International Biodeterioration & Biodegradation. Vol. 39. No.

4 (1997) 253-285
(I 1997 Elsevier Science Limlted
All rights reserved. Printed in Great Britain
PII: SO964-8305(97)00021-8 0964~R305/97 $17.00 + 0.00
ELSEVIER

Bioremediation of Organic Waste Sites: A Critical

Review of Microbiological Aspects

Ann-Sofie Allard & Alasdair H. Neilson

Swedish Environmental Research Institute, Box 21060, S-100 31 Stockholm, Sweden

(Received 14 November 1996; revised version received 25 January 1997; accepted 2 February 1997)

An overview is presented of the potential for bioremediation of a range of

contaminated sites. Most of the compounds belong to groups that are widespread
and are generally persistent or toxic. Although attention has been focused on
microbiological aspects of their application, it is pointed out that a successful
programme requires integrated input from geologists, engineers, chemists and
microbiologists. It is emphasized that a protocol must be available for evaluating
the success of the procedures that have been implemented. Attention is directed
to critical issues, including partial degradation, formation of metabolites, and
recalcitrance of specific components in compiex mixtures. An attempt is made to
discuss the basic aspects of the biodegradation of components specific to the
various sites and to illustrate the outcome of experiments in bioremediation in
laboratory-based, pilot-scale or full-scale field operations. Brief discussion is
given of some less commonly perceived contaminants that may be present
simultaneously. c) 1997 Elsevier Science Limited

INTRODUCTION The range of compounds in non-domestic

Orientation
There is increasing concern over the increasing
volume of solid waste-both industrial and
domestic. There are several reasons for this:
(1) Some sites occupy valuable land in urban
areas and it is therefore imperative that its
reuse for commercial or domestic buildings
must be insured against present or future
health hazards.
(2) There is environmental concern over long-term
effects of landfills used for industrial waste on
account of hazard from the transformation of
the components and discharge of leachate into
water courses.
(3) The volume of domestic waste is huge, although
its heterogeneity will be diminished with the
adoption of source-separation by households.
It should also be emphasized that the sludge from
industrial water treatment facilities may be sent to
landfills, which therefore may contain both
undegraded-and presumably more recalcit-
rant-substrates and include terminal metabolites
that may have been produced.
landfills is extremely wide and includes not only
industrial waste from the manufacture of chemicals
but also substantial volumes of military waste
containing a number of explosives and chemical
warfare agents. It should also be noted that
continuous atmospheric precipitation will have
occurred, and therefore virtually any established
atmospheric contaminant may also be present.
Indeed, the atmospheric input into Lake Michigan
has been found to exceed that from landfills
(Hornbuckle et al., 1995), so that important inter-
phase partitions should be taken into account. The
range of relevant compounds to which attention
should be directed is dependent on the availability
of analytical procedures both for the original
compounds and for their possible transformation
products. An illustrative example is provided by the
development of chromatographic procedures that
have been applied to carbon black and coal tar and
that have revealed the presence of PAHs with up to
nine rings (Bemgard et al., 1993).
On account of the toxicity of most of the
compounds that are discussed here, there is
increasing demand for remediation of such sites
that involves removal of the offending
compounds. In this overview, the concept will be

2.53
 A.-S. Allard, A. H. Neilson

enlarged to include remediation of riverine developed in simulation experiments with

sediments in the vicinity of industrial production microcosms, although all of them necessitate
and contamination of groundwater by low- maximal appreciation of the microbiological
molecular-mass compounds that have penetrated issues. It should be emphasized that all
the overlying cover. The emphasis is placed on procedures, by necessity, will be site-specific since
microbiological aspects, whereas engineering they must take into account both the physical
considerations that are of cardinal importance lie environment and microbiological issues. They will
beyond the expertise of the authors. An attempt is also be contaminant-specific, and take into
made to summarize: (1) a range of sites that have account both the pathways and the regulation
attracted attention; (2) the contaminants involved; mechanisms for degradation of the generally
(3) the results of full-scale and laboratory model complex range of contaminants. For a given
experiments; (4) the relevant microbiological substrate, the induction and maintenance of the
aspects, including the organisms, the pathways of degradative enzymes will depend on the
biodegradation and biotransformation; and (5) environmental conditions (Guerin & Boyd,
determinants such as oxygen concentration and 1992a): the presence of readily degradable
bioavailability. Biological treatment of waste substrates will enhance the bacterial population
water and industrial exhaust gases is excluded: although not necessarily those required for
there is, moreover, an increasing tendency for degradation of recalcitrant compounds. In
recovery rather than destruction of valuable general, the principles that have emerged from the
components of these discharges. study of aquatic systems can be adapted to the
For the sake of completeness, attention is drawn terrestrial environment-with two important
to some alternative procedures: (1) supercritical changes of emphasis: the cardinal role of
carbon dioxide extraction of hydrocarbon association of xenobiotics with organic
contaminants (Schleussinger et al., 1996); (2) components of the soil (Bollag & Loll, 1983) and
extraction by vapour stripping (Siegrist et al., 1995); the importance of the water content of the soil.
(3) thermal blanket in situ removal and destruction In this overview, no account is given of the
of PCBs (Iben et al., 1996); (4) the use of higher procedure for genetic manipulation to improve
plants (Schnoor et al., 1995); and (5) the application the effectiveness of bacteria, and the account is
of enzymes (Bollag, 1992). A valuable review devoted to indigenous organisms or to
(Hamby, 1996) has been given that summarizes the inoculation with naturally occurring organisms
chemical and physical treatments of soils and of established metabolic relevance. Procedures
contaminated ground and surface waters. for the improvement of microorganisms for their
Application of higher plants, however, should be applicability to treatment programmes have been
preceded by a more thorough understanding of presented (Timmis et al., 1994). In many
their metabolic capacity for xenobiotics, and the situations, the waste will have lain for
extent to which they may contribute to discharge considerable lengths of time with the result that
of metabolites to the atmosphere. As an a natural flora of degradative microorganisms
illustration, it has been shown that: (1) increased will have developed through enrichment under
degradation of trichloroethene to CO2 took place natural conditions. It is, therefore, highly
in soil slurries from a site with a cover of desirable to optimize the activity of such
Paspalum notatum, Lespedeza cuneata, Solidago organisms.
sp. and Pinus taeda (Walton & Anderson, 1990); There is an enormous amount of literature in
and (2) prairie grasses may stimulate the treatment this area. Only a small fraction of this (only
of PAH-contaminated soil (April1 & Sims, 1990). primary publications) is cited, although as far as
The review by Bollag (1992) also addresses the possible, references to review articles have been
important issue of fungus-mediated poly- given where extensive literature citations may be
merization reactions of phenols and anilines, found.
which are noted again in Sections 2.3 and 2.4.4. A number of contaminated sites have been used
Three different strategies have been employed for illustration, though the extent to which
that involve treatment in situ, on-site or off-site, bioremediation has received practical application
and each has both advantages and disadvantages. is variable. For a number of reasons, emphasis is
The last two offer a more controlled approach placed on basic microbiological issues that have
that may be based upon a scale-up of procedures emerged from controlled laboratory experiments.
 Bioremediation of organic waste sites
(1) These illustrate many cardinal issues that
should be taken into consideration in scale-up
to filed exploitation.
(2) They reveal fundamental issues, such as the
reasons for using biphenyl to facilitate degra-
dation of PCBs, or of phenol or toluene to
induce enzymes for the degradation of tri-
chloroethene.
(3) Mixtures of a number of compounds are often
involved, and among groups such as PAHs
and PCBs, there are wide variations in the
biodegradability of individual components.
Microbiological considerations
Although a valuable review on microbiological
aspects of bioremediation of sites contaminated
with organdchlorine compounds has been given
(Morgan & Watkinson, 1989b), it is convenient to
provide a short summary of some cardinal issues
of significance for all xenobiotics.
(1) In most natural systems, many different
microorganisms will be present, and the
degradation of a xenobiotic is dependent upon
the activity of several microorganisms,
although sequential reactions may be carried
out by only a single member of the con-
sortium. Illustrative examples include the
degradation of parathion (Daughton & Hsieh,
1977) and chloropropionamides (Reanney et
al., 1983) and the important hydrogen-trans-
fer reactions and syntrophic associations
among anaerobic bacteria (Schink, 1992).
(2) It is very seldom that in natural ecosystems
only a single substrate is present. It is
therefore important to examine how the
regulation of degradative pathways may be
affected. The regulatory pathways for mono-
cyclic aromatic compounds, such as phenol,
benzoate, and hydroxybenzoates, have been
reviewed in detail (Ornston & Yeh, 1982).
Whereas biodegradation of a substrate is
frequently induced by growth with an early
metabolite, for example of biphenyl (Furuka-
wa et al., 1983) and naphthalene (Barnsley,
1976) by salicylate, the metabolites in the
lower pathway may suppress induction of
essential degradative enzymes. For example,
although the enzymes for the degradation of
toluene in Pseudomonas putida strain pWW0
are induced during succinate-limited growth,
they are suppressed when succinate is in
255
excess (Austen & Dunn, 1980; Duetz et al.,
1994). Where plausible and comparable de-
gradation pathways exist, however, e.g. in the
biodegradation of PAHs, a single organism
may display wide versatility. For example,
Pseudomonas paucimobilis strain EPA505 is
capable of degrading fluoranthene, pyrene,
benz[a]anthracene, chrysene, benzo[a]pyrene
and benzo[b]fluoranthene (Ye et al., 1996).
For halogenated substrates, the situation is
more complex. Whereas degradation of PCBs
is often carried out by organisms enriched by
growth with biphenyl itself (Hernandez et al.,
1995) naphthalene is a much less effective
inducer (Pellizari et al., 1996). The degrada-
tion of toluene and chlorobenzene generally
proceeds by respective extradiol (2,3) and
intradiol (1,2) fission of the initially formed
catechols. Since, however, 3-chlorocatechol
inhibits the activity of the 2,3_dioxygenase,
the degradation of toluene and chlorobenzene
is generally incompatible, except in strains
such as Pseudomonas sp. strain JS150 that
have developed a strategy for overcoming
this limitation (Haigler et al., 1992). Compar-
able incompatibility due to the synthesis of
toxic metabolites is also displayed in the
degradation of chlorobenzoates (Reineke et
al., 1982).
(3) In the absence of molecular oxygen, a number
of alternative electron acceptors may be used:
these include nitrate, sulphate, selenate, carbo-
nate, chlorate, Fe(III), Cr(V1) and U(V1). In
the succeeding sections, attention is directed
primarily to the role of nitrate, sulphate and
Fe(III), with only parenthetical remarks on
Cr(V1) and U(V1). The role of nitrate and
sulphate as electron acceptors for the degrada-
tion of monocyclic aromatic compounds is
discussed in Section 2.6.1; the wide metabolic
versatility of sulphate-reducing bacteria should
be noted.
(4) One of the cardinal issues is that of ageing
(Hatzinger & Alexander, 1995). This term is
applied to processes-whose mechanisms are
largely unestablished-whereby organic com-
pounds become associated with polymeric
components of the soil matrix with the result
that these initially monomeric compounds are
no longer accessible to the relevant degrada-
tive microorganisms. This implies that evalua-
tions of biodegradability that do take this
phenomenon into account may seriously un-
 A.-S. Allard, A. H. Neilson
derestimate the persistence of the compound.
A critical issue is the degree of reversibility of
these complexes since slow release by leaching
could introduce the xenobiotic into the aqu-
eous phase and thereby contaminate ground-
water. Although this process has been
suggested as a mechanism whereby, in the
short run, their adverse effect may be dimin-
ished, it seems unwise to conclude that this is
an acceptable long-term solution to diminish-
ing their environmental impact (Alexander,
1995b). This is discussed in the context of the
biodegradation of PAHs in Section 2.1.
(5) In both aquatic and terrestrial systems, bacter-
ia may be present in the form of biofilms that
are formed by the production of polymeric
products from the organisms themselves, and
this may serve both to increase their penetra-
tion of the matrix, and to localize the
organisms, thereby preventing their loss from
the system (Wolfaardt et al., 1995). Their role
in waste water trickling lilter treatment sys-
tems is, for example, well established.
(6) The significance of metabolites is a recurring
theme in this overview, and it may be valuable
to consider the various ways in which this may
be deleterious, either for the organism itself or
for other organisms in the ecosystem. Atten-
tion has been drawn above to the role of
potentially toxic metabolites in the regulation
of biodegradation, but metabolites may under-
go further purely chemical reactions to com-
pounds that are terminal products. Examples
include the formation of 5hydroxyquinoline-
2-carboxylate from 5-aminonaphthalene-2-sul-
phonate (Nortemann et al., 1993), or of
benzo[b]naphtho([ 1,2-d]thiophene from ben-
zothiophene (Kropp et al., 1994). Microbial
metabolites may be toxic to higher organisms,
and illustrative examples include the toxicity
of 3,4,3,,4-tetrachloroazoxybenzene produced
from 3,4-dichloroaniline (Lee & Kyung, 1995),
and of chloroveratroles produced by microbial
methylation of the guaiacols to fish (Neilson
et al., 1984). Metabolites may also play a role
in the association of the substrate with humic
and fulvic acid components. For example,
naphth-l-01, which is an established fungal
metabolite of naphthalene, may play a role in
the association of naphthalene with humic
material (Burgos et al., 1996), and further
examples involving chlorophenols are given in
Section 2.3.
(7) Many substrates contain N, P or S, and
degradative organisms may utilize one or more
of these leaving the major part of the substrate
intact. This is particularly important for
munitions-related compounds with a high N/C
ratio, and the addition of carbon sources in
these circumstances may lead to the favour-
able development of anaerobic or facultatively
anaerobic microorganisms.
(8) There are no organisms or groups of organ-
isms universally applicable to bioremediation.
Their compatibility with the site of applica-
tion should be considered, as well as their
physiology and biochemical potential. How-
ever, it should be noted that some groups of
organisms are metabolically versatile and are
capable of degrading a wide spectrum of
substrates. Examples of these include bacteria
using Ci substrates, such as methane and
methanol, and examples of their application
to trichlororethene degradation are cited in
Section 2.6.2. Increasing attention also needs
to be directed to Gram-positive bacteria
belonging to the genera Rhodococcus and
M_vcobacterium. Whereas, in aquatic systems,
bacteria are generally considered to be the
major agents of biodegradation, the role of
fungi in terrestrial systems may be equal or
greater. Among fungi, considerable attention
has been devoted to the metabolically versa-
tile white-rot fungi (Shah et al., 1992; Barr &
Aust, 1994) and several examples are cited
later.
(9) There is substantial evidence from the aquatic
environment for the existence of threshold
concentrations, below which rates of degrada-
tion of xenobiotics are slow or even negligible
(Alexander, 1985). A number of hypotheses
have been put forward, including the critical
concentrations required for induction and
maintenance of the degradative enzymes
(Janke, 1987). Whether this also occurs in the
terrestrial environment has been only super-
licially explored, but may be important when
only low concentrations of the substrate are
available by desorption.
Few of the compounds that are the object of
bioremediation programmes are readily degradable,
and although many have been shown to be
degradable in controlled laboratory experiments,
the cardinal question is whether they are degraded
in the specific environment. Biodegradation implies
 Bioremediation of organic waste sites
an appreciable degree of mineralization and not
merely biotransformation. General aspects of
bioremediation have been discussed by Thomas &
Ward (1989) Morgan & Watkinson (1989a, b),
Edgehill (1992) and Alexander 1995a). Discussion
of the pathways followed during the biodegradation
and biotransformation of a wide range of
xenobiotics by both bacteria and fungi has been
given by Neilson ( 1994).
An essential component of any bioremediation
programme is the evaluation of its success. This is
not as readily accomplished as might be assumed,
for a number of reasons (Madsen, 1991; Heitzer &
Sayler, 1993; Macdonald & Rittman, 1993;
Shannon & Unterman, 1993). A valuable and
critical review (Dott et al., 1995) discusses the
need for an assessment that takes into account all
the phases involved, and suggests that the
difficulty of demonstrating in situ bioremediation
requires extensive long-term studies. Some of the
cardinal issues include:
(1) diminution in the concentration of the sub-
strates is not an acceptable measure of
degradation since loss may occur by volatiliza-
tion or by transformation with the formation
of transient or terminal metabolites;
(2) the environment is highly heterogeneous so
that representative sampling generally presents
a serious problem; and
(3) the extent of leaching may be difficult to
evaluate since few of the systems are sufti-
ciently enclosed to make a convincing balance
of the concentrations of the substrates and
their metabolites, including CO;! or CH4 that
are lost to the atmosphere.
A number of strategies have been proposed as
potentially attractive solutions. Application of
PCR to specific degradative organisms has been
reviewed (Steffan & Atlas, 1991) and it has been
applied to hydrocarbon-degrading Mcyobacterium
sp. (Wang et al., 1996b). Metabolically related
parameters have also been used, and these include
establishing:
(1) the presence of metabolites determined from
laboratory experiments of degradation path-
ways: examples are given for PAHs (Section
2.1) PCBs (Section 2.4.2) and BTEX (Section
2.6.1); or
(2) specific enzymatic activity towards the relevant
substrates: examples for PAHs include toluene
2,3-dioxygenase (Eaton & Chapman, 1995)
257
naphthalene dioxygenase (Ensley et al., 1983)
and manganese peroxidase (Bogan et al.,
1996).
REPRESENTATIVE SITES
Various sites that involve a range of contaminants
and environments have been used as illustrations.
These sites have been the subject of either full-
scale operation or microcosm experiments that
simulate the conditions that will be encountered.
For each site, an account is also given of
microbiological issues with the emphasis on
metabolic aspects that are relevant both to
appreciating the design of the strategies and also
for assessing their success. It is worth pointing out
that agrochemicals may contain structures that are
not discussed here: for example, the diphenyl ether
acifluorfen contains both aromatic nitro- and
trifluoromethyl groups. By-products from the
manufacture of such compounds could present a
potentially serious problem.
Coal-distillation products
Introduction
Coal gas for illumination, and later for heating, was
produced by the destruction of coal and resulted in
the production of large volumes of tar. This is used
in the form of creosote for wood preservation, so
that both gas-works sites and impregnation facilities
became heavily contaminated with coal-tar products.
The range of compounds involved is enormous, but
attention has been directed to groups with
established toxicity or persistence: PAHs, azaarenes,
thiaarenes and phenolic compounds-mainly
cresols, xylenols and 2,3,5_trimethylphenol (Mueller
et al., 1989). It should be emphasized that on
account of analytical problems resulting from a lack
of authentic samples (Biggs & Fetzer, 1996) the
frequency and concentrations of PAHs with more
than seven rings have probably been seriously
underestimated. The principal reasons for
attempting remediation of such sites is the
established human carcinogenicity of important
PAHs. including benzo[u]pyrene, dibenz[a,h]anthra-
cene, and dibenzo[a,h]-, dibenzo[a,& and
dibenzo[a,l]pyrenes. PAHs with five and six rings
therefore should be the primary target in
bioremediation programmes.
Several reviews that cover various aspects have
258 A.-S. Allard, H. Neilson

