Journal of Environmental Sciences 2010, 22(1) 116126

A review: Advances in microbial remediation of trichloroethylene (TCE)

Prabhakar Pant1,, Sudhakar Pant2

1. Applied Research Center, Florida International University, Miami, Florida 33174, USA. E-mail: [email protected]
2. Department of Biology, St. Xaviers College, Maitighar, Post Box 7437, Kathmandu, Nepal

Received 02 March 2009; revised 25 August 2009; accepted 01 September 2009

Abstract
Research works in the recent past have revealed three major biodegradation processes leading to the degradation of trichloroethylene.
Reductive dechlorination is an anaerobic process in which chlorinated ethenes are used as electron acceptors. On the other hand,
cometabolism requires oxygen for enzymatic degradation of chlorinated ethenes, which however yields no benet for the bacteria
involved. The third process is direct oxidation under aerobic conditions whereby chlorinated ethenes are directly used as electron
donors by microorganisms. This review presented the current research trend in understanding biodegradation mechanisms with regard
to their eld applications. All the techniques used are evaluated, with the focus being on various factors that inuence the process and
the outcome.

Key words: biodegradation; trichloroethylene; reductive dechlorination; cometabolism; direct oxidation

DOI: 10.1016/S1001-0742(09)60082-6

Introduction eective remedial process will be highly valuable.

Several methods are available for remediation of TCE
Trichloroethylene (TCE) is a halogenated volatile organ- pollution. Physical processes, such as soil excavation and
ic compound (VOC), which is believed to be carcinogenic venting, and ground water extraction techniques, all of
and mutagenic (USEPA, 1997). It is considered a prima- which have been used in remediation of TCE pollution in
ry pollutant with 5 g/L maximum contamination level the subsurface environments, have been found relatively
allowed in drinking water (ASTDR, 1997; Spitz and slow, costly, and inecient, along with being environmen-
Moreno, 1996). Most of the hazardous-waste sites in the tally disruptive in nature (Bankston, 2002). The complex
United States listed in the National Priority List (NPL) nature of aquifers, contaminant sorption onto solid media,
are contaminated with VOC, including TCE (Zachritz et and entrapment of contaminants in the porous media also
al., 1996). It is quite alarming that almost all the states attribute to the limited eectiveness of pump and treat
in the USA have problems related to TCE and/or other technologies in groundwater remediation (Beeman and
chlorinated ethenes in soil and groundwater (Moran et Bleckmann, 2002). Several other chemical and physical
al., 2007; http://www.epa.gov/superfund/sites/npl). Most pollution control methods have been found to require
of these TCE contaminants in the NPL sites originated toxic chemicals or are expensive (Han et al., 2002). In
from usage of chlorinated degreasing solvents during nu- this context Monitored Natural Attenuation (MNA) has
clear reactor operations. emerged as a promising technique for TCE pollution
TCE is also widely used in textile processing, refrigera- remediation. The term MNA refers to the reliance on
tion, lubricants and adhesives, along with the production of natural processes to achieve site-specic cleanup objec-
vinyl chloride, pharmaceuticals, and insecticides (Schettler tives within a reasonable time frame (USEPA, 1997).
et al., 1999). Due to inappropriate disposal methods that These in-situ natural processes include biodegradation,
are carried out, soil and groundwater contamination by dispersion, dilution, sorption, volatilization, and chemical
TCE has become widespread (Doucette et al., 2007; Riley or biological stabilization, transformation, or destruction
and Zachara, 1992). Reports are also available document- of contaminants (USEPA, 1997). Of these processes, biore-
ing contamination of the surface water as a result of TCE mediation technology is preferred over other technologies
release from the groundwater sources (Brewer and Fogle, because it allows for complete mineralization of TCE to
2001; Brigmon et al., 2001). The toxic eects associated harmless chemical forms, including carbon dioxide, water
with TCE have raised a serious public concern over the and chlorine (Russell et al., 1992). Other processes, such as
extensive contamination problems, and knowledge of its chemical reaction, sorption, dispersion, and volatilization,
are either slow or unable to degrade TCE, thus not resulting
* Corresponding author. E-mail: [email protected] in a net degradation of TCE (Lorah et al., 1997).
No. 1 A review: Advances in microbial remediation of trichloroethylene (TCE) 117

