Annals of Applied Biology (2005), 146:217–221*Corresponding Author Email: jim.lynch@forestry.gsi.gov.uk© 2005 Association ...
J M LYNCH & A J MOFFAT218or temporarily until the degree of contaminationfalls below a danger threshold. The source-pathwa...
219Bioremediationpotential reasons why bioremediation fails is thatthe microbes bringing about the remediation do nothave ...
J M LYNCH & A J MOFFAT220process of the potassium cyanide added to thesystem. It was also demonstrated by D Redmondand J M...
221Bioremediationuses that can be used safely by the public, and couldit help to advance the time when public access isper...
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Bioremediation - prospects for the future application of innovative applied

  1. 1. Annals of Applied Biology (2005), 146:217–221*Corresponding Author Email: jim.lynch@forestry.gsi.gov.uk© 2005 Association of Applied Biologists217Bioremediation – prospects for the future application of innovative appliedbiological researchJAMES M LYNCH* and ANDREW J MOFFATForest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UKSummaryIt has been estimated that there may be as much as 300 000 ha in the UK where contamination fromprevious industrial land use has occurred. The ‘Source-Pathway-Receptor’ model is used to evaluatethese risks. Traditional engineering approaches have dominated remediation technology, but biologicalmethods have become increasingly important in recent years. ‘Bioremediation’ has been defined as‘the elimination, attenuation or transformation of polluting or contaminating substances by the use ofbiological processes’. Techniques to treat soil materials include biopiling, windrowing, landfarmingand bioventing, all of which depend on microbiological degradation. However, increasingly it alsoincludes the use of vegetation to take up and/or degrade contaminants (phytoremediation) or restrictcontaminant movement (phytostabilisation). Phytoremediation can be encouraged by manipulation ofthe rhizosphere, using selected fungal isolates in a process now defined as phytobial remediation.Key words: Bioremediation, brownfield, contamination, landfill, phytoremediation, rhizosphere,trees, TrichodermaIntroductionAs well as the use of the land for agricultureand forestry, the land surface has had to supporta growing world population for housing, industryand other needs of the urban infrastructure. Here,pressures on the soil have been intense, and mosthas been destroyed or severely degraded physicallyor nutritionally. However, contamination withinorganic and organic compounds as a result ofindustrial (and transport) pollution is probably themost serious issue to contend with today, because ofthe proximity of the urban population and the needto maintain its health and well being. In addition,economic pressures are increasingly driving theneed for ‘land recycling’, so that a new use can bemade of old land in advantageous positions withinthe town or city. As well as hard-end uses, soft-enduses involving vegetation are of particular interestin the urban context (Moffat, 2001), to providegreenspace for recreation, landscape enhancementand to encourage inward investment.Land remediation is an essential response tothe principle of sustainable development. Mostdefinitions embrace the need to be equitable notjust to people alive today, but to future generations.So it is important not just to cease or minimisethe degradation of land – in a world of ever-largerpopulation,landremediationwillbeincreasinglyseenas an appropriate measure. In developed countries,remediation activity is currently focussed in urbanareas for reasons given above. What follows is abrief assessment of the scope for land remediation,a brief definition of bio/phytoremediation rather thana review of the subject, and the outline of the novelconcept of phytobial remediation.Land Remediation in the UKThe UK has a large legacy of land detrimentallyaffected by industrial use. In addition, it is one ofthe most heavily populated countries in the world,and a large proportion (15%) of the land is definedas urban. In the UK, the term ‘brownfield land’is used as a synonym for ‘previously developedland’ (Department of Environment, Transportand the Regions, 2000a), and there is no mentionof potential contamination (in contrast to the USdefinition). Nevertheless, it is clear that many UKbrownfield sites have suffered contamination byprevious industrial activity and may be regarded as‘contaminated land’ (Department of Environment,Transport and the Regions, 2000b). Some estimatessuggest there may be as much as 300 000 ha in theUK where contamination from previous industrialland use has occurred (ENDS, 1999).In order to raise land quality to meet newexpectations, remediation is increasingly requiredon brownfield land. Remediation differs from‘restoration’ and ‘reclamation’ because it involvesaddressing the issue of potential or actual risk ofharm from ‘sources’ of chemical contaminants todefined‘receptors’. Thesemayincludehumanswholive on or visit the land, water draining into surfaceor groundwaters, or sensitive ecosystems wherespecies could be lost. Remediation can take severalforms: dealing with the source of contamination,preventing or breaking the ‘pathway’ betweensource and receptor, or removing the sensitivereceptor, either permanently by changing land-use,