been devoted to this topic. These include: (1) a
discussion of the design of different systems and
factors such as temperature, pH, nutrients and
oxygen supply (Thomas & Lester, 1993); (2) a
summary of the maximum concentrations that
must be attained at contaminated sites, and
discussion and exemplification of the various
strategies that may be used--in situ, on-site and
bioreactors (Wilson & Jones, 1993); and (3) an
evaluation of a range of remediation strategies
(Ram et al., 1993). All of these provide valuable
summaries of the various technologies that have
been exploited and cover important engineering
and geological aspects. A desirable procedure,
using extrapolation of data from laboratory
experiments, has been given (Findlay et al., 1995).
The mobility of bacteria and substrates at a coal-
tar contaminated site has been described (Madsen
et al., 1996a), and involved the ingenious use of
sorbents to trap both the bacteria and the
analytes. It also drew attention to the important
issue of the extent to which microorganisms
penetrate the contaminated matrix. The regulation
of metabolism has been noted above in Section
1.2, although the deliberate introduction of
inducers such as salicylate to enhance
biodegradation of the naphthalene (Ogunseitan &
Olson, 1993) does not seem attractive as a
generally applicable strategy. The extensive
background on the biodegradation and bio-
transformation of PAHs has been reviewed and
should be consulted for more detailed metabolic
and enzymatic aspects (Neilson & Allard, 1997).
An attempt is made to illustrate some of the
complex factors that influence the degree of
success of bioremediation programmes.
On-site or off-site treatment
On-site treatment in batch reactors using refinery
and wood-preserving waste added to soil resulted
in a highly variable loss of PAHs over a year
(April1 et al., 1990). An extensive study (Ellis et
al., 1991) examined a number of design features
and the relative merits of on- and off-site
treatments of a creosote-contaminated site.
Preliminary experiments were carried out in liquid
culture and in soil pots, and showed only a limited
advantage in adding strains isolated from the site
and a rather variable success in removal of most
PAHs except naphthalene. Two kinds of large-
scale experiments were also carried out: (a) on-site
by recirculation of groundwater and addition of
surfactants, hydrogen peroxide, inorganic
nutrients; and (b) off-site in concrete-lined basins
with recirculation of water and mixing by surface
ploughing. There were differences between in situ
and on-site removal, and for most PAHs studied,
the degree of removal was variable for PAHs with
up to four rings (Table 1). Data for the important
group of PAHs with more than four rings were
unfortunately not reported.
The physical state of the contaminant
Although the bioavailability of xenobiotics and its
decrease with ageing have been considered as
limiting factors in their biodegradation (Hatzinger
& Alexander, 1995) laboratory studies on the rate
of degradation of phenanthrene using samples
from a contaminated site showed that this
depended critically on the source of the inoculum
within the site (Sandoli et al., 1996). Most
contaminated sites are highly heterogeneous in the
concentration of the contaminant, and it has been
shown that, for naphthalene contained in coal-tar
globules, the area-dependent mass transfer
coefficient for globules was lo3 times greater than
when the substrate was coated on microporous
silca beads, and that this was an important factor
in determining the rate of mineralization (Ghoshal
& Luthy, 1996). The sorptiondesorption of PAHs
has been extensively investigated, and the role of
desorption in determining their biogradability in
aged sediments has been widely accepted
[references in Carmichael et al. (1997)]. A

Table 1. Comparison of the Efficiency of In Situ and Off-Site Procedures for the Removal of Selected PAHs

Substrate Mean percentage removal

In situ Off-site

Naphthalene 100 100

Acenaphthalene 3 87
Phenanthrene 8 74
Anthracene 36 69
Fluoranthene 53 57
Pyrene 37 53
Chrysene 76 50
 Bioremediationof organic wastesites 259

