Sustainable Environment Research 28 (2018) 149e157

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Review

A review in the current developments of genus Dehalococcoides, its

consortia and kinetics for bioremediation options of contaminated
groundwater
Donamel M. Saiyari a, b, Hui-Ping Chuang c, Delia B. Senoro a, Tsair-Fuh Lin d, e, *,
Liang-Ming Whang c, e, Yi-Ting Chiu e, Yi-Hsuan Chen e
a
School of Civil, Environmental and Geological Engineering, Mapua Institute of Technology, Manila 1002, Philippines
b
Department of Civil Engineering, Adamson University, Manila 1000, Philippines
c
Sustainable Environment Research Laboratories, National Cheng Kung University, Tainan 70955, Taiwan
d
Tainan Hydraulics Laboratories, National Cheng Kung University, Tainan 70955, Taiwan
e
Department of Environmental Engineering, National Cheng Kung University, Tainan 70101, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: This article reviews the current developments in genus Dehalococcoides as key dechlorinating bacteria in
Received 6 June 2017 chlorinated ethene contaminated sites. The presence of chlorinated ethenes in environment had been a
Received in revised form concern for more than five decades as it represents significant threat to human and ecological health due
4 December 2017
to its extreme toxicity. This review elucidates the kinetics of Dehalococcoides spp. growth and compound
Accepted 22 January 2018
Available online 31 January 2018
utilization in dechlorination of chlorinated ethenes compounds. The metabolic pathways in physiology of
Dehalococcoides spp. are important in the transformation of chlorinated species. The potential of isolates
and its reductive dehalogenase genes are seen to infer activities of Dehalococcoides spp. that would be
Keywords:
Dechlorinating consortia
used to the development of engineered systems. This system is helpful in making decision on biore-
Dehalococcoides mediation option to treat the contaminated groundwater. Hence, the role of Dehalococcoides spp. in
Reaction kinetics chlorinated ethene biodegradation is controlled by kinetics in complex ways. Therefore, intensive in-situ
characterization and understanding the microbial growth on dechlorination evaluation are essential to
develop a consistent and rational engineered system. This is to achieve a successful bioremediation
strategies for sites contaminated with chlorinated ethenes.
© 2018 Production and hosting by Elsevier B.V. on behalf of Chinese Institute of Environmental
Engineering, Taiwan. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Chlorinated ethenes have been widely used in various industrial
applications as shown in Table 1. They were chosen for their low
cost, easy availability, excellence as solvents, chemical stability and
fire safety [1]. Chlorinated ethenes are soluble, dense non-aqueous
phase liquids that moderately sorbs in soil due to both low
octanolewater partitioning coefficients (Log Kow) and soil-sorption
coefficients (Log Koc in soil) as shown in Table 2. With its density
higher than water, it penetrates through the water table until it
* Corresponding author. Tainan Hydraulics Laboratories, National Cheng Kung
University, Tainan 70955, Taiwan
E-mail address: tflin@mail.ncku.edu.tw (T.-F. Lin).
Peer review under responsibility of Chinese Institute of Environmental
Engineering.
reaches the bottom of aquifer. Also, the half-lives of these solvents
are usually longer than the other volatile organic compounds [2].
This implies that it may persist for a long period of time in soil and
groundwater. Its deposition to subsurface brought toxicity and
carcinogenic effects in the environment. Hence, various countries
and international organizations have set standard (summarized in
Table 3) for these chemicals from industrial sources and human
dwellings to protect the environment and public health. Although
various technologies have been applied to remediate the sites
contaminated with chlorinated ethenes, bio-engineered technolo-
gies [3], based on detoxification of organic contaminants through
microorganisms, are being developed and become popular for
cleaning-up the sites.
Bio-engineered technologies such as in-situ bioremediation
(ISB) techniques are the innovative solutions to treat chlorinated
ethene through microbial reductive dechlorination process. ISBs

https://doi.org/10.1016/j.serj.2018.01.006
2468-2039/© 2018 Production and hosting by Elsevier B.V. on behalf of Chinese Institute of Environmental Engineering, Taiwan. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
150 D.M. Saiyari et al. / Sustainable Environment Research 28 (2018) 149e157

Table 1
Applications of common chlorinated-ethene compounds [70].

Common or alternative name Predominant Other uses as parent compound or additive

use group (or source)

Vinyl chloride (VC), chloroethylene Organic synthesis PVC (plastics)

1,1-dichloroethylene (DCE)
cis-1, 2-dichloroethylene (cis-1,2-DCE)
Perchloroethylene, tetrachloroethylene (PCE)
1,1,2-trichloroethylene (TCE)
trans-1,2-dichloroethylene (trans-1,2-DCE)
Organic synthesis PVC (plastics); adhesives; refrigerants
Solvent Refrigerant; pharmaceutical manufacture; artificial pearl manufacture; extraction of fats
from fish and meat; organic synthesis
Solvent Chemical intermediate; dry cleaning; textile processing
Solvent Caffeine extractant; dry-cleaning; paint and ink formulation; rubber processing
industries
Solvent Refrigerant; pharmaceutical manufacture; artificial pearl manufacture; extraction of fats
from fish and meat; organic synthesis

Table 2
The physico-chemical properties of common chlorinated-ethene compounds.