  2. 2. J M LYNCH & A J MOFFAT218or temporarily until the degree of contaminationfalls below a danger threshold. The source-pathway-receptor model is now the cornerstone ofBritish contaminated land remediation policy andpractice.Until recently, the most popular method of landremediation was to remove contaminated materialsto licensed landfill premises, i.e. to remove the‘source’. However, this ‘dig and dump’ procedurehas been criticised as exporting the problemrather than dealing with it more fundamentally.Another common approach has been to ‘contain’the source of the contamination, by sealing it fromreceptors using engineered impermeable caps orwalls, or to combine it physically or chemicallyto reduce its reactivity or mobility, i.e. break thepathway. However, these technologies have alsobeen attacked as taking too short a view of thelikely long term fate of contaminants. Remediationtechnologies are increasingly focussing on ways inwhich contaminants can be destroyed, or removedsensitivelywithlowriskofsecondaryenvironmentalproblems, taking a holistic and life cycle analysisapproach. In the past, these have been largelybased on physical and chemical technologies.Bioremediation‘Bioremediation’ has been defined as ‘theelimination, attenuation or transformation ofpolluting or contaminating substances by the useof biological processes’ (BBSRC, 1999). Thesubject has been a fallow area for the applicationof the principles of microbial ecophysiology andmolecular biology. For example, a classic study oncatabolism of the herbicide Dalapon in soil showedthat microbial communities formed to bring aboutthe catabolism and that catabolic enzymes wereproduced across the members of that community(Senior et al., 1976). This principle probably appliesto the catabolism of many organic pollutants whichenter the soil environment. Similarly, some of thefirst studies on soil molecular biology showed howplasmid DNA segments specified hydrocarbondegradation as a bioremediation target (Chakrabartyet al., 1978). A multitude of such studies followedwith the characteristic feature of using pure ordefined mixture culture studies of microorganismsin vitro. Subsequently some investigators developedmicrocosms, which more closely mimicked thenatural environment, but the value of these inrelation to true in vivo applications of bioremediationhas been questioned. In more recent years, therehave been attempts to practice bioremediation insitu (Crawford & Crawford, 1996). Sometimesinnundative approaches have been used by theapplication of microbial cultures to soils and water,but the common approach has been to elevate theuseful native populations by management of theecosystem with such approaches as forced aerationor nutrient additions. Most commonly the targethas been pollutants such as ‘BTEX’ hydrocarbons,petroleum, polycyclic aromatic hydrocarbons,microaromatics, PCBs, chlorinated phenols andaliphatics. Much less attention has been paid tometals. Considerable emphasis has been paidto measurement of bioavailability, and newchemical and sensor technologies (Valdes, 2000).Biogeochemists have focussed particularly onmineral interactions and the remediation of metals,including radionuclides (Hattori, 2003).Today, bioremediation technologies such aswindrowing, biopiling, land farming and bioventinghave secured a significant proportion of the marketfor remediation. They are mainly concernedwith microbial processes to break down organiccontaminants, and the technologies involve theprovision of ideal environmental conditions forthe growth and activity of the microorganismsconcerned. However, the useful introduced or nativeorganisms are frequently overtaken or outgrown byother soil microbes, especially when glucose or othernutrients are added. This may necessitate their usein a bioreactor. In addition, introduced organismsmay be pathogenic to plants or produce toxicantsand this seriously limits their usefulness if the landis to be returned to a soft end-use involving theestablishment of vegetation.Asasubtypeofbioremediation,‘phytoremediation’uses living plants to remove, degrade, immobiliseor contain the contaminants in situ (Nathanail &Bardos, 2004). The greater emphasis has been onmetal as opposed to organic remediation. Reed-bedsystems to clean and polish mine and quarry effluentsare a good example