Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200577909915Review ArticleBioremediation

by bioaugmentationI. P. Thompson, C. J. van der Gast, L. Ciric

and A. Singer

Environmental Microbiology (2005) 7(7), 909–915 doi:10.1111/j.1462-2920.2005.00804.x

Minireview

Bioaugmentation for bioremediation: the challenge of

strain selection

Ian P. Thompson,1* Christopher J. van der Gast,1 tation in bioremediation. Some see it as an expensive
Lena Ciric2 and Andrew C. Singer1 and ineffective equivalent to voodoo medicine, while
1
Environmental Biotechnology Section and others extol its virtues and even derive a living from its
2
Molecular Microbial Ecology Section, NERC Centre for commercialization. The rationale underlying bioaugmen-
Ecology and Hydrology – Oxford, Mansfield Road, Oxford tation in bioremediation is simple: the augmentation of
OX1 3SR, UK. catabolically-relevant organisms to hasten remediation.
But despite its apparent simplicity there have been many
failures, and these have been well detailed and reviewed
Summary
(Goldstein et al., 1985; Stephenson and Stephenson,
Despite its long-term use in bioremediation, bioaug- 1992; Bouchez et al., 2000; Vogel and Walter, 2001;
mentation of contaminated sites with microbial cells Wagner-Döbler, 2003). Often, failures have been simply
continues to be a source of controversy within envi- due to choosing bioaugmentation as the remediation
ronmental microbiology. This largely results from its approach in the first place. In these cases the absence of
notoriously unreliable performance record. In this relevant catabolic genes within the indigenous microbial
article, we argue that the unpredictable nature of the community is not the factor limiting contaminant degrada-
approach comes from the initial strain selection step. tion. Biodegradation can be inhibited by a plethora of
Up until now, this has been dictated by the search for factors, such as pH and redox, the presence of toxic co-
catabolically competent microorganisms, with little or contaminants, concentration, or the absence of key co-
no consideration given to other essential features that substrates, to name but a few (Barriault and Sylvestre,
are required to be functionally active and persistent 1993; Harms and Zehnder, 1995). The purpose of this
in target habitats. We describe how technical article is not to review the many instances where bioaug-
advances in molecular biology and analytical chemis- mentation failures could have been predicted by undertak-
try, now enable assessments of the functional diver- ing microbial and chemical analyses to identify possible
sity and spatial distribution of microbial communities constraints to degradation. Our focus is on those more
to be made in situ. These advances now enable micro- challenging cases where, after appropriate analysis, there
bial populations, targeted for exploitation, to be dif- are no obvious constraints to microbial degradation, yet
ferentiated to the cell level, an advance that is bound bioaugmentation still fails. We believe that the route of
to improve microbial selection and exploitation. We these failures is summarized by Vogel and Walter (2001),
argue that this information-based approach is already ‘Although microbial ecology issues are among the most
proving to be more effective than the traditional important in bioaugmentation approaches, unfortunately,
‘black-box’ approach of strain selection. The future they are rarely addressed’. The authors argue that, as with
perspectives and opportunities for improving selec- indigenous populations, a broad range of environmental
tion of effective microbial strains for bioaugmentation parameters, together with the phenotypic characteristics
are also discussed. of the strains and procedures for introduction, culminate
to determine the activity, persistence and performance of
bioaugmented strains. It is the lack of consideration of
Introduction
such determinants and the complete and sole preoccupa-
There are few issues in environmental biotechnology that tion with the search for vigorous degradative strains that
generate more controversy than the use of bioaugmen- is the root of many failures.
In this article, we argue that ecological considerations
should be a priority, even before the application of inoc-
Received 23 September, 2004; accepted 20 January, 2005. *For
correspondence. E-mail ipt@ceh.ac.uk; Tel. (+44) 0 1865 281630; ula: that is at the strain selection stage. Vital to these
Fax (+44) 0 1865 281696. ecological considerations is the relative spatial and tem-