Common or alternative Molecular Molecular weight Henry's Law constant Water solubility Density (g cm
3 Octanol/water Soilesorption Approximate
name [70] Formula [71] Mw (g mol
1) [71] (kH) (x 10
3 atm m3 (mg L
1 at at 20  C) [70] partition coefficient half-lives (yr)
mole1 at 25  C) [71] 25  C) [71] coefficient (Log Koc in
(Log Kow) [71] soil) [70]

Vinyl chloride (VC), C2H3Cl 62.5 79.2 2700 0.910 1.38 1.75 0.55 [40]
chloroethylene
1,1-dichloroethylene (DCE) C2H2Cl2 96.9 23.0 3400 1.213 2.13 2.18 NAa
cis-1,2-dichloroethylene C2H2Cl2 96.9 7.4 3500 1.284 1.86 1.56e1.69 1.1 [40]
(cis-1,2-DCE)
Perchloroethylene, C2Cl4 165.8 26.3 150 1.623 2.88 2.37 0.7 [2]
tetrachloroethylene (PCE)
1,1,2-trichloroethylene (TCE) C2HCl3 131.4 11.7 1100 1.464 2.53 2.00 0.7 [2]
trans-1,2-dichloroethylene C2H2Cl2 96.94 6.8 6260 1.256 1.93 1.56e1.69 NAa
(trans-1,2-DCE)
a
NA e not applicable.

Table 3
Drinking water regulation and guidance values of the chlorinated ethene compounds [72].

Compound Maximum Contaminant Level in mg L
1

Taiwan regulation (2015) USA regulation (2012) Canada guidance (2014) EU guidance (2007) WHO guidance (2008)

PCE e 0.005 0.03 0.01 0.04

TCE 0.005 0.005 0.005 0.01 0.02
1,1-DCE 0.007 0.007 0.014 e e
cis-1,2-DCE e 0.07 e e 0.05
trans-1,2-DCE e 0.1 e e
VC 0.002 0.002 0.002 0.0005 0.0003

are cost-effective technologies for bioremediation of contaminated
soil, sediments and groundwater [4]. These technologies convert
contaminants into non-toxic by-products through biological pro-
cess. The microorganisms, in anaerobic reductive dechlorination,
gain energy and grow as one or more chlorine atoms are replaced
with H2 in anaerobic environment. This process is illustrated in
Fig. 1. During chlorinated ethene degradation, tetrachloroethene
(PCE) and trichloroethene (TCE) with high oxidative state are easy
to decompose under anaerobic condition. This condition generates
Fig. 1. Anaerobic reductive dechlorination pathway for PCE and TCE detoxification [3].
more energy than the degradation of cis-1,2-dichloroethene (cis-
1,2-DCE) and vinyl chloride (VC) as both have low oxidative state.
Microbes, having enzyme that catalyzed the reduction of cis-1,2-
DCE/VC to ethene or ethane, have significant role in complete
dechlorination. The biological dechlorinating metabolism is
considered to remediate chlorinated ethene pollution which be-
comes the focus of current bioremediation techniques. These
dechlorinating metabolisms are successfully implemented at
chlorinated ethene contaminated sites through biostimulation and
bioaugmentation approaches [5,6].
As early as 1990s, there were several PCE dehalogenating bac-
teria found from different metabolic groups such as halorespirators,
acetogens, methanogens, sulfate reducers and facultative anaer-
obes [7,8]. In recent years, various bacteria populations were found
that can respire chlorinated ethenes. These include genera Geo-
bacter [9e13], Desulfitobacterium [14e16], Desulfuromonas [17e19],
Sulfurospirillum [20,21], Dehalobacter [22e24], and Dehalococcoides
[13,25,26]. However, knowledge on degradation kinetics, site con-
ditions, microbial consortia populations, and the consortium roles
in the dechlorination process to develop engineered remediation
strategies are not fully understood. Among various bacteria, the
abundance of Dehalococcoides spp. is believed to play an important
 D.M. Saiyari et al. / Sustainable Environment Research 28 (2018) 149e157 151

