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bioremediation
Bioremediation of organic
compounds - putting microbial
metabolism to work
Edward J. Bouwer and Alexander J. B. Zehnder
Microorganisms
can
metabolize
many
contaminants, either to obtain carbon and/or energy for growth, or as cosubstrates, thus converting them to products such as carbon dioxide, water,
TIBTECHAUGUST1993(VOL11)
1993,ElsevierSciencePublishersLtd(UK)
361
bioremediation
anaerobes: both types of organisms attack the molecule
initially by hydration 2.
There are a number of biological reaction mechanisms that can deal with chlorinated aliphatic compounds. In aerobic organisms, nucleophilic displacements, hydrolysis, oxidation by a monooxygenase,
intramolecular substitutions and hydration reactions
have been reported to be responsible for removal of
the halogen 4. Anaerobically, the halogen is generally
removed by reductive processes (C. Holliger, PhD
Thesis, Agricultural University, Wageningen, The
Netherlands, 1992).
Aerobic
degradation
Anaerobic
degradation
H3C-(CH2)n-CH2-CH 3
H3C-(CH2)n-CH=CH 2
~~H20
NADH2
~1802
NAD ~ ; "~H2180
H3C-(CH2)n-CH2-CH218OH
HaC-(CH2)n-CH2-CH2OH
NADH2-- ;
~2[H]
TIBTECHAUGUST1993(VOL11)
362
bioremediation
Aerobic conditions
a
Eukaryotic
R
1- ~
"
".
non;-.
-~ " ~ "H
%/
/
:>%-->7>:0n--
~H
" ~ - OH
=
,.....
enzymatid-.
acid # ~"~"'~
non- . /
H ,/
orth~-
.
fission
enzymatic.,"
[]
~
~
.-
"~ ~"~.,..~OH
~
i~'-Zl
~
meta-
~ T . , ~ O~ /
COOH_._Aco,y, OoA
'--r-' ~. ~',~/COOH
,.~
CliO
I-
COOH---~-'~
~t"~,"& OH
Succinate
Acetaldehyde
^ + -
vyruvate
fission
c/s-Diol
Prokaryotic
Anaerobic conditions
/ - - - ~ r COOH
COOH
2-oxocyclohexanecarboxylate
Be~zoate
--/At~
~ ,,',~
. / " t~CO2
"~OH
Phenol
. . . .
~
OH
Catechol
Pimelate
COOH
4~]=_@COOH~ =-@~--~-'-~COOH
3132
=
~ J
"~
CO2
~
~
Cyclohexanone
Methylcyclo-
CZooV
Heptanoate
COOH
hexanoate
""~
2[H]
1420
.~...~ . . . ~
. CocO%
6coo .
COOH
Caproate
C'CcOO
Adipate
Figure 2
(a) General degradation pathways for several classes of aromatic compounds under aerobic conditions. Catechol is a key intermediate prior to ring cleavage. Multiple arrows indicate that several reaction steps are involved between the major intermediates and the products shown. Molecular oxygen (0 2) is
required to initiate the aerobic degradation. (b) Specific mechanism for conversion of an aromatic compound to catechol under aerobic conditions. R denotes
a functional group that may or may not remain on the molecule. For example, if R is a hydrogen atom, then the pathway is for benzene degradation. The
non-enzymatic reactions shown can occur in the absence of microbial activity (chemical processes). (c) General degradation pathways for benzoate, phenol and catechol under anaerobic conditions. Cyclohexanone is a key intermediate prior to ring cleavage. (d) Specific mechanism for conversion of benzoate under anaerobic conditions.
Co-substrate biotransformations
For some important pollutants, such as trichloroethylene (TCE) is, dichlorodiphenyltrichloroethane
(DDT) 16, and polychlorinated biphenyls (PCBs) 17,
biotransformation occurs as co-substrate utilization.
