Monografia Microbial Degradation of Chloroform

Microbial Degradation of Chloroform

Case Study: Bioaugmentation with Distinct Dehalobacter

Strains Achieves Chloroform Detoxification in Microcosms

Prof. Cristina Viegas

Ângela Neves, 79247

Mafalda Cavalheiro, 79229

Sara Gomes, 79230

24th March of 2014

MASTER DEGREE IN BIOTEHCNOLOGY

Environmental Biotechnology

2013/2014



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Index

Abstract ......................................................................................................................................... 3

Abbreviations................................................................................................................................ 4

1. Introduction .......................................................................................................................... 5

1.1. Physical and chemical properties of chloroform .......................................................... 5

1.2. Former and recent applications of CF ........................................................................... 6

1.3. Chloroform sources and release to the environment ................................................... 6

1.3.1. Natural and anthropogenic CF sources ..................................................................... 6

1.3.2. CF production and contaminated sites ..................................................................... 7

1.4. Chloroform toxicity and regulation ............................................................................... 8

2. Aerobic degradation of CF .................................................................................................... 9

3. Anaerobic degradation of CF .............................................................................................. 10

3.1. Case study: Bioaugmentation with Distinct Dehalobacter Strains Achieves Chloroform

Detoxification in Microcosms .................................................................................................. 12

3.1.1. Culture Dhb-CF (“CF-to-DCM-respiring Dehalobacter strain”) ........................... 13

3.1.2. Dehalobacter sp. strain RM1 ............................................................................... 13

3.1.3. Results ................................................................................................................. 13

4. Other bacterial strains involved in CF biodegradation ..................................................... 15

4.1. Desulfitobacterium sp. strain PR ................................................................................. 15

4.2. Pseudomonas stutzeri KC ............................................................................................ 15

5. Critical analysis ................................................................................................................... 16

6. Conclusion ........................................................................................................................... 17

7. References .......................................................................................................................... 18



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Abstract

Chloroform (CF) is a volatile organic compound (VOC) largely produced by both

anthropogenic and natural sources. The fact that CF is detected in ground and surface water

leads to both environmental and human health concerns. In order to achieve CF detoxification,

there is a need to develop new strategies, not only to degrade CF but also other CF-derived

toxic compounds. One possible strategy is the microbial degradation of CF, which occurs under

both aerobic and anaerobic conditions. Regarding the aerobic process, it involves a particular

type of enzyme family (monooxygenases) which catalyzes CF together with other substrates by

co-metabolism. Under anaerobic conditions, CF can be biodegraded by three main pathways:

co-metabolic reductive dechlorination, hydrolysis followed by oxidation to CO2 and

dehalorespiration.

In this review, we focus on a case study related to anaerobic CF-dehalorespiration

performed by two distinct Dehalobacter consortiums, Dhb-CF and RM. The first consortium is

able to respire CF to dichloromethane (DCM), while the second one is capable of degrading

DCM into acetate and inorganic chloride by fermentation. The results of this study are

promising since full detoxification was achieved in microcosms bioaugmented with both

strains. However, there are aspects that need to be improved before implementing this

strategy, in particular, overcoming the inhibitory effect of carbon tetrachloride (CT) on

organohalide respiration.



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Abbreviations

CT Carbon Tetrachloride

CAHs Chlorinated Aliphatic Hydrocarbons

CF Chloroform

CM Chloromethane

DCM Dichloromethane

HCFC-22 Monochlorodifluoromethane

MOs Monooxygenases

NADH Nicotinamide Adenine Dinucleotide

qPCR quantitative real-time Polymerase Chain Reaction

TCA 1,1,1-Trichloroethane

TCE Trichloroethene

THMs Trihalomethanes

VOCs Volatile Organic Compounds

US EPA United States Environmental Protection Agency

ZVI PRB Zero-Valent Iron Permeable Reactive Barrier



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1. Introduction

1.1. Physical and chemical properties of chloroform

Chloroform (usually abbreviated as CF), also known as trichloromethane or formyl

trichloride, is a compound included in the VOCs group. Its molecular formula is CHCl3 and its

molecular geometry is tetrahedral (Figure 1) 1.