This article reviews the types of microbial processes that it inltrates easily into the aquifer where it forms a dense
are available for TCE pollution remediation, and evaluates non-aqueous liquid phase (DNAPL) at the bottom (Bourg
dierent biological and physical factors that may aect et al., 1992). Furthermore, the diusion rate of TCE in
the successful biodegradations of TCE in the environ- water has been cited as 9.0105 m2 /day by Chiao et al.
ment. This review also reports other compounds, including (1994), based on their study with the molar volume of
dichloroethylene (DCE) and vinyl chloride (VC), which 107 cm3 /mol at 25C. The lower rate of TCE diusivity
are often considered as the daughter products of TCE in water can reduce the mobility, and hence its transport
biodegradation. in a slow owing groundwater system. Parker et al. (2004)
conducted a simulation study on TCE diusion in aquifer,
1 Physical and chemical aspects of TCE and reported that TCE migration is diusion-dominated
within aquifer. Further, adsorption of TCE to both organic
TCE and its daughter products, such as DCE and VC, and mineral components in groundwater matrix such as
are double bonded between two carbons, and thus are in sediments and rocks may also retard the movement of
grouped together with other chlorinated alkenes (Fig. 1). TCE in groundwater. Extensive research has already been
TCE is a colorless, volatile liquid with a sweet smell done to establish a phenomenon of TCE adsorption with
similar to that of chloroform (Jacoby et al., 1998). TCE respect to various minerals and organic carbon (Aggarwal
is relatively insoluble in water and thus commonly cate- et al., 2006; Li and Werth, 2001; Lin et al., 1994; Smith
gorized as a hydrophobic compound. Table 1 lists a few et al., 1990). In general, the adsorption coecient (Kd ),
important physico-chemical parameters pertaining to its which is a measure of TCE partitioning between water
biodegradation potential. and sediment/soil phases, has been reported to increase
While all the characteristics are important in understand- with organic carbon content in the soil or sediment (Lee
ing the TCE behavior, its density plays a crucial role in et al., 2007; Poulsen et al., 2000). Adsorption not only
groundwater contamination, even when the discharge of hinders the mobility of TCE, it also inuences the process
TCE occurs at the surface. Agarwal and Singh (2004) have of TCE biodegradation by aecting TCE availability in
experimentally determined the density of TCE as 1.4557 aqueous phase. Studies by Alvarez-Cohen et al. (1993)
g/mL at 298.15 K (25C), which compares well with an and Sheremata et al. (2000) have indicated that organic
earlier reported value of 1.4514 g/mL (Venkatesulu et al., contaminants, such as TCE, are not available to bacteria for
1997). Jacoby et al. (1998) also reported a similar value: degradation when they are adsorbed in soil or sediments.
1.46 g/mL at 25C. Given the fact that water has a density Similarly, as shown in Table 1, TCE also has a higher
of 0.9970479 g/mL (calculated) at the same temperature, Henrys law constant (ability to volatilize) resulting from
it is clear that TCE is much denser than water and thus its high vapor pressure and low solubility in water (Marrin
and Kerfoot, 1988). As such, some TCE in water phase
may volatilize into head space or inter-particular-space that
are available in unsaturated soil above the water table, thus
becoming less bioavailable for bacterial degradation. The
same scenario can be expected in case of unsaturated zone
or surface soil polluted by TCE. Although volatilization
of TCE is not a predominant process in groundwater, it
certainly is in the case of surface waters, as documented
by Pant et al. (2007), Rathbun (2000), and Stocking and
Kavanaugh (2000), who have reported an increase in TCE
volatilization from surface waters with the increase of
velocity of water and wind.
Fig. 1 Molecular structures. (a) TCE with three chlorine atoms and one
hydrogen atom bonded to carbon atoms; (b) 1,1-DCE; (c) cis-1,2-DCE;
(d) trans-1,2-DCE; (e) vinyl chloride.

Table 1 Chemical and physical properties of TCE

Description Symbol Common value

Molecular weight (g/mol)a MW 131.39

Liquid density at 25C (g/mL)a 1.46
Water solubility (mg/L)c S 1000
Melting point (C)a Tm 86.5
Boiling point (C)a Tb 87.3
Vapor pressure (Pa)b VP 9700
Octanol-water partition coecientc Kow 320
Sorption coecient (organic carbon)c Koc 2.42
Henrys law constant ((Pam3 )/mol)b H 890
Diusion coecient in pure air (m2 /day)b Dair 0.68 Fig. 2 Reductive dechlorination mechanisms (modied from DeBruin et
Diusion coecient in pure water (m2 /day)b Dwater 9.00 105 al., 1992; Freedman and Gossett, 1989; Maymo-Gatell et al., 1999; Mohn
a Jacoby et al., 1998 ; b Chiao et al., 1994; c Russell et al., 1992. and Tiedje, 1992).
118 Prabhakar Pant et al. Vol. 22