definitive study using i4C-phenanthrene and 14C- Supplementation with fungi

chrysene showed that, in contaminated soils, their
rates of mineralization were much lower than the
rates of desorption from spiked sediments. By
contrast, for aged substrates, desorption rates
were essentially comparable to rates of
mineralization. This suggests that the indigenous
microflora may have become adapted to the low
substrate concentrations available by desorption
(Carmichael et al., 1997). This would be consistent
with the existence of bacteria capable of using low
substrate concentrations, but would imply the
limited effectiveness of adding bacteria that,
although metabolically active, were not adapted to
use low substrate concentrations.
Environmental parameters
Two aspects of general significance have been
examined: the water concentration and the oxygen
concentration. Extensive studies using microcosm
experiments with creosote-contaminated
revealed losses in surface soil samples that
exceeded those from the sediment, and that the
use of soil slurries was more effective than solid-
phase systems. The removal of benz[a]anthracene,
benzo[b/k]fluoranthene and indeno[ 1,2,3,-
c,d]pyrene was poor, even using the slurries,
although the heteroarenes-quinolines, acridine
and carbazole-were partially removed (Mueller
et al., 199 la, b). The physical design of any system
therefore requires careful consideration. Whereas
oxygen must be accessible, it has been shown that
the degradation of pyrene by a strain of
Mycobacterium sp. can occur at low oxygen
concentrations (Fritzsche, 1994), and this is
consistent with the observation that toluene-
degrading bacteria isolated from sites such as
aquifer sand or groundwater with
concentrations of oxygen have lower values of K,
for 02 and 3-methylcatechol than other strains
(Kukor & Olsen, 1996). The biodegradation
naphthalene in a subsurface site was oxygen-
limited and was not facilitated by the addition of
nitrate as an alternative electron acceptor
(Madsen et al., 1996b). This is in contrast to the
effectiveness of nitrate as an electron acceptor for
the degradation of monocyclic aromatic
hydrocarbons in groundwater (Section 2.6.1). It is
important to note that, under oxygen limitation,
cultures of even strictly anaerobic methanogenic
bacteria may coexist with aerobic bacteria
(Gerritse & Gottschal, 1993). Aeration may be
expensive, but obligatory.
There has been considerable interest in the use of
fungi in a bioremediation programme, and some
illustrative examples are given here. The
supplementation of indigenous bacteria with
Phanerochaete chrysosporium increased the loss of
phenanthrene in a former oil gasification site,
although there was apparently a significant
contribution from polar metabolites (Brodkorb
& Legge, 1992). Phenanthrene is not, however,
representative of PAHs of toxicological
significance, and the presence of unidentified
metabolites may be plausibly interpreted as
transformation products. Application of the
white-rot fungus Phanerochaete sordida to treat
creosote-contaminated soil has been described,
but, again, had limited success with PAHs having
more than four rings (Davis et al., 1993). Not only
white-rot fungi have been examined; experiments
in microcosms supplemented with inorganic N
soil and P sources, using the non-lignolytic fungus
Cunninghamella echinulata var. elegans, showed
not only the expected loss of three- and four-ring
PAHs but also partial loss of
benzo[b]fluoranthene and benzo[g,h,z]perylene
(Cutright, 1995). In this study, hydrogen peroxide
was used as source of oxygen, although in natural
systems, the presence of bacteria with catalase
activity would probably remove this effectively.
Several important conclusions may be drawn
from the results of these studies:
(1) Determination of degradation, as opposed to
transformation to metabolites, or loss by other
processes, such as volatilization, is difficult to
assess.
(2) The collective evidence suggests that, although
PAH components with less than four rings are
low
probably lost, those with more than four rings
are persistent.
(3) The extent to which bioavailability is a limit-
of
ing factor and may be circumvented by the
addition of surfactants remains unresolved.
Details are given below.
(4) Oxygen limitations may be a seriously limiting
factor in deep subsurface sites, and there is an
impression that degradation in the solid phase
is less effective than in slurries.
The fate of azaarenes seems not to have been
examined directly in this context, whereas the fate
of thiaarenes has attracted attention mainly in
connection with the desulphurization of coal and
coal-oil (Wang et al., 1996a).
260 A.-S. Allard, A. H. Neilson

A brief summary of the metabolism of PAHs,
azaarenes and phenols by bacteria and fungi is
presented below. Emphasis is placed upon those
aspects that have greatest general relevance, and
that may be used to rationalize the results of the
bioremediation studies that have been presented
above.
Aerobic bacterial degradation
PAHs
Bacterial degradation under aerobic conditions is
almost invariably accomplished by dioxygenation
to cis-dihydrodiols, dehydrogenation to 1,2-
dihydroxy compounds and ring fission to
compounds that enter metabolic pathways.
Although monooxygenation is dominant among
eukaryotic microorganisms, it has also been
observed in some bacteria.
Naphthalene dioxygenase has a broad substrate
specificity and displays monooxygenase activity at
aromatic and benzylic carbon sites, and is able to
transform indole to indigo (Ensley et al., 1983)
whereas the dioxygenase from 3-methylbenzoate
carries out a comparable reaction with indole-2-
carboxylate and indole-3-carboxylate (Eaton &
Chapman, 1995). These have been used as
markers for the dioxygenases and have wider
application. The cis-dihydrodiols that are the
initial products of dioxygenation have also been
used to indicate in situ degradation of pyrene in a
marine sediment (Li et al., 1996) and of
naphthalene in leachate from a contaminated site
(Wilson & Madsen, 1996). Only incomplete
degradation, however, may occur, for example, in
the formation of chrysene dicarboxylate from
benzo[a]pyrene (Schneider et al., 1996) and
phenanthrene carboxylates from benz[a]anthra-
cene (Mahaffey et al., 1988).
Bacteria vary considerably in their capacity to
degrade a range of PAHs. Some degrade only the
more readily degradable naphthalene
phenanthrene, whereas Pseudomonas paucimobilis
strain EPA 505 can degrade fluoranthene, pyrene,
benz[a]anthracene, chrysene, benzo[a]pyrene and
benzo[b]fluoranthene (Ye et al., 1996). Gram-
positive mycobacteria have attracted increasing
attention; for example, a strain of Mycobacterium
sp. degrades pyrene, benz[a]anthracene
benzo[a]pyrene (Schneider et al.,
Extrapolation of results from the more readily
degraded naphthalene and phenanthrene,
however, should be carried out with caution.
The accessibility of the substrates at sites with a
long history of contamination is a serious
limitation to bioremediation, and attempts have
been made to increase this by addition of
surfactants (Grimberg et al., 1996). This is a
complex issue, and significant differences have
been found among different organisms (Guerin &
Boyd, 1992b; Cracker et al., 1995), and indeed,
incorporation of PAHs into micelles may make
them less accessible (Volkering et al., 1995). There
is also evidence that, as for alkanes, biosurfactants
may be produced during growth with PAH
(Deziel et al., 1996) although the practical
implementation of this may be limited by the
requirement for a suitable monomer such as
glucose or mannitol. A lipoprotein surfactant is
produced during the growth of Pseudomonas
marginalis strain PD-14B with succinate and
prevented flocculation of cells in media containing
anthracene, acenaphthylene, naphthalene and
chrysene. The relation between surfactant
concentration and the stimulation of biodegra-
dation is, however, complex and not so far
understood (Burd & Ward, 1996).
Finally, it should be emphasized that the
development of suitable chromatographic columns
for GC analysis has begun to reveal the presence
of PAHs with molecular masses greater than 300.
Those with more than seven rings are probably
ubiquitous, although their determination is made
difficult by the inaccessibility of reference
compounds and by their long retention times on
GC columns (Biggs & Fetzer, 1996). PAHs with a
molecular mass greater than 300 that occur in
both carbon black and the NIST SRM 1597 coal-
tar extract include dibenzo[a&oronene (M, 400)
with nine rings, dianaphtho[2,1 -a:2,1-hlanthracene
(M, 378) and benzo[a]coronene (M, 350) with
eight rings (BemgHrd et al., 1993). The
degradability of such compounds is unknown, and
they may possibly contain carcinogenic
and representatives.
Heteroarenes
By comparison with their carbocyclic analogues,
much less is known about the biodegradation of
azaarenes, although the biochemistry of the
aerobic degradation of quinolines (including
and methyl- and carboxyquinolines) has been
1996). extensively investigated by Lingens and colleagues
(Schwartz & Lingens, 1994). As a broad
generalization, it may be stated that the initial
reaction frequently involves formal hydroxylation
 Bioremediation of organic waste sites 261

(addition of the elements of HZ0 followed by considerable attention has been directed to the
dehydrogenation) in the hetero ring, whereas application of white rot fungi, several important
fission of the carbocyclic ring is accomplished by issues should be noted. Most attention has
dioxygenation. It is worth noting that these hitherto been given to species of Phanerochaete,
organisms may be quite restricted in their ability especially Ph. chrysosporium and Ph. sordida,
to use other N-heterocylic compounds: for although other species and other taxa (Field et
example, a strain of Rhodococcus, sp. that was al., 1992) may be of greater interest in view of
isolated by enrichment of pristine samples with their greater biodegradative capability. Whereas
quinoline was unable to use isoquinoline or transformation has generally been demon-
acridine, and supported only poor growth with a strated-often to quinones-only relatively low
series of pyridines (OLoughlin et al., 1996). The levels of mineralization to CO2 have been
degradation of acridine and phenanthridine seems, frequently observed, for example in Pleurotus
however, to have been examined only ostreatus (Bezalel et al., 1996a), and it has been
parenthetically. The degradation of carbazole has shown that a substantial fraction of the
been examined more extensively, and an substrates may bind to fungal mycelia (Bezalel
exhaustive study has revealed the production of et al., 1996b).
several terminal metabolites formed by cyclization
of the fission products from the carbocyclic ring
(Gieg et al., 1996). The relevant benzocarbazoles Anaerobic degradation
seem not to have been examined. (1) Hydrocarbons-Until now, degradation of
The mechanism of the degradation of aromatic hydrocarbons has been limited to
dibenzofurans has been established in detail and is monocyclic representatives-particularly tol-
accomplished by angular dioxygenation with uene, and there is only circumstantial evidence
fission of the furan ring, whereas the so far of the degradation of naphthalene and
biodegradation of benzothiophenes is complicated phenanthrene coupled to sulphate reduction
by oxidation at the sulphur atom. under anaerobic conditions (Coates et al.,
1996a). Discussion of the anaerobic degrada-
Phenols tion of monocyclic aromatic hydrocarbons is
The aerobic degradation of alkylated phenols is discussed in Section 2.6.1 in the context of
accomplished by either of two pathways: (a) BTEX remediation.
hydroxylation to catechols, followed by extradiol (2) Phenols-The anaerobic degradation of phe-
ring fission or (b) oxidation of the methyl group nol generally proceeds by carboxylation, fol-
to carboxyl followed by hydroxylation to lowed by dehydroxylation and fission of the
hydroquinones and fission by the gentisate ring after partial reduction (Brackmann &
pathway (Bayly et al., 1988). Fuchs, 1993). Degradation of o-cresol (Bisail-
lon et al., 1991) and m-cresol (Roberts et al.,
Fungal transformation 1990) has been observed under methanogenic
It is important to emphasize that, in contrast to conditions, and a summary of the possibilities
bacterial degradation, most fungi carry out only under denitrifying conditions has been given
transformation without ring fission. An (Rudolphi et al., 1991). An alternative path-
important consequence is that demonstration of way involving oxidation of the methyl groups
substrate loss during bioremediation under denitrifying conditions has also emerged
experiments is equivocal. A monooxygenase (Bonting et al., 1995).
produces an epoxide that is either hydrolysed to (3) Heteroarenes-Although the anaerobic degra-
a trans-dihydrodiol or rearranged to a phenol dation of azaarenes containing several N
that is often conjugated. The most atoms-particularly pyrimidines and purines
comprehensive studies have been made using -has been extensively studied, less is known
Cunninghamella sp. [references in Sutherland et about those with only a single hetero atom.
al. (1995)]. It has been suggested that naphth-l- Evidence for the loss of pyridine in sulphate-
01, which is one of the expected transformation reducing and methanogenic aquifer slurries
products of naphthalene, is the form in which at and of 2-methylpyridine in the former has
least part of naphthalene is associated with soil been presented (Kuhn & Suflita, 1989),
matrices (Burgos et al., 1996). Although although no details of the degradative path-
262 A.-S. Allard. A. H. Neilson