of a phytoremediation systemthat has been in operation for several decades. Inmore recent times, short rotation coppice systemsof willow or poplar have been investigated andreviewed (Bardos et al., 2001; Britt & Garstang,2002) for their potential to take up and remove usefulamounts of some ‘heavy metals’, and other interesthas focussed on so-called ‘hyperaccumulators’suchas some ferns and brassicas. In all these systems,scientific investigation has proceeded largely in theignorance of the plant–microbial interface.Phytobial RemediationPhytobial remediation aims to combinephytoremediation and bioremediation usingmicrobes (Harman et al., 2004b). Plants are grownwhose roots are colonised by symbiotic microbesthat efficiently create stable microbial communitiesthat degrade toxicants and assist plants in taking uptoxic materials. It thus combines the best of bothtraditional bio- and phyto-remediation. One of the
  3. 3. 219Bioremediationpotential reasons why bioremediation fails is thatthe microbes bringing about the remediation do nothave an energy source to promote their growth otherthan the toxicant which in itself may not be an energysource but instead be a transformation substrate.The whole concept of the phytobial process is thatit utilises the rhizodeposition products of the plantroot as the energy source for the microorganisms tofunction. In quantitative terms this can account forup to 40% of the photosynthate produced or the drymatter production by the plant (Lynch & Whipps,1990). In order to make maximum use of that carbonand energy pool the microorganisms need to berhizosphere competent. Symbionts such as rhizobiaand mycorrhiza are the best illustrations, but otherfree-living saprophytic organisms can also formstable associations with roots which approach thesymbiotic relationship. One such organism whichhas been identified by several research groups assatisfying the conditions of rhizosphere competenceis Trichoderma harzianum. However, there areonly specific strains which exhibit this qualityand some of them have been developed using thetechnique of protoplast fusion. One such strain of T.harzianum is T22 which has already been exploitedas a biocontrol agent against root diseases (Harmanet al., 2004a). During the registration process forthat product developed by a spin-out companyfrom Cornell University known as Bioworks, thetoxicology and pathogenicity was evaluated andwas shown to have no deleterious effects on plants,animals (including humans) or to the environmentif used at recommended rates. It therefore has fullclearance of the regulatory authority in the USA,the Environmental Protection Agency (EPA). Itis rhizosphere competent, stable and robust. It iseffective on all plants tested, ranging from ferns toconifers, monocots to dicots. It is effective acrossa wide range of soil pH (acidic to alkaline) and soilconditions (sandy to organic to gravel to very finesoils). The use of its effectiveness in biocontrolimproves its competitive ability in the rhizosphereand its robustness. It has also been through anextensive process of production and deliverancesystems which give the product a long shelf life forwhich a very satisfactory quality control procedurehas been developed. An added advantage of thisstrain of the fungus is that it is one of the few strainsof Trichoderma which increase seedling vigourand translate to an improvement of plant growthby as much as 30% (Lynch, 2004) (Fig. 1). It alsoincreases deep rooting long after application.The phytobial remediation concept is of course notlimited to Trichoderma spp. As already mentionedwe can expect the symbiotic groups to be within theremit of phytobial remediation. However, there maybe other organisms which could enhance the activityof the small defined microbial communities whichcan develop in such rhizosphere ecosystems.As an initial target for the catabolic potential ofTrichoderma spp. in remediation, cyanides andmetallocyanides, which are produced by industrialsources, were studied. These toxicants can comefrom the electroplating industries, manufactured gasplants, recovery of precious metals, synthetic fibreproduction, processing of cyanogenic crops andpaint manufacturing industries. It was found thatcertain strains were both resistant to cyanide andcould in fact detoxify it. They could be resistant toas much as 2000 ppm cyanides and could catabolisethe cyanide utilising its formamide hydrolase or itsrhodanese. The characterisation of the enzymesinvolved has been published in two papers (Ezzi& Lynch, 2002, 2003). The microbiology of thedecomposition process has also been reported (Ezzi& Lynch, 2004). In some hitherto unpublishedobservations by M Ezzi and J M Lynch, soil wastreated with 50 or 100 ppm cyanide and it was foundthat wheat and pea seeds failed to germinate. Whenthe seeds were coated with T. harzianum the seedsgerminated and the plants grew the same extent asin untreated soil (Fig. 2). This clearly showed thecapability of the detoxification by the phytobialFig. 1. Stimulation of lettuce growth by Trichodermaharzianum (WT) compared to untreated control(cont).Fig. 2. Detoxification of potassium cyanide byTrichoderma harzianum. A, Uninoculated with50 ppm CN; B, Uninoculated with 0 ppm CN; C,Inoculated with 50 ppm CN; D, Inoculated with 100ppm CN.A B C D