2 TCE biodegradation processes and mecha- able, environmental conditions become important for the
nisms initiation and termination of this process. McCarty (1997)
has reported that the major environmental requirement in
Over the past two decades, several laboratory and eld aquifers of concern is the presence of sucient concentra-
studies have shown that subsurface microorganisms can tions of other organics, which can act as electron donors for
degrade a variety of hydrocarbons including chlorinated energy metabolism. Also, sucient amount of organic co-
solvents. The works of Bouwer et al. (1981), Harker and contaminants, other than TCE or its intermediate products,
Kim (1990), Hartmans and de Bont (1992), and Wilson and is required to reduce other electron acceptors. Further, the
Wilson (1985), for example, have indicated the importance presence of other electron acceptors can negatively impact
of microorganisms in the attenuation process of organic TCE dechlorination. Byl and Williams (2000) have provid-
contaminants. In line with these ndings, the biodegrada- ed a list of such electron acceptors, which could interfere
tion potential of TCE and its intermediate products have in or delay the process of dechlorination. Those electron
been thoroughly investigated. Studies have revealed that acceptors that interfere in TCE dechlorination are oxygen,
three types of metabolic processes are generally involved nitrate/insoluble manganese, insoluble ferric iron, sulfate
in the biological degradation of chlorinated ethenes. Mc- and carbon dioxide, respectively, in the order of bacterial
Carty (1994) has provided an overview of the reductive preference. Noell (2009) also reported the presence of
dechlorination process for chlorinated solvents. This is an competing electron acceptors such as dissolved oxygen, ni-
anaerobic process in which chlorinated ethenes are used as trate, ferrous iron, and sulfate, during TCE bioremediation
electron acceptors. Similarly, Alvarez-Cohen and McCarty pilot test conducted in contaminated groundwater at the
(1991) and Hanson and Brusseau (1994) have reported Naval Weapons Industrial Reserve Plant in Dallas, Texas.
another biodegradation method known as co-metabolism. 2.2 Co-metabolism of TCE
In this process chlorinated ethenes are anaerobically de-
graded as a result of fortuitous biochemical interactions, The possibility of aerobic co-metabolism of TCE be-
which yield no benet to bacteria. The third method is came clear from the work of Wilson and Wilson (1985).
direct oxidation, an aerobic or anaerobic process in which They reported that aerobic biodegradation of TCE is
sparsely chlorinated ethenes are used as electron donors carried out by methanotrophic bacteria in soil pre-exposed
(Bradley and Chapelle, 1996; McCarty and Semprini, to methane. Later, the study by Little et al. (1988)
1994). Any of these processes could be occurring at a given and Alvarez-Cohen and McCarty (1991) made it clear
site depending on the form of chlorinated ethenes and the that it is the methane monooxygenase (MMO) produced
site environmental characteristics. There is a decrease in by methanotrophs that was actually responsible for the
the rate of reductive dechlorination process as the number aerobic degradation of TCE. Little et al. (1988) also
of chlorine atoms in the chlorinated molecules decreases reported that 3.4% to 4.0% of the nal products were
(Bouwer 1994; Mohn and Tiedje, 1992; Vogel and Mc- cell bound materials, 40.1% to 42.7% was carbon dioxide,
Carty, 1985). This might be the reason that several studies and the remaining 53.5% to 56.2% was water-soluble
have indicated the accumulation of dichloroethylene and products. Similar ndings were made available by Henry
vinyl chloride at various TCE contaminated sites that and Grbic-Galic (1994), who proposed a mechanism of
has anaerobic condition (Freedman and Gossett, 1989; TCE degradation by methanotrophs. According to these re-
Grin et al., 2004; Mohn and Tiedje, 1992; Murray searchers, MMO converts TCE into a TCE epoxide, which
and Richardson, 1993). However, aerobic environment fa- breaks down into carbon monoxide, formate, glyoxylate,
vors oxidative biodegradation of less chlorinated ethenes. and chlorinated acids in aqueous environment. These com-
Aerobic biodegradation goes more rapid than reductive pounds are subsequently metabolized by methanotophs
dechlorination. and heterotrophs either collectively or individually, form-
ing the nal products (i.e., carbon dioxide and cell mass).
2.1 Reductive dechlorination of TCE It is not only TCE, but also its intermediates (DCE and
DeBruin et al. (1992), Freedman and Gossett (1989), VC) produced from reductive dechlorination that may be
and Maymo-Gatell et al. (1999) have pointed out that TCE co-metabolized.
undergoes complete sequential reductive dechlorination Besides methane-oxidizing bacteria, other bacteria, such
under anaerobic conditions, yielding ethene or ethane as as toluene oxidizers and phenol oxidizers, have also
nal products. According to Mohn and Tiedje (1992), chlo- received tremendous attention. These bacteria produce
rine atom is replaced by a hydrogen atom at each reaction oxygenases in response to phenol or toluene, which initiate
step during reductive dechlorination, producing hydrochlo- the oxidative degradation and mineralization of TCE. Fan
ric acid (HCl) as byproducts (Fig. 2). The various and Scow (1993) in their study with TCE biodegradation
intermediates have been reported as cis-1,2-dichloroethene by indigenous soil microbial populations reported that
(cis-DCE), trans-1,2-dichloroethene (trans-DCE), 1,1- 60%75% of TCE was degraded when both TCE and
dichloroethene (1,1-DCE) and vinyl chloride. According toluene were present at 1 g/mL and 20 g/mL, respective-
to Bouwer (1994), cis-DCE is a more prevalent interme- ly. The ratio of TCE and co-metabolites, such as toluene or
diate than trans-DCE, and 1,1-DCE is the least prevalent phenol, has been found to control co-metabolism process
intermediate during reductive dechlorination. by inuencing microbial growth. Mu and Scow (1994) re-
Although the reductive dechlorination option is avail- ported an increase in population of TCE/toluene degraders
No. 1 A review: Advances in microbial remediation of trichloroethylene (TCE) 119