role in the chlorine cycle and biogeochemical cycles [27]. Hence,
this paper aims a comprehensive review including overviewing of
the recent advances in Dehalococcoides spp. its isolates evolution,
microbial cooperation, and reaction kinetics as aid to molecular
tools. This will be helpful for biological controls of degradation
evaluation in chlorinated ethenes contaminated site.
2. Characteristics of Dehalococcoides community
Dehalococcoides spp. are one of the most important microbial
groups in groundwater remediation for their capability of complete
reductive dechlorination. The Genus Dehalococcoides belongs to
class Dehalococcoidia and obtain its energy via the oxidation of H2.
It is also an organohalide-respiring bacteria which act as electron
acceptors to halogenated compounds via dehalorespiration [28].
Dehalococcoides favors neutral pH and are usually disc-shaped non-
pigmented, non-spore forming and strict anaerobes with non-
motile cells. Dehalococcoides is strictly hydrogenotrophic (convert
H2 to other compounds as part of the metabolism activity) with
multiple reductive dehalogenase genes (pceA, tceA, vcrA, bvcA,
mbrA, cbrA and pcbA genes) [29]. These are the enzymes that ca-
talyse the chemical reactions.
“Dehalococcoides ethenogenes”, now Dehalococcoides mccartyi
strain 195 is the first discovered member of Dehalococcoides that
were described by Maymo
-Gatell et al. [30]. Lately, strains within
Dehalococcoides became more versatile and diverse, as shown in
Table 4. Each strain has one or more reductive dehalogenase genes
that correspond to particular chlorinated electron acceptors.
Reductive dehalogenase genes are complex molecules with iron/
sulfur clusters and a cobamide coenzyme [31] and also oxygen-
sensitive enzymes which catalyze the reduction of carbone
halogen bond. Conventionally, vcrA reductive dehalogenase genes
pathway dechlorinates cis-1,2-DCE and VC compounds [25].
Strains 195, BAV1 and 11a can utilize much more chlorinated
compounds as energy sources than other strains [32]. Dehalo-
coccoides is the only microorganism for complete degradation of
chlorinated compounds except for strain MB [33,34] (trans- or cis-
DCE as end-product), and strains CG1, CG4 and CG5 (TCE as end-
product) [35]. Furthermore, based on the free energy of dechlori-
nation reactions, isolates form Dehalococcoides community, which
act for stepwise reduction of chlorinated species, at low H2 pressure
under strict anaerobic condition [36].
Discovery of Dehalococcoides spp. brought significant environ-
mental contribution in detoxification of anthropogenic haloge-
nated compounds (e.g., chlorinated ethenes) in the contaminated
soil and groundwater. Its presence is considered as a biomarker to
assess possible options of bioremediation in chlorinated ethenes
contaminated sites [37e39]. Furthermore, effective bioremediation
entails high abundance of Dehalococcoides spp. and electron sub-
strate concentrations [40].
3. Dehalococcoides isolates for bioaugmentation options
In onsite biodegradation, some complex microbial community
information are provided by several molecular tools, summarized
in Table 5, such as specific activity through gene expression data

Table 4
The functional genes of various D. spp. strains for its dechlorinated compounds and end products.

Strain Functional genes Dechlorinated compounds End products References

D. mccartyi 195 pceA, tceA PCE, TCE, cis-DCE, 1,1-DCE Ethene [30,56,73,74]
1,2-dichloroethane VC, Ethene
1,2,3,4-tetrachlorodibenzo-p-dioxin 1,2,4-trichlorodibenzo-p-dioxin,
1,3-dichlorodibenzo-p-dioxin
Hexachlorobenzene 1,2,3,5-tetrachlorobenzene,
1,3,5-trichlorobenzene
2,3,4,5,6-chlorobiphenyls (CB) 2,3,4,6-CB, 2,3,5,6-CB, 2,4,6-CB
2,3-DCP, 2,3,4-TCP, 2.3,6-TCP Lower chlorinated phenols
D. mccartyi CBDB1 cbrA 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, 1,3-DCB, 1,4-DCB, and 1,3,5-TCB [75e77]
1,2,3,4-TeCB, 1,2,3,5-TeCB and 1,2,4,5-TeCB
D. mccartyi VS vcrA cis-DCE & VC Ethene [43,44,46]
D. mccartyi BAV1 bvcA cis-DCE, trans-DCE, 1,1-DCE, VC, Ethene [45,46]
Vinyl bromide, 1,2-dichloroethane
D. mccartyi FL2 tceA TCE, cis-DCE & trans-DCE VC & Ethene [45,78]
D. mccartyi KB1/VC tceA TCE, cis-1,2 DCE & VC Ethene [59,63]
D. mccartyi GT vcrA TCE, cis-DCE, 1,1-DCE, VC Ethene [64]
D. mccartyi DCMB5 cbrA 1,2,3,4-tetrachlorodibenzo-p-dioxin 2-monochlorodizenbo-p-dioxin [79,80]
D. mccartyi MB mbrA PCE & TCE trans-DCE, cis-DCE [33,34]
D. mccartyi BTF08 pceA, tceA, vcrA PCE, TCE, cis-DCE, & VC Ethene [41,80]
D. mccartyi ANAS1 tceA TCE, 1,1-DCE, & cis-DCE VC & Ethene [33]
D. mccartyi ANAS2 vcrA TCE, cis-DCE, 1,1-DCE, & VC Ethene [56,66]
D. mccartyi 11a vcrA TCE, trans-DCE, cis-DCE, 1,1-DCE, 1,2-DCA, & VC Ethene [56]
D. mccartyi 11a5 tceA TCE, trans-DCE, cis-DCE, & 1,1-DCE VC & Ethene [56]
D. mccartyi IBARAKI vcrA cis-DCE & VC Ethene [47]
D. mccartyi UCH007 pceA, tceA, vcrA TCE, cis-1,2-DCE & VC Ethene [42]
D. mccartyi CG1 pcbA1 PCE, 234-234-CB, 234-24-chlorinated TCE, 24-24-CB, 24-25-CB, [35]
biphenyls (CB) 235-24-CB,236-24-CB
D. mccartyi CG4 pcbA4 PCE, 2345-, 2346-, and 245-CB, 23456-, TCE, 24-24-CB, 24-25-CB [35]
2345-, 245-, and 234-CB
D. mccartyi CG5 pcbA5 PCE, 2345-, 234-, 235-, 236-, and 245-CB, TCE, 24-24-CB, 24-25-CB, [35]
2345-, 2346-, and 245-CB 25-26-CB, 235-24-CB, 236-24-CB, 245-24-CB
D. mccartyi JNA pcbA4, pcbA5, pceA, mbrA Pentachlorophenol 3,5-dichlorophenol (DCP) [81]
2,2,4,6-tetrachlorophenol, 2,4,6-(TCP)
2,4,5-trichlorophenol (TCP) 2,4-DCP, 3,4-DCP,
2,3-DCP 3-chlorophenol (CP)
D. mccartyi GY50 NAa NAa NAa [42,82]
D. mccartyi SG1 NAa NAa NAa [35,42]
a
NA e not applicable.
152 D.M. Saiyari et al. / Sustainable Environment Research 28 (2018) 149e157