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd

910 I. P. Thompson, C. J. van der Gast, L. Ciric and A. Singer
poral abundance of potential source populations and their
ability to tolerate the prevailing conditions in target habi-
tats. Until recently, initial strain selection has been based
on a single key criterion, degradation ability, with no con-
sideration of the potential ability of strains to proliferate
and be active in sites to which they were applied. We
propose that strains selected from populations that have
low abundance, or are transient within the source or tar-
get habitats, are less likely to succeed as inocula, when
compared with those that are spatially and temporally
ubiquitous. We discuss the challenge of selecting strains
for reliable exploitation in bioaugmentation applications: a
key stage, but sparsely dealt with in most relevant papers.
We briefly summarize some of the more recent technical
advances that now enable strain selection to be based on
an in situ understanding of organism abundance, func-
tional activity and population dynamics in the habitat from
which they are derived. We go on to provide evidence as
to why a more informed approach is already proving to be
more effective, than the traditional ‘black-box’ attitude to
selecting ‘good strains’. The future opportunities these
technological advances provide for selecting well-adapted
and catabolically competent strains, for degradation per
se, as well as their potential as vehicles for delivering and
maintaining degradative genes in the target habitat are
discussed.
The challenge
Successful application of bioaugmentation techniques is
dependent on the identification and isolation of appropri-
ate microbial strains, and their subsequent survival and
activity, once released into the target habitat. Sourcing
microbial strains for bioaugmentation has typically been
achieved, for at least the past 100 years, by selective
enrichment (Beijerinck, 1901). This involves strains from
polluted samples being enriched to grow, relative to the
background community, in culture, using the target con-
taminant as the sole enriching carbon or nitrogen
source. The procedure results in the selection of strains
that express the required degradation ability: in the spe-
cific conditions of the enrichment culture, at least. How-
ever, such enriched populations are not necessarily
typical or representative of indigenous communities in
the target habitat and could equally, by chance, be
derived from transient populations. It seems likely that
this latter point is the crux of many bioaugmentation fail-
ures. The problem is that the enrichment procedure is
unlikely to have any influence on other traits that are
also required for strains to be competitive and effective
in the target environment. These additional traits are
required to survive prevailing, often fluctuating, environ-
mental conditions (e.g. moisture, nutrients, redox, pH
and osmotic factors) and, of course, competition from
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 909–915
indigenous microbial populations, among many others
stresses (Recorbet et al., 1992; England et al., 1993).
We believe it is this initial selection step, and specifically
poor knowledge of the composition and population
dynamics of source communities, as well as those in the
target habitats (competitors in waiting), that underlie
many inocula failures.
An alternative approach, which would probably increase
success rates, in terms of persistence and activity, would
be to base the initial selection step on a priori knowledge
of the ubiquity, population dynamics and spatial and tem-
poral distribution of microbial communities in sampled
habitats. It seems logical that a strain, derived from a
population that is temporally and spatially prevalent in a
specific type of habitat, is more likely to persist as an
inoculum when reintroduced, than one that is transient or
even alien to such a habitat. Once abundant populations
have been identified, the second phase of the selection
procedure should then be to identify strains which can
degrade the target contaminant. Indeed, there is evidence
to suggest that such a strategy would aid inocula survival.
Belotte and colleagues (2003) isolated bacteria from a
range of locations in a forest soil. When vigorous growers
were cultured and reintroduced into the environment, they
tended to colonize the sites from which they originated,
providing strong evidence for local adaptation. The
authors estimated that isolates were 50% fitter when rein-
troduced into their home sites, and that fitness diminished
exponentially with distance from their origin (Belotte et al.,
2003).
We are not advocating that the selective enrichment
method should be made redundant. The approach is
essential in order to derive ‘superior strains’ and so will
continue to be an essential step for obtaining exploitable
strains (Singer et al., 2005a). Nor are we suggesting that
intensive microbial community analysis be taken for every
site targeted for bioaugmenation. Tailoring inocula for
every site and application would just not be practicable.
However, we are suggesting that selection of strains
should be taken on the basis of some understanding of
the kind of microbial communities present in the source
habitat, and preferably with some knowledge of the type
of organisms that are common in the target habitat. With
the introduction of superior methods for undertaking more
thorough assessments of microbial communities, this
knowledge-based approach is becoming increasingly
more feasible.
Identification of key populations and genes in situ
In recent years technological advances in molecular
microbial ecology and analytical chemistry have been
brought together, enabling us to identify in situ popula-
tions, and even individual cells, responsible for undertak-