by more than 4 orders of magnitude when 20 g of toluene cis-DCE but only 68.88%71.83% of trans-DCE within
and 1 g of TCE were added to 1 mL of soil slurry. seven days of incubation. When both DCE isomers were
However, a retarding eect on the microbial population presented in a mixture, trans-DCE showed a greater degra-
size and TCE degradation was reported for increasing dation (45.74%63.63%) than cis-DCE (32.93%46.45%)
concentrations of TCE (> 1 g/mL). Also, under stagnant over the same incubation period, indicating a microbial
conditions, Ely et al. (1997) have reported a substrate preference towards trans-DCE during direct oxidation
competition (e.g., TCE vs. DCE) for microbial utilization, processes. The authors reported that trans-DCE is more
together with substrate toxicity leading to inhibition and readily utilized as sole carbon source than cis-DCE, an
inactivation of enzymes that are responsible for degrading observation also reported by other researchers (Federle et
TCE or other chlorinated VOCs. Competition for the al., 1990; Hopkins et al., 1993). cis-DCE is more toxic
active site on the oxygenases among natural substrates (for than trans-DCE (Olaniran et al., 2008). It remains to be
example, CH4 , NH3 or toluene) and chlorinated solvents determined if the dierence in oxidative degradation and
has been documented by Semprini et al. (1991). supporting bacterial growth between cis-DCE and trans-
Other environmental factors, such as temperature and DCE is attributable to their toxicity dierence.
soil moisture, also inuence the co-metabolism activities.
Fan and Scow (1993) reported an increase in lag period in 3 Microbiology of TCE degradation
microbial activities with decreasing temperature, although
the initial rates of degradation were similar for toluene and The reductive dechlorination also depends on the mi-
TCE. They also reported the role of moisture contents in crobial populations. The microorganisms that are present
the soil in TCE degradation. The degradation was found in TCE contaminated sites can dier signicantly in their
to be below detection when moisture content was 2.5% ability and potential to dechlorinate TCE. It has been found
5%, while the degradation was observed at 16%30% that not all TCE dechlorinating organisms can degrade
soil moisture contents. Additionally, contact time contact TCE completely. Maymo-Gatell et al. (1997) reported
between the microorganisms and co-solvent has been Dehalococcoides ethenogenes strain 195 that is capable
found to play an important role in expression of oxygenase. of reducing TCE to ethane. Bacteria related to D. etheno-
Han et al. (2007) studied TCE co-metabolism in a slurry genes were found in sites that showed complete TCE
microcosm by supplementing toluene as a co-solvent. They dechlorination (Fennel et al., 2001; Hendrickson et al.,
reported a decreased degradation constant (0.5 day1 ) for 2002). However, other bacteria only partially dechlroinate
TCE with only 46% biodegradation eciency, which was TCE, producing intermediate product, such as cis-DCE.