ways are available. Whereas indole and quino- accidents at sea. In many important respects, the
line were degraded anaerobically under ni- basic issues are similar, and the strategy for all
trate-reducing, sulphate-reducing and experiments on bioremediation may be viewed
methanogenic conditions, neither benzofuran against the background provided by a valuable
nor benzothiophene was degraded (Licht et review by Atlas (1981). The broad classes of
al., 1996). compounds involved (alkanes, cycloalkanes,
aromatic hydrocarbons and heteroarenes) that are
Conclusion formed by transformation of plant and algal
Since the indigenous microflora have been exposed residues were summarized, together with the
to the substrates, the relevant degradative range of microorganisms capable of carrying out
pathways often will be present in populations of their degradation. It is worth pointing out that
these microorganisms, and induction of the the biodegradability of cycloalkanes has attracted
appropriate degradative enzymes will have taken less attention than the other components and that
place. Indeed, many of the most versatile some present potential recalcitrance. Important
organisms have been isolated from such environmental determinants of biodegradation
contaminated sites. Therefore, if organisms with were outlined, including the addition of nutrients,
the relevant degradative capacity have been the concentration of oxygen, the temperature, the
demonstrated, the prognosis for remediation is salinity, and the role of the hydrocarbon-water
good. Additional readily degradable carbon interface as the site of active degradation.
sources are probably counterproductive to Reviews on the microbial metabolism, including
maintaining the required degradative capacity, biochemical aspects, are available, and include
although addition of inorganic nutrients, those of Britton (1984) on alkanes and of Morgan
provision of suitable oxygen concentrations and & Watkinson ( 1994) which also includes
water are necessary. There are, however, several cycloalkanes and some aromatic compounds.
potentially serious problems: Virtually all the issues that are discussed in these
recur in the examples that will be used as
(1) The collective evidence suggests that com-
illustrations. Some broad generalizations are
pounds with five or more rings may be
therefore justified.
recalcitrant, and it is these compounds that
arouse the greatest concern.
(1) Biodegradation of all hydrocarbons requires
(2) There may be incompatibilities between the
access to electron acceptors, generally oxygen
degradation of PAHs and azaarenes that are
in natural situations, added hydrogen peroxide
often simultaneously present.
in terrestrial systems, or nitrate or sulphate
(3) If high concentrations of phenols are present,
under anaerobic conditions that prevail at
these may be toxic to the flora.
deeper levels of the soil.
(4) The most serious limitation is probably the
(2) The biodegradation of n-alkanes and the path-
degree to which the substrates are accessible to
ways whereby it is accomplished are well
the relevant microorganisms, and the advan-
established. The range of compounds includes
tage of adding surfactants is equivocal.
branched-chain compounds such as pristane,
On the basisof these limitations, a cautious view of although branching may present an obstacle
the effectiveness of bioremediation of PAHs seems (Pirnik, 1977). The biodegradation of aromatic
justified. hydrocarbons has been noted above, and it is
necessary only to repeat that, whereas
Refinery waste and stranded oil: petroleum naphthalene and phenanthrene are readily
hydrocarbons degradable, PAHs with more than live rings
may be more recalcitrant.
There are several different situations in which (3) Many bacteria produce surfactants in response
petroleum hydrocarbons pose a problem that has to exposure to hydrocarbons, and these have
attracted solution by bioremediation: (1) oil- been demonstrated both for those that degrade
retinery waste and contamination of surrounding alkanes and for PAHs (Deziel et al., 1996).
soil, (2) leakage from oil pipelines and (4) Occasionally, biotransformation in the absence
underground storage tanks or basins, and (3) spill of substantial biodegradation may be accepta-
of crude oil in the marine environment after ble. Dicyclopentadiene is produced pyrolyti-
 Bioremediation of organic waste sites
tally in petrochemical plants and has a
nauseating and penetrating odour. Although it
was degraded by a mixed bacterial culture, the
major part was transformed into a series of
oxygenated compounds that are presumed to
be less malodorous (Stehmeier et al., 1996).
Terrestrial habitats
A number of reviews cover different aspects of the
bioremediation of soil contaminated with refinery
waste. The relative merits of in situ and on-site
procedures, the role of electron acceptors and
addition of inorganic nutrients have been discussed
by Dott et al. (1995), who also showed that
inoculation, even with organisms isolated from the
sites and putatively able to degrade the
contaminants, had only a very limited effect. The
important determinants were, therefore, provision of
oxygen as O2 or H202 and of an inorganic source of
nitrogen. The modes of dissipation and a summary
of remedial techniques, including designs for solid-
phase systems and bioreactors (biopiles), have been
given in Bossert & Compeau (1995): the authors
conclude that the potential of bioremediation has
not been fully exploited, and that the important
issues of bioavailability and means of evaluating
effectiveness should be addressed.
Marine habitats
General aspects of the degradation of oil in the
marine environment have been given (Van der
Linden, 1978), and this discusses important issues,
including the effect of adding nutrients and the
significance of temperature. A comprehensive
study (BIOS) to determine both the environmental
effect and the effectiveness of remediation
strategies was carried out in the Canadian Arctic
where oil was deliberately discharged (Owens et
al., 1987). The original reports in a special issue of
Arctic should be consulted for details of the
conclusions drawn from this investigation, but the
following summarize the basic requirements that
an assessment should provide:
a procedure for determining remedial action in
the event of an oil spill;
an evaluation of the advantage or disadvantage
of using oil dispersants;
a method for estimating the persistence of oil
washed ashore; and
an assessment of the effectiveness of alternative
procedures for removing stranded oil from
beaches.
263
Experience with some major spills of crude oil has
been summarized (Atlas, 1995; Swannell et at.,
1996) and attention drawn to a number of
important general issues. The rates of
biodegradation are generally low compared with
the speed and ease of mechanical removal, and are
strongly dependent on whether the beaches are
sandy or rocky. The form in which nutrients are
administered, e.g. granulated or slow-release, is
important, and a periodic monitoring programme
should be implemented to determine the need for
addition of nutrients in view of the possible
adverse effects resulting from excess addition of
nutrients, e.g. abnormally extensive algal growth
and high bacterial activity leading to oxygen
limitation. It is important to be able to assess
biodegradation of the oil relative to that of its
most recalcitrant components.
Conclusion
There seems little doubt that biodegradation of
many components of petroleum may be facilitated
by the addition of inorganic nutrients-primarily
nitrogen-and the provision of suitable oxidant(s).
It seems that inoculation with exogenous
organisms-ven when these have been isolated
from the same site-is not generally effective, and
that indigenous organisms are effective, provided
that suitable nutrients and a supply of oxygen (as O2
or H202) are available. The greatest effectiveness
probably would be achieved by optimizing the
capacity of these indigenous hydrocarbon-degrading
organisms, which are virtually ubiquitous. The
limitations that have already been noted for PAHs
are equally relevant here.
Wood preservation sites: chlorophenolic compounds
Creosote contamination has been noted in Section
2.1, and in this section, only contamination with
chlorophenolic compounds-particularly penta-
chlorophenol-will be discussed. Concern with
their production, use and disposal is motivated by
their widely distributed toxic effects (Ahlborg &
Thunberg, 1980) and their possible persistence.
The degradation of chlorophenols has been
extensively investigated by a number of workers,
and the pathways have been reviewed (Haggblom,
1990). The application of specific strains to
remediation of a wood-preservation site in
Finland and elsewhere has been presented in detail
(Haggblom & Valo, 1995). Contamination of
surrounding groundwater may also have occurred,
264 A.-S. Allard, A. H. Neilson

and fluidized-bed reactors inoculated with (3) There are important requirements that may be
activated sludge from a pulp mill producing necessary: an additional substrate may be
chemothermomechanical pulp have been used to required either to promote cell growth or to
treat ground water highly contaminated with serve as a reductant, and degradation is
chlorophenols. An important aspect of this study functional under low oxygen concentrations
is that the organisms were fully functional at the and may be facilitated by sorption of the cells
relatively low temperature of 5-7C (Jarvinen ej onto polyurethane films.
al., 1994). Evaluation of white-rot fungi for (4) O-Methylation to chloroanisoles may occur,
treatment of pentachlorophenol-contaminated soil and under aerobic conditions, these may be
has been made (Lamar & Dietrich, 1990; Lamar et terminal metabolites which, on account of
al., 1993), and two issues have emerged: the their lipophilic character, may be transported
production of pentachloroanisole and the into higher biota and hence into the food
association of the substrate with the soil matrix chain via earthworms (Palm et al., 1991).
that presents a potentially serious problem that is
noted below. Both 2,4-dichlorophenol and 2,4,5-trichlorophenol
Only some general microbiological issues are may be degraded by the white-rot fungus
briefly summarized here. Phanerochaete chrysosporium by a complex
pathway involving oxidative displacements of
chloride and O-methylation with the formation of
Aerobic conditions
1,2,4,5-tetrahydroxybenzene before ring fission
The aerobic bacterial degradation of phenols with
(Valli & Gold, 1991). Although pentachloro-
less than three chlorine atoms occurs by initial
anisole is formed from pentachlorophenol by a
hydroxylation, followed by ring fission and
number of species of Phanerochaete, it is
elimination of chloride from the acyclic
subsequently mineralized. The formation of
intermediates. For compounds with more than three
products by oxidative coupling with loss of
chlorine atoms, a different mechanism has been
chloride mediated by lactase or peroxidase also
elucidated for the degradation of pentachlorophenol
should be noted (Dee & Bollag, 1994) since these
by Mycobacterium chlorophenolicus strain PCPl
reactions may be the basis of associations between
(Apajalahti & Salkinoja-Salonen, 1987a), and for a
chlorophenols and humic substances (Rtittimann-
strain of Flavobacterium sp. (Steiert & Crawford,
Johnson & Lamar, 1996).
1986; Xun & Orser, 1991; Xun et al., 1992): this
involves hydrolytic loss of chloride, followed by
reductive and hydrolytic elimination to produce Anaerobic conditions
1,2,4_trihydroxybenzene or 2,6-dichlorohydro- A review of anaerobic dechlorination has been
quinone. Details may be found in a review given by Mohn & Tiedje (1992). Under these
(Htiggblom, 1990). Several other pentachloro- conditions-and in contrast to aerobic con-
phenol-degrading organisms, including the Gram- ditions-the more highly substituted compounds
negative Sphingomonas chlorophenolica strain RA2, are more readily transformed. Partial dechlori-
have also been examined. In the application of such nation of pentachlorophenol has been observed,
strains to bioremediation of contaminated sites, a although the resulting chlorophenols are
number of important considerations must be taken degradable under aerobic conditions, so that,
into consideration. under suitable conditions, complete
mineralization may be accomplished. Pure
(1) The tolerance of the strains to high concentra- cultures of Desulfomonile tiedjei are able to
tions of pentachlorophenol: S. chlorophenolica carry out dechlorination: strain DCB-1 of
appears to be less sensitive than M. chlorophe- polychlorinated phenols (Mohn & Kennedy,
nolicus (Miethling & Karlson, 1996). 1992), and strain DCB-2 of 2,4,6-
(2) The longevity of the strains in the environ- trichlorophenol and dichloro-phenols (Madsen
ment: this is dramatically illustrated by the & Licht, 1992).
fact that an initial inoculum of lo* cellsg- of It is convenient to add here some brief
S. chlorophenolica fell to zero within 7months, comments on chlorophenoxyalkanoic acids. The
whereas for M. chlorophenolicum, 106-lo7 pathway for their degradation involving the
cells g- could be recovered from a similar intermediate formation of phenols is well
inoculum (Miethling & Karlson, 1996). established, and several groups of organisms able
 Bioremediation of organic waste sites
to do so have been isolated. Kinetic parameters for
the rate of degradation and for sorption of 2,4-D
(Estrella et al., 1993) have been carried out and
showed higher values for the maximum growth
rate constants in column experiments than in
batch experiments. Experiments on
bioremediation of 2,4-dichlorophenoxyacetic acid-
contaminated soil using Pseudomonas cepacia
strain BRI 6001 were promising and indicated the
critical role of the cell density of the inoculum
added to non-sterile soil (Comeau et al., 1993). On
the other hand, the bacterial degradation of 2,4-D
sorbed to sterile soil has been shown to be
extremely slow (Ogram et al., 1985) and the rates
in soil with a low organic content were greater
than in an organic-rich soil (Greer & Shelton,
1992). The biodegradation of phenoxypropion-2-
oic acids is more complex, and mecoprop 2-(2-
methyl-4-chlorophenoxy)propionic acid has been
consistently found in municipal landfill leachates
(Gintautus et al., 1992). Whereas in pure cultures
of bacteria, only one enantiomer of 2-(2-methyl-4-
chlorophenoxy)propionic acid-the (s)-(-)-
enantiomer-may be degraded (Tett et al., 1994),
in agricultural soil treated with a herbicide
mixture containing 2-(2,4-dichlorophenoxy)pro-
pionic acid, both enantiomers were degraded with
the opposite preference (Garrison et al., 1996).
The same preference for degradation of racemic 2-
(2-methyl-4-chlorophenoxy)propionic acid was
observed in Sphingomonas herbicidovorans
Zipper et al. (1996) who made the important
observation that cells grown with either of the
pure enantiomers preferentially degraded that
enantiomer. Current formulations, however,
contain only the biologically active and more
readily biodegradable enantiomer.
Conclusion
There is evidence for the success of bioremediation
of chlorophenol-contaminated sites by on-site
processes, using a number of aerobic micro-
organisms as inocula, although their activity
depends on a number of factors that should be
investigated in each case (Miethling & Karlson,
1996). A relatively high degree of mineralization is
generally observed, although the possibly
undesired formation of chloroanisoles might
present a potential hazard, together with the
fungal-catalysed polymerization of chlorophenols.
The extent to which this is applicable to
chlorophenols containing methyl or nitro
substituents is apparently unknown.
265
Chemical production waste
The range of compounds in waste or in by-
products from chemical production is enormous,
and includes compounds containing fluorine,
the chlorine, bromine, iodine, nitro, sulphonate, and
thiophosphate groups. A useful structural listing
of a wide range of chemicals has been compiled
(Hartter, 1985; Swoboda-Colberg, 1995) and
attention is drawn to two less-prominant groups
containing the CFs group (Section 2.4.3) and
organic iodo compounds, such as the herbicide
ioxynil and X-ray contrast agents. Chlorophenols
and phenoxyalkanoic acid herbicides have been
discussed in Section 2.3, nitro compounds will be
discussed in Section 2.5.1, and phosphorothioate
esters briefly noted in Section 2.5.2. The present
discussion therefore will be concentrated on
halogenated aromatic and alicyclic hydrocarbons,
although attention is directed to other structural
groups such as benzothiazoles (Reemtsma et al.,
1995; Kumata et al., 1996) that have wide
industrial application but about which little is
known of their biodegradability (De Vos et al.,
1993).
Chlorinated alicyclic hydrocarbons
Hexachlorocyclohexanes (HCHs) are among the
organochlorine compounds at sites contaminated
with former pesticide production (Lang et al.,
by 1992) and although evidence has been presented
for the existence of a rich soil microflora at such
sites, it appears that many of the substrates
remain, even after 30years (Feidieker et al., 1994).
This indicates the existence of one or more
limiting factors, of which the degree of
bioavailability is probably of major significance:
this is consistent with the established difficulty of
chemical recovery of HCH from aged
contaminated soil (Westcott & Worobey, 1985).
The situation is additionally complicated by the
existence of various isomers of HCH (r&y,@ each
of which behaves differently.
The aerobic degradation of y-HCH has been
accomplished with a strain of Pseudomonas
paucimobilis UT26 (Nagasawa et al., 1993) and
involves a complex chain of elimination,
hydrolytic displacement of chlorine and ring
fission reactions. It should be noted that the
biotransformation to chlorobenzenes as terminal
metabolites presents an undesirable alternative.
The aerobic degradation of c(-HCH by indigenous
bacteria in soil slurries has been demonstrated
266 A.-S. Allard, A. H. Neilson