  4. 4. J M LYNCH & A J MOFFAT220process of the potassium cyanide added to thesystem. It was also demonstrated by D Redmondand J M Lynch (unpublished), that Trichodermagrown in a solution of Prussian blue (a classicexample of a metallocyanide which could enter theecosystem) could be rapidly absorbed by the fungusand spread throughout the cytoplasm with eventualdetoxification using the cyanide metabolisingenzymes.In some recent unpublished experiments at CornellUniversity (G Harman), it has also been shown thatthe arsenic hyperaccumulating fern Pteris vittatainoculated with T22 have larger roots and moreroot hairs than non-treated plants. They also takeup more heavy metals and arsenic, and for arsenicthere is 140% increase in uptake. In other work ithas also been shown that the uptake of nitrates canbe promoted by the phytobial system which couldbe valuable in land where nitrate leaching is aproblem. In studies at the University of Naples by MLorito (unpublished), it has been demonstrated thatTrichoderma can remove phenolic pollutants fromolive oil waste water.These results suggest a potential opportunity touse the phytobials technology to help remediate bothinorganic and organically contaminated materials,though more work is obviously needed to extendthe range of contaminants tested, especially morecomplex organics.In the most recent work by P Adams, F DeLeij and J M Lynch (unpublished) that has beendeveloping between the University of Surrey andForest Research atAlice Holt Research Station usingwillow, the Trichoderma has given a substantial, upto 30%, increase in tree growth. We would expectthe increased tree growth to contribute to the landremediation qualities of the tree. In the work onbrownfield sites such as former landfills where treeshave been planted generating the concept of urbanand peri-urban forestry, this quality would seem tobe eminently exploitable.Whereas the initial description and illustrationof the phytobial remediation concept has useda rhizosphere-competent free-living fungus(Trichoderma harzianum), there are likely to beapplications with symbiotic fungi (mycorrhizae).In this respect it is interesting to note that theendophytic arbuscular mycorrhizae show potentialin bioremediation (Oliveira et al., 2001; Gonzalez-Chavez et al., 2002a,b). With trees, it might beexpected that ectotrophic mycorrhizae would alsobe useful.DiscussionOpportunities for further development and useof phytobial remediation technologies are likelyto increase in the future. There is already a movetowards forms of remediation that themselvespose a small risk of environmental damage,and this favours bioremediation over chemicaltechnologies. One traditional method of dealingwith contamination, the ‘dig and dump’ approach,is likely to be reduced significantly in the UK asa result of recent changes in landfill legislationfollowing the EU Landfill Directive (1999/31/EC)which has reduced the number of British licensedlandfills permitted to accept hazardous materialto a very small number. Nevertheless, in situbioremediation technologies remain unprovenin some quarters, partly because there remainsuncertainty over the degree of homogeneityachieved by bioremediation treatment and thusresidual risk. Bioremediation requires that the soilconditions (chemical, physical and biological) areabove a minimum threshold to support the chosenvegetation and desired microbial communities,and this is not always easily achievable across thewhole site. In addition, there is the perceptionthat working with plants, remediation can only beachieved to the depth of effective rooting, and thusto about 1 m in many circumstances. However,certain tree species may permit greater depths tobe remediated.Further research, both fundamental and applied,is therefore necessary. Microbiological ecologicalresearch is needed to understand more fully theability of microbes to degrade toxicants within therhizosphere, and when, how and over what timeperiodssuchprocessestakeplace. Thedevelopmentof further rhizosphere competent symbionts, andtheir testing on a range of host plants and types ofcontaminated substrate is necessary to extend theusefulness of this technology and help to define theappropriateness of its use. The ability of phytobialsystems to degrade complex organic compoundsneeds to be established, given that industriallycontaminated land usually