Figure 1. Tridimensional structure of Chloroform. Adapted from Grostern, et al. (2010)

CF is a clear and colorless liquid that is highly refractive and heavy volatile. Its boiling

point is 61,17⁰C and its melting point is -63,41⁰C. As for its solubility, CF is soluble in carbon

disulfide and miscible with alcohol, ether, benzene, carbon tetrachloride, fixed and volatile

oils. In addition, CF is highly soluble in water, 7,95g/L at 25⁰C 2. Another important propriety of

CF is its octanol/water partition coefficient which is 1,97. These properties indicate that CF

bioconcentration should have low relevance and that this compound should be mostly present

in aquatic ecosystems.

CF is known to be reactive with acetone, powdered aluminum, sodium, dinitrogen

tetraoxide, fluorine, sodium metal and alcohols, nitromethane, and triisopropylphosphine 3.

Other proprieties of CF can be observed in Table I 4.

Table I – Chloroform physical properties (EXP – experimental values).



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1.2. Former and recent applications of CF

On 4 November 1847, the Scottish obstetrician James Young Simpson discovered the

anesthetic qualities of CF when he and his friends were experimenting with different

substances on themselves in search of a replacement for ether as a general anesthetic. He was

so astounded by the success of his own trial that the next morning he hired a chemist and

within the next few days was administering it to his patients during childbirth. The use of CF

during surgery expanded rapidly thereafter in Europe. In the 1850s, CF was used during the

birth of Queen Victoria's last two children 5.

CF was banned from consumer products in 1976 by the Food and Drug Administration

due to the finding that CF was carcinogenic in laboratory animals. However, its use as an

industrial solvent almost doubled from 1980 to 1990 6.

Nowadays, CF still has a variety of applications. The mainly application is the use of CF

to produce monochlorodifluoromethane (HCFC-22) and in the production of fluoropolymers 7.

HCFC-22 has different uses, such as in refrigerant, fire-fighting material, foam blowing agent,

and it is an ozone depleting substance. The use of HCFC-22 has been regulated under the

Copenhagen Amendment (1992) to the Montreal protocol, for Europe8.

CF has other applications such as production and extraction solvent, especially in the

pharmaceutical industry for the extraction of penicillin and other antibiotics. CF is also used as

a degreasing agent and as a chemical intermediate for the production of different substances

such as dyes or pesticides 8.

1.3. Chloroform sources and release to the environment

1.3.1. Natural and anthropogenic CF sources

An estimated 700 Gg of chloroform are released per year, and only 9,5% is originated

from anthropogenic sources 9. These sources include pulp and paper manufacturing, water

treatment, biomass burning (e.g. wood and charcoal burning), biogas pits, incineration, cooling

water treatment, and other types of industries like the pharmaceutical industry 9,10 . In 1998,

the most recent year with production data, about 46 million gallons of CF were produced by

industry 11.

Natural sources of chloroform include termite mounds, landfills and ruminants, forest

soil, volcanism, gas emittion by the Earth’s crust, deciduous mosses, drill wells, mine gas and

http://en.wikipedia.org/wiki/Obstetrics

http://en.wikipedia.org/wiki/James_Young_Simpson

http://en.wikipedia.org/wiki/Diethyl_ether

http://en.wikipedia.org/wiki/General_anaesthesia

http://en.wikipedia.org/wiki/Childbirth

http://en.wikipedia.org/wiki/Surgery



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minerals, and even some terrestrial plants and fungi capable of producing chloroform from

inorganic chloride 9,10. Soil processes generate around 200 Gg yr-1 of CF, and fungi might be the

main source of chloroperoxidase in the soil, an enzyme capable of producing CF in the

presence of hydrogen peroxide and the chloride ion 10. Oceans have been estimated to

account for about half of the annual emission of CF into the atmosphere (340 Gg yr-1), and

micro and macroalgae (e.g. brown, red and green seaweed) have been found to release CF into

the surrounding water 9. Besides these natural sources, CT abiotic and biotic transformation

also contributes to CF formation and release 12. The interaction of CT with electron transfer-

active biomolecules can yield the trichloromethyl radical, which leads to unspecific

transformation reactions and, consequently, CF formation 13.

1.3.2. CF production and contaminated sites

Chloroform and other trihalomethanes (THMs) are commonly generated by the

haloform reaction during water chlorination when chlorine interacts with organic material

dissolved in water 11. In addition, sodium hypochlorite, gaseous chlorine, and chloramines

(commonly used in public systems as disinfectants) may react with organic matter in water to

generate CF and CT 11.