(Bachmann et al., 1988), and in soil, is apparently hexachlorobenzene has been described in anae-
limited by rates of soil desorption and robic mixed cultures supplemented with elec-
intraparticle mass transfer (Rijnaarts et al., 1990). tron donors, including lactate, ethanol or
Although the p-isomer is highly persistent, its glucose (Holliger et al., 1992): successive and
susceptibility to degradation under methanogenic partial dechlorination produced 1,2,4- and
conditions in a laboratory column reactor has 1,3,5-trichlorobenzenes, whereas the 1,2,3-tri-
been studied (Middeldorp et al., 1996): chlorobenzene was further dechlorinated.
chlorobenzene and benzene were terminal (b) PCBs-There is an enormous amount of
metabolites plausibly produced from an literature on the degradation of PCBs, and it is
intermediate 6-2,3,4,5-tetrachlorocyclohex-1-ene. not intended to attempt a coverage of this.
The anaerobic transformation of the structurally Attention is directed to reviews on anaerobic
related y-hexachlorocyclohexene and y-1,3,4,5,6- dechlorination (Bedard & Quensen, 1995) and
pentachlorocyclohexene by Clostridium rectum to those that include a summary of aerobic
produced 1,2,4-trichlorobenzene and 1,4- degradation (Bedard, 1990; Sylvestre, 1995).
dichlorobenzene, respectively (Ohisa et al., 1980). The last also includes valuable comments on
It should also be noted that considerable attention the regulation of PCB degradation and the
has been directed to the highly toxic polychlorinated significance of chlorobenzoate degradation. As
bornanes (toxaphene) and derivatives of a background to a discussion of bioremediation
hexachlorocyclopentadiene (chlordane) that were technologies, a few simplistic remarks are given
used as pesticides. Chlordane has been recovered here on the metabolic pathways used for the
from biota (Buser et al., 1992) and toxaphene degradation of PCBs. It should be emphasized,
components from sediments (Stern et al., 1996) so however, that there are considerable differences
these are highly persistent contaminants. For both, among the congeners, both in their suscept-
the commercial products contained a large number ibility to degradation and in the details of the
of congeners, and the analysis of chlordane is mechanisms whereby this is accomplished and
complicated additionally by the occurrence of among the various organisms. No attempt is
enantiomers, some of which have undergone made to discuss the details of these important
degradation and transformation after discharge. differences.
Nevertheless, little attention appears to have been
directed to waste from locations at which these A number of aerobic organisms are able to degrade
products were manufactured. various congeners of PCB. Most have been isolated
by enrichment with biphenyl as the sole carbon and
Chlorinated and brominated aromatic hydrocarbons energy source. These include both Gram-negative
The structural range of industrially important strains belonging to the genera Alcaligenes and
representatives is enormous, covering chloro- Pseudomonas and Gram-positive strains of
benzenes (solvents) to polychlorinated biphenyls Rhodococcus. Aerobic metabolism is initiated by
(PCBs) (hydraulic and insulating fluids) and dioxygenation of the ring bearing the least number
polybrominated biphenyls and biphenyl ethers of chlorine substituents, followed by ring fission
(flame retardents). There is widespread concern either to chlorobenzoates or to chloro-
over both the persistence and the potential toxicity acetophenones (Bedard et al., 1987). Either
of all these compounds, so sites contaminated biphenyl-2,3-dioxygenase or the 3,4-dioxygenase
during their production represent a threat both to may be involved. In an ingenious application, the
the environment and to human health. recovery of chlorobenzoates has been used to
demonstrate aerobic degradation under natural
(a) Chlorobenzenes-Aerobic organisms cap- conditions (Flanagan & May, 1993).
able of degrading chlorobenzenes with less than Anaerobic dechlorination has been
live chlorine atoms have been described and demonstrated under a number of conditions, and
the pathways elucidated: the metabolism may an extremely complex pattern of dechlorination
be initiated by dioxygenation without elimina- among the congeners has been observed (Bedard
tion of a chlorine atom from 1,2,4-trichloro- & Quensen, 1995). There are also varying and
benzene or with elimination of a single chlorine sometimes conflicting views on the role of
atom from 1,2,4,5-tetrachlorobenzene (Sander sulphate.
et al., 1991). The anaerobic dechlorination of A valuable overview (Adriaens & Vogel, 1995)
 Bioremediation of organic waste sites
includes a range of chlorinated aromatic
compounds and provides illustrative engineering
designs. In the light of the microbiological
background that has been presented, a number of
different strategies may be distinguished, and all
of these have been applied to and evaluated in
contaminated soils or sediments.
(1) Contaminated soil has been inoculated with
bacteria of established capacity for the degra-
dation of chlorobenzoates (Hickey et al.,
1993). In the presence of added biphenyl,
mineralization of PCBs was shown, although
it was pointed out that there may exist an
incompatibility between the production
chlorocatechols from chlorobenzoates
their inhibition of dihydroxybiphenyl-2,3-diox-
ygenase that catalyses the ring fission of many
PCBs.
(2) Experiments using Pseudomonas testosteroni
strain B-356 in microcosm systems revealed
the necessity of adding biphenyl to encourage
degradation of the tetrachlorinated congeners
of Arochlor 1242 and the effectiveness of
repeated inoculation (Barriault & Sylvestre,
1993).
(3) In situ stimulation of aerobic PCB degradation
has been shown in Hudson River sediments
(Harkness et al., 1993). The biodegradative
capacity of the indigenous organisms was
enhanced by the addition of biphenyl and
inorganic nutrients, although repeated inocula-
tion with an organism, having established an
ability to degrade PCBs, did not apparently
improve the degree of biodegradation.
(4) Anaerobic strategies are complicated by the
different susceptibilities of the various con-
geners to dechlorination and by the effect of
electron acceptors, especially sulphate. Inge-
nious alternatives have used the addition of
specific PCB congeners that are more readily
dechlorinated to prime dechlorination
specific positions (Bedard & Quensen, 1995),
and the use of dibrominated biphenyls in the
presence of malate to stimulate dechlorination
of the hexachloro- to nanochlorobiphenyls
(Bedard & Quensen, 1995).
PBBs
Highly brominated biphenyls and biphenyl ethers
have been manufactured as flame-retardants, and
have apparently become environmental
contaminants. One of the reasons for concern is the
267
formation of polybrominated dibenzofurans and
dibenzo-1,6dioxins (though not 2,3,1,8-
tetrabromdibenzo-1,4-dioxin) during incineration
(Buser, 1986). Mixed cultures of organisms isolated
from sediments contaminated with PCBs and PBBs
were shown to debrominate PBBs under anaerobic
conditions (Morris et al., 1992) and the dominant
congener, 2,4,5,2,4,5-hexabrombiphenyl, could be
successively debrominated to 2,2-dibrombiphenyl.
On the other hand, in sediments from the most
heavily contaminated site containing contaminants
in addition to PBBs, very little debromination
occurred, and the recalcitrance was attributed to the
toxicity of the other contaminants (Morris et al.,
of 1993).
and
Chlorinated dioxins
These are probably ubiquitous in the environment,
and their biodegradability has been extensively
examined. This will not be discussed in any detail
here, and attention will be drawn merely to a few
investigations that illustrate important new
aspects.
Chlorinated dioxins have been recovered from
sediments contaminated with both industrial
discharge and atmospheric deposition
(Macdonald et al., 1992; Evers et al., 1993; Gotz
et al., 1993) and attention has therefore been
directed to anaerobic dechlorination processes
(Adriaens et al., 1995; Beurskens et al., 1995). It
has been suggested that microbial dechlorination
of cell-partitioned 2,3,7,8-tetrachlorodi-
benzo[ 1,4]dioxin in aged sediments was as
effective as that in freshly spiked sediment
(Barkovskii & Adriaens, 1996). This suggests
that the kinetics of transport into the cells is a
cardinal determinant, although the extent to
which hydrophobic xenobiotics in sediments are
partitioned into the indigenous microflora has
not been generally established.
Chlorinated dioxins occur in atmospheric
at deposition (Koester & Hites, 1992) and thereby
will enter the terrestrial environment. The
degradation of tetrachloro- to octachloro-
dibenzo[l,4]dioxins has been examined in low-
nitrogen medium by Phanerochaete sordida YK-
624 (Takada et al., 1996). All the compounds were
extensively degraded, and the ring-cleavage of
2,3,7,8-tetra- and octachlorodibenzo[ 1,4]dioxin
produced 4,5-dichlorocatechol and tetrachloro-
catechol. These results establish important new
evidence for the biodegradability of even highly
chlorinated dioxins.
268 A.-S. Allard, A. H. Neilson