contains a large range.Modelling phytobial systems may be an importantway of exploring their further potential, once morebasic ecological research has been undertaken.Applied research is required to establish howeffective phytobial systems are in remediation atfield scale, and what the operational constraints are.To what extent is phytobial remediation likely torely on its use in conjunction with more establishedclean up technologies? Comparison with traditionalremediation approaches should be undertakento establish relative clean up effectiveness andeconomic cost. Such work would benefit frominvolvement with waste regulatory authoritiesin order to maximise the confidence with whichthese novel technologies are viewed. Phytobialremediation should also be placed in the context ofthe urban greenspace agenda. To what extent canthis type of remediation be used to create soft-end
  5. 5. 221Bioremediationuses that can be used safely by the public, and couldit help to advance the time when public access ispermitted? In fact, can it be used for remediationat the same time as public access is allowed? Andwill the public be content, or even supportive ofbioremediation techniques over more traditionalones which have a larger ecological footprint?From a multitude of communication networksit is clear that the feasibility of bioremediation ofcontaminated land has been established. Reliability,however, is more questionable and in addition themagnitude of the remediation needs to be fullyevaluated. There is a massive impetus to developthese concepts further.This paper has focussed on our own researchactivity. The various approaches to bioremediationcan be accessed through a variety of texts (e.g.Crawford & Crawford, 1996; Valdes, 2000; Hattori,2003). In addition, following an excellent EUInterCOST meeting on bioremediation in Sorrento,Italy in November 2000, a selection of the presentedpapers was published in Minerva Biotechnology(Journal of Biotechnology and Molecular Biology)Vol. 13, pp. 1–152.AcknowledgementsWe are grateful for inputs to this work fromGary Hartman (Cornell University), Matteo Lorito(University of Naples), Muffadel Ezzi, PaulAdams,David Redmond and Frans De Leij (University ofSurrey) and Tony Hutchings (Forest Research).ReferencesBardos P, French C, Lewis A, Moffat A, Nortcliff S. 2001.Marginal Land Restoration Scoping Study: InformationReview and Feasibility Study. exSite Research Project Report1. Nottingham: LQM Press.BBSRC. 1999. A Joint Research Council Review ofBioremediation Research in the United Kingdom. Swindon:BBRRC, EPSRC and NERC.BrittC,GarstangJ.2002. BioenergyCropsandBioremediation– a Review. Final Contract Report to Defra. http://www.defra.gov.uk/farm/acu/research/reports/NF0417.pdfChakrabartyAM, Friello DA, Bopp LH. 1978. Transpositionof plasmid DNA segments specifying hydrocarbondegradation and their expression in various microorganisms.Proceedings of the National Academy of Sciences of the USA75:3109–3112.Crawford R L, Crawford D L. (Eds) 1996. BioremediationPrinciples and Applications. Cambridge: CambridgeUniversity Press.Department of Environment, Transport and the Regions.2000a. Housing. Planning Policy Guidance Note 3. London:The Stationery Office.Department of Environment, Transport and the Regions.2000b. Contaminated Land: Implementation of Part IIAof the Environmental Protection Act 1990. DETR Circular02/2000. London: The Stationery Office.ENDS. 1999. April targets for new contaminated land regime inEngland. ENDS Report 297:45–48. London: EnvironmentalData Services.Ezzi M I, Lynch J M. 2002. Cyanide catabolising enzymesin Trichoderma spp. Enzyme and Microbial Technology31:1042–1047.Ezzi M I, Pascual J A, Gould B J, Lynch J M. 2003.Characterisation of the rhodanese enzyme in Trichodermaspp. Enzyme and Microbial Technology 32:629–634.Ezzi M I, Lynch J M. 2004. Biodegradation of cyanide byTrichoderma spp. and Fusarium spp. Enzyme and MicrobialTechnology (In Press).Gonzalez-Chavez C, D’Haen J, Vangronsveld J, Dodd J C.2002a. Copper sorption and accumulation by the extraradicalmycelium of different Glomus spp. (arbuscular mycorrhizalfungi) isolated from the same polluted soil. Plant and Soil240:287–297.Gonzalez-Chavez C, Harris P J, Dodd J, Meharg A A.2002b. Arbuscular mycorrhizal fungi confer enhancedarsenate resistance on Holcus lanatus. New Phytologist155:163–171.Harman G E, Howell C R, Viterbo A, Chet I, Lorito M.2004a. Trichoderma species – opportunistic, avirulent plantsymbionts. Nature Reviews Microbiology 2:43–56.Harman G E, Lorito M, Lynch J M. 2004b. Uses ofTrichoderma spp. to alleviate soil or water pollution.Advances in Applied Microbiology 56:313–330.Hattori T. (Ed.) 2003. Biogeochemical Aspects of EarthSystem and Bioremediation of Polluted Environments. 16thInternational Symposium on Environmental Biogeochemistry,Sendai, Japan