Most CF entered the environment as a consequence of improper handling, storage,

and disposal practices 12. Nowadays, CF is a common contaminant found at many superfund

sites and has been detected in urban air and in rainwater 11. Industrial effluent, municipal

waste treatment plant discharge, hazardous waste site, sanitary landfills and spills are the

major source of CF to the environment 14. In urban areas, it is presumed that released treated

drinking water or wastewater, moves through the unsaturated zone to deeper groundwater

supplying public wells, thus contaminating them with CF. In a study where the presence of

THMs in various samples of untreated groundwater in public and domestic wells across the

United States was analyzed, CF was detected in groundwater beneath a broad range of land-

use settings, including agricultural, mixed, undeveloped, and, more frequently, urban 11. For

the same study, when no assessment level was used, CF was detected in 44,4% of public-well

samples and 24,2% of domestic-well samples. Although most of the THMs concentrations

encountered did not pose a substantial human health hazard, these results show how

widespread CF is.



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1.4. Chloroform toxicity and regulation

Under the subject of eco-toxicity, CF is much more of a risk to aquatic organisms than

for soil organisms, since its concentration is higher in aquatic environments. However, it is

considered that the general concentrations of CF do not present a real problem for these

organisms 10.

Several acute and prolonged toxicity tests were conducted for aquatic vertebrates,

such as fish and amphibians. In these studies, the LC50 range was from 18 mg CF/L, for

Oncorhynchus mykiss, to 171 mg CF/L, for Pimephales promelas 8. Also, a relevant study for

chronic toxicity of CF was done in Japanese Medaka Fish. This fish had continuous exposure to

CF, in concentration levels similar to the ones found in drinking water, for 9 months. The

results revealed hepatocellular proliferation, gallbladder and bile duct abnormalities, and

chronic hyperplasia, for a concentration of 1,463 mg CF/L 15. Since the concentration of CF in

water is generally not so high, it is possible that CF may not present a greater problem for

these aquatic organisms. However, since there are not many valid chronic studies, adverse

chronic effects may still occur.

CF is also a potent inhibitor of several microbial processes such as methanogenesis and

detoxification of other chlorinated organic compounds 12.

As for humans, CF is mainly absorbed by inhalation and can cause different effects,

such as general anesthesia, changes in structure or function of salivary glands, nausea,

vomiting, anorexia, hallucinations, and others 4.

As a result of the potential hazard of CF, this compound has regulation to prevent its

negative effects. CF regulation differs for each country. In the United States, CF is considered

as a possible carcinogenic substance to humans. The United States Environmental Protection

Agency assessed a maximum contaminant level for CF equal to 80 µg/L for drinking water, a

value that has been applied since 1979 11,16. In Europe, the maximum value for CF, which is

2,5μg/L, has been established in the framework of water policy (Directive 2008/105/EC) 17.

The present review provides an overview of CF microbial degradation. The first part

focuses on CF aerobic biodegradation with details on the different enzymatic systems involved

in CF co-metabolism. The second part describes the CF anaerobic transformation with special

attention on a particular case study.



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2. Aerobic degradation of CF

Usually, CF aerobic degradation requires a co-metabolic process, with the only partial

exception to this rule being the aerobic Bacillus sp. 2479. This strain showed the capacity to

dechlorinate CF as the sole energy and carbon source, although no consistent data on

microbial growth and CF degradation were reported 18.

Co-metabolism is the transformation of a non-growth substrate (co-substrate) that can

be metabolized after the microorganism has grown on a primary substrate (carbon source)19.

Growth on the physiological substrate induces the production of enzymes that catalyze the

degradation of the co-substrate and that provide energy to drive reactions to transform it. The

degradation of chlorinated aliphatic hydrocarbons (CAHs) by co-metabolism involves the

activity of nonspecific oxygenases which utilize O2 and reducing power (NADH, nicotinamide

adenine dinucleotide) to oxidize both the growth and the co-metabolic substrates 20. CF co-

oxidation is catalyzed by monooxygenases (MOs) which introduce one oxygen atom into the

substrate that is oxidized, while another oxygen atom is reduced to H2O utilizing NADH as a

reducing agent (Figure 2) 21.

