Environmental Pollution 107 (2000) 179185

www.elsevier.com/locate/envpol

Means to improve the eect of in situ bioremediation of

contaminated soil: an overview of novel approaches
M. Romantschuk*, I. Sarand, T. Petanen, R. Peltola, M. Jonsson-Vihanne,
T. Koivula, K. Yrjala, K. Haahtela
Helsinki Biocenter, Department of Biosciences, PO Box 56 (Viikinkarig), FIN-00014 University of Helsinki, Finland

Received 29 August 1998; accepted 22 May 1999

``Capsule'': How to increase the eciency and reproducibility of bacterial degradation of organic pollutants is reviewed.

Abstract
Dierent aspects of bacterial degradation of organic contaminants in soil, and how to improve the eciency and reproducibility
is discussed in this review. Although bioremediation in principle includes the use of any type of organism in improving the condi-
tion of a contaminated site, most commonly bacteria are the degraders and other organisms, such as soil animals or plant roots,
play a role in dissemination of bacteria and, indirectly, plasmids between bacteria, and in providing nutrients and co-substrates for
the bacteria active in the degradation process. There are a number of dierent procedures that have been tested more-or-less suc-
cessfully in attempts to improve reliability, cost eciency and speed of bioremediation. The methods range from minimal inter-
vention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or bacterial inocula or
improvement of physico-chemical conditions, all the way to excavation followed by on site or ex situ composting in its dierent
varieties. In the past the rule has been that more intervention (leading to higher costs) has been more reliable, but novel ideas are
continuously tried out, both as a means to come up with new truly functional applications and also as a line of studies in basic soil
microbial ecology. Both approaches generate valuable information needed when predicting outcome of remediation activities,
evaluating environmental risks, deciding on cleaning-up approaches, etc. The emphasis of this review is to discuss some of the novel
methods for which the value has not been clearly shown, but that in our view merit continued studies and eorts to make them
work, separately or in combination. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Bioremediation; Bacteria; Novel approaches; Contaminated soil; Genetic engineering

1. Introduction The capacity to degrade a great variety of recalcitrant

The microbial ora at a specic site is dependent on a
multitude of environmental variables. By studying the
indigenous microbial ora at a contaminated site, and
taking into account a sucient number of the physical
parameters there is hope for ability to predict how nat-
ural attenuation will proceed at such a site. If external
action to improve the purication seems essential, the
environmental and ecological knowledge is required to
choose the right, maximally non-intruding method.
Methods being developed should provide tools to eval-
uate and remediate when possible by biological means
various contaminated sites.
* Corresponding author. Tel.: +358-9-708-59219; fax: +358-9-
708-59262.
E-mail address: martin.romantschuk@helsinki. (M. Romantschuk).
0269-7491/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0269-7491(99)00136-0
compounds are particularly harboured in bacteria.
Although the degradation capacity of an organism or
consortium of organisms is required, their mere exis-
tence is not enough. Also, the conditions have to favour
ecient degradation and here, apparently, is where
active intervention may bring the best and most cost
ecient result the circumstances promoting degra-
dation should be optimized while still minimizing
contaminant evaporation, leaching, etc.
A contaminated site may be relatively stable but may pose
a future threat if not remediated. If cleaning of such a site is
attempted by excavation followed by, for example, mixing
with a suitable matrix material and nutrients for compost-
ing, there is a risk of mobilizing the contaminant by volati-
lization or ushing. Therefore, remediation in situ by
improving the conditions and/or the degradation potential
in the contaminated soil layer should be preferred.
180 M. Romantschuk et al. / Environmental Pollution 107 (2000) 179185
2. Factors limiting eciency of in situ bioremediation
Soil contaminated with various organic recalcitrant
compounds is a very widespread problem throughout
the world, particularly in industrialized areas. There are
many reasons for organic compounds being degraded
very slowly or not at all in the soil environment, even
though they are per se biodegradable. Among those are:
1. Low temperature. In soil, particularly in northern
industrialized countries in Europe and North
America, the soil temperature during a large part
of the year is too low for ecient microbial
degradation of soil contaminants. The same may
be true also for deeper soil layers in other parts of
the world.
