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2005 TIBTECH

2005 TIBTECH

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Published by Andrew Singer
Perspectives and vision for strain selection in bioaugmentation
Perspectives and vision for strain selection in bioaugmentation

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Published by: Andrew Singer on Jul 01, 2008
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Perspectives and vision for strainselection in bioaugmentation
Andrew C. Singer, Christopher J. van der Gast and Ian P. Thompson
Environmental Biotechnology Section, Centre for Ecology and Hydrology – Oxford, Oxford OX1 3SR, UK
Notwithstanding the phenomenally large and ever-increasing resource of pollutant-degrading microbialisolates in laboratories around the globe, inoculumsurvivalremainsthe‘Achillesheel’forbioaugmentationof contaminated land. Considerable effort has beeninvested into inoculum strain selection to facilitatepollutant biodegradation, ranging from the isolation of‘superbugs,’ which are microorganisms highly resilientto environmental stresses, harboring catabolicallysuperior pollutant-degrading enzymes, to the otherextreme in ‘priming’, where pollutant degradation iscarried out through the addition of soil enriched withan undefined consortium of pollutant-degradingmicroorganisms.
The past four years (2001–2004) have seen 172 scientificpapers including the keyword ‘bioaugmentation’ (ISI Webof Science) – nearly double that of the previous four-yearperiod. Nevertheless, this increase has yet to correlatewith an increase in the implementation of field-scalebioaugmentation. In this article, we discuss our perspec-tive on the pinch-point for bioaugmentation strainselection. We suggest that many of the challenges of bioaugmentation can be attacked with greater efficacythrough the manipulation of a select subset of the‘superbugpopulation. We propose that the future of bioaugmentation rests in the coordinated efforts of theinternational scientific community to harness the unrea-lized potential of the superbugs, rescuing them from adestiny of serial ‘proof of concept’ studies. We alsohighlight two strain-selection techniques that showgreat promise for further enhancing the efficacy andapplicability of bioaugmentation in the field.Bioaugmentation is an approach rooted in the ancientart offood preparation. Most notably, cheese, yoghurt andbeer production employ small quantities of the previousbatch of fermented product to initiate the process onceagain, thereby reproducing with high precision the sameproduct with each successive batch. For millennia, the‘starter culture’ for milk fermentation was supplied fromthefortuitousstorageofcoworgoatmilkinananimalskinbag. It was not until insights provided by Louis Pasteur inthe later half of the 19th century, that dairy science wasable to transform from the black-box art form mastered byfew, to a six billion dollar (US), scientifically preciseindustry, mastered by many.Despite our long cultural history of bioaugmentation, itis only recently that we have attempted scientifically toengineer many of these processes. Bioaugmentation of polluted soil is an approach whereby microorganisms areamended to a contaminated site to hasten the degradationof pollutants. It is through the introduction of excess,active, degrader microorganisms that the pollutant ismineralized, thereby providing remediation at a fasterrate than would otherwise occur by means of theindigenous microorganisms. We direct the reader to van Veen
et al.
, for an excellent, comprehensive review of bioaugmentation techniques[1].Strain selection for bioaugmentation is contingent onour understanding of not only microbial ecology butsociety. Clearly, recent developments in molecularmicrobial ecology have greatly assisted our ability totrack strains, interrogate environmental systems andappreciate the complexity of soil microbiology in waysthat only five years ago were impossible. If a plentifulsupply of superbugs are available for exploitation, and ourunderstanding of the target system is increasing rapidly,why are there so few examples offield-scale bioaugmenta-tion? In this paper, we propose that much progress can begained through a coordinated exploitation of the currentknowledge base, molecular biology techniques and globalculture collections.