Organojluoro compounds component of lignin complexes is mineralized by

Attention is directed primarily to groups of Phanerochaete chrysosporium slowly-although as
compounds containing the CF3 group that include effectively as the free compound (Arjmand &
agrochemicals, such as the diphenyl ether Sandermann, 1985). Negative aspects of fungal
herbicides (Lee et al., 1995), and pharmaceuticals. metabolism include the transformation of 3,4-
A single example of the existence of fluorinated dichloroaniline by white-rot fungi (Pieper et al.,
organic compounds is used as an illustration of a 1992) which may produce the toxic 3,3,4,4-
problem area. Sediments containing waste from tetrachloroazobenzene (Lee & Kyung, 1995) and
the production of 4-chloro-(trifluoro- association with humic and fulvic acids.
methyl)benzene contained a number of The prognosis for bioremediation of residues
compounds containing chloro and CF3 containing polychlorinated anilines and their
substituents: these included benzophenone, a impurities is therefore somewhat discouraging,
difluorodiphenylmethane and several biphenyls although removal from soil of 2-chloro-N-
(Jaffe & Hites, 1985), although the more isopropylacetanilide by a strain able to degrade
widespread occurrence of these compounds is this has been reported (Martin et al., 1995). This
unknown. This is important since, whereas strain was able to dechlorinate the substrate,
pathways for the degradation of fluorophenols although it was unable to grow with either aniline
and fluorobenzoates are well established, the CF, or phenol: evidence for bioremediation based on
group appears to be resistant to both chemical loss of the initial substrate therefore should be
and microbiological degradation. A combination evaluated critically, even when production of
of photolytic and microbiological reactions, C02-presumably from the alkyl substi-
however, brings about the degradation of 3- and tuents-has been demonstrated.
4-trifluoromethylbenzoate via 7,7,7-trifluoro-
hepta-2,4-diene-6-one carboxylate that was sub- Conclusion
sequently degraded photochemically with the loss There is, therefore, unequivocal evidence for the
of fluoride (Taylor et al., 1993). success of in situ strategies for bioremediation of
PCB in soils and sediments using indigenous
Chlorinated anilines microorganisms. The major problem arises from
These are the starting compounds for the the complex mixture of compounds in PCB
production of a range of agrochemicals commercial preparations that include not only a
(insecticides, herbicides and fungicides), including range of congeners but also polychlorinated
anilides, carbamates and ureas. Their hydrolysis terphenyls (Wester et al., 1996). Current research
products-halogenated anilines, particularly is therefore directed to understanding the basis of
chloroanilines-are widely distributed and the biodegradability of specific congeners, and to
notorious for association with, and incorporation the possibility of developing strategies to
into, humic substances (Bollag & Loll, 1983). The overcome this. Before implementation of
mechanism of this interaction has been examined bioremediation of sites contaminated with PBBs,
in model enzyme-mediated reactions with ferulic PCDDs, aromatic compounds containing CFs
acid (Tatsumi et al., 1994), and it has been shown groups, and chloroanilines can be successfully
that oligomerization of 4-chloroaniline mediated implemented, however, important unresolved
by oxidoreductases may produce 4,4-dichloroazo- issues remain.
benzene and 4-chloro-4-aminodiphenyl, as well as
trimers and tetramers (Simmons et al., 1987). Military waste
From a purely microbiological view, there are
some important issues. Explosives
Although bacteria that degrade chloroanilines A range of nitro compounds has been used as
have been isolated, their effectiveness towards military explosives, including nitrotoluenes, nitrate
dichloroanilines appears to have been less esters and nitramines. There is concern over the
explored (Brunsbach & Reineke, 1993). destruction of these dangerous and potentially
Biotransformation to the acetanilides has been explosive products, so considerable effort has been
demonstrated, although a strain of Pseudomonas made to develop microbiological processes of
putida was able to degrade 3,4-dichloroaniline destruction (Kaplan, 1990): a review gives details
(You & Bartha, 1982a). The chloroaniline of the degradation pathways of the relevant
 Bioremediation of organic waste sites
compounds (Walker & Kaplan, 1992). Compared
with the wastes that have been considered above,
these compounds are distinguished by a higher N/
C ratio, so, although they may serve as sources of
both C and N, only the N is used by some
organisms: in this case, an additional carbon
source must obligatorily be added.
Only limited data are available on full-scale or
pilot-scale operations and may present an overly
simplified view. Indigenous microorganisms from
a munitions-contaminated site reduced a single
nitro group of 2,4_dinitrotoluene, 2,6-dinitro-
toluene (DNTs) and 2,4,6_trinitrotoluene (TNT)
to amines, whereas TNT was mineralized to ca
10% (Bradley et al., 1994). A preliminary
experiment, using soil contaminated with both
TNT and nitramines, was carried out by adding
phosphate and starch to the surface: the anaerobic
flora that developed reduced the nitro groups of
TNT, and, subsequently, the reduced compounds
were removed, although their fate was not
established (Funk et al., 1993). One important
additional observation was that, under aerobic
conditions, or under anaerobic conditions at
alkaline pH, polymerization of the intermediate
amines took place. This experiment has since been
extended to an evaluation of an in situ bioreactor
(Funk et al., 1995).
Nitrotoluenes
Reviews of the biodegradation and biotrans-
formation of nitroaroamatic compounds have
been given (Crawford, 1995; Spain, 1995a) to
which reference should be made for details, so
only a very brief summary is justified here. Three
principal reactions are found among aerobic
bacteria: (1) oxidative elimination of nitrite, (2)
partial or complete reduction of the nitro group,
and (3) reduction of the aromatic ring.
(1) Dioxygenase-mediated loss of nitrite with
formation of a catechol. For 2,4-DNT, degra-
dation hy Pseudomonas sp. strain TNT is
initiated by the formation of 4-methyl-5-
nitrocatechol, whereas loss of the second nitro
group results in the formation of an inter-
mediate 1,4-quinone before ring fission occurs
(Haigler et al., 1994). Alternative pathways for
ring fission have been presented (Spain,
1995a).
(2) Reduction of the nitro group has been
observed under aerobic conditions, and a
number of alternative pathways have emerged.
269
The biodegradation of nitrobenzene by a
strain of Pseudomonas pseudoalcaligenes pro-
ceeds by rearrangement of the initially formed
hydroxylamine to 2-aminophenol before de-
gradation by extradiol ring cleavage (Nishino
& Spain, 1993). The situation for more highly
nitrated toluene is more complex. A range of
metabolites is formed from TNT by a strain of
Pseudomonas sp. and includes both hydroxyla-
mino-dinitrotoluenes and tetranitroazoxyto-
luenes formed by condensation of the partially
reduced hydroxylamine and nitroso com-
pounds (Haidour & Ramos, 1996). The biode-
gradation of 2,4-DNT by Phanerochaete
chrysosporium involves initial reduction of one
of the nitro groups followed by a series of
reactions (Valli et al., 1992) similar to those
involved in the degradation of 2,4-dichloro-
phenol-oxidation, O-methylation and further
oxidation to 1,2,4_trihydroxybenzene (Valli &
Gold, 1991). TNT is also transformed by this
organism and partially mineralized, although
it was shown that 4-hydroxylamino-2,6-dini-
trotoluene was a potent inhibitor of lignin
peroxidase activity (Bumpus & Tatarko, 1994).
Partial reduction of 2,4-D to the dinitroazo
compound has been observed in a strain of the
fungus Mucrosporium (McCormick et al.,
1978) and in a strain of Pseudomonas sp.
(Duque et al., 1993). Consistent with the
presence of three strongly electron-withdraw-
ing groups and the established formation of
charge-transfer complexes involving trinitroar-
enes, a strain of Mycobacterium sp. forms a
hydride-TNT complex in addition to products
from reduction of the nitro groups (Vorbeck
et al., 1994). Although of minor current
interest as an explosive, it is noted that a
similar complex is formed from 2.4,6-trinitro-
phenol, although the intermediate is further
degraded (Lenke & Knackmuss, 1992). Reduc-
tion by anaerobic bacteria has been widely
observed, and a sulphate-reducing organism
that uses TNT as a nitrogen source carries out
sequential reduction to 2,4,6-triaminotoluene
(TAT) (Preuss et al., 1993) although the
pathway for the further metabolism of this
compound from which NH: is released was
not elucidated. Incubation of TNT under
anaerobic conditions was shown to produce
TAT and methylphloroglucinol (Funk et al.,
1993) and it is attractive to hypothesize that
further degradation of the latter is analogous
270 A.-S. Allard, A. H. Neilson

to that used for the degradation of 3,4,5- Nitramines

trihydroxybenzoate by Pelobacter acidigallici Of the nitramines used as explosives, greatest
that proceeds via 1,3,5_trihydroxybenzene be- attention has been directed to the degradation of
fore ring cleavage (Brune & Schink, 1992). 1,3,5-trinitro-hexahydro-1,3,5-triazine (RDX).
Transformation of TNT by Clostridium bifer- This is degraded by several species of
mentens produced TAT via a number of Enterobacteriaceae (Kitts et al., 1994) and is used
partially reduced amino- and hydroxylamino by Stenotrophmonas maltophilia strain PBl as a
compounds (Lewis et al., 1996), together with source of N, and produced metabolites with loss
a compound produced by condensation of of two nitro groups and a single N-atom from the
TAT with pyruvaldehyde. triazine: their structures were tentatively put
(3) Terminal metabolites may be formed by forward (Binks et al., 1995), although an
reduction of the aromatic ring. 4,6-dinitrohex- alternative anaerobic pathway, involving
anoate is produced from 2,4-dinitrophenol reduction of the nitro groups to nitroso and a
(Lenke et al., 1992) and 2,4,6-trinitrocyclohex- series of transformations yielding hydrazine,
anone from 2,4,6_trinitrophenol (Lenke & dimethylamine and 1,2-dimethylhydrazine, has
Knackmuss, 1992). been presented (McCormick et al., 1981).
In addition to military uses, it should be noted
Nitrodiphenylamines that aromatic nitro compounds are of
These are no longer used as explosives, but they are considerable industrial value for the production of
formed from diphenylamine, which is used as a anilines (Hartter, 1985) that are components of
stabilizer for nitrocellulose explosives. A number agrochemicals, some of which have been briefly
of nitrated diphenylamines are then formed by noted in Section 2.4.4.
reaction with the NO, that is produced. The
metabolism of nitrodiphenylamines has been Conclusions
examined under anaerobic conditions, and The possibility of bioremediation of sites
phenazine and 4-aminoacridine, which are contaminated with nitrotoluene waste is clearly
cyclization products of the initially formed 2- possible. Important issues, however, should be
aminodiphenylamine, have been identified clearly appreciated, including: (a) that addition of
(Drzyzga et al., 1996). additional carbon sources which may be necessary
may encourage partial or complete reduction of
Nitrate esters nitro groups; (b) under aerobic conditions, dimeric
A review (White & Snape, 1993) has been azo compounds that are terminal metabolites may
devoted to this topic, and only very brief be formed; and (c) that aromatic amines may be
comments can be added to this relatively incorporated into humic material and thereby
unexplored area. The biotransformation of resist further degradation.
glycerol trinitrate by strains of Bacillus
thuringiensislcereus or Enterobacter agglomerans Chemical warfare agents
involves the expected successive loss of nitrite A few brief notes are added on phosphoro-
with formation of glycerol (Meng et al., 1995). fluoridates, although their destruction by microbial
The transformation of pentaerythritol tetra- activity, though clearly possible, may be limited by
nitrate by Enterobacter cloacae, which uses this their toxicity to microorganisms. One of the
as a source of N, proceeds comparably with motivations for their inclusion is the fact that the
further metabolism of the two hydroxymethyl hydrolytic enzyme(s) responsible for
groups produced by hydrolysis to the dialdehyde defluorination+rganophosphorous acid anhydrase
(Binks et al., 1996). The enzymes that produce (OPAbis widespread, and is found in a number of
nitrite from the nitrate esters are apparently bacteria (Landis & DeFrank, 1990). The microbial
reductases, and the enzyme from E. cloacae is hydrolysis of organophosphorus pesticides and
strongly inhibited by steroids, and is capable of cholinesterase inhibitors is accomplished by several
reducing cyclohex-2-ene-l-one (French et al., distinct enzymes, all of which are termed
1996). However, the metabolism of glyceryl organophosphorus acid anhydrolases (OPAs). These
trinitrate by Phanerochaete chrysosporium have been reviewed (DeFrank, 1991) so that only a
(Servent et al., 1991) involves production of the few additional comments are justified.
physiologically important nitric oxide. The enzymes from Pseudomonas putida strain
 Bioremediation of organic waste sites 271