The MOs able to co-oxidize CF can typically catalyze the same reaction on a large

number of other CAHs, although the same MO can show different activities for different

chlorinated compounds 6. Regarding CF degradation, all the studies that investigated chloride

release reported a complete conversion of organic chlorine to chloride ion (Figure 2) 22. This

conversion of organic chloride to CO2 and HCl (or chloride ion) represents a full detoxification

of chloroform to innocuous products, a strategy which might be applied to aerobic

contaminated sites.

Compounds such as methane, propane, butane, hexane, toluene, ammonia and

acetone can support CF co-metabolism. On the other hand, phenol oxidizers showed poor

ability to co-metabolize CF, while they were effective in transforming chlorinated ethenes 6.

The chemical mechanism of CF oxidation is, thus, variable among the various MOs that show

different degradation abilities and co-substrate ranges 23.

The rate of CF co-metabolism by oxygenase-expressing bacteria is affected by: (1)

enzyme competition between primary and co-substrate, and between different co-substrates

when they are contemporarily present; (2) toxicity of CF degradation intermediates on cellular

metabolism; and (3) reducing energy consumption resulting from CF transformation (Figure

2)24.



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Figure 2. CF microbial degradation under aerobic conditions. The co-metabolic reactions performed by the MO on

both the growth substrate and CF are shown along with the hypothetical steps leading to the production of

phosgene from CF. Adapted from Cappelletti et al. (2012).

3. Anaerobic degradation of CF

Under anaerobic conditions, CF is mainly degraded through co-metabolic processes.

Methanogens, sulfate reducers, fermenting bacteria such as Clostridium spp. and Enterobacter

spp., and one homoacetogenic strain, Acetobacter woodii, can co-metabolically degrade CF.

Recently, CF dechlorination was linked to the growth of bacterial strains belonging to

Dehalobacter genus through a CF dehalorespiration process 1,25,26. As shown in Figure 3, three

main CF anaerobic biodegradation pathways are reported in the literature: co-metabolic

reductive dechlorination, hydrolysis followed by oxidation to CO2 and dehalorespiration.

CF co-metabolic dechlorination (Figure 3 pathway 2) often resulted in the

accumulation of DCM, as reduction of DCM to CM (chloromethane) and methane was

observed at very low rates. For example, in a study related to CF dechlorination by

Methanosarcina spp., 65 % of the theoretical DCM was obtained 27, while a Clostridium sp.

completely degraded CF with the accumulation of 20% of the theoretical DCM, which was not

further degraded 28.

Pathways 3a and 3b in Figure 2 represent oxidative routes in which CF, acting as the

electron donor, is oxidized to CO2. Indeed, CO2 was found to be the product of CF

transformation performed by methanogens, acetogenic and fermenting bacteria 6. As several

studies reported the formation of CO along with CO2, it was suggested that oxidation of CF to

CO2 can proceed either via net hydrolysis of CF to CO (Fig. 2 pathway 3a) or via the formation



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of an intermediate (mono- or dichlorocarbene) that can be hydrolyzed and finally oxidized to

CO (Figure 3 pathway 3b) 29. In both cases, CO was suggested to be further oxidized to CO2 6.

Dehalorespiration of CF (Figure 3 pathway 1) was observed in two mixed cultures

containing mainly Dehalobacter species, when H2 and acetate were added as electron donor

and carbon source, respectively. These microbial communities utilized CF as the final electron

acceptor, transforming it into DCM 1,25. DCM was further fermented to H2, CO2, and acetate by

another microbial community also containing Dehalobacter species 13,25.

In all the above listed CF degradation pathways, the products of CF transformation

depend on the type of reducing environment, growth substrate, organisms present, CF

concentration and coenzymes 6. For instance, the addition of methanol stimulated DCM

production from CF transformation by Methanosarcina barkeri 30, while vitamin B12 stimulated

the conversion of CF to CO2 in methanogens and acetogens 29.

Figure 3. CF microbial degradation pathways under anaerobic conditions. 1 – dehalorespiration (CF is utilized as the

final electron acceptor when an electron donor and a carbon source are supplied); 2 - co-metabolic reductive

dechlorination; 3a - direct CF hydrolysis to CO; 3b - hydrolysis of the mono- or dichlorocarbene hypothetically

deriving from CF dechlorination. Square brackets show unstable intermediates. Adapted from Cappelletti et al.