2. Anaerobic conditions. Degradation in anaerobic
contiditions is slow; some compounds are not
degraded anaerobically and some are degraded
only partly and may give rise to toxic compounds.
3. Low levels of nutrients and co-substrates. A con-
taminated site usually has a sub-optimal nutrient
balance. If the contaminant is a hydrocarbon, e.g.
oil contamination, there is likely to be a shortage
of at least nitrogen, but each site has to be eval-
uated case by case, taking into account also mat-
ters such as solubility of the contaminant in order
not to over-fertilize.
4. Bioavaliability. Spatial distribution of contam-
inants in relation to degrading organisms and
solubility of the contaminant; these variables are
in part interrelated and are both separately and in
combination major factors aecting degradation
velocity.
5. Absence of degradation potential. A biological
degradation pathway for synthetic, xenobiotic
compounds may not exist precluding biodegrada-
tion, or genes encoding enzymes that may be
active on the compound are not induced by the
contaminant. Suitable pathways are, however,
likely to evolve, either naturally or accelerated in
laboratory conditions.
Some of the approaches to tackle these problems will be
discussed below.
3. Evolution of degradation capacity in contaminated soil
Given sucient time and suciently favourable con-
ditions the capacity to degrade any organic compound
is likely to evolve or immigrate to a contaminated site,
even if the compound(s) are completely synthetic/xeno-
biotic, and no natural degradation pathway originally
would exist. On the other hand, bacterial cells/clones
tend to limit the amount of genetic coding capacity to
only what it presently needs, but as a population the
genetic capacity of a certain bacterium is wider so that a
trait that gives a selective advantage in changed condi-
tions rapidly becomes common in the population, either
by giving the carrier bacterium better growth velocity or
by being transferred, e.g. as a conjugative plasmid. Par-
ticularly the latter phenomenon, which has been docu-
mented to be ecient on plant root and leaf surfaces
(Bjorklof et al., 1995, and references therein; Top et al.,
1998; Sarand, unpublished), greatly increases the plasti-
city of the plant-associated soil microbial ora com-
pared to the bulk soil microbes.
There are at least four principally dierent routes that
result in bacteria (or other microbes) capable of degra-
dation of a certain compound or group of compounds
at a certain site
1. The indigenous microbial ora has been exposed
to the xenobiotic contaminant long enough for
genetic evolution to create a capacity to degrade
the compound(s). This type of evolution takes
place constantly, but is relatively slow. As a con-
sequence the microbial community possesses the
degradative pathways, but degradation may be
inecient because of low cell number or low
activity level.
2. The indigenous microbial ora, which is adapted
to the local conditions, is exposed to a con-
taminating xenobiotic compound(s). The bacteria
acquire genes and degradation pathways from
bacterial cells immigrating from elsewhere
(McGowan et al., 1998). Transfer of genetic mate-
rial can take place through conjugation, transduc-
tion or transformation; and all these have been
shown to take place in environmental conditions
(van Elsas et al., 1988; Miller et al., 1990; Bjorklof
et al., 1995). From a bioremediation point of view,
this type of evolution is also relatively slow, but
can be enhanced (see below).
3. As point 2, but the indigenous well-adapted micro-
bial ora is articially supplied with the required
degradative capacity. Once the contaminant is
known, gene-clusters (e.g. in a conjugative broad
host range plasmid) may be supplied. If no natural
gene clusters are available, these may be constructed.
`Laboratory strains' can be used as donors, either to
transfer the capacity to `wild type' strains newly iso-
lated from the site, or by introducing the donors into
the site and letting gene transfer occur. The presence
of suitable recipients can be tested.