Table 5
Recent molecular tools for biological characterization of dechlorinating microbial communities.

Technique Obtainable Information References

Direct and nested Polymerase Chain Reaction (PCR)
Quantitative Polymerase Chain Reaction (qPCR)
Clone library (Phylogenetic Analysis)
Next-Generation Sequencing (NGS)
Phospholipid Fatty Acid (PFLA)
Denaturing Gradient Gel Electrophoresis (DGGE)
Fluorescent In Situ Hybridization (FISH)
Catalyzed Reporter Deposition-Fluorescence
In situ Hybridization (CARD-FISH)
Qualitative: presence/absence of 16S rRNA genes of interest. [83]
Quantitative: presence of specific organisms of interest (i.e., 16S rRNA gene, mRNA, [83]
functional genes).
Qualitative: data on gene biodiversity (i.e., 16S rRNA gene, functional genes). [83]
Qualitative: data on gene biodiversity (i.e., 16S rRNA gene, functional genes, genome sequence). [83]
Determination of total biomass composition, quantitation of individual groups of organisms, and [83]
analyses of nutritional status and metabolic activity combination with other technologies.
Qualitative: presence/absence of 16S rRNA and/or functional genes of interest. [83]
Quantitative: information on activity, spatial distribution and the whole biodiversity of the sample. [83]
Quantitative: information on activity, spatial distribution and the whole biodiversity of the sample. [39,84]

[37]. On the establishment of comprehensive microbial informa-
tion, microbial morphology, identification, quantification and dy-
namics are discussed. In microbial morphology, fluorescent in-situ
hybridization and catalyzed reporter deposition-fluorescence in
situ hybridization (CARD-FISH) are applied for detection of the
targeted microorganisms. These are used to quantify the rate of
targeted microbes per total bacterial or archaeal community. CARD-
FISH can further be used to investigate the substrate metabolism in
combination with isotopic technology. In microbial identification,
four common tools, such as polymerase chain reaction (PCR), clone
library, next-generation sequencing (NGS) and phospholipid fatty
acid (PFLA), are widely applied. The PCR is used for determination
of targeted genes existence, while clone library and NGS are applied
to clarify the community composition, and primarily estimate the
ratio of each microbe per entire community. PFLA technology is to
investigate the biomass structure composed by different types of
microorganisms, and further analyze the metabolic activity com-
bined with other technologies. Additionally, quantitative PCR
(qPCR) method is used to quantify the amounts of targeted mi-
crobes and the abundance of concerned functional genes. In the
microbial dynamics during wastewater treatment or contaminant
bioremediation, denaturing gradient gel electrophoresis (DGGE) is
applied to scan the microbial diversity of samples taken from
different time or location points in combination with fragment
database.
When amounts of targeted microbes are lower than 103 copies

1
L groundwater in the contaminated site, bio-augmentation is a
good strategy for bioremediation. Dehalococcoides is the key factor
for success of bioaugmentation in the contaminated sites. In the
application of bioaugmentation in the different contaminated sites,
strains 195 [30], BTF08 [41] and UCH007 [42] are considered if PCE
were main pollutants. However, strains having tceA and vcrA
reductive dehalogenase are the options for TCE-contaminated sites.
In preventing cis-1,2-DCE or VC accumulation in the field, strains VS
[43,44], BAV1 [45,46] and IBARAKI [47] were selected to be the
mixed culture with the strains specific for degradation of PCE or
TCE. Therefore, community of Dehalococcoides spp. in bio-
augmentation is the appropriate option for detoxification.
4. Dehalococcoides community controls in biodegradation
Microbes in the environment often form commensal or
competitive relationships with other bacteria in the system. A
consortium of bacteria often performs better as an inoculum since
the bacteria are already with a community of other bacteria that
synergistically support the activity of interest such as pollutant
degradation. Also, these bacteria possess the functional genes
which have a significant role in the reductive dechlorination pro-
cess of chlorinated ethenes contaminated sites. Thus, the metabolic
pathways [48] of horizontal gene transfer for mixed substrate uti-
lization is essential in the success of dechlorination as they provide
specific nutrients needed in the dechlorinating consortia.
It is interesting to know that Dehalococcoides is not capable of
synthesizing a compound essential for growth but relies on other
organisms to provide the nutrients [48e50]. The complicated
community composition works for the dechlorination are sum-
marized in Table 6. The activity of fermentative communities is
linked to Dehalococcoides spp. [28,51]. Syntrophic fermenters like
Firmicutes and d/ε-Proteobacteria divisions often coexist with
Dehalococcoides community [37,49]. These syntrophs produce H2