very low as compared to over 90% eciency observed dur- For example, Dehalospirillum multivorans (Neuman et al.,
ing an in-situ pilot study. According to these researchers, 1994), Enterobacter agglomerans (Sharma and McCarty,
toluene in the microcosm was detectable for only a day, 1996) and Desultobacterium sp. strain PCE1 (Gerritse et
while in case of in-situ experiments toluene was detected al., 1996) only dechlorinate tetrachloroethylene (PCE) and
for 3 days. TCE to cis-DCE.
2.3 Direct oxidation of TCE There are several aerobic bacteria that can co-
metabolize TCE using their oxygenases, thus giving rise
With regard to direct oxidation of TCE, studies have to complete mineralization of TCE. Lau et al. (1994) and
indicated that the less chlorinated reductive dechlorination Wackett and Gibson (1988) reported that toluene-oxidizing
products can serve as electron donors (primary substrates) bacteria (e.g., Pseudomonas putida F1) can produce
for some respiratory bacteria. McCarty and Semprini toluene a dioxygenase that mediates co-metabolization of
(1994) have reported that VC can serve as a primary TCE. Radway et al. (1998) and Takami et al. (2000)
substrate for some microorganisms under aerobic condi- have also reported co-metabolization of TCE by toluene-
tions. During the direct oxidation process, microorganisms oxidizing bacteria and recombinant cells. In an earlier
obtain energy and organic carbon from the chlorinated study, Winter et al. (1989) reported the production of a
ethenes that undergo oxidative biodegradation. In anoth- toluene para-monooxygenase by Pseudomonas mendocina
er study with VC conducted in iron-reducing aquifer KR1 when vapor-phase toluene is supplied as a sole carbon
sediments, Bradley and Chapelle (1996, 1997) noted a source. According to this study, P. mendocina KR1 can de-
complete mineralization of VC in the presence of Fe(III), grade TCE completely with monooxygenase initiating the
which was attributed to the direct oxidation of chlorinated oxidative process. Sun and Wood (1996) carried out an ex-
ethenes. Apart from environmental factors, the types of tensive study to examine the TCE degrading capability of
substrates have also been found to play an important role Pseudomonas cepacia G4, Pseudomonas cepacia G4PR1,
in direct oxidations of chlorinated ethenes. Olaniran et al. Pseudmonas mendocina KR1, Pseudomonas putida F1,
(2008) found that the bacterial growth pattern was greatly and Methylosinus trichosporium OB3b. Their results in-
inuenced by the substrate types. The peak bacterial cell dicated that, although diering in degradation rates, all
densities ranged from 5.1 106 to 2.2 107 colony the microorganisms were capable of degrading TCE. Just
forming units (CFUs)/mL in the presence of cis-DCE, as in the case with Methylosinus trichosporium OB3b
while 8.9 106 to 2.6 107 CFUs/mL in the presence mentioned above, which is a methanotroph oxidizing
of trans-DCE. According to the same authors, the bac- methane, Chang and Alvarez-Cohen (1997) noted that a
terial isolates were able to degrade 69.24%75.00% of mixture of methanotrophic bacteria in a methanotrophic
120 Prabhakar Pant et al. Vol. 22

