360

bioremediation

Bioremediation of organic
compounds - putting microbial
metabolism to work
Edward J. Bouwer and Alexander J. B. Zehnder
Microorganisms

can

metabolize

many

aliphatic and aromatic organic

contaminants, either to obtain carbon and/or energy for growth, or as cosubstrates, thus converting them to products such as carbon dioxide, water,

chloride and biomass. These biotransformations can be exploited for treatment of

contaminated soils and ground water.
Contamination of ground water and soil by industrially derived organic chemicals is common. C o n ventional decontamination methods often involve
flushing the subsurface with water to dissolve the
contaminants which can then be pumped to the surface for treatment. Such pump-and-treat systems are
generally inefficient, slow, and involve physico-chemical processes that do not result in complete destruction of the contaminants. Stimulation of microbial
growth in situ offers the prospect of converting a wide
variety of dissolved and sorbed contaminants to lessharmful products. This approach entails perfusion of
chemicals (nutrients, electron donors, and/or electron
acceptors) through the contaminated subsurface to
encourage microbial growth 1. This article addresses
key issues concerning the transformation of organic
contaminants that can be applied to the problem of
environmental contamination, as well as to the development of engineered treatment processes for subsurface clean-up.

Biotransformations that provide carbon and/or

energy for microbial growth
For organisms to grow, the presence of electron
donors and acceptors, a carbon source, and nutrients
is essential. In addition to compounds that are the
natural substrates, many contaminants can also satisfy
these growth requirements. Most organic contaminants can be classed as either aliphatic or aromatic
compounds that contain different functional groups,
such as - O H , -Cl, -NH2, - N O 2 and -803. As electron donors, these contaminants undergo oxidation as
a result of microbial metabolism; in the best case, they
are mineralized. Some o f the breakdown intermediates
may be assimilated as a carbon source for microbial
growth. Functional groups (e.g. -NH2, - N O 2 and
E. j . Bouwer is at the Department of Geography and Environmental
Engineering, The Johns Hopkins University, Baltimore, MD 21218,
USA. A. J. B. Zeknder is at E A W A G / E T H , CH-8600
Dfibendo~ Switzerland.

TIBTECHAUGUST1993(VOL11)

-SO3) may either be used as nutrients, or cleaved from

the carbon skeleton when the compound is reduced
or oxidized. Oxidation can occur aerobically or anaerobically, and the fate of aliphatic and aromatic c o m pounds that undergo biological oxidation is governed
primarily by the presence or absence of oxygen. O x y gen serves two distinct functions. It can act as the terminal acceptor of electrons that are released during the
oxidation of electron donors, or it can react directly
with the organic molecule. As an electron acceptor,
oxygen can be replaced by other oxidized inorganic
compounds such as nitrate, metal ions, sulphate, or
carbon dioxide, although the energy gains are then
smaller. However, no other compound can substitute
for its function as a direct reactant 2.

Degradation of aliphatic compounds

The general strategy is to convert the alkane chain
into a fatty acid. In the presence o f oxygen, the alkane
chain is attacked by a monooxygenase, which introduces one atom of oxygen from molecular oxygen
into the molecule, thus forming a fatty alcohol. This
alcohol is oxidized to an aldehyde, and finally to a
carboxylic acid. The carbox-ylic acid is channelled into
central metabolism for further oxidation by [3-oxidation (Fig. 1). Obviously, the initial oxidation step
with molecular oxygen is not possible under anoxic
conditions, and there is increasing evidence that alkanes (with the exception of methane) are anaerobically
recalcitrant 3.
Unsaturated aliphatic compounds (alkenes and
alkynes) are degraded aerobically by mechanisms similar to those by which saturated compounds (alkanes)
are degraded. However, the double and triple bonds
are more chemically reactive and can undergo additional reactions, such as epoxidation and hydration 3.
There are some indications that the double bond of
alkenes can be hydrated anaerobically (Fig. 1) to form
an alcohol, which is then further converted as in the
aerobic pathway 2. Alkynes, such as acetylene, are used
as both carbon and energy source by aerobes and

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]