Table 6
Microbial community composition works for the dechlorination.

Group Taxonomy Functions in the dechlorinating community References

Syntrophic fermenter phylum Firmicutes
Supply corrinoid cofactors and methionine to D. spp. [37,49]

It also scavenge the oxygen.
Syntrophic fermenter phylum d/ε-Proteobacteria
Degrades organic matter to produce H2 and acetate [49]
Syntrophic fermenter family Geobacteraceae
Helps in dechlorination of PCE and TCE. [12]

Provides corrinoid cofactor to D. spp.
Organohalide respiring bacteria (OHRB) genus Dehalococcoides
Performs the stepwise decomposition of PCE, TCE, [30]
three-types DCEs to VC, and ethene as end-product.
Other OHRB genus Dehalobacter
Helps in dechlorination of PCE and DCA. [14,36]
Other OHRB genus Sulfurospirillum
Helps in dechlorination PCE to cis-DCE. [21,36]

Competes for H2 utilization.
Other OHRB genus Desulfitobacterium
Helps in dechlorination PCE to cis-DCE. [21,85,86]

Competes for H2 utilization.
Sulfate-reducing bacteria genus Desulfuromonas
Cooperates to degrade PCE/TCE and competes for H2 utilization [17,30]
Sulfate-reducing bacteria family Desulfobacteraceae
Helps in dechlorination of PCE and TCE. [10]