(2012)

Although transformation of CF as a result of abiotic and co-metabolic anaerobic processes

has been reported, these reactions are generally slow and difficult to manipulate in situ 12.



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3.1. Case study: Bioaugmentation with Distinct Dehalobacter Strains

Achieves Chloroform Detoxification in Microcosms

The main objective of this study was to explore the feasibility of achieving total CF

detoxification with a Dhb-CF culture and RM consortia. For this purpose, a microcosm

treatability study was conducted with aquifer materials collected from a CF-contaminated site

and with phosphate-buffered mineral salts medium. The treatability study presented herein

was accompanied by the development and implementation of molecular biological tools that

served to track the two distinct organohalide respiring and organohalide fermenting

Dehalobacter populations contributing to CF detoxification. Particularly, a quantitative real-

time PCR (qPCR) approach involving the measurement of the abundance of Dehalobacter 16S

rRNA gene copies was applied. This approach allowed the detection of exclusively the strains

of interest since it involved the design of primer/probe combinations that differentially

amplified a 16S rRNA gene fragment of the CF-to-DCM-respiring Dehalobacter strain and the

Dehalobacter sp. strain RM1.

To control and remediate a large CT plume, a zero-valent iron permeable reactive

barrier (ZVI PRB) was installed (Figure 4). This barrier allows the removal of CT by creating a

physical barrier permeable to water downstream of the plume of contamination that aims to

"filter" contaminants that pass through it and promote treatment through chemical reactions.

CT is rapidly degraded (t1/2 = 2 to 4 hours) in the presence of ZVI via two reaction pathways: (1)

one-electron transfer yielding dichlorocarbene intermediates (phosgene) followed by

hydrolysis to form CO and H2O; and (2) a two-electron reductive pathway yielding CF and DCM

as the major chlorinated end products 31.

Figure 4. Representation of the strategy implemented by the authors. A ZVI PRB was installed in order to remove

CT. The groundwater was bioaugmentated with two different Dehalobacter-containing consortia (culture Dhb-CF

and consortium RM) to evaluate the biodegradation of both CF and DCM present in increasing concentrations

downgradient of the PBR. Adapted from Justicia-Leon et al. (2014).



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3.1.1. Culture Dhb-CF (“CF-to-DCM-respiring Dehalobacter strain”)

CF-to-DCM-respiring Dehalobacter strain, derived from subsurface soils, revealed to be

capable of achieving complete dechlorination of CF at a rate of 40 µM/day 25. The final product

of this process is DCM, indicating that this strain is not capable of degrading DCM. Although it

cannot degrade DCM, this compound is not inhibitory to CF reductive dechlorination at

concentrations below 200 mg/L 12.

The process of complete CF dechlorination by this culture starts by the respiration of

CF to DCM, with the presence of an electron donor, H2. The observations made from a study

on this culture suggest that the dechlorination process requires syntrophic partners to

maintain low hydrogen partial pressures 25.

Another study revealed that this Dehalobacter population was capable of reductive

dechlorination of 500mM CF to DCM in 29 days. The authors also observed that this

population can respire TCA, probably using the same reductive dehalogenase 1.

3.1.2. Dehalobacter sp. strain RM1

In order to achieve full detoxification, there is a need to use a second strain capable of

degrading DCM. The authors utilized consortium RM, which was derived from pristine Rio

Mayemes sediment and harbors Dehalobacter sp. strain RM1.This strain is capable of

degrading DCM, generating acetate, methane and biomass as products of DCM degradation 12.

In a previous study, DCM degradation was achieved at a rate of 4.0 mg liter-1 per day

and the culture tolerated at least 200 mg liter-1 of DCM without apparent inhibition, however,

CF inhibited both DCM degradation and methane formation even with the lower CF

concentration of 5 mg liter-1, suggesting a high susceptibility of the DCM-degrading

population(s) to CF 13. In this study, Dehalobacter 16S rRNA gene-specific qPCR assays

confirmed that the growth of the Dehalobacter culture was linked to DCM fermentation.

3.1.3. Results

Regarding the detection, differentiation and quantification of both Dehalobacter

strains used in this study, the authors concluded that the native strains present in the

microcosms did not interfere with the monitoring of the CF-dechlorinating and the DCM-

fermenting Dehalobacter strains. Also, both strains could be independently enumerated in the



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microcosms, since the Dehalobacter 16S rRNA gene-specific qPCR assays allowed the two

strains to be distinguished.