ing specific processes. Using molecular approaches, such
as denaturing gradient gel electrophoresis (DGGE)
(Muyzer et al., 1993), terminal restriction fragment length
polymorphism (tRFLP) (Liu et al., 1997) and length-heter-
ogeneity polymerase chain reaction (LH-PCR) (Suzuki
et al., 1998), we are now in a better position to obtain a
more comprehensive assessment of the composition and
structure of microbial communities in the environment. In
addition, other complementary techniques can provide
more direct assessments of microbial community compo-
sition. For example, fluorescent in situ hybridization
(FISH) (Delong et al., 1989) uses a fluorochrome-labelled
oligonucleotide to identify specific populations in situ, and
obtain direct measures of their relative abundance. More
recently stable isotope probing (SIP) (Boschker et al.,
1998; Radajewski et al., 2000) has emerged as a poten-
tially powerful tool, which employs stable isotopes such
as 13C, to determine exactly which organisms assimilate
specific contaminants. Extraction of labelled fatty acids,
DNA and/or RNA, allows accurate identification and dif-
ferentiation of the active members of the community. With
further development this should become the most power-
ful of the new techniques discussed; although as yet, it
does not provide a reliable estimate of the relative abun-
dance of the catabolically active forms (Manefield et al.,
2002). However, SIP has already been applied in the field,
where it successfully identified the populations that drive
the remedial process (Jeon et al., 2003). In an exciting
recent development, this approach has been combined
with Raman confocal microscopy, lending insight into
those populations responsible for contaminant catabolism
to be differentiated, at the individual cell level (Huang
et al., 2004; Singer et al., 2005b).
Better selection gives better performance
The techniques described, in particularly SIP, are new and
not yet routinely and widely used to scrutinize communi-
ties, so it is really too early to predict how they might
revolutionize the selection, detection and exploitation of
strains for bioaugmentation. However, several studies are
provided as evidence of how systematic and careful selec-
tion process for competitive bacterial strains might be
used to improve bioaugmentation. For instance, in an
extensive field study 690 characterized isolates of fluores-
cent pseudomonads from a single site were analysed to
determine the genetic composition using RFLP rRNA
(ribotyping) analysis, and dynamics of the population over
several seasons (Goddard et al., 2001). The population
was found to be highly heterogeneous: the 690 character-
ized isolates consisted of 385 ribotypes, and notably most
were transient, being detected only once. Approximately
26 ribotypes were detected more frequently (spatially and
temporally); one isolate, ribotype A, was ubiquitous in the
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 909–915
Bioremediation by bioaugmentation 911
samples analysed. Ribotype A was subsequently geneti-
cally tagged (lacZY with kanamycin resistance), inocu-
lated in lab-based field soil and found to persist
significantly better than randomly selected (transient)
ribotypes, similarly tagged. This demonstrates that differ-
ent strains, even when genetically very similar, have dif-
ferent competencies, in terms of survival, when introduced
into the environment as inocula. Interestingly, ribotype A
not only proved to have exceptional survival ability, but
when co-inoculated, improved the persistence of the
poorly surviving transient isolate. In an attempt to deter-
mine which traits distinguished competent strains from
transient forms, the authors examined a range of pheno-
types that had previously been associated with rhizo-
sphere competence, including the presence of flagella
and lipopolysaccharides (de Weger et al., 1987a,b). How-
ever, the only trait found to distinguish the two populations
was their organic acids utilization characteristics. Contrary