Provides H2 and essential corrinoid cofactors.
Sulfate-reducing bacteria family Desulfobulbaceae
Provides H2 and essential corrinoid cofactors. [10,87]
Sulfate-reducing bacteria family Desulfovibrionaceae
Supply essential corrinoid cofactors and scavenge oxygen. [49]
Sulfate-reducing bacteria family Syntrophobacteraceae
Supply essential corrinoid cofactors and scavenge oxygen. [49]
Methanogens genus Methanospirillum
Competes for H2 utilization. [53]
 D.M. Saiyari et al. / Sustainable Environment Research 28 (2018) 149e157
and acetate which are then consumed by D. mccartyi and metha-
nogens. Since Dehalococcoides spp. are non-fermentative microor-
ganisms; it depends on H2 supply from other microorganisms such
as Firmicutes and d/ε-Proteobacteria [52]. Other microorganisms in
the consortium also maintain low H2 concentration which is a
suitable environment for Dehalococcoides spp. However, the
organohalide-respiring bacteria usually compete with other mi-
croorganisms (i.e., methanogens and sulfate-reducing bacteria) for
H2 supply [30], e.g., Methanospirillum hungatei competes with
Dehalococcoides spp. for H2 [53]. Further, H2 competition occurs
among methanogens and other dechlorinating bacteria like Deha-
lobacter restrictus and Dehalospirillum multivorans [36]. Apart from
Dehalococcoides spp. depending on other microorganisms for H2,
other microorganisms in the consortium may also create a better
environment for Dehalococcoides spp. to grow. For instance, some
bacteria may act as reducing agent to scavenge oxygen and thus to
provide a strictly anaerobic condition and some may supply nu-
trients such as corrinoids cofactors (norpseudo-B12) for dehaloge-
nation reaction [31]. Moreover, other bacteria groups such as
methanogenic archaea (i.e., family Methanosaetaceae), v-proteo-
bacteria (i.e., family Geobacteraceae, family Desulfobacteraceae,
and family Desulfobulbaceae) also perform in the partial dechlo-
rination but they are not as prevalent as Dehalococcoides spp. of
family Dehalococcoidaceae within phylum Chloroflexi.
Furthermore, several studies also showed that populations of
Dehalococcoides spp. grow more slowly and become more
fastidious as they are purified from other organisms. Only few
laboratories worldwide have succeeded in obtaining and main-
taining Dehalococcoides spp. Nutritional requirement of Dehalo-
coccoides spp. becomes more complex when isolation process
starts. It will only grow when specific nutrients are included in
their diet. In some cases, incomplete degradation are observed, it
is suggested that bacteria are inactive or even dormant. These
findings in the dechlorinating consortia further help to under-
stand the behaviors and interactions in the metabolic pathways
of the microorganisms in the dechlorinating communities for the
development of biomarker trends. This implies that prior to any
biostimulation or bioaugmentation strategies, it is necessary to
identify the indigenous populations at contaminated sites and be
able to characterize the dechlorinating populations and their
abundances.
5. Microbial growth kinetics for chlorinated ethene
degradation
Microbial kinetics can tell how fast the biomass is produced and
the rates pollutants are degraded. In biodechlorination (or reduc-
tive dechlorination), the active microorganisms metabolically
catalyze the removal of chlorinated pollutant or degrading it into a
lesser hazard form. Previous works have applied comparative
dechlorination and microbial kinetics approaches for degradation
rate determination as shown in Table 7. First-order kinetics (Eq. 1
shown in Table 7), a simplified version of Monod kinetics, is
applicable for substrate concentration that is much lower than the
half velocity constant [54]. Majority of the published papers in
dechlorination used Monod kinetics because it has been demon-
strated to be able to describe the growth (Eq. 2) of Dehalococcoides
spp. which are known to be involved in the sequential degradation
of chlorinated ethene compounds [55]. On the other hand, the mass
removal rate of chlorinated ethene is considered for the impartial
comparison of dechlorination and growth kinetics. For dechlori-
nation, mass removal rate has been compared in several ap-
proaches, included based on per microcosm [56], per unit of total
protein [41], per unit of total biomass [57], and per unit liquid
volume [58].
153
In a mixed culture, proteins and biomass of both dechlorinating
and non-dechlorinating organisms are usually present. Relative to
this, Cupples et al. [59] developed a dilution procedure model (Eq.
3) embedded to Monod kinetics to determine only the dechlori-
nating organism concentration for the maximum growth and
dechlorination rates. The dechlorination rates using dilution
model [59] were better than the rates in growth of Dehalococcoides
spp. enumerated by competitive PCR [43]. The model shown in Eq.
4 relates the microbial growth rate to the changes in microbial
concentrations. However, this model is limited probably because
of the electron donor (i.e., H2) non-inclusion and the enzyme
competition of the different chlorinated ethenes [59] not consid-
ered. But then, total organism or protein may include both active
and inactive dechlorinating organisms which may affect the
degradation prediction in some aspects. Hence, caution must be
taken into consideration when comparing dechlorination rates.
Some techniques addressed the gap for active and inactive cells
which account only for active organisms such as considering
mRNA and the use of photoreactive DNA binding dye called pro-
pidium monoazide (PMA). When mRNA was compared to protein
abundance, poor correlations were observed [37]. On the other
hand, PMA was used in selective detection of live/dead bacteria
[60,61] in DGGE, microarray and qPCR but not in Dehalococcoides
spp. in particular.
If either microbial growth or decay is insignificant and has a low
to moderate concentrations of chlorinated ethene compounds,
MichaeliseMenten kinetics can be adapted as shown in Eq. (5). This
kinetic model is commonly used for short duration (i.e., hours,
days) wherein steady state of biomass is assumed [62]. In cases
when the dechlorinating bacteria desired only to degrade a
particular compound even several electron acceptors are present
especially at high concentration (~4 mM) of chlorinated ethenes
(i.e., PCE, TCE), Haldane inhibition model in Eq. (6) fits this kind of
experiment. On the other hand, Eq. (7) shows the downside model
which cannot account for abrupt decline in the dechlorinating ac-
tivity [55].
Self-inhibition of TCE occurs at 1 mM and complete inhibition at
concentration of about 4 mM and above. In this light, TCE con-
centrations from 0.04 to 8.4 mM were used to develop an effective
concentration (EC50) model [55] as a log-logistic doseeresponse
model that can accurately simulate competitive inhibitions as
presented in Eq. (8). In the considered EC50 model, the highest
biodegrading activity was at 0.3 mM TCE but no activity at 4e8 mM
of TCE concentrations [55]. This EC50 can model the specific
degradation of the species; however, information on biomass
concentrations of the dechlorinating bacteria is unavailable which
can only be provided by Monod kinetic model. Neither first order
nor MichaeliseMenten kinetic models can describe inhibition.
From the current reaction kinetic models used, Monod kinetics
delivers a sound prediction of the dechlorination reactions taken at
the bioremediation site. The chloroethene dechlorination rate de-
pends on the targeted microbial amount and the activity of func-
tional gene for the particular compound.
Reported dechlorination rates, growth rates and doubling time
of Dehalococcoides spp. under optimal conditions in the laboratory
are shown in Table 8. For instance in TCE dechlorination, strain 11a
has the highest dechlorination rate of 81 mM d
1 [56] compared to
strain KB1/VC (60 mM d
1) [59,63], strain ANAS2 (47 mM d
1) [56],
strain GT (40 mM d
1) [64], and strain FL2 (27.5 mM d
1) [45].
However, the doubling time of strain 11a (31.2 h) is 1.6-fold longer
than strain ANAS2 (19.9 h). This suggests that strain 11a has higher
activity for TCE degradation than strain ANAS2. In the field study,
amounts of Dehalococcoides spp. are the key factor for the success of
chlorinated compounds degradation. The threshold values for
application of Dehalococcoides spp. are listed as follows. Population
154 D.M. Saiyari et al. / Sustainable Environment Research 28 (2018) 149e157

Table 7
Review of dechlorination kinetics.

