenating microbial communities, which may contribute to observed inhibitory effects on reductive dechlorination at contaminated

sites and in other environments (Chap. 14). In many of the described anaerobic

dehalogenating microbial communities, more than one OHRB population is stably maintained. Fermenting and acetogenic bacteria are also critical for providing essential electron donors, co-factors, and nutrients. Moreover, other groups of

anaerobes, such as iron-reducing and sulfate-reducing bacteria, have been found



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