Strain selection
Strain selection is founded on the principle that certainmicroorganisms are better suited for particular (catabolic)tasks and environments than others. Traditional bioaug-mentation has achieved its greatest results throughrepeated application of highly competent pollutant-degrading bacteria[2,3]. Arguably, the high frequency of bioaugmentation events is necessary owing to poorsurvival and activity of the inoculum, which, among amyriad of other reasons, is ultimately founded on strainselection.Whendesigning abioaugmentationregimen,forobvious reasons, strain selection is confined to thosespecies that are not ‘linked’ to human pathogens(i.e. share the same genus and species name); however,it is often the case that a ‘superbug’ is closely related to ahuman pathogen, precluding its field implementation[4].This is exemplified by
Pseudomonas aeruginosa
, a versatile Gram-negative bacterium that grows in soil,marshes and coastal marine habitats and has beenutilized as a plant growth-promoting rhizobacterium.Specifically,
Pseudomonas aeruginosa
strain IE-6 was
Corresponding author:
Singer, A.C. (acsi@ceh.ac.uk). Available online 24 December 2004
Opinion
TRENDS in Biotechnology 
Vol.23 No.2 February 2005
www.sciencedirect.com0167-7799/$ - see front matter
Q
2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.12.012
 
shown to suppress the root-infecting fungi
 Macrophomina phaseolina
,
Fusarium oxysporum
,
Fusarium solani
and
 Rhizoctonia solani
in tomato plants[5]. However, a rangeof different strains of 
P. aeruginosa
are also known toinfect the lungs of cystic fibrosis sufferers, where it causesinflammation and episodes of intense breathing problemswhich can be potentially fatal[6]. Hence, strain selectionmust not be based on performance alone.
Heirloom strains 
The search for novel microorganisms with exceptionalcatabolic or survival abilities is an ongoing pursuit of many laboratories worldwide. This effort has generatedmany hundreds of thousands of ‘ordinary’ microorgan-isms, along with a notable few with exceptional abilities(i.e. superbugs). A subset of the superbug population,denoted as‘Heirloommicroorganisms, aresuperbugs thatare maintained and handed down from generation togeneration within a research group – with each successivetransfer adding phenotypic and genotypic details andgenetic modifications to its meta-dossier. These Heirloomsuperbugs are particularly attractive for bioaugmentationfor several reasons: they are usually easily cultured, fastgrowing, thoroughly characterized and accessible world-wide through one of the many culture collections(e.g. American Type Culture Collection (ATCC), BelgianCo-ordinated Collections of Microorganisms (BCCM),Deutsche Sammlung von Mikroorganismen und Zellkul-turen GmbH (DSMZ) and Czech Collection of Micro-organisms (CCM)). Arguably, one of the most exemplary Heirloom micro-organisms is
Deinococcus radiodurans
. This bacteriumhas been under increasing scientific scrutiny for nearlyfivedecades,accruingathickphenotypic[7]andgenotypicdossier[8]. The bacterium touts a dizzying array of talents, ranging from its natural resistance to genotoxicchemicals, oxidative damage, high levels of ionizing andultraviolet radiation and dehydration to its geneticallyengineered acquisitions of toleranceto hightemperatures,ability to biodegrade toluene and detoxify a range of potentially hazardous metals [e.g. mercury (II), iron (III),uranium (VI) and chromium (VI)][9,10].Ultimately,
 D. radiodurans
is being groomed for one of the mostdifficult of pollutant challenges – bioaugmentation intohigh-temperature radioactive mixed wastes (i.e. organicand metal pollutants). Despite its awesome potential toremediate among the most challenging and hazardouspolluted sites, it is not likely to be readily adopted byproblem holders worldwide because of its genetic modifi-cations and unknown impact on the environment. Hence,this superbug could very well find itself in Heirloompurgatory, where it might remain indefinitely, foreverstymieing the interests of researchers, funding bodies andproblem holders[11,12].One solution to this problem, in principle supported byBarac
etal.
[13]and Blumenroth and Wagner-Dobler[14], is that the scientific community coordinates its researchefforts on Heirloom microorganisms. The pool of Heirloomspecies would be chosen based on their amenability tomanipulation, exceptional catabolic ability, currentknowledge base, tolerance to environmental stresses,technical ease of use and lack of public health threat(e.g.