Here, enzymes involved in the metabolism of the
growth-supporting substrate are also able to transform
the organic contaminant (co-substrate). The organic
products of co-substrate metabolism may be resistant
to further biotransformation, may be transformed
further by another co-substrate reaction, or may be
used by other microorganisms as substrate for generating carbon and energy 18. In some instances, cosubstrate metabolism stops when the metabolites
formed inhibit further degradation 19. In order to
TIBTECH AUGUST 1993 (VOL 11)
exploit co-substrate biotransformation for bioremediation, one or more exogenous substrates must be
added: T w o important examples o f co-substrate
utilization are detailed below: (1) aerobic co-oxidation
o f chlorinated solvents; and (2) anaerobic reductive
dcchlorination of 1,2-dichloroethane by corrinoids
and factor F430in methane-producing bacteria.
363
bioremediation
methane monooxygenase (MMO), has an unusually
broad substrate-specificity. M M O can transform a
variety of other compounds, such as halogenated
alkanes and alkenes, alcohols, cyclic compounds and
aromatic compounds is. Methanotrophs must expend
energy in this first step, catalysed by M M O , that results
in oxidation of the coenzyme N A D H to NAD +. In
subsequent oxidation steps, energy is derived for
growth and maintenance through N A D H regeneration. The transformation oftrichloroethylene (TCE)
and trans-l,2-dichloroethylene (t-DCE) by M M O is
also shown in Fig. 4. These compounds do not appear
to be used by methanotrophs as a source of carbon or
energy, but are oxidized in a reaction secondary to
methane oxidation. The initial step is an epoxidation
to form either the TCE-epoxide or t-DCE epoxide
(Fig. 4) 2. The epoxides are unstable and react chemically to yield aldehydes and acids. These products are
easily mineralized by many bacteria to chloride, water
and carbon dioxide. Little et al. 21 proposed a general
pathway for T C E transformation by methanotrophic-heterotrophic mixed cultures. This pathway
involved the epoxidation o f T C E by methanotrophs,
abiotic hydrolysis o f the epoxide to nonvolatile products, and the subsequent heterotrophic degradation of
the products to carbon dioxide, chloride and water.
Methanotrophs co-oxidize less-halogenated compounds more rapidly than highly halogenated compounds 22. Compounds saturated with chlorines, such
as carbon tetrachloride and tetrachloroethylene (PCE),
do not appear to be transformed. The ratio of methane
mass used to the mass of T C E oxidized was found to
vary between 100:1 and 1000:1 (IKeE 20). Consequently, large quantities o f methane need to be
injected into a contaminated.soil system for treatment
of even a relatively small amount of T C E contamination.
Bacteria utilizing isoprene instead of methane were
also found to be highly efficient co-oxidizers o f T C E
and other chlorinated solvents23. The isoprene-utilizing bacteria were able to tolerate bigher concentrations o f T C E than methanotrophs.
Anaerobic reductive &chlorination of 1,2-dichloroethane
In the absence o f molecular oxygen, microbial
reduction reactions involving organic contaminants
increase in significance as environmental conditions
become more reducing. Anaerobic biotransformation
of halogenated aliphatic compounds has been
observed in field studies, in continuous-flow fixedfilm reactors, and in soil, sediment and aquifer microcosms under conditions of denitrification, sulfatereduction or methanogenesis. The initial step in the
anaerobic biotransformation is usually reductive
dehalogenation, where the halogenated compound
becomes an electron acceptor, and in this process, a
halogen is removed and is replaced with a hydrogen
atom (Fig. 3).