to expectation, ribotype A utilized a narrower range of
organic acids than the transient form, but grew faster on
compounds they could assimilate (e.g. citric and lactic
acid). The importance of nutrients in determining inocula
performance, in the rhizosphere at least, is not surprising,
in particularly organic acids, as they are significant con-
stituents of the root exudates (Rovira, 1969). However, it
is surprising that the range of organic acids utilized by
ribotype A was narrower than the less competitive strains.
Indeed, the ability to utilize a diverse range of nutrients
has previously been correlated to competitive ability in the
rhizosphere (Bakker et al., 1993; Oresnik et al., 1998).
The authors concluded that, in some cases at least, the
ability to grow rapidly on a more narrow range of sub-
strates can confer a competitive advantage, if that com-
pound is abundant in the habitat. However, the contrasting
conclusions of these studies highlights the difficulty of pin-
pointing the specific traits that confer fitness on inocula,
which are very much dependent on the characteristics of
the habitat and strains, and their interactions, which in turn
are unlikely to remain constant.
In a similar study, the authors also noted that
pseudomonad strains had different rhizosphere compe-
tencies, and similarly used a dual selection criterion. In
this instance numerical dominance in the target habitat
(rhizosphere) and the ability to degrade a contaminant
(naphthalene) were used to select strains for long-term
phytoremediation applications (Kuiper et al., 2001). The
authors demonstrated a 100-fold increase in strain sur-
vival after two enrichment cycles within the Lolium multi-
florum rhizosphere, in addition to naphthalene
degradation. The exceptional root-colonization ability and
excellent performance of the selected strain was attrib-
uted to its ability to effectively utilize the nutrients specific
to the plant cultivar selected, and the careful and system-
atic targeting of the strain.
912 I. P. Thompson, C. J. van der Gast, L. Ciric and A. Singer
In a series of studies, a systematic selection/isolation
programme was undertaken whereby bacterial inocula
were developed to treat spent metal working fluids (MWF)
in bioreactors (van der Gast et al., 2003a; 2004), also
based on significant criteria that reflected key features of
the microbial habitat. However, in this instance the authors
used a triple-selection criterion. These were: (i) the rela-
tive abundance of source populations in the target habitat
(waste MWF), (ii) tolerance to co-contaminants (MWF are
chemically mixed) and (iii) the ability to degrade compo-
nents of MWF. Extensive analysis of the microbial com-
munity composition and structure of a single MWF
formulation, both temporally and spatially at a worldwide
scale, were undertaken to identify ubiquitous populations
in waste MWF (van der Gast et al., 2002; 2003b). Subse-
quent screening cycles were based on the ability of iso-
lates to tolerate the toxicity of co-contaminants and to
catabolize individual chemical constituents. A consortium
of four isolates was generated that proved to be 85%
more effective at processing waste MWF in bioreactors
than undefined inocula from sewage. It also demonstrated
exceptional persistence in the chemically dynamic biore-
actors. Perhaps even more importantly, in terms of appli-
cations, the consortium performance was more consistent
and predictable than those from uncharacterized microbial
communities (activated sludge), a feature of great value
to chemical engineers and technicians who design and
manage such systems.
Improved exploitation of robust strains
Over a decade has passed since it was first proposed that
inoculation of strains containing mobile genetic elements
(MGE) might provide a mechanism for the introduction of
specific traits into microbial communities (Brokamp and
Schmidt, 1991; Fulthorpe and Wyndham, 1991). For
instance, the transfer of 2,4-dichlorophenoxyacetic acid