bioreactor which was seeded with landll soil could also dation in the mesocosm lled with aquifer materials, in
co-metabolize TCE. comparison to the native microbial community. Also, the
As discussed earlier, along with reductive dechlorination maximum degradation of VC was achieved between 30
and cometabolism of TCE, direct oxidation of TCE has and 37C. Schaefer et al. (2009) in a separate study eval-
also been noted, but yielded complete mineralization of uated Dehalococcoides sp. for its application during the
TCE. However, this process requires microorganisms that bioaugmentation for chlorinated ethenes. They performed
are capable of utilizing chlorinated ethenes as their sole batch and column experiments using groundwater and soil
source of carbon. Kitayama (1997) has reported Pseu- that were obtained from the TCE contaminated area at
domonas aeruginosa JI104 as a potential microbe that US Air Force Facility in Ft. Worth, Texas. The transport
can use TCE as a sole carbon source and degrade it and the growth of Dehalococcoides bacteria in saturated
aerobically. On the other hand, Olaniran et al. (2008) porous media, together with dechlorination eciency were
reported seven bacterial isolates that were able to de- measured. The study showed higher concentrations of
grade cis- and trans-DCE directly by using them as their Dehalococcoides bacteria in the aqueous phase compared
energy source. According to these authors, two of the to that of the soil phase, which suggested a greater degree
isolates were closely related to strains of Acinetobacter of bacterial mobility in porous media. This was conrmed
species. Other two isolates belonged to Bacillus species, with the nding that the dechlorination did occur through-
mainly Bacillus subtilis and Bacillus cereus. Other three out the length of the column in this study. Additionally,
isolates each were similar to the strains of Achromobacter it was observed that the aqueous phase concentrations of
xylosoxidans, Klebsiella and Pseudomonas aeruginosa, Dehalococcoides bacteria increased uniformly across the
respectively. These bacterial strains had been isolated from column with respect to the time after the injection but
the contaminated sites in Nigeria and South Africa. not in relation to the distance from the column inuent.
It was also observed that the batch experiments indeed
4 Microbial bioaugmentation for TCE reme- underestimated the outcomes from the column studies,
diation thus the authors expected a higher rate of dechlorination in
eld conditions than that suggested through the laboratory
Many contaminated sites undergoing natural remedi- batch experiments. In this study, the dechlorination activity
ation under the inuence of indigenous microorganisms was reported almost 200-times greater for the columns
have been found to accumulate cis-DCE and VC, which than for the batch experiments.
can be attributed to the lack of microbial community that is Hood et al. (2008), in their laboratory study and a
capable of complete dechlorination of TCE either through pilot test at Launch Complex 34, Cape Canaveral Air
reductive or oxidative pathways. In this regard, bioaug- Force Center, observed a signicant increase in the rate of
mentation technique, which involves the introduction of dechlorination after bioaugmentation with a dechlorinating
specic microorganisms in the contaminated aquifer to culture (KB-1; SiREM, Guelph, Ontario, Canada). KB-1
enhance the biodegradation of chlorinated ethenes, has is a natural microbial culture containing Dehalococcoides
been found very successful (Ellis et al., 2000; Major et al., organisms that was proven to be highly ecient in dechlo-
2002; Lendvay et al., 2003). Bioaugmentation can be used rination of TCE and other chlorinated ethenes. The authors
to treat a range of TCE concentrations in groundwater, utilized the bioaugmentation technique in conjunction with
including source areas where high concentration of TCE the amendments (methanol, ethanol, acetate, and lactate)
is often seen as advantage in reducing the competitive in microcosm that contained indigenous microorganisms.
role of other electron donors (DiStefano et al., 1991; They noted a half life (t1/2 ) as short as 1 day for TCE
Yang and McCarty, 2000). Over the years many bacterial biodegradation, which was signicantly shorter than that
strains have been isolated and studied for their ecacy in those microcosms with indigenous microorganisms that
in biodegradation of TCE, most of them have been found were either amended (t1/2 = 17 day) or unamended (t1/2
capable of dechlorinating only specic molecules (TCE, = 92 day) without bioaugmentation. Similar trend was
DCE or VC), while only one bacterial strain (Dehalo- observed during a pilot study, in which TCE concentra-
coccoides sp.) has been reported to dechlorinate TCE to tion declined almost to the limit of detection (20 g/L)
ethene/ethane completely (Cupples et al., 2003; Sung et al., after bioaugmentation. However, the accumulated VC was
2006). Accordingly, bioaugmentation for TCE remediation ultimately dechlorinated to ethene only after a 5-months
may utilize the combination of dierent bacterial strains lag period. It was observed that the ethene concentrations
or just a population of Dehalococcoides species. However, in groundwater increased signicantly to an average value
most of the bioaugmentation eorts have relied on mixed of 67 mg/L at the end of the demonstration (249 day
cultures of bacteria including Dehalococcoides sp. for bioaugmentation). Kane et al. (2005) also reported a
Fathepure et al. (2005) studied Mycobacterium sp. for successful use of KB-1 culture in TCE bioremediation.
its potential role in bio-augmenting TCE biodegradation in They utilized KB-1 culture that included Dehalococcoides
aquifer materials. They isolated Mycobacterium sp. strain ethenogenes for bioaugmentation in the groundwater areas
TRW-2 from a chloroethene-degrading enrichment culture. near the TCE source zone that had high TCE concen-
This strain was found to utilize VC or ethene as the sole tration (approx. 700,000 g/L). The contaminated aquifer
carbon source. According to the authors, bioaugmentation was located in the Caldwell Trucking Superfund Site in
of Mycobacterium sp. yielded a higher rate of VC degra- Essex County, NJ. Also, Duhamel et al. (2002, 2004) in
No. 1 A review: Advances in microbial remediation of trichloroethylene (TCE) 121