4. A bacterium that is thought to be competitive at
the contaminated site is chosen. This may be a
strain that is known to degrade the contaminating
compound, or one that is specically constructed
for this purpose. If genetic engineering is involved,
special considerations apply. Thus, if containment
of the modied genes is required, suicide functions
 M. Romantschuk et al. / Environmental Pollution 107 (2000) 179185
may be inserted (Molina et al., 1998). In this case
the strain has to be constructed to be as stable as
possible, precluding any type of genetic exchange.
Therefore, the strain itself needs to be able to
compete with the indigenous ora of the site to be
remediated.
4. The use of genetic engineering for strain construction
Bacteria can be modied genetically for a multitude of
purposes. One obstacle when designing genetically
modied organisms (GMOs) for dierent environ-
mental purposes is the bureaucracy involved in obtain-
ing permission for environmental release, rather than
limitations to what can be constructed. These type of
restrictions are not equal in all parts of the world, and it
seems likely that they will be lifted or eased in the future
as more experience is accumulated.
Among the purposes for which GMOs have been
designed are:
1. monitoring presence of added bacteria;
2. measuring bioavailability of contaminants; and
3. use of bacteria with improved/altered degradation
capacity.
In the rst case, the bacteria are simply tagged with a
suitable gene construct which is easily detected in
extracts done from the monitored site, with or without
cultivation prior to detection of the microbes (Jansson
et al., 2000). In basic experiments of this type the bac-
teria may or may not be designed to perform a certain
degradation task, as long as they provide information
on survival, spread, and possibly metabolic activity
(Ford et al., 1999). Bacteria carrying a certain desired
degradation capacity may also be tagged independently
with a neutral tag to be able to correlate putative
increases in degradation activity with the presence and
density of the added microbe (Sarand, unpublished).
In the second case, bioavailability of a contaminating
compound is a prerequisite for any type of biological
treatment. Observations of bioavailability can be done
with biosensors, a promoter inducible by the compound
to be tested linked to a gene with a product that is easy
to observe and quantify. The most commonly used
reporter genes are the bioluminescent (luc, lux) genes
which are described elsewhere in this issue (Jansson et
al., 2000), and gfp (Cale et al., 1994) and its deri-
vatives encoding green uorescent proteins, or novel
colour and stability variants of this protein (blue uor-
escent protein, redshifted gfp, less stable gfp3, etc.)
(Tsien, 1998). Bacteria have been developed that are
particularly suited for testing in a specic environment.
Such is the case for Pseudomonas uorescens OS8
pPTP11 or pPTP21 strains, that are good rhizosphere
colonizers and produce light upon exposure to mercury
181
or arsenite in the presence of the luc substrate luciferin
(Petanen, unpublished). These strains are used to deter-
mine bioavailability of heavy metal contaminants in
dierent soil types and samples.
In the third case, bacteria have been genetically mod-
ied to be able to degrade contaminating compounds
that would not otherwise be degraded (Brazil et al.,
1995). Also, bacteria have been modied to recognize
and become induced by compounds that naturally
would not induce the degradation operon. Once induced
the gene products degrade the contaminant (Pavel and
Shingler, 1994). One of the novel approaches used in the
former case is to enhance degradation capacity and
broaden the substrate range by a form of directed evo-
lution called DNA shuing (Stemmer, 1994) which has
been used to improve degradation of polychlorinated
biphenyls and other aromatics (Kumamaru et al., 1998).
In the latter case there are two alternative approaches,
only one of which relies on genetic engineering. Co-
metabolism can be used to degrade compounds that do
not themselves induce the required genes (Semprini,
1997). Phenol or toluene have been used in situ to
induce indigenous bacteria to degrade trichloroethylene
(TCE) and other chlorinated ethenes (Hopkins and
McCarthy, 1995). One of the problems then is of course
that readily biodegradable, but as such contaminating,
compounds are used to add to the contaminant burden
at a site. Although principally functional, there is no
guarantee that neither the original nor the added con-
taminant is degraded enough to meet the requirements.