(2,4-D) degradation plasmids pEMT1 from an introduced
donor strain to the indigenous soil microbial community
resulted in the complete degradation of 2,4-D after
19 days, while the contaminant persisted in the non-
inoculated soil after 89 days (Dejonghe et al., 2000).
Enhanced 2,4-D degradation was attributed to the emer-
gence of transconjugants (105 colony forming units per
gram soil), as the donor was undetectable before degra-
dation started. It might be argued that this approach would
negate the requirement to take the trouble to screen for
competitive strains, as we are advocating, as the donor is
no longer required, once catabolic activities are trans-
ferred and expressed in the indigenous bacteria. However,
it can be equally reasoned that introduction of robust
strains, containing desired genes, that persist long-term
in a habitat, would have greater opportunities to transfer
the catabolic genes. Indeed, plasmids-borne catabolic
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 909–915
genes (e.g. 3-chlorobenoate and 4-chlorobiphnyl) have
been successfully introduced, by conjugation, into strains
specifically selected for their tolerance (Top et al., 2002).
For instance, introduction of catabolic genes into
desiccation-tolerant strains resulted in good degradation
of the contaminants (biphenyl, isopropylbenezene and 3-
chlorobenzoate) by the tranconjugants in desiccated soil
conditions, and the strains demonstrated excellent shelf
life when stored in a dried form (Weekers et al., 1999).
Enhanced degradation rates of carefully selected
adapted strains have also been achieved by recombinant
approaches. Watanabe and colleagues (2002) screened
a bacterial community in an activated sludge plant for
phenol degradation, and selected the dominant (most
abundant) population Comamonas sp. rN7. Phc genes
encoding for phenol hyroxylase, from the parent strain C.
testosteroni R5, were integrated into the chromosome of
the Comamonas sp. rN7. The specific phenol-oxidizing
activity of the resultant transformant, designated
rN7(R503), was three times greater than the activity of the
original strain R5. Quantitative PCR revealed that the phc
genes were retained in the transformant, at 108 copies per
ml of liquor, for more than 35 days, while the parent R5,
the original source of the phc genes, was undetectable.
Furthermore, inoculation of phenol containing activated
sludge with the transformant resulted in high phenol-oxy-
genating activity and improved resistance to phenol shock
loading, compared with inoculation with the parent.
Taking a similar approach the catabolic genes were
genetically engineered into persistent and adapted
Deinococcus radiodurans strains, specifically selected for
their natural tolerance to high doses of radioactivity, and
modified to express the cloned mercury (II) resistance
genes (merA), derived from Escherichia coli (strain
BL308) (Lange et al., 1998). The resultant was a strain
able to grow in the presence of radiation, a natural char-
acteristic of this organism, and ionic mercury, which it
transformed to the less toxic elemental mercury (II). Sim-
ilarly, the same radiation-tolerant strain was modified to
express toluene dioxygenase (tdo) activity. Cloning of the
tdo genes into the chromosome of D. radiodurans
imparted the ability to oxidize toluene, chlorobenzene and
indole. The recombinant was also capable of growth and
functional synthesis of tdo in highly irradiated conditions
(Lange et al., 1998). This approach puts us in very strong
position not only to select persistent and active strains,
but to add functionality to well performing strains.
An alternative but complementary approach, success-
fully used to maintain the catabolic activity of introduced
inocula, is to manipulate key environmental parameters
selected specifically to favour catabolic expression and
survival of introduced cells. Again this approach requires
good understanding of the ecology of the selected strains.
For instance, Pseudomonas stutzeri KC was selected for