MG and Flavobacterium sp. [ATCC 275511 are systems and the role of the indigenous microflora
carried on plasmids (Mulbry et al., 1986), and are (Salanitro, 1993); and (3) general procedures,
membrane bound. Although they hydrolyse a including provision of oxygen and comments on
range of phosphorothioate esters at rates greatly anaerobic treatment (Bowlen & Kosson, 1995).
exceeding those for chemical hydrolysis and have The pathways for the aerobic biodegradation of
been proposed for application to destruction of alkylated benzenes have been elucidated in
these (Munnecke, 1976) they hydrolyse extensive investigations, and only some of the
phosphorofluoridates much more slowly (Dumas salient features need to be briefly summarized.
et al., 1989). This, together with the fact that they The genes for the degradation of toluene may be
are membrane bound, makes them of somewhat either chromosomal or plasmid-borne. The latter,
limited desirability for the destruction of therefore, may be lost in the absence of selection
phosphorofluoridates. Two other enzymes have pressure. The degradation of toluene may take a
attracted considerable attention for the hydrolysis number of different pathways: (1) involving either
of phosphorofluoridates: one from Bacillus dioxygenation or successive monooxygenation of
stearothermophilus is also membrane-bound, the substrate with subsequent ring cleavage
whereas the other fram Alteromonas sp. strain JD leaving the the alkyl group intact, and (2)
6.5. is a soluble enzyme. OPA anhydrolase activity involving oxidation of the alkyl group to a
has also been observed in a number of other carboxylic acid before ring cleavage. For xylenes,
organisms (DeFrank et al., 1993). The enzyme it appears that there are significant differences in
designated OPA-2 has been purified from the degradability of the isomers, o-xylene being
Alteromonas strain JD 6.5, and has a molecular apparently more recalcitrant.
mass of 60kDa (DeFrank & Cheng, 1991). There In many situations, such as in deep groundwater,
has been considerable speculation on the natural however, oxygen concentration may be severely
substrate for OPA anhydrolase activity, and a limiting because of its consumption by surficial
surprising fact emerged from a detailed aerobic organisms that lower its rate of transport.
comparison of the amino acid sequence. This There has therefore been great interest in the
revealed a high degree of homology with an anaerobic degradation of BTEX. Although this has
aminopeptidase from Escherichia coli and that the been demonstrated for benzene under anaerobic
OPA anhydrolase had high prolidase activity conditions by methanogenic consortia, sulphate-
(Cheng et al., 1996). reducing bacteria, and Fe(III)-reducing bacteria,
All of these observations underscore the greatest attention has been directed to laboratory
potential for application of appropriate OPAs to studies on the anaerobic degradation of toluene in
the destruction of organophosphorus compounds the presence of nitrate. This is supported by the fact
with anticholinesterase activity (Cheng & that the appropriate organisms are widely
Calomiris, 1996). Since, however, hydrolysis distributed (Fries et al., 1994).
results in release of fluoride, the possibility of its Some of the important principles may be
subsequent incorporation into organic substrates illustrated from two groups of experiments. The first
to produce fluoroacetate and 4-fluorothreonine used a laboratory microcosm and a field experiment
(Reid et al., 1995) may be worth consideration. with a contaminated aquifer (Barbaro et al., 1992).
This clearly showed the effectiveness of adding
Groundwater contamination nitrate, but there were significant differences in the
extent of degradation of the substrates: for example,
Benzeneltoluenelethylbenzenelxylenes (BTEX) benzene appeared resistant, the xylenes and ethyl
All these substrates can be degraded under aerobic benzene were less degradable than toluene. Nitrate
conditions, although there appear to be significant was apparently consumed by indigenous organisms
differences among the xylene isomers. Reviews at the expense of other carbon substrates, so this
have covered different aspects of this problem: (1) was removed from the system. In this
biodegradation in gas-condensate-contaminated experiment-and possibly in general-sorption of
groundwater with emphasis on the role of these substrates to organic carbon was not a limiting
inorganic nutrients and oxygen (Morgan et al., factor. In the second example, the effect of nitrate or
1993); (2) the determinative factors for treating sulphate additions was examined by adding either of
aquifer plumes after accidental spillage of these together with samples of BTEX to a
automobile fuel with discussions of the design of contaminated aquiifer in the form of slugs of
272 A.-S. Allard, A. H. Neilson
purified ground water (Reinhard et al., 1997). For
nitrate additions, the rates of removal of the
substrates were in the order, benzene co-
xylene < ethylbenzene < toluene = m-xylene, the last
three being removed within 6days. For sulphate
additions, removal of toluene and all of the xylenes
occurred within 50 days. Metabolites characteristic
of degradation (Beller et al., 1995) were isolated as
confirmation of biotransformation. The important
observation was made that, although the results
were broadly in agreement with those from
laboratory microcosm experiments, the rates of
degradation of toluene and m- and p-xylenes seemed
to be lower. Benzene and o-xylene seem to be
considerably more recalcitrant than other
components of BTEX.
These experiments should be viewed against the
results of extensive laboratory investigations of
the anaerobic degradation and transformation of
the individual components of BTEX. The
degradation of ethylbenzene and n-
propylbenzene-though not xylenes-has been
shown in pure cultures of denitrifying bacteria
(Rabus & Widdel, 1995). In crude oil, toluene,
ethylbenzene and m-xylene, but neither o-xylene
nor p-xylene, were degraded (Rabus & Widdel,
1996). Degradation of toluene by the sulphate-
reducing anaerobe Desulfobacula toluolica has also
been shown (Rabus et al., 1993), and by another
sulphate-reducing bacterium with a novel
16SrRNA gene sequence (Beller et al., 1996). The
mechanism of these anaerobic degradations is
complex, and only a very brief summary using
toluene as an illustration is justified, since reviews
have been given by the pioneers in this area
(Evans & Fuchs, 1988; Fuchs et al., 1994). In
summary, there are three cardinal reactions:
(1) oxidation of the methyl group to carboxyl by
a number of different pathways;
(2) partial reduction of the aromatic ring of the
CoA ester of benzoate before ring fission; and
(3) fission of the ring of cyclohexa-1,4-diene
carboxylate (or possibly also the cyclohex-l-
ene carboxylate) by a series of hydroxylation,
and reductive and elimination reactions to
produce 3-hydroxypimelate (or pimelate) that
then enters the metabolic system.
It should be noted that benzylsuccinates and
benzylfumarates have been suggested as
intermediates during the oxidation of methyl to
carboxylate and that, as noted above, these
compounds, together with the analogous
compounds that would be produced from xylenes,
have been used in support of the active in situ
anaerobic degradation of toluene and xylenes
(Beller et al., 1995).
Choroethenes
A substantial effect has been devoted to the
bioremediation of groundwater contaminated with
trichloroethene (TCE) that has been used
extensively as a solvent and degreasing agent.
Determinative issues are the relatively low
substrate concentrations and the large volumes of
groundwater that are involved. There has
therefore been greater interest in the application
of in situ procedures than more conventional
pump and treat on-site methods. In order to
appreciate the microbiology of the procedures, a
brief summary of the relevant aspects is given.
Reviews have been given, and provide further
details (Ensley, 1991; Arp, 1995).
TCE is unable to support the growth of a single
organism, but it is susceptible to co-oxidation by
different oxygenases elaborated by organisms
during growth with structurally unrelated
substrates. There are several bacterial oxygenases
that can bring about co-oxidation of TCE after
induction on the relevant substrates: methane
monooxygenase in Methylosinus trichosporium
strain OB3b (Jahng & Wood, 1994) propane
monooxygenase in Mycobacterium vaccae
(Vanderberg et al., 1995) and ammonia
monooxygenase (Arciero et al., 1989; Vannelli et
al., 1990). Although the methane monooxygenase
system involves the epoxide as an intermediate
that may form CO that toxilies the organism, this
may be overcome by addition of a suitable
reductant such as formate (Henry & Grbic-Galic,
1991). A review of methanotrophic bacteria
(Hanson & Hanson, 1996) contains a useful
account of their application to bioremediation of
TCE-contaminated sites.
Pseudomonas putida strain Fl elaborates a
dioxygenase for the degradation of toluene; this is
able to co-oxidize TCE, which may also be carried
out during growth with phenol (Nelson et al.,
1988). A similar situation prevails in the
isopropylbenzene-degrading Rhodococcus erythro-
polis strain BD-2 (Dabrock et al., 1994). Increased
biodegradation of TCE has been observed in a
constructed strain of Escherichia coli carrying the
genes for the large subunit of toluene terminal
oxygenase (from Pseudomonas putida Fl), and the
small subunit of biphenyl terminal oxygenase and
 Bioremediationof organic wastesites
the ferredoxin and ferredoxin reductase (from P.
pseudoalcaligenes KF707) (Furukawa et al., 1994).
Monooxygenase systems may also be involved in
the degradation of toluene and bring about the
co-oxidation of TCE under suitable growth
conditions: in P. ceppciu strain G4 which carries
out ortho-hydroxylation, in P. mendocinu KR-1,
which carries out pcEYu-hydroxylation, and in P.
pickettii, which carries out metu-hydroxylation.
Two different types of experiments on the
bioremediation of sites contaminated with TCE
have been carried out, and have been preceded by,
and taken advantage of the valuable results
obtained in microcosm experiments.
(1) The first procedure used an indigenous micro-
flora enriched by the addition of substrates that
induced the appropriate oxygenases. Microcosm
experiments (Hopkins et al., 1993b) demon-
strated the differential effects of phenol, toluene,
methane and ammonia: whereas the first two
substrates were equally effective in removing
both TCE and trans- 1,2-dichloroethene,
methane was only marginally effective in remov-
ing TCE but more effective in removing truns-
1,2dichloroethene, and ammonia was less effec-
tive in removing all of the substrates. It was
shown that in bioreactors fed with phenol under
various conditions, maximum removal of TCE
occurred when phenol was supplied as a pulse
every 24 h: in this reactor, there was a greater
diversity of microorganisms and higher numbers
that degraded phenol and TCE (Shih et al.,
1996). Field evaluation of this procedure has
been carried out, using either phenol or toluene
or methane as oxygenase inducers (Hopkins et
al., 1993a; Hopkins & McCarty, 1995). The
effectiveness of the various substrates for re-
moval of a number of chloroethenes is given in
Table 2 from which it is clear that all of the
chloroethenes, except l,l-dichloroethene, were
Table 2. Influence of Growth Substrates on the Removal of Chloroethenes
Growth
substrate
Concentration TCE
Methane (mg$:I 19
Phenol 12.5 94
Toluene 9 93
TCE
l,l-DCE
Cis-DCE
Truns-DCE
MCE
213
effectively removed. There remains, therefore,
no serious concern over the persistence of
these-and particularly vinyl chloride-pro-
duced by partial anaerobic dechlorination of
TCE. In this experiment, the concentrations of
residual phenol and toluene were below the
standards set for drinking water.
(2) In the second procedure, organisms with an
established degradation potential for TCE
were injected at the site.
(a) Cells of Methylosinus trichosporium strain
OB3b were added and became attached to the
sediments to form an in situ bioreactor: the
concentration of TCE in samples withdrawn
through the biofilter region fell rapidly, and after
40days, the concentrations had reached back-
ground levels (Duba et al., 1996). The major
remaining unresolved issue is, therefore, how the
degradative activity can be maintained during the
time required for effective bioremediation.
(b) In the light of possible concern over the
introduction of phenol into aquifers, mutant
strains of Burkholderia (Pseudomonas) cepucia
strain G4, in which toluene 2-monooxygenase is
constitutive, have been examined in microcosm
experiments. Effective removal of TCE could be
demonstrated (Krumme et al., 1993; Munakata-
Marr et al., 1996), although in the first experi-
ment, a serious loss of viability of the cells
occurred during the lo-week incubation; this
presents, therefore, a further challenge.
It may be noted also that the anaerobic
dechlorination of chloroethenes has received some
attention. These have been encouraged by concern
that partial dechlorination could result in the
production of vinyl chloride as a terminal
metabolite. After induction by growth of Desulfi-
monile tiedjei DCB- 1 with 3-chlorobenzoate, partial
dechlorination of tetrachloroethene to
Percentage removal of substrate
l,l-DCE Cis-DCE Trans-DCE MCE
43 90 95
54 92 73 >98
>98 75
Trichloroethene
1,l-Dichloroethene
Cis-1,2_dichloroethene
7ra~l,ZdichIoroethene
Chloroethene (vinyl chloride)
214 A.-S. Allard, A. H. Neilson