Recent work demonstrated the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethane
and chloroethane (CA) by concentrated cell suspen-
Hydrogenolysis
RX+2H ++2e-~
Dihalo-elimination
I I
--C--C-+2H++2e ~/"C
RH+HX
= C/\+2HX
X x
examples :
examples :
CI
I
CI--C-CI
CI
+2H++2e ~
H - C--Cl +HCI
H H
H
H
I
I
H-- C--C--H + 2H++ 2e- ~ ~.C= C." + 2HCI
I
Cl
Cl
C/
/CI
C/
/(31
/C=C% + 2 H + + 2 e = ~ /C=CKcI+HCI
Cl
Cl
H
CI
C l , ~ CI
H,'-,..~(,~ H
H
+ 2H++ 2e ~
H
C k . ~ CI
H---,~i,~ H
H
H"
\ H
CI
CI
CIT~C[
+2H++ 2e - - C I T J ~ + 2HCI
Cl-'-',-,r~ Cl
Cl,--.h,/
Ci
Cl
+ HCl
Hydrolytic reduction
Coupling
I
2RX + 2H+ + 2e ---~ R- R + 2HX
example :
Ht
2H-C-CI
CI Cl
H ~ 2HX
2 ~
RO
RXn+ 2H + + 2e ~
[:RXn.2] /
22X
2H20~2HX ROOH
example :
+2H++2e ~
HI HI
. - C - - C - - H +2.CJ
'
H H
CI
CI
H~
~12~''
2HCI
1/~C0
;C( + 2 H + + 2 e - - ~ ' l : C ( ~ / ~
c, ,
.)I:
2HCI
~'.J~.COOH
Lr12u
"
2HCI
Figure 3
Reductive dehalogenationmechanisms and examples using chlorinated compounds
that are commonly detected as contaminants in the subsurface. The hydrogenolysis
reaction replaces a halogen substituent by a hydrogen. In dihalo-elimination,two halogens are eliminated from the compound, and at the same time a double bond is
formed. The coupling reaction occurs when free radicals are involved. The fourth
mechanism, hydrolytic reduction, involves a two-electron reduction of a polyhalogenated carbon to a carbenoid followed by hydrolysis. Hydrogenolysis and dihaioelimination reactions are more common. (This figure was redrawn, with permission,
from C. Holliger, PhD Thesis, Agricultural University, Wageningen, The Netherlands,
1992.)
Most pathways discussed so far have been investigated in bacteria. Far fewer metabolic studies for
organic contaminants have been performed with
fungi, especially in the context of bioremediation.
Several reasons for this exist: generally, bacteria are
easier to culture and grow more quickly than fungi;
they are more amenable to straightforward molecularbiology manipulation techniques; they appear to be
able to metabolize chlorinated organics and other
organic contaminants better; and they mineralize these
TIBTECH AUGUST 1993(VoL 11)
364
bioremediation
Assimilation into biomass
H20
o2
E2
CH 4
NADH
CI
~'~ C H 3 O H ~
NAD
CI
E4
HCHO"~I~'HCOOH
XH NAD
CI
"C=C "
CI "
~H
"---'I~CO 2 + H20
N A D H NAD
NADH
CI
TCE ~
NADH
H
E3
TCE epoxide
NAD
Cl
~'C=C "
CI"
~'H
t-DCE ~
NADH
CI
~C--C
CI s NO/ ~H
t-DCE epoxide
NAD
Figure 4
Example of co-substrate biotransformation. Methanotrophic bacteria oxidize methane
aerobically for growth according to the pathway at the top of the figure. The
initial enzyme is nonspecific and co-oxidizes trichloroethylene (TCE) and trans-l,2dichtoroethylene (t-DCE)to TCE and t-DCE epoxides as shown by the two lower pathways. The epoxides are unstable and form products that are good substrates for
growth of heterotrophic bacteria. The combination of the methanotrophic and heterotrophic bacteria yields complete mineralization of the TCE and t-DCE.
E1 = methane monooxygenase; E2 = methanol dehydrogenase or alcohol oxidase;
E3 = formaldehyde dehydrogenase; E4 = formate dehydrogenase; X = a proton and
electron carrier.
influence biotransformation rates include pH, temperature, concentration and redox condition.