In terms of CF degradation, the authors determined that there was no increased CF

removal in microcosm established with site material and groundwater, which indicates that

the indigenous microbes do not have the capability to degrade CF. This hypothesis was

supported by the absence of Dehalobacter 16S rRNA gene sequences in the microcosms. It is

also important to mention that although the PRB significantly reduced the CT concentrations,

groundwater gradient of the PRB contained up to 35,5 µM (5 mg L-1) CT.

The results of the addition of the Dhb-CF Dehalobacter strain to microcosms

established with a completely synthetic, defined mineral salts medium were encouraging,

since stoichiometric transformation of CF to DCM was observed within 32 ± 11 days (Figure

5A). In accordance, qPCR assays indicated that the Dhb-CF strain grew in the medium

(measured via 16S rRNA gene copy increase). Regarding the augmentation of the microcosms

with the DCM-degrading Dehalobacter sp. strain RM1, DCM degradation was observed, but

only after amending the microcosms with 10 mM NaHCO3. DCM was completely degraded

within 106 ± 34 days of bicarbonate addition (i.e., 211 ± 34 days after bioaugmentation) in

microcosms that had been initially amended with CF. The qPCR assay results also suggest that

this strain grows via DCM fermentation (Figure 5B).

Figure 5. Sequential bioaugmentation with the Dhb-CF and the RM consortia in microcosms prepared with

phosphate-buffered mineral salts medium and amended only with CF leads to complete detoxification. A second CF

feeding was added between day 25 and 50; 10 mM NaHCO3 were added near day 125; the first of multiple DCM

feedings was added between day 175 and 200. Augmentation with consortium RM (3% inoculum, v/v) was

performed near day 50. (A) Increase in 16S rRNA gene copies of the CF-to-DCM-dechlorinating Dehalobacter strain

concomitant with reductive dechlorination of CF. (B) Increase in the 16S rRNA gene copies of the DCM-

dechlorofermenting Dehalobacter sp. strain RM1 concomitant with DCM degradation. Adapted from Justicia-Leon

et al. (2014).

A B



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In contrast to the previous results, the addition of the Dhb-CF Dehalobacter strain to

the microcosms prepared with site groundwater indicated that this strain is CT sensitive, due

to the fact that no CF removal was detected, and CF persisted even after an extended 7-month

incubation period. Also, no dechlorination occurred in medium amended with 2.5 mg L-1 (17,75

µM) CT. Since the groundwater contained 5 mg L-1 CT, the authors concluded that CT inhibition

prevented CF reductive dechlorination. These results lead to an important conclusion: CT

removal is a prerequisite for the implementation of anaerobical biological remedies using

culture Dhb-CF.

4. Other bacterial strains involved in CF biodegradation

4.1. Desulfitobacterium sp. strain PR

A recent study announced the discovery of a novel chloroform-respiring isolate,

Desulfitobacterium sp. strain PR 32. Besides chloroform, this strain reductively dechlorinates

1,1,1-trichloroethane (TCA) to monochloroethane, both being growth-supporting electron

acceptors for strain PR. The results obtained by the authors of this study indicate that strain PR

has high dechlorination activity on chloroform. In pyruvate-amended medium, strain PR

dechlorinated ∼ 1,2 mM of chloroform stoichiometrically to predominantly DCM (∼ 1,14 mM)

and a trace amount of monochloromethane (∼ 0,06 mM) within 10 days. Residual chloroform

was ∼ 0,9 μM on day 10 32. The authors stated that strain PR possesses the highest specific

dechlorination rates among known dechlorinators including various Dehalococcoides and

Dehalobacter strains.

4.2. Pseudomonas stutzeri KC

Pseudomonas stutzeri KC is a denitrifying bacterium that co-metabolically degrades

tetrachloride (CT), without producing chloroform (CF), and that can be used for biodegradation

activity, utilizing bioaugmentation and pH control. This strain can mediate rapid CT

degradation to CO2 and non-volatile products. To make sure that no formation of CF is

observed, this strain must be adequately stimulated, especially by the increase of pH.