their studies with microcosms have reported a complete pounds. Before the onset of biostimulation process, the
dechlorination of chlorinated ethenes when bioaugmented groundwater was anaerobic in nature and had iron reduc-
with KB-1 culture. ing conditions, which transformed into sulfate reducing
conditions after biostimulation. It was observed that the
5 Sequential anaerobic-aerobic processes for biostimulation enhanced the reductive dechlorination of
TCE remediation TCE, cDCE, and VC, and there was an accumulations of
ethane, which implied that there were a complete sequence
As discussed earlier, when the number of chlorine of anaerobic biodechlorination of TCE occurring in the
atoms in the chlorinated molecules decreases, the reductive groundwater that was biostimulated. However, owing to
dechlorination process slows down; and aerobic environ- the fact that the rate of degradation of TCE is faster
ment favors the oxidative biodegradation of the resulting than its daughter products under anaerobic environment,
less chlorinated ethenes. Therefore, the oxidation rates accumulations of cDCE and VC were observed. Thus,
for 1,2-DCE and VC are relatively faster compared to when biosparging was conducted to create nitrate-reducing
that of TCE (Pfaender, 1990). Researchers have utilized conditions in the groundwater downgradient from the
these concepts in developing sequential anaerobic-aerobic biostimulation site, there was an enhancement of biodegra-
processes in order to biodegrade TCE more eciently. dation of cDCE and VC, however TCE degradation was
Lorah et al. (1997) reported both anaerobic and aero- not observed. It has also been reported that the rate of
bic biodegradation of TCE and other chlorinated ethenes biodegradation for cDCE and VC during aerobic condi-
occurring in a fresh water tidal wetland in Maryland. tions were greater than that under anaerobic conditions.
In that study, the contaminated groundwater discharged Similarly, it was also noticed that the biodegradation of
into the wetland passed through the lower sediment layer VC was much faster than that of cDCE under aerobic
rst, which had iron-reducing conditions, followed by an conditions. It was noted that there was an increase in
upper layer of peat, which had methanogenic conditions. the concentrations of TCE during the aerobic conditions,
They noted a tremendous decrease in the concentration of which was attributed to the processes like desorption and
TCE in the groundwater after being transported upward leaching from soils/sediments that were less permeable
through these two layers with dierent redox potentials. earlier. In another study with groundwater, Devlin et al.
The original groundwater TCE concentration of 1002000 (2004) reported dierent kinds of in-situ anaerobic-aerobic
g/L declined to a very low or undetectable concentration sequential biodegradation of TCE. In this study, instead of
in the recipient wetland water, while 1,2-DCE and VC water owing through an anaerobic and aerobic environ-
were detected in the same recipient water. According ment, a section of aquifer (Borden aquifer, Canada) was
to these researchers, 1,2-DCE and VC concentrations isolated and maintained anaerobically with the supply of
became undetectable near the surface in the wetland, nutrient solution until the daughter products of anaerobic
which was attributed to the aerobic biodegradation of these dechlorination of TCE were observed; later on the same
low molecular chloroethenes. The microcosm experiments groundwater was biosparged with oxygen to stimulate
conducted using the wetland sediments and water further aerobic biodegradation. Both the processes were found to
conrmed the aerobic biodegradation process in near sur- enhance microbial biodegradation of chlorinated ethenes
face regions of the wetland that showed methanotrophic and their daughter products.
activities. In a similar study, Bankston et al. (2002) also Reports are also available on anaerobic-aerobic sequen-
investigated the possibility of anaerobic/aerobic biodegra- tial TCE degradation in engineered systems like reactors.
dation of TCE in wetland environment. Based on their Tartakovsky et al. (2003) studied TCE degradation under
ndings from the wetland microcosm in which the min- sequential anaerobic-aerobic conditions using a biolm
eralization of TCE occurred in concurrence with the reactor that was fed with ethanol and was oxygenated
oxidation of methane, they suggested a role of methan- using hydrogen peroxide. In order to seed the reactor,
otrophs in TCE mineralization. Further, they revealed the microbial consortium from an anaerobic digester was used
importance of wetland vegetation in aerating the root zone. as the inoculums. During operational phase of the reac-
In a separate study, Noell (2009) has reported anaerobic tor, two major groups of microorganisms, methanogenic
biodegradation of chlorinated ethenes in the upgradient and methanotrophic, were observed. These microorgan-
portion of the aquifer followed by aerobic biodegradation isms were able to degrade TCE almost completely into
in the downgradient segment of the aquifer. These obser- ethenes/ethanes. In a similar study with reactors, Guiot
vations were made during a pilot test that was conducted et al. (2008) used a single-stage laboratory scale reactor
in a section of a larger TCE plume located in the ground- by coupling anaerobic/aerobic conditions that favored both
water at the Naval Weapons Industrial Reserve Plant in reductive dechlorination and oxidative mineralization of
Dallas, Texas. The pilot test constituted biostimulation of chlorinated ethenes such as PCE, TCE and other daughter
indigenous microbes with amendments (sodium acetate products. A unique feature of this reactor was the use of
and ammonium phosphate) for 163 days to anaerobically electrolysis-generated hydrogen as an electron donor for
biodegrade chlorinated ethenes in the upgradient areas reductive dechlorination as well as for the formation of
of the aquifer, followed by air-sparging of the downgra- methane. The authors have reported over 98% reductive
dient aquifer for another 69 days in order to stimulate dechlorination of PCE to DCE, with TCE as intermediate
aerobic biodegradation of the remaining chlorinated com- product. Similarly, a maximum oxidative DCE mineraliza-
122 Prabhakar Pant et al. Vol. 22