Therefore, a better approach, whenever possible, may
be genetic or natural modications to increase the sub-
strate range of the contaminant-degrading strains. The
phenol degradation pathway (dmp) found in the large
plasmid pVI150 of Pseudomonas CF600 (Shingler et al.,
1992) is induced by, for example, 2-methyl phenol, but
not by 4-methyl phenol (Shingler and Moore, 1994).
Using a genetic construct a strain was generated where
the mutant DmpR regulator responds to both these
aromatic co-regulators/substrates (Pavel and Shingler,
1994), which should provide the mutant strain with a
selective advantage in 4-methyl phenol-polluted soil.
This also seems to be the case, but only for a short period
of time, since spontaneous mutants with a broader sub-
strate specicity arise rapidly in natural soil conditions
(Sarand, unpublished). Although this is an isolated case,
it is an example of natural selective conditions generating
desired results faster and technically easier than going
via genetic engineering.
5. Introducing degradation capacity on conjugative plasmids
The capacity to degrade various more-or-less recalci-
trant compounds is often carried on large plasmids that
may be conjugative or mobilizable. When such plasmids
182 M. Romantschuk et al. / Environmental Pollution 107 (2000) 179185
and their host bacteria are studied, it is found that similar
bacteria-carrying plasmids with high homology can be
isolated from widely dierent locations, both measuring
by geographical distance and type of sample source. This
is the case for Sphingomonas sp. strain HV3 (formerly
Pseudomonas), for example, that was isolated from
contaminated Finnish eld soil (Kilpi et al., 1980).
The aromatics degradation genes carried on the plasmid
pSKY4 (Yrjala et al., 1994) share homology with genes
of various other xenobiotics degrading strains of
Sphingomonas (Yrjala et al., 1997). The strain HV3 is
a relative of certain Sphingomonas species, but dier-
ent enough not to be included in any existing species
(Yrjala et al., 1998). Although pSKY4 has not been
shown to be conjugative, it is tempting to speculate,
based on analogy with other similar plasmids that it is
at least mobilizable.
Neither degradation plasmids nor resistance plasmids
are necessarily stably maintained in the cells in the
absence of selective pressure, and segregate rapidly from
bacterial cells growing in more easily degradable sub-
strates or in the absence of antibiotic selection, respec-
tively. However, they are maintained in bacterial
populations at levels that is, in part, relative to con-
jugation frequency (Bjorklof et al., 1995) and may
rebound rapidly due, in part, to their capacity for hor-
izontal transfer if the selective pressure is reintroduced.
When a plasmid is benecial it will be maintained in the
bacterial ora, but not necessarily harboured by the
bacterium in which it was introduced. In the case of soil
contaminated with a recalcitrant organic compound,
indigenous bacteria colonizing the site may not possess
the capacity to degrade the compound, whereas an
added bacterium carrying this capacity in a plasmid
may not be optimally t in other respects. If the plasmid
is conjugative and has a suciently broad host range,
which is often the case (Sayler et al., 1990), and if it
provides a selective advantage, it will be disseminated
into suitable recipients. The resulting bacterialplasmid
combination which in each micro-environment has the
highest local tness will prevail. Such is apparently the
situation for 2,4-dichlorophenoxyacetic acid (2,4-D)-
degrading plasmids in 2,4-D-contaminated agricultural
soil (Top et al., 1998) and toluene-degrading plasmids in
meta-toluate-contaminated humus and pine mycor-
rhizosphere soil (Sarand, unpublished). In a hetero-
genous environment like the soil, a high number of
dierent recipients will ensure maximal spread into the
dierent micro-environments present, and thereby
hopefully maximal degradation eciency.
6. Natural attenuation/intrinsic bioremediation
Natural attenuation could cynically be looked upon
as doing nothing, but what it does involve is monitoring
the natural attenuation process and the self-cleaning
potential. The process is monitored and if non-func-
tional abandoned in favour of more direct and drastic
measures. The minimum is to monitor the site so that
further spread of contaminating compounds is limited.
Among the approaches in this context is to ensure that
the genetic potential (Stapleton et al., 1998) or actual
capacity of the intrinsic microora (Solano-Serena et
al., 1998) for degradation is present in the soil.