Type of kinetics Kinetics equations Advantages Disadvantages References

First order kinetics Raten ¼
k1 ; n Cn (1) - Very simple. - Applicable only when [54]
Where: substrate concentration
Raten is the rate of reductive dechlorination ðmM d
1 Þ; are below half velocity
constant.
k1 is the first order degradation rate constant ðd
1 Þ;
- Limit electron donor and
Cn is the aqueous concentration of the chlorinated
electron acceptor.
ethene compound, ðn mMÞ.
- Cannot demonstrate
microbial growth or
decay.
Monod kinetics Raten ¼
kmax ; n Cn Xn
(2) - Demonstrate microbial - Limit electron donor and [55]
Ks; n þ Cn
growth. electron acceptor.
Where:
- Can describe two or more
kmax ; n is the maximum growth rate ðmM cell
1 d
1 Þ; limiting substrates.
Xn is the microbial concentration ðcell L
1 Þ;
Ks; n is the half velocity constant mM of the compound, n.
Monod kinetics X0 ¼ DYSm
or Xo ¼ DS - Distinguish - Limit electron donor and [59]
Vi (3)
m
Vi Y
with dilution model Where: dechlorinating and non- electron acceptor.
dechlorinating
X 0 is the initial organism concentration (cells L
1);
microorganisms.
D is the dilution factor where X mL of the inocula is transferred
to 60 mL of media in the batch bottles equals to X/60;
YSm =Vi is the concentration of the dehalogenating microbe (mM);
Sm is the mass of the compound consumed (mM);
Vi is the volume of the culture (mL).
Whereas, the microbial growth rate is related to the change in
biomass concentration as described in Eq. (4) below.
m1 ¼ dX=dt
X or dX X
Y ¼ m1 Y dt (4)
Where:
m1 is the maximum growth rates (d
1);
X is the dechlorinating cells concentration (cells L
1);
Y is the yield.
Michaelis Menten kinetics Raten ¼
kmax ; n Cn
(5) - Electron donor can be - Cannot demonstrate [62]
Ks; n þ Cn
considered. microbial growth or
- Can describe two or more decay.
limiting substrates. - Steady state condition is
- Can be used for short assumed.
duration.
Haldane Inhibition model Raten ¼
kmax

; n Cn
Xn  (6) - Can be used for high - Cannot demonstrate [55]
Ks; n þ Cn 1þ Cn
KH;n concentration of limiting microbial growth or
8   substrate. decay.
>
< kmax ; n Cn Xn 1
Cn if Cn  Cmax; n - Cannot be used to for
Raten ¼ K s; n þ C n C max; n (7)
>
: abrupt decline in
0 if Cn > Cmax; n
dechlorination activity.
EC50 model Raten ¼
kmax ; n
Cn Xn
 (8) - Can be used to model the - Cannot demonstrate [55]
Ks; n þ Cn 1þexp bn log Cn
EC50;n specific degradation of microbial growth or
Where: the species at high decay.
EC50;n describes the concentration at which kmax ; n concentration.
is half the concentration of the
uninhibited level of the compound ðmMÞ
kmax; n is the
maximum degradation rate
on a unit cell basis with cell growth related to degradation
activity ðmM cell
1 d
1 Þ;
n is half of the uninhibited level;
Cn is the aqueous concentration of the chlorinated
ethene compound, (n mMÞ.
Xn is the microbial concentration ðcell L
1 Þ;
Ks; n is the half velocity constant ðmMÞ of the compound, n.
bn is the parameter for slope of the doseeresponse curve ðmMÞ.

of Dehalococcoides spp. higher than 107 copies L
1 groundwater is
good for natural attenuation. Whereas, extra addition of nutrients
(biostimulation) and Dehalococcoides spp. (bioaugmentation) are
required for bioremediation when amounts of Dehalococcoides spp.
are lower than 107 copies L
1 groundwater and 103 copies L
1
groundwater, respectively. In comparison with laboratory data,
growth rates of Dehalococcoides spp. in the field are 10 times higher
than that of its cultured in the laboratory. Therefore, microbial
growth rates, corresponding to total amounts of Dehalococcoides
spp. play the key role on the estimation of biodegradation duration
and bioremediation strategy.
6. Future perspectives
The paradox of substrates threshold concentrations is possible
perspective in the research field. Among Dehalococcoides spp., only
strain 195 possesses nitrogenase encoding operon [65] and is
capable of fixing atmospheric N2. Nitrogen increment results in
Dehalococcoides spp. exponential growth [66]. Also, all Dehalo-
coccoides spp. show evidence of carbon source metabolic re-
strictions [67] and strong correlations of 16S rRNA gene copies in
Dehalococcoides spp. with concentration of dissolved organic car-
bon [68]. Additionally, in the electron transport chain, the
 D.M. Saiyari et al. / Sustainable Environment Research 28 (2018) 149e157 155

Table 8
The doubling time, growth and dechlorination rates of Dehalococcoides isolates.