Degradation of aromatic compounds

H3C-(CH2)n-CH2-CH180
H3C-(CH2)n-CH2-CHO
Aerobic biodegradation of many classes of aromatic
compound is common and proceeds through the key
intermediate, catechol (Fig. 2a). The mechanism of
2[H] ~
O
catechol formation and subsequent ring cleavage
under aerobic conditions is shown in Fig. 2b.
B-oxidation
H3C-(CH2)n-CH2-COOH
Eukaryotic organisms produce catechols from singlering aromatic compounds via an epoxide and a transHSCoA
diol using a monooxygenase (upper sequence in Fig.
2b). Prokaryotes introduce the entire oxygen molecule by a dioxygenase reaction forming first a cis-diol
H3C-(CH2)n-CH2-GO-SCoA
(lower sequence in Fig. 2b). In both types of organism, the aromatic ring of the catechol is opened by a
further dioxygenase reaction by either an ortho- or
~2[H]
recta-fission (Fig. 2b). Halogenated aromatic compounds are most often degraded in this manner and
H3C-(CH2)n_I-CH=CH-CO-SCoA
the chlorine is, in general, eliminated after ring cleavage by mechanisms that are not yet fully understoodS, 6.
H20
There are examples where at least one chlorine is
eliminated hydrolytically. The resulting phenol is
further oxidized by a monooxygenase to a catechoF.
Anaerobically, the aromatic ring is not oxidized but
2[H]
reduced, as illustrated for benzoate, phenol, and catH3C-(CH2) n_ 1-CO-CH2-CO-SCoA
echo1 (Fig. 2c). The key intermediate in this pathway
is a cyclohexanone 8. The ring is opened through
hydration of the cyclohexanone. Details of the
anaerobic biodegradation pathway for benzoate are
HSCoA T
CH3-CO'SCA
provided in Fig. 2d. Little is known about the metabolic potential of anaerobes for the biodegradation
HaC-(CH2)n_ 1-CO-SCoA
of aromatic compounds. In the absence of oxygen,
substituted aromatic compounds appear to be more
Figure 1
easily degraded than nonsubstituted ones. Current
Aerobic degradation of an alkane yields a fatty acid (top left). The appearance of 180 in
knowledge suggests that benzene is recalcitrant under
the intermediatesduring aerobic biodegradationconfirms that the one atom of oxygen
all anaerobic conditions, whereas substituted aromatic
introduced into the aliphatic compound comes from molecular oxygen, Aerobic degra
compounds [(PAHs) including some polyaromadc dation of alkenes and alkynes follows the same sequence. Anaerobic degradation of
hydrocarbons] are degraded, depending on the func- alkenes also leadsto fatty acids (top right). Here the oxygen introduced into the aiiphatic
tional group and the terminal electron acceptor compound comes from H20. Anaerobic degradation of alkynesfollows the same path
present. Aromatic compounds with more than one way. Akanes appear to be anaerobicallyrecalcitrant. The fatty acids formed by either
chlorine (polychlorinated) are reductively dehal- the aerobic or anaerobic processes are further oxidized by ~-oxidation,a common pathogenated before the
ring is reduced 9,1. way for both aerobic and anaerobic microorganisms. [H] indicates reducing equivalents
Monochlorobenzene was found to be inert under that are either required or formed in each reaction.
anaerobic conditions 11.
methanogenic conditions, chlorinated compounds are
Reductive dehalogenation
dehalogenated in a metabolic side-reaction, apparently
Halogenated compounds may be reductively without any profit for the organisms TM. Halogen atoms
dehalogenated during their degradation, or be used can be reductively removed from the molecule by
as a terminal electron acceptor for the growth of hydrogenolysis, dihalo-elimination, a coupling and a
microorganisms 12,13. In some cases, particularly under hydrolytic reaction (Fig. 3).

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.

Aerobic co-oxidation of chlorinated solvents

Methanotrophs are aerobic bacteria capable of initiating the oxidation o f chlorinated solvents while
using methane as a co-substrate. The pathway of
methane oxidation by methanotrophs to gain energy
for growth is shown in Fig. 4. The initial enzyme
responsible for the oxidation of methane to methanol,

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.)

sions of four strains of methanogenic bacteria (C.