site inoculation because of its ability to transform carbon
tetrachloride to carbon dioxide (Dybas et al., 1998). Pre-
vious studies revealed that the inoculum was favoured by
anoxic conditions and demonstrated an exceptionally high
affinity for iron (Criddle et al., 1990). By manipulating the
pH in the field to pH 8, which lowered iron availability, the
strain was able to persist and actively degrade the con-
taminant, by out-competing indigenous populations that
were unable to obtain adequate concentrations of iron.
Future perspectives
It would be naive to believe that by simply picking the
‘right’ organisms or manipulating the right field parameter,
bioaugmentation will suddenly become as reliable and
predictable as engineered systems. Inevitably, as with
most biological systems, there will always be an element
of unpredictability. Such biotic factors as inoculum density,
preparation and modes of introduction are known to
greatly influence performance (Vogel and Walter, 2001).
It is not practical to tailor consortia specifically to each
habitat and eventuality. However, it is clear that members
of the same genera/species do not all have equal fitness
and some are likely to be competitive in a broader range
of scenarios, while others may be more suited to specific
conditions and habitats. With improving knowledge we
should become more effective at determining and target-
ing the kind of organisms or traits (e.g. resistance to low
pH or high heavy metal concentrations, poor nutrient con-
ditions or high salinity) that are likely to suit specific
conditions and remedial requirements. This should then
allow us to develop a suite or culture collection of well-
characterized organisms which maintain exceptional cat-
abolic ability and tolerance in a broad range of chemical
and environmental stresses. These superbugs are
referred to as an Heirloom strains – named after the
microorganism’s often long lineage within research
groups, where they acquire an ever-increasing dossier of
desirable traits from an equally long list of researchers
(Singer et al., 2005a). For instance, bioaugmentation of
soils co-contaminated with organics and metals may see
greater success with the application of microorganisms
selected specifically for their dual catabolic competence
and tolerance of toxic metals. Furthermore, such a collec-
tion would avoid the impractical requirement of painstak-
ingly selecting and tailoring inocula for each specific site.
The notion that different types of microorganisms have
different survival strategies and opportunities is far from
new, but seems to have been largely forgotten. The great
pioneering microbiologist Winogradsky distinguished
between two classes of microbes in terms of their survival
strategy (Winogradsky, 1924). He coined the term ‘zymog-
enous’ microbes (e.g. Pseudomonas), for those microor-
ganism that exist mostly in a resting phase, which
© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 909–915
Bioremediation by bioaugmentation 913
demonstrate brief periods of activity, stimulated by the
appearance of an available substrate, returning to quies-
cence, when resources were exhausted. In contrast, the
‘autochthonous’ forms were defined as those organisms
that demonstrate slow but continuous activity (e.g. Arthro-
bacter). Later, Hirsch and colleagues (1979) utilized the
term ‘r-strategist’ to refer to an organism typified by a high
Km and Vmax, suggesting the need for a large food source
to induce growth followed by a high metabolic rate and
subsequent short-lived but large increase in population
size. ‘K-strategists’ are typified by a low Km and Vmax, and
an equivalent metabolic and population growth response
to both large and small nutrient introduction (Hirsch et al.,
1979). On this basis, if a strain for bioaugmentation was
required that could survive long-term in a habitat, slowly
and continuously degrading a contaminant, an autochth-
onous candidate would be most suitable. Zymogenous
forms would be ideal when there were occasional pollution
episodes. When the contaminant was present, the zymog-
enous strain would explode in activity and growth rate,
their numbers then rapidly diminishing when the contam-
inant was completely degraded. These scenarios may
seem far fetched, and they probably are, but they serve
to stress the point that it is essential to select the right
‘strategist’ for a specific task. However, what is described
above may not be too far from the real world. In a long-
term study into the persistence of bacterial inocula, the
performances of Arthrobacter (A109) and Flavobacterium
strains (P25), selected specifically to represent the two
contrasting classes of microorganisms defined by Wino-
gradsky, were evaluated in the field. As Winogradsky
might have predicted, after 87 days, counts of the zymog-
enous form (P25) fell below detectable limits, while those
of the autochthonous form (A109) were detected in
excess of 300 days after introduction, when monitoring
ceased.
Even though Winogradsky’s theory of survival may now
seem simplistic and may not survive the test of time, or
the scrutiny of modern techniques, it does at least provide
some theoretical basis on which we can more systemati-
cally select strains for exploitation for specific tasks in
bioaugmentation. With the introduction of new technolo-
gies, we are in a much better position to understand the
bewildering extent of microbial diversity, and to develop
ecological theories and models (Curtis et al., 2003) that
will inevitably help target the best microorganisms for any
one task. Moreover, by minimizing the size of the ‘black
box’, we are better able to respond to changes in system
and environmental parameters – ultimately leading
towards a more robust and reliable bioaugmentation.
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