Strain Doubling time (h) Growth rate (d
1) Dechlorination rate according to compound (mM d
1) References

D. mccartyi 195 19.2 NAa NAa [30]

D. mccartyi CBDB1 NAa 0.41 NAa [75e77]
D. mccartyi VS NAa 0.42e0.49 ± 0.02 VC ¼ 19.9 [43,44,46]
D. mccartyi BAV1 52.8 2.2 VC ¼ 54 [45,46]
D. mccartyi FL2 57.6 NAa TCE ¼ 27.5, cis-DCE ¼ 30.4, trans-DCE ¼ 18.8 [45,78]
D. mccartyi KB1/VC 0.28 ± 0.01 TCE ¼ 60, VC ¼ 43 [59,63]
D. mccartyi GT 48e60 NAa TCE ¼ 40, 1,1-DCE ¼ 62, cis-DCE ¼ 41, [64]
VC ¼ 127
D. mccartyi DCMB5 NAa NAa Dioxin ¼ 16 [79,80]
D. mccartyi MB 24.0 NAa PCE ¼ 44.5, TCE ¼ 78.6 [33,34]
D. mccartyi ANAS2 19.9 NAa TCE ¼ 47, 1,1-DCE ¼ 53 cis-DCE ¼ 55 [56,66]
VC ¼ 89
D. mccartyi 11a 31.2 NAa TCE ¼ 81, 1,1-DCE ¼ 54, cis-DCE ¼ 73, [56]
trans-DCE ¼ 69, 1,2 DCA ¼ 25, VC ¼ 407
a
NA e not applicable.

Table 9 in the metabolic pathways for rapid degradation of chlorinated

Hydrogen threshold concentrations for reduction of different chlorinated-ethene
compounds.
Process in mixed culture/field studies H2-threshold (nM) References
PCE and TCE reduction 0.6e0.9 [36,88]
cis-1,2-DCE reduction 0.1e2.5 [36,88]
VC reduction 2e24 [36,88]
concentration thresholds of H2 as electron donor used for reduction
of PCE/TCE, cis-1,2-DCE and VC are summarized in Table 9, and the
detail information of electron transformation has been considered
[69].
On the other hand, perspectives in developing a more compre-
hensive dechlorination kinetic model that accounts for the growth
rates of active dechlorinating microorganisms, interspecies supply
and demand of different substrates in the consortia and the use of
alternative abiotic substrates even in extreme chlorinated ethenes
concentrations are promising research directions. Microbial growth
rates and degrading activities of the concerned compounds in the
Dehalococcoides spp. are the important factors for prediction of
biodegradation duration. These are also important in making de-
cision for bioremediation strategy options to remediate contami-
nated site. Finally, microbial interaction, competition and synergy
between Dehalococcoides spp. and co-existing microbes in the
entire community grown in the field need to be understood and
require more studies.
7. Conclusions
Recent biological system developments in bioremediation of
chlorinated ethene contaminated site reveal that Dehalococcoides
spp. are the most important group of microorganisms for their
capability of complete reductive dechlorination. Dehalococcoides
spp. favor anaerobic, neutral pH, and reducing conditions, and has
multiple reductive dehalogenase genes. These genes metabolically
use H2 which are essential in the reductive dechlorination of
chlorinated ethenes. In the dehalogenation processes, chlorinated
ethene compounds serve as electron acceptors while H2 is the
electron donor at low concentrations.
The increasing amount of Dehalococcoides spp. provides new
information in the functional diversity of this key dechlorinating
bacterium. Dechlorinating consortia of Dehalococcoides spp. eluci-
date a complex and diversified microbial community. This requires
a deep understanding of various systems (i.e., hydrogeology,
geochemistry, biochemistry, microbiology and ecology). The other
microorganisms in the dechlorinating consortia perform a vital role
ethenes. These microorganisms offer services to growth kinetics of
Dehalococcoides spp. especially on its nutritional requirements.
However, there is a need to fully understand its nutrient re-
quirements in order to prosper isolation related activities. Reduc-
tive dehalogenase genes in Dehalococcoides spp. are considered
biomarker, being able to infer the specific activity of every strain for
the particular compound being dechlorinated.
The expanding knowledge from recent developments in
biomolecular tools divulges interaction of Dehalococcoides spp.
and adaptation to the environment. Further, the microbial
growth kinetic gives information on how Dehalococcoides spp.
respond within its consortia and to bioremediation treatment.
Therefore, gene quantification coupled with reaction kinetics
are acceptable predictive model for both natural and engi-
neered conditions. Hence, the previous works on reductive
dechlorination of chlorinated ethenes in Dehalococcoides spp.
provide an integrated approach to properly estimate, evaluate,
and stimulate parameters to enhance degradation with pre-
dictable outcomes for rapid bioremediation and other possible
bioremediation options.
Acknowledgement
The authors gratefully acknowledge the scholarship to Donamel
M. Saiyari provided by the Engineering Research and Development
for Technology (ERDT) of the Department of Science and Technol-
ogy (DOST) through Mapua Institute of Technology in the
Philippines, and the Global Water Quality Research Center of the
Department of Environmental Engineering of National Cheng Kung
University in Taiwan where part of the research was carried out.
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