Holliger, op. cit.). The methanogenic cultures
required H2/CO2, acetate or methanol as electron
donor (co-substrate). The same reductive dechlorination reactions were catalysed by cobalamin, and the
native and diepimeric form of factor F430 in a buffer
with Ti(III)citrate as electron donor, which indicates
that the dechlorination of 1,2-DCA to ethene and CA
is a co-metabolic reaction. The predominant reaction
mechanism with cobalamin was dihalo-elimination of
1,2-DCA to ethene. The native F430- and 12,13-diepi-F430-catalysed transformations produced ethene
and CA in a ratio of 2 : 1 and 1 : 1, respectively, indicating hydrogenolysis as a second reaction mechanism.
Bacteria versus fungi

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

'~ C--C ~.,

C I / NO/ H

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.

chemicals and use them, usually as carbon and/or

energy sources. N o studies with anaerobic fungi have
yet been carried out under anoxic conditions; the discovery o f strict anaerobic fungi is relatively recent
compared with that o f anaerobic bacteria 24.
Some aromatic contaminants can be partially oxidized by lignin peroxidase and manganese peroxidase,
the major components o f the lignin-degradative system of the white-rot basidiomycetes 2s. However, a
primary growth substrate, such as cellulose or glucose,
is required for the co-oxidation o f aromatic contaminants. The products o f the co-oxidation are generally
not metabolized further by the fungi. Consequently,
a mixed population o f fungi and bacteria is usually
required to provide all the metabolic capabilities for
complete mineralization o f the organic contaminant.
Fungi might prove valuable should molecular structures, which are otherwise not easily transported into
bacterial cells or metabolized by bacteria (e.g. dioxins) 26, have to be oxidized.
E n v i r o n m e n t a l factors
Important environmental factors that determine
whether or not biotransformation takes place, and

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)

pH for growth lies between 6.5 and 7.5, and relatively

few species can grow at pH values less than two or
greater than ten.
Temperature
The rates of enzymatically catalysed reactions are
influenced by temperature and can be quantified by
the Arrhenius relationship27:
k = A exp(-u/RT)
where k is the reaction rate constant, A is a constant,
u is the activation energy, R is the gas constant, and T
is the absolute temperature. The values o f A and u can
be determined from a plot ofln(k) versus 1/T. The
rates o f many microbial reactions typically double for
each 10C rise in temperature. Above a certain temperature cellular components become irreversibly
inactivated. Below 10C, many microbial reactions are
slow, which translates to long clean-up times for
bioremediation in cold climates.
Concentration
Many xenobiotic compounds tolerated at low concentrations become toxic to microorganisms at high
concentrations (compilation of toxicity data given in
Ref. 28), and this inhibitory influence on specific
growth rate can be described by the following expression29:

/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

Mn(IV) and Fe(lll)

reduction

Denitrification

Sulfatereduction

Methanogenesis

Organic contaminants transformed

Toluene*
[with Fe(lll)]
m-xylene*
p-cresol
Napthalene*
Acenaphthalene
Tetrachloromethane*
Bromoform
Hexachloroethane

Chlorobenzenes*
Chlorophenols
Petroleum compounds*
PAHs
Chlorobiphenyls
Biphenyls
Chloroanilines

Several halogenated
aliphatics*
Chlorophenols
Toluene*

Toluene*

Many halogenated aliphatics*

PCBs*
Chlorophenols
Trichlorobenzenes*

b
cs

Chemical species in bulk groundwater

Mn(ll)

CH 4

NO3-

Distance in the direction of groundwater flow

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.

a desired biotransformation. The compounds listed in

Fig. 5 are frequent contaminants at waste sites. Successful bioremediation strategies have been reported
for petroleum hydrocarbons, chlorinated phenols and
chlorinated solvents 3.
Bioavailability
Microorganisms and pollutants are distributed
among the solid, liquid and gas phases within the subsurface (Fig. 6) 31. Many organic contaminants are
hydrophobic and tend to sorb onto soil, such that only

a small fraction of the compound may actually be in

the bulk-water phase. Over long contact time, sorbing pollutants slowly diffuse into the inorganic and
organic matrix and may also form bound residues.
Most evidence indicates that the uptake o f compounds
by bacteria proceeds via the liquid phase. Consequently, a process such as sorption or volatilization that
reduces the solution concentration tends to reduce the
biotransformation rate. Furthermore, the accumulation of contaminants in fissures and cavities (Fig. 6)
renders them inaccessible to microorganisms and their
TIBTECHAUGUST1993(VOL1i)

366

bioremediation
C (in soil gas)