A bioaugmentation study was performed in a CT impacted aquifer at Schoolcraft with

P. Stutzeri KC. The results of this study revealed a 98% removal of CT over 4 years 33.



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5. Critical analysis

The results obtained by the authors suggest that an augmentation approach that

combines culture Dhb-CF and consortium RM, or other CF-degrading cultures, may be effective

to remediate CF-contaminated sites 12. However, as already mentioned, the CF-respiring

Dehalobacter strain is sensitive to CT leading to an important prerequisite in order to achieve

anaerobic bioremediation of CF, which is CT removal. As it is usual to come across contaminant

mixtures in groundwater (and not CF exclusively), it is important to take this issue in

consideration before implementing a bioaugmentation strategy.

To solve this matter, the implementation of Pseudomonas stutzeri KC strain (capable of

degrading CT) might be helpful in solving the presence of CT in groundwater sites, which

precludes the bioremediation of CF 32.

Another strategy would be to replace culture Dhb-CF by Desulfitobacterium sp. strain

PR, which is capable of degrading CF as well and showed high tolerance on TCE

(trichloroethene) 32. Since strain PR has a higher dechlorination activity when compared to

culture Dhb-CF, this strain might be a better candidate for chloroform degradation in

contaminated plumes. One other positive aspect is the residual substrate concentrations at

the end of dechlorination (0,9μM for chloroform and 1,0μM for TCA), which are close to or

even lower than the drinking water standards set by US EPA 32. However, further studies are

needed in order to determine if this strain is more CT tolerant than culture Dhb-CF and to

validate strain PR as a feasible candidate.

Besides the possibility of microbial inhibition, another great drawback of

implementing bacterial strains as bioremediators are the long periods of time necessary to

achieve full detoxification (several months or even years). In this sense, the development of

chemical treating methods may be a better approach to deal with CF contaminated sites. In a

study where the potential of low frequency ultrasound was evaluated for the remediation of

CF contaminated waters in batch and flow cell treatment, about 7mg/L of CF was degraded

within 60 minutes 14. Although these results show how chemical treatment might be more

effective and less time consuming, few studies related with this topic have been reported.

To sum up, this case study demonstrates how laboratory studies do not always

represent what may happen in real groundwater contaminated sites, as there are many factors

which may influence the results (e.g. presence of inhibitory compounds, competition with

native bacterial strains, lack of nutrients, pH). In particular, we can conclude that the



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phosphate-buffered mineral salts medium prepared by the authors is not a feasible lab-scale

model for CF contaminated groundwater.

6. Conclusion

In the last decade, new knowledge has been acquired related with microbial treatment

of CF, since it is a toxic and highly widespread compound. Different strains capable of

biodegradating CF by anaerobic and aerobic processes have been discovered. While abiotic

and co-metabolic anaerobic processes are generally slow and difficult to manipulate in situ,

successful cases of CF dehalorespiration have been reported. In contrast, chemical treatment

studies have not been as much reported.

The case study described herein reported the usage of two different Dehalobacter

strains in order to achieved complete degradation of CF. As the contaminated groundwater

native strains were not capable of degrading this compound, a bioaugmentation strategy was

necessary. Although the results obtained were promising, these methods cannot be applied

yet, due to microbial dehalorespiration inhibition concerns. In the future, we predict that this

process will be developed enough to be applied to wastewater and groundwater CF-

contaminated sites.



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7. References

1. Grostern, A., Duhamel, M., Dworatzek, S. & Edwards, E. A. Chloroform respiration to

dichloromethane by a Dehalobacter population. Environ. Microbiol. 12, 1053–1060 (2010).

2.http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6212&loc=ec_rcs#x27,

consulted in 15th March of 2014.

3. http://cameochemicals.noaa.gov/chemical/2893, consulted in 16th March of 2014.

4. http://chem.sis.nlm.nih.gov/chemidplus/rn/67-66-3, consulted in 17th March of 2014.

5. Gordon, L. Sir James Young Simpson and Chloroform (1811-1870), University Pass of the

Pacific, 2002, pp 104 – 107.

6. Cappelletti, M., Frascari, D., Zannoni, D. & Fedi, S. Microbial degradation of chloroform.

Appl. Microbiol. Biotechnol. 96, 1395–1409 (2012).

7. Agency for Toxic Substances and Disease Registry (ATSDR) (2011) CERCLA priority list of

hazardous substances. URL http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id053&tid016

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Monografia Microbial Degradation of Chloroform

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