trichloroethene and cis- 1,l -dichloroethene has been
observed (Cole et al., 1995; Townsend & Suflita,
1996). Dehalogenases for both trichloroethene and
tetrachloroethene have been examined in a strain of
Dehalospirillum multivorans (Neumann et al., 1994)
and it has been shown that the dechlorination of
tetrachloroethene in anaerobic aquifer slurries was
stimulated by C3 carboxylic acids or ethanol
(Gibson & Sewell, 1992).
Conclusion
In situ procedures, using indigenous organisms
induced for the aerobic degradation of
chloroethenes, have been successful, particularly
in broadening the range of substrates to include
those that might be produced by partial anaerobic
dechlorination. The application of introduced
organisms is currently limited by the relatively
short period during which their activity can be
maintained, but this could be possibly achieved by
designing more effective in situ bioreactors.
Methyl t-butyl ether
There has been concern over possible adverse effects
of the oxygen additive methyl t-butyl ether (MTBE)
that has been used as an octane booster in petrol and
to minimize automobile discharge in cold weather. A
survey has revealed its widespread distribution in the
USA, although in no case did concentrations exceed
those set for drinking-water standards (Squillace et
al., 1996). Contrary to initial fears, this compound
was not associated with the existence of BTEX, so its
source remains unestablished. For the sake of
completeness, attention is drawn to different aspects
of its biodegradability.
Under aerobic conditions, a mixed bacterial
culture was able to degrade the substrate with
intermediate formation of t-butanol (Salanitro et
al., 1994). Even under prolonged incubation under
a variety of anaerobic conditions, however, the
substrate was resistant to degradation under
sulphate-reducing conditions and very poorly
degraded under nitrate-reducing conditions
(Mormille et al., 1994). Bioremediation
groundwater contaminated with MTBE therefore
seems problematic.
Metabolic interaction of metal cations and organic
compounds
Although discussion of strategies for remediation
of base-metal contaminated sites lies beyond the
scope of this review, it has already been noted that
Cr(V1) and U(VI) may serve as alternative
electron acceptors to oxygen, and it therefore
seems appropriate to mention examples in which
their metabolic interaction with organic
degradation has been utilized. The reduction of
metals at higher oxidation levels can be
gratuitously carried out by many bacteria, but in
the present context, the cardinal issue is its
coupling to the oxidation of an organic substrate
under anaerobic conditions.
(1) Whereas the gratuitous aerobic reduction of
chromate by bacteria is widely distributed
(Lovley, 1993), the reduction of chromate Cr(V1)
to Cr(II1) anaerobically at the expense of the
oxidation of benzoate to CO2 has been accom-
plished in cultures that were initially enriched
using nitrate as an additional electron acceptor
(Shen et al., 1996). In the absence of nitrate,
there was good agreement between the removal
of benzoate and the reduction of Cr(VI), and
this could be an attractive procedure in view of
the low toxicity and ready degradability of
benzoate if this were used in excess.
(2) The organism now known as Geobacter metallir-
educens is a strictly anaerobic organism that can
couple the oxidation of acetate, toluene or
phenol to the reduction of Fe(II1) supplied as an
insoluble oxide (Lovley, 1991) and strains of the
genus have been isolated from a variety of
sediments (Coates et al., 1996b). In addition, this
organism can carry out the oxidation of acetate
to the reduction of U(V1) to insoluble U(IV)
(Lovley et al., 1991), and this opens the
possibility of simultaneous remediation of sites
containing organic contaminants and U(V1)
residues. For the sake of completeness, it is
noted that the acidophilic organisms Thiobacillus
ferrooxidans and T. thiooxidans are able to carry
out a formally equivalent reduction of pertech-
nate Tc(VI1) to insoluble Tc(V) and Tc(IV)
(Lyalikova & Khizhnyak, 1996).
of Municipal waste
Only a few parenthetical remarks are given for
completeness. In view of the volumes that may be
involved in municipal landfills (Suflita et al.,
1992) it is not generally practical to attempt
remediation of the highly heterogeneous solid
phase. The potential adverse effect of leachate that
may reach water courses justifies serious concern,
and this is underscored by the range of
 Bioremediation of organic waste sites 275

compounds that have been recovered from (1) Linear styrene dimers have a wide spectrum
leachates from municipal landfills. These may of use, including lubricants and plasticizers.
include virtually any organic compound, together Those without methyl substituents on the
with their transformation products: some rings or with only one were biodegradable,
illustrative examples include: 2-(4-chloro-2- whereas those with methyl substituents in
methylphenoxy)propionic acid, N-n-butylbenzene- both rings were recalcitrant (Higashimura et
sulphonamide, camphor, 2-hydroxybenzothiazole, al., 1983).
and 2,2- and 4,4-dihydroxydiphenylmethane (2) Detergents comprise surfactants combined in
(Oman & Hynning, 1993). Attention already has commercial preparations with compounds that
been directed to the biodegradability of function as chelating agents, and both surfac-
phenoxyalkanoic acids in Section 2.3. A range of tants and their degradation products may be
wood-related products may be expected, and encountered in domestic waste. Attention is
considerable attention has been directed to the directed to a review of the bacterial degrada-
biodegradation of lignin and hemicellulose that tion of anionic surfactants comprising alkyl
are complex polymers ubiquitous in higher plants. sulphates, aromatic sulphonates and alkyl
This has been accomplished particularly by fungi ethoxy sulphates (White & Russell, 1994) from
(Kirk & Farrell, 1987; Jeffries, 1994), although it which it is clear that complete mineralization
appears that the variability in the structures of of some groups may be difficult to achieve.
these polymers may result in significant There has been increasing interest in thermally
recalcitrance, and that there are significant stable fluorinated surfactants (Dams, 1993)
incompletely resolved issues on the role of and perfluoropolyethers (Gurarini et al.,
bacteria and the regulation of ligninases in white- 1993). The biodegradability of some surfac-
rot fungi. The biodegradability of leachate under tants has been examined (Remde & Debus,
methanogenic conditions has been examined, 1996) and the apparent recalcitrance of an
although there was evidence of toxicity at high anionic fluorinated sulphonic acid is note-
concentrations (OConnor et al., 1990). worthy.
Landfills containing municipal waste are (3) 2-Mercaptobenzothiazoles have wide industrial
extremely heterogeneous both in texture and in the uses as surface-active biocides (Reemtsma et
range of compounds that have been deposited, and al., 1995) and (4-morpholino)-2-benzothiazole
they may be used to illustrate a potentially serious is used in rubber vulcanization and has been
problem of wider significance-the chemical encountered in a range of environmental
interaction of microbial metabolites that may samples (Kumata et al., 1996).
include, for example, aromatic amines, benzthiazole (4) Silicones have found diverse and increasing
sulphones, quinones, and aldehydes. These may uses and are generally considered to be
react with each other, or with reactive substituents highly stable. Polydimethylsiloxanes are,
of humic and fulvic acids to produce stable however, at least partially degraded in soil
compounds that may be recalcitrant to microbial to dimethylsilanediol (Carpenter et al.,
degradation or of limited bioavailability. Indeed, 1995) and the mineralization in soil of the
these condensation products may not be readily monomer dimethylsilanediol has been shown
detected using conventional analytical protocols, (Sabourin et al., 1996). Interesting observa-
particularly when these are limited to a restricted list tions revealed that, in liquid cultures, degra-
such as those designated priority pollutants. dation could be obtained with Fusarium
oxysporium growing concurrently with pro-
Miscellaneous contaminants pan-2-01 or aceto, and by a strain of
Arthrobacter sp. growing concurrently with
Brief attention is drawn to a number of dimethylsulphone.
compounds that have received less publicity.
These are not generally related to specific
industrial activity but may be revealed by careful A HIERARCHICAL STRATEGY
analysis of landfills and sediments. In view of their
general recalcitrance, their removal during It is obvious that there are several basic questions
application of conventional procedures is that must be answered before considering
questionable. bioremediation:
276 A.-S. Allard. A. H. Neilson
l What compounds are present and which have
to be removed?
l By what means is success to be evaluated?
l Are indigenous or exogenous organisms to be
used?
In virtually all the examples that have been used as
illustrations, three stages may be discerned, and it
is convenient to assemble these into a hierarchical
system: (1) basic laboratory studies, (2) microcosm
studies using site material, and (3) evaluation in
large-scale systems: on-site pilot-plants or in situ
installations. It is essential that geological,
engineering, microbiological and chemical
interactions be set up at the beginning and
maintained throughout.
Laboratory experiments
For both exogenous and indigenous organisms:
examination of pathways, including formation
of intermediate and terminal metabolites;
use of the substrates as sources of C, N, P and
S;
physiological optima of temperature, pH and
oxygen concentration;
toxicity of substrates;
induction of catabolic enzymes by growth with
a structurally-related substrate;
degradation of a compound during growth
with a metabolically compatible substrate (co-
metabolism or concurrent metabolism);
the existence of metabolically incompatible
mixtures of substrates; and
evaluation of relevant probes for the presence
of the relevant microorganisms or their activ-
ity.
For many exogenous organisms, this will already
have been established so that only
complementation will be required for indigenous
organisms: determination of their presence,
number, and metabolic activity by conventional
laboratory procedures. It may be worthwhile to
set up enrichment cultures using site material to
provide suitable material for laboratory and
microcosm studies.
Microcosm experiments
These should be constructed from material
collected from the relevant site and simulate
aerobic, microaerophilic or anaerobic conditions.
They should provide the kinetic data necessary for
large-scale operation, and specifically address the
following:
0 rates of substrate loss;
a data on existence and stability of metabolites,
and their toxicity to other biota;
l existence of threshold substrate concentration
below which rates of degradation are low or
negligible;
l effectiveness of analogue substrates and meta-
bolites in promoting degradation; and
l stability of the system under prolonged opera-
tion.
It is appropriate at this stage to evaluate
procedures that will be used to assess the
effectiveness of bioremediation. These may
include: (1) use of radiolabelled substrates (that
will not generally be permitted in field operation),
(2) evaluation of metabolites, and (3) evaluation
of markers such as specific enzymes.
Large-scale operations
Apart from the preceding issues, it is particularly at
this stage that very close collaboration with
geologists and engineers is necessary: putatively
optimal solutions may not be realistic for the site,
and alternatives may have to be adopted. The
choice then may depend on returning to Stage 2
or even Stage 1 to evaluate these new constraints.
A good example is provided by the reduced
permeability of substrata encountered during
application of a pump-and-treat system: this was
due to several causes, including mobilization of
colloidal material in the system, and subsequent
deposition and fouling at injection sites and
infiltration zones (Wiesner et al., 1996). It is
perhaps inadvisable to evaluate alternative
strategies in large-scale systems. All the intrinsic
problems of scale-up will clearly have to be
addressed and solved, and these include: (1) the
mobility of microorganisms, substrates, nutrients
and metabolites; (2) oxygen transport, or
maintenance of another electron acceptor, e.g.
nitrate; and (3) the stability of the system.
CONCLUDING COMMENTS
Whereas the potential of bioremediation is
substantial, its application has important
limitations that are apparent from the many
 Bioremediation of organic waste sites 211

Dechlorination and para-hydroxylation of polychlorinated

examples used here as illustrations. The authors
phenols by Rhodococcus chlorophenolicus. Journal of
feel that these limitations can be overcome only Bacteriology 169, 675-68 1.
when adequate attention is directed to Apajalahti, J. H. A. & Salkinoja-Salonen, M. S. (1987)
fundamental microbiological, chemical and Complete dechlorination of tetrachlorohydroquinone by
cell extracts of pentachloro-phenol induced Rhodococcus
engineering issues. Only then can the barriers to chlorophenolicus. Journal of Bacteriology 169, 5 125-5 130.
achieving success in full-scale operation be Aprill, W. & Sims, R. C. (1990) Evaluation of the use of prairie
surmounted, since there are signiticant problems grasses for stimulating polycyclic aromatic hydrocarbon
treatment in soil. Chemosphere 20, 253-265.
with in situ or on-site programmes that have not
Aprill. W., Sims, R. C., Sims. J. L. & Mattews, J. E. (1990)
taken advantage of experiments under the Assessing detoxification and degradation of wood preser-
controlled conditions in microcosms or small-scale ving and petroleum wastes in contaminated soil. Waste
bioreactors. Of the several unresolved Management Research 8, 45-65.
Arciero, D.. Vannelli, T., Logan, N. & Hooper. A. B. (1989)
microbiological issues, it is suggested that the degradation of trichloroethylene by the ammonia-oxidizing
following deserve particular consideration: bacterium Nitrosomonas europaea. Biochemical and Biophy-
sics Research Communications 159, 640-642.
The degree to which the compounds are Arjmand, M. & Sandermann, H. (1985) Mineralization of
accessible to the appropriate microorganisms. chloroaniline/lignin conjugates and of free chloroanilines
by the white rot fungus Phanerochaete chrysosporium.
The extent to which the population of degrada-
Journal of Agriculture & Food Chemistry 33, 105551060.
tive organisms can be maintained and in- Arp, D. J. (1995) Understanding the diversity of trichlor-
creased. oethylene co-oxidations. Current Opinions in Biotechnology
The relative biodegradability of components of 6, 352-358.
Atlas, R. M. (1981) Microbial degradation of petroleum
complex mixtures, such as PAHs and PCBs, hydrocarbons: an environmental perspective. Microbiologi-
since some of the less readily degraded compo- cal Reviews 45, 18&209.
nents may be the least desirable from an Atlas, R. M. (1995) Bioremediation of petroleum pollutants.
International Biodeterioration and Biodegradation 35, 3 17-
environmental and toxicological viewpoint. 321.
Even when removal of, say, 90% of the Austen, R. A. & Dunn, N. W. (1980) Regulation of the
contaminant has been achieved, it may not be plasmid-specified naphthalene catabolic pathway of Pseu-
domonas putida. Journal of General Microbiology 117, 521-
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528.
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even negligible. Biodegradation of alpha- and beta-hexachlorocyclohexane
in a soil slurry under different redox conditions. Applied
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