II

tributed and trapped globules. In contrast, hydrophilic

contaminants with low Kow , such as ketones and
alcohols, have high wettability and flow freely in the
KZ
subsurface.
Water table
Biodegradation rate-constants tend to decrease with
increasing Kow (Ref. 33), because Kow and aqueous
solubility are inversely related, with the latter controling bioavailabiliW and biodegradation rate. Density
determines the tendency for the immiscible phase to
float or sink in the subsurface. Volatilization can occur
in the vadose zone for compounds with a high Henry's
constant (H). Physico-chemical data for some organic
compounds commonly encountered in ground-water
contamination problems are provided in Ref. 34.
Biotransformations of contaminants during bioremediation attempts are most often controlled by
environmental conditions or availability of the substrate, and not by the presence, or absence, or the size
of appropriate microbial populations Is,35,36. Chemicals
Figure 6
Distribution of microbial cells and contaminant (substrate) in the subsurface. The solid (e.g. nutrients and oxygen) are often added to encourmineral phases in the subsurface are usually aggregated and ground water flows age growth of the best-adapted microbial population.
mainly in the channels between aggregates. The shaded areas depict crevices within Such chemical manipulations are perhaps not difficult
an aggregate that are filled with immobile water along with organic carbon that coats in the case of surface contamination, but the effects of
the mineral surfaces. A contaminantdissolved in the ground water (C) can be removed geological complexity and heterogeneity, such as strata
from solution by biodegradation (cell uptake), volatilization to soil gas above the of gravel, sand, silt and clay, and fractured layers, along
ground water, and by sorption to the organic-carbon and mineral surfaces within the with the difficulty of locating the sources of subsuraggregate (S; circle is a label to represent the sorbed phase). Sorption can occur at face contamination, can severely hamper the supply of
the surface of the aggregate or within the crevices of the aggregate. Contaminant chemicals throughout subsurface contamination. If the
sorbed and trapped within the aggregate is not easily transported back out and into appropriate, naturally occurring microbial populations
the ground water. Sorption and volatilization decrease the ground-water contaminant are not present, introduction of a variety of microbial
concentration (C), which decreases the amount biodegraded (reduces bioavailability). strains carrying genes encoding the requisite metabolic
pathways may be effective37,38. Such microorganisms
enzymes. Ri.jnaarts et al. 32 demonstrated that aerobic may be natural or genetically engineered with adbiodegradation of ci-hexachlorocyclohexane in soil ditional catabolic genes to degrade contaminants.
slurries was limited by mass transport and desorption However, the latter type of organisms will, in general,
from soil aggregates. The important conclusion from not persist in the subsurface without specific selection
this work is that the overall reaction rate is controlled pressure 38. Furthermore, the use of genetically modiby the desorption rate and not by the activity of the fied organisms for bioremediation raises public
degrading microorganisms. The practical effect of such concerns. Another difficulty is the transport of the
slow diffusion from within soil aggregates, and other introduced microorganisms to the place of need. The
kinetic limitations to desorption, is to decrease the rate subsurface is an efficient filter medium that generally
of removal of the contaminant, thereby increasing the restricts microbial transport in ground-water systems.
time required to achieve clean-up and the amount of Movement for short distances before deposition makes
chemicals that must be added to sustain microbial it difficult to disseminate introduced microorganisms
widely in soil and ground water. Nevertheless, such
activity.
The physico-chemical properties of the contami- approaches merit investigation as they will improve
nant play a role in determining its bioavailability. For our knowledge of how to exploit microbial
example, low water-solubility is an indication of the metabolism for control of contaminants.
potential to form a separate, nonaqueous phase that may
be too toxic for direct biodegradation. Wettability Towards practical application
influences the movement and availability of such an
Experience with large-scale bioremediation efforts
immiscible phase. Hydrophobic contaminants, such as is limited. The best approach might be to apply biopetroleum mixtures, PCBs, and some chlorinated sol- remediation at sites that contain readily degradable
vents, tend not to wet the hydrophilic surfaces in the contaminants and are relatively simple hydrogeologiground. Hydrophobicity is characterized by the cally. Exploiting anaerobic microbial processes for
n-octanol-water partition coefficient (Kow). Kow indi- bioremediation is still at an early stage, but is potencates the tendency for the compound to partition into tially advantageous for overcoming the difficulty in
soil organic matter. Compounds with high Kow tend supplying oxygen for aerobic processes in the subsur=
to sorb strongly to aquifer solids, which retards their face. The complete reductive dechlorination of 1,2movement and decreases their availability for bio- DCA to ethene (C. Hollinger, op. cit.) and the contransformation. This often results in irregularly dis- version of PCE to ethane or ethene, reported by
Vadose zone

TIBTECHAUGUST1993(VOL11)

367

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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