Environmental pH
Most soil environments have pH values between
five and nine, the range that is favorable for growth of
many microorganisms. For most species, the optimum
TIBTECHAUGUST1993 (VOL11)
/x=
~[~maxS
K s + S + S2/Ki
where/x is the specific growth rate, S is the compound
concentration, [/"maxis the maximum specific growth
rate, K s is the Monod half-velocity constant, and K i
is the inhibition constant. At low concentrations, often
in the range of micrograms to nanograms per liter,
insut~cient energy and carbon may be available for
growth and maintenance. Consequently, biodegradation ofxenobiotic compounds may be hampered at
both high and low concentrations.
Redox conditions (availability of electron accepto0
Since microorganisms are often specific with regard
to the electron acceptors that they are able to use, the
availability o f particular electron acceptors in the environment will determine which microorganisms
flourish. This, in turn, will determine which organic
contaminants may be degraded. The coupling o f mass
transport and microbial reactions in the subsurface
results in spatial gradients o f electron acceptors and
redox conditions (Fig. 5). Some compounds are only
transformed under aerobic conditions, others require
strongly reducing conditions, and still others are transformed in both aerobic and anaerobic environments.
This knowledge is important for identifying environmental conditions conducive to biotransformation o f
a particular organic contaminant, and in establishing
how to manipulate the medium chemically to achieve
365
bioremediation
a
Vadosezone
Aerobic
Denitrification
Sulfatereduction
Methanogenesis
Chlorobenzenes*
Chlorophenols
Petroleum compounds*
PAHs
Chlorobiphenyls
Biphenyls
Chloroanilines
Several halogenated
aliphatics*
Chlorophenols
Toluene*
Toluene*
b
cs
CH 4
NO3-
Figure 5
(a) Cross-section of a typical ground-water plume in the subsurface with the source of contamination shown on the left and ground water
flowing from left to right. The zone between land surface and the top of the ground water is called the vadose zone. As the chemicals in
the source region move with the ground water, the concentrations of chemical species will change due to physico-chemicaland biological
processes. The kinds of chemical changes that can occur for a mixture of organic contaminants, oxygen, ammonia, nitrate and sulfate are
illustrated in (b). Microbial processes wilt consume the availableelectron acceptors and will create a sequence of different redox regimes
within the plume. Oxygen provides the most free-energyto microorganisms during electron transfer and is consumed first. Use of nitrate,
Mn(IV), Fe(lll),sulfate and carbon dioxide during anaerobic biodegradationtypically yield decreasingamounts of free energy during electron
transfer according to the order listed. Possible redox condition changes are shown immediatelybelow the plume in (a) and include aerobic, denitrification, manganeseand iron reduction, sulfate reduction, and methanogenesis.The size of each redox regime within the plume
will depend on the relative initial concentrations of the chemicals. Below each redox regime is a list of organic contaminantsthat are known
to be biotransformed under those conditions. Consequently, an organic contaminant is likely to biodegrade in only a portion of the plume
with favorable redox conditions. The compounds marked (*) are among the 35 most prevalentcontaminantsin disposal-siteground water31.
PAH = polyaromatic hydrocarbon, PCBs = polychlorinatedbiphenyls.
366
bioremediation
C (in soil gas)
II
TIBTECHAUGUST1993(VOL11)
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bioremediation
De Bruin et al. 39 and DiStefano et al. 4 are potentially
very useful reactions to employ for site clean-up.
Through improved understanding of organiccontaminant biotransformation, the prospect for
successfully stimulating and exploiting microbial
metabolism in the environment appears very good.
Acknowledgements
We are grateful for financial support from the
Netherlands Organization for Scientific Research,
The Netherlands Integrated Soil Research Programme, Agricultural University Wageningen, and
Cooperative Agreement ECD-8907039 between the
National Science Foundation and Montana State University. We thank ChristofHolliger for his courtesy in
allowing the authors to use a figure from his PhD
dissertation.
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