The presence of a particular gene or degradation
capacity in a soil can be traced by molecular genetics
methods, once homologous genes have been isolated or
suitable primers designed. This potential gives a possi-
bility to test a contaminated site for the potential of
intrinsic remediation (self cleaning) once the conditions
support the growth of the carrier microbe. If this is the
case, no addition of degraders or of degradation poten-
tial in the form of, for example, conjugative plasmids
may be needed. Also, an evaluation of the site and the
conditions there is needed. Are the ambient natural
attenuation mechanisms biodegradation, dilution and
sorption eciency enough to meet regulatory require-
ments? Is intrinsic bioremediation, the deliberate use of
natural attenuation mechanisms, sucient, or are more
drastic measures required (Chapelle, 1999)? Further, is
the localization of the contaminants of a kind that
spreading of the contaminant plume to and in, for
example, groundwater does not pose an increased
environmental threat? If there is little or no risk for the
situation to worsen, and natural events more or less
eciently improve the situation, then possibly all that is
needed is monitoring (Chapelle, 1999).
7. Utilizing plants to improve soil contaminant
biodegradation
One way to achieve truly in situ bioremediation is by
utilizing plants to perform rhizosphere bioremediation.
The roots of plants penetrate into the soil, changing
locally the conditions in the rhizosphere and mycor-
rhizosphere. The roots themselves are colonized by
bacteria (Suominen et al., 2000) or mycorrhiza-forming
fungi, in which the fungal hyphae are more of less den-
sely colonized by a bacterial biolm (Sarand et al.,
1998). Plants growing at a contaminated site themselves
improve the appearance of the site, their roots prevent
erosion and they stabilize and increase the microbial
population density and diversity of the soil. Plant root
exudates may also provide co-substrates for the bacteria
which can improve degradation on certain contaminat-
ing compounds by inducing the right set of genes. The
roots also change the porosity of the soil, improving the
aeration.
The presence of a large diversity of bacteria in close
proximity may also aid the sequential degradation of
 M. Romantschuk et al. / Environmental Pollution 107 (2000) 179185
compounds through the concerted activities of dierent
bacterial species rather than individual strains. On the
other hand, a key question is whether the bacteria, in
the presence of more easily degradable substrates, utilize
the contaminating compound. Microcosm studies in our
laboratory indicate that upon availability of higher
levels of substrate, the bacterial cell density increases. In
the conditions used, availability of carbon and energy
source remains the growth-limiting factor for the bac-
teria, thus ensuring ecient contaminant-degradation.
When the carbon and energy source is the growth-lim-
iting factor, also the sub-optimal substrates will be used,
as long as the concentration is high enough to induce
the promoters of the degradation pathway genes.
In all, the perceived advantages of rhizosphere bior-
emediation approach include:
1. maintenance of high numbers of target bacteria in
the nutrient-rich root zone;
2. high energy conditions allowing the expression
and functioning of the degradative enzymes;
3. likely association of degradative bacteria with
other benecial microorganisms such as root sym-
biotic mycorrhizal fungi that may be able to co-
degrade the organic pollutants or their inter-
mediates;
4. the plant roots may in some cases produce com-
pounds that function as natural co-substrates,
inducing the degradative genes of the bacteria also
when the contaminant level is low;
5. perennial plants promote survival of the associated
bacteria over the years;
6. plant roots reach dierent layers of the soil
thereby distributing microbes without need for soil
mixing;
7. plants bind the surface soil and prevent erosion
and ooding, while mixing may actually lead to
worsened leaching of toxins into the ground water;
8. plants take up water, counteracting the downward
ow of possibly contaminated soil water towards
the groundwater level, or into the waterways;
9. plants may take up and enrich heavy metals from
the soil; and
10. plants also have an aesthetic function by improving
the appearance of the site.
In our microcosm studies using Suillus bovinus
mycorrhized pines the model compound meta-toluate
was rapidly degraded, even from high initial concentra-
tions (Sarand et al., 1999). Furthermore, degradation-
active bacteria were able to protect and improve the
growth of the mycorrhizal fungus present in the system.
