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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Microbial Degradation of Chloroform
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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
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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.
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