 Pseudomonas
sp. strain ADP,
 Pseudomonas
sp. NCIB9816–4,
Burkholderia xenovorans
LB400,
Phanerochaetechrysosporium
strain RP78,
Methylococcus capsulatus
(Bath),
Geobacter sulfurreducens, Deinococcus radio-durans
). The Heirloom collection would constitute adynamic pool of superbugs, thoroughly tested for theirsuitability for field use as well as optimized for enhancedsurvivalandactivity intheenvironmentthrough(genetic)manipulation. The proposal would establish the microbialequivalent of open-source coding (e.g. LINUX), focusingtheinternationalresearcheffortintoprovidingatoolboxof workhorse microorganisms upon which all field releasescould be carried out, affording the greatest likelihood of achieving the desired objectives while providing the bestopportunitytoaddressthesafetyconcernsofthepublic.Inthe end, the approach would impart greater value formoney to the research-funding body, provide theproblem holder with a scientifically sound, cost-effective technology to remediate polluted sites andultimately inspire creative and innovative science, inlarge part owing to
a priori
knowledge that thefinished product stands a reasonable chance of contribut-ing to the remediation of contaminated land.
In rhizo-directed strain selection 
In a majority of cases, superbugs are isolated through astandard procedure of batch-enrichment culture, whichrelies on the ability of the isolate to grow rapidly on aspecific carbon source in defined chemical and environ-mental laboratory conditions. Although highly effective ingenerating superbugs, the approach operates in a vacuumfrom the natural environment. In an effort to advance thetechniques of strain selection beyond traditional batchenrichment, researchers have turned to a relatively newapproach, which incorporates the benefit(s) of plants aswitnessed in phytoremediation with optimized biodegra-dative strains[15], called
in rhizo
-directed strain selec-tion. The approach utilizes a microorganism with thecapacity to grow on one or more root exudates within therhizosphere in addition to degrading the relevant pollu-tant. The microorganisms, once inoculated into theappropriate plant rhizosphere, would express the pollu-tant-degrading enzymes, thereby decontaminating thesoil while growing on the plant root exudates to sustain itspopulation and metabolic activity. An added benefit of including plants in the bioaugmentation approach is thesupply of additional secondary metabolites and rootexudates, which might also fortuitously stimulate andmaintain the induction of pollutant-degrading enzymes
in situ
[16,17]. The inoculum, in return, can provideprotection to the plant against phytotoxic chemicals andplant pathogens. The synergistic relationship between theplant and the inoculum established by this approach isarguably more resilient, sustainable and cost-effectivethan the standard bioaugmentation approach. Reliance of the inoculum on plant exudates for survival precludes itsunintended dispersal to nontarget soils, thereby providinga mechanism for addressing public safety concerns.Kuiper
et al
.[18]were the first to methodically select astrain indigenous to a host plant’s rhizosphere that
Opinion
TRENDS in Biotechnology 
Vol.23 No.2 February 2005 75
 
exhibited preferential growth on the plant root exudates,good survival in the rhizosphere and enhanced pollutant-degrading ability. To a similar end, Narasimhan
et al.
[19]engineered a bacterium to utilize the predominant rootexudates of 
Arabidopsis
for the remediation of polychlori-nated biphenyl (PCB) in soil. This nutritional bias for theinoculum provides a selective advantage for the strainwhile in the
Arabidopsis
rhizosphere. The inoculum, aPCB-degrading
Pseudomonas putida
PML2, was thencapable of degrading PCBs upon augmentation into the
 Arabidopsis
rhizosphere, while growing on phenylpropa-noids, the main root exudates, as a ‘preferential’ growthsubstrate[14]. This study highlights one of the mostpromising approaches for the future of bioaugmentation:the combination of a sustainable, semiselective nutrientsource in plant exudates with the rhizosphere competenceof a pollutant-degrading ‘superbug’[17]. Additional techniques can be employed
in situ
toachieve superior strains for bioaugmentation. de Weert
 et al.
[20]demonstrated a novel method of enhancingcompetitive root-tip colonization in a
Pseudomonas fluor- escens
strain through repeated cycling of the strain andmutant derivatives on the plant root
in situ
. The authorsobserved a 100- to 1000-fold increase in the strain’s root-tip-colonizing ability in tomato and grass plants. Theauthors concluded that enhanced competitive root-tipcolonization was a result of the accumulation of nonlethalmutations, which enabled the strain to rapidly adapt tothe plant rhizosphere. This approach to strain selection isparticularly attractive because it is rapid, inexpensive,does not require genetic engineering and is readilyapplicable to Heirloom strains.