tion to CO2 was reported to be 89%. The values reported 6.3 Duluth, USA
above were based on the experimental study utilizing 6
Semer and Banerjee (2001) conducted a study on anaer-
days of hydraulic retention time and loading rate of 4.3
obic bioremediation of TCE in groundwater at a former
mol PCE/L of reactor per day.
landll site near the Duluth International Airport. This
area was earlier used by the US Air Force Air Combat
6 Case studies Command (ACC) for disposing of dierent types of waste
materials that included aircraft parts, empty drums, and
6.1 Bitterfeld, Germany drums containing chemicals. As the groundwater recharge
in the site was primarily through inltration from the
Imfeld et al. (2008) studied a contaminated site located surface, this could have easily aided in contamination of
in the city of Bitterfeld in eastern Germany. This site the groundwater over the years. The bioremediation study
covering an area of approximately 2 km2 was originally focused on enhancing the dechlorination of TCE, DCE,
contaminated with PCE and TCE. However, cis-DCE and and VC by injecting polylactate ester (HRC ) that released
VC were also found later in the same site and were lactic acid. The authors reported a signicant reduction
believed to be products of biodegradation of PCE and (> 99.5%) in TCE concentration over a period of nine
TCE. The authors used carbon stable isotope technique and months. The decrease in TCE concentration accompanied
biomarkers to conrm the occurrence of biodegradation an increase in anaerobic biodegradation products, with
and the presence of microbial consortia likely involved concentrations of cis-1,2-DCE and VC increasing from
in the process of chloro-ethene biodegradation. The study 50 to 750 g/L and 10 to 20 g/L, respectively. Over
revealed the occurrence of biodegradation and also the the same time period, TCE decreased from 400 g/L
presence of Dehalococcoides species that is capable of to less than 2 g/L, thus corroborating the fact that the
degrading TCE to ethene. According to the authors, the degradation products present in groundwater were derived
presence of cis-DCE in the aquifer was suggestive of from reductive dechlorination of TCE.
the premise that a reductive dechlorination process was
occurring at the site. Also, the isotopic data and the results 6.4 Savannah River Site, South Carolina, USA
from microbial proling using taxon-specic molecular The Savannah River Site (SRS) that belongs to the US
analyses supported that notion. The chlorinated ethene Department of Energy is located in South Carolina. This is
concentrations were reported to inuence the bacterial one of the Superfund Sites and is included in the National
community composition. The authors also observed a Priority List. Brigmon et al. (1999) conducted a TCE biore-
positive relationship between the presence of Dehalococ- mediation study on a Sanitary Landll (SLF) in the SRS
coides-like bacteria and the ethane concentration in some area. This site had been used to dispose of materials con-
of the wells studied. taminated with TCE and PCE, including sanitary waste,
construction material, and disposable batteries. Due to
6.2 Connecticut, USA
leaching process, TCE migrated into groundwater beneath
Chapman et al. (2007) investigated a TCE plume, which the SLF. A project was initiated for the biodegradation of
originates from a DNAPL source zone in a shallow sand TCE by injecting gaseous nutrients to stimulate microbial
aquifer and its discharges into a river, at a former indus- populations that would degrade TCE and the products of
trial facility in Connecticut. Earlier, this site was used TCE. The nutrient injection mix contained methane (4%),
for manufacturing metal related articles. TCE was used nitrous oxide (0.07%) and phosphate (Triethyl phosphate,
as a primary solvent for degreasing and was stored in 0.007 to 0.01%) and was blended with air. Helium was
underground tanks that probably leaked forming a DNAPL also added to the nutrient mix to a nal concentration
into the bottom of the aquifer and contaminating even the of 0.1% as a tracer. The results revealed an increase
underlained aquitard. The authors found minimal degra- in methanotrophic bacterial population in groundwater,
dation of TCE within the distance of 280 m from the which was attributed to selective biostimulation during
source, as indicated by the low concentration of cis-DCE the nutrient injection test. Concurrent with the increas-
and the undetectable VC. However, cis-DCE was observed ing methanotrophic counts, concentrations of chlorinated
further downstream, with a maximum cis-DCE concentra- hydrocarbons decreased. The authors maintained that the
tion reaching 500 g/L. It was also reported that in most processes involved in TCE and cis-DCE biodegradation
of the samples the cis-DCE concentration exceeded that could be of cometabolic nature, but metabolic evidence
of TCE. The presence of cis-DCE at high concentrations was not available from their study.
in groundwater, along with carbon isotopic results and
hydrogeochemical information available for the site have 7 Concluding remarks
all conrmed the existence of natural attenuation process
at the site. The authors also maintained that dilution and Studies have shown various microbial machineries that
volatilization might also accounted greatly for the TCE are capable of degrading TCE and its intermediate forms.
reduction observed in the groundwater. Given the fact that there are hundreds of sites contaminated
No. 1 A review: Advances in microbial remediation of trichloroethylene (TCE) 123

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