An important nding was that when the contaminant
level rose high enough to kill the plant, all degradation
even by the degradation-active bacteria appeared to
seize, while degradation in microcosms with the bacteria
183
alone in the same contaminant concentration remained
ecient (Sarand et al., 1999). This eect appeared to be
due to killing of the bacteria by plant-derived com-
pounds. It is therefore important to make sure that
plants are used only when they are likely to survive,
since otherwise they may only worsen the situation.
8. Increase of bioavailability by using surfactants
One of the reasons for recalcitrance of many organic
soil and groundwater contaminants is their hydro-
phobicity low solubility in water and thereby poor
bioavailability (Van Loosdrecht et al., 1990; Weissenfels
et al., 1992). Some bacteria produce biosurfactants that
help them access hydrophobic compounds as carbon
and energy sources (Francy et al., 1991; Volkering et al.,
1998). Bacteria with this capacity may in themselves be
good candidates for bioremediation purposes (Falatko
and Novak, 1992; Banat, 1995), but both synthetic and
biosurfactants have, in many studies, been added to con-
taminated sites or in bench-scale test as a means of accel-
erate biodegradation (Volkering et al., 1998). As with
addition of nutrients, one diculty is then the dissemina-
tion of the surfactant during in situ bioremediation.
9. Electrokinetic extraction in combination with
bioremediation
Electrokinesis and electro-osmosis is a relatively new
in situ method that has been used to mobilize con-
taminants from ne-grained soil types such as clay. The
mobilization is based on direct ionic migration of dis-
solved charged contaminants but, more importantly, on
movement of the positively charged liquid layer at the
surface of the negative-charged soil particles towards
the cathode. The latter form of migration, electro-
osmosis, creates a liquid ow transporting both neutral
and positively charged dissolved contaminants that can
then be collected at the cathode. Both inorganic con-
taminants (heavy metals, radionuclides, etc.) and selec-
ted organics can be extracted this way. This process has
recently been reviewed (Alshawabkeh et al., 1999).
During ongoing extraction, organic contaminants in the
soil are also degraded, and also this biological portion
of the clean-up process may be improved by the elec-
trokinesis. Electro-osmosis apparently helps spreading
both indigenous bacteria or added inoculum around
and into the contaminated site. But even more sig-
nicant for biological degradation is perhaps that other
limiting factors such as low temperature and nutrient
limitions can be alleviated by using an electric eld.
Either way, the results in preliminary application-scale
tests have been promising (Romantschuk, unpub-
lished).
184 M. Romantschuk et al. / Environmental Pollution 107 (2000) 179185
10. Soil fauna
The role of soil fauna in decontaminating soil is in
most cases indirect, but still important in that they
redistribute microbes or help reintroduce them from less
contaminated soil layers. Particularly, worms of various
sizes also mix the soil and makes it more porous, and
thereby improve aeration. Dierent aspects of soil fauna in
relation to bioremediation is reviewed in depth elsewhere
in this issue (Haimi, 2000).
11. Conclusions
Many dierent approaches have been tested for soil in
situ bioremediation, and success stories can be found in
the literature; but also many attempts have failed, and
these are much more likely not to be reported in the sci-
entic press. It should be possible, however, building on
both positive and negative experiences, to come up with
new ideas that perhaps combine advantages from two or
more methods. Dissemination of various components into
the site, and bioavailability of the contaminant are two of
the key questions for workable in situ bioremediation.
From the examples given above and from the reviews and
papers presented elsewhere in this issue it should be easy
to come up with an as yet untested, but worthwhile, com-
bination to test in order to achieve higher reliability, pre-
dictability and eciency in in situ soil bioremediation. In
upcoming years cost-ecient, safe and environmentally
sound methods that are good enough to develop into
marketable products will hopefully evolve.
Acknowledgements
This work was supported by grants from the Maj and
Tor Nessling Foundation and the Academy of Finland.
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