Non-traditional strain selection
Uptothispoint,wehaveprovidedourvisionforthefutureof bioaugmentation: Heirloom species and
in rhizo
-directed strain selection. However, we recognize thatsignificant potential rests in a less traditional approach,termed ‘priming’. Priming is generally described aspredisposing an isolate or population of microorganismsto future conditions in which they are designed to performa function. Priming is routinely used in probiotic yogurtculture preparation, where
Lactococcus
or
Bifidobacter-ium
cultures are subjected to the mildly acidic environ-ment of the yogurt, ‘priming’ the inoculum for the harsh,acidic conditions within the stomach[21,22]. This pre-treatment, albeit unnecessary for acid-tolerant strains, isessential for the viable transit of nonacid-tolerant strainsthrough the stomach into the intestines[21].The priming approach has also been effectively demon-strated in environmental systems, whereby a portion of clean soil is enriched for pollutant-degrading microorgan-isms by repeated biostimulation with the relevant pollu-tant(s). The resulting soil itself, with its highly competentdegrader microbial community, is then used as theinoculum for the target polluted soil[23].This approach has severaladvantages:(i)theinoculumisa consortiumof indigenous microorganisms which is potentially moreresilient to stress than a single isolate; (ii) the ‘primed’consortium is maintained within its native soil, poten-tially enhancing its survival in the target soil; and(iii) inclusion of unculturable microorganisms, whichmight contain one or more highly competent pollutantdegraders. In addition, the approach is theoreticallyexpandable to solving the issue of co-contaminated soil(i.e. one with more than one pollutant), which mightnecessitate the combination of singly primed soils or onesoil primed to degrade multiple contaminants.Takeuchi et al.[24]were among the first to applypriming to contaminated groundwater. The authorsinitially demonstrated cometabolism of trichloroethylene(TCE) by methanotrophs from a borehole in a contami-nated aquifer. They subsequently amended a poorlyperforming borehole with groundwater from the TCE-degrading borehole, resulting in a rapid decline in theTCE groundwater concentration. Treatment success wasattributed to the activity of the bioaugmented indigenousmethanotrophs within the ‘primed’ TCE-degradinggroundwater. Albeit a return to the ‘black box’ method of remediation, instinctively avoided by scientists, thisapproach is attractive from a practical perspective; it istechnically accessible, easily implemented, inexpensiveand environmentally friendly. It remains to be seen howeffective different matrices respond to priming (e.g. clay-primed soil augmented into sandy target soil, or high pHprimed soil augmented into netural or low pH target soil).Moreover, the approach has not been suitably tested forpotential scale-up to address large contaminated sites;however, priming clearly warrants a more thoroughinvestigation to answer these and other practicalquestions.
Conclusion
In time, it is hoped that bioaugmentation can fulfill itspotential to remediate contaminated soils and water,
in situ
. Many of the limitations to its applicability in thefield are the result of an uncoordinated effort to addressthe social concerns of bioremediation along with thescientific interests. To this end, we propose that exploita-tion of Heirloom species should be coordinated betweenresearch laboratories, providing both scientists andregulators with a predictable, (globally) tested selectionof microorganisms, which would facilitate pollutantremediation as well as its public acceptance. In addition,we highlight two non-traditional approaches to strainselection, termed
in rhizo
-directed strain selection andpriming, which demonstrate great promise for remedia-tion of polluted matrices but remain largely on theperiphery of the discipline.
Acknowledgements
WewishtothanktheRefereesfortheirvaluablefeedbackandsuggestionsin preparing this manuscript, as well as Stephanie Hunter and PennyCarter for their assistance.
References
1 van Veen, J.A.
et al
. (1997) Fate and activity of microorganismsintroduced into soil.
Microbiol. Mol. Biol. Rev.
61, 121–1352 Gilbert, E.S. and Crowley, D.E. (1998) Repeated application of carvone-induced bacteria to enhance biodegradation of poly-chlorinated biphenyls in soil.
 Appl.Microbiol.Biotechnol.
50, 489–494
Opinion
TRENDS in Biotechnology 
Vol.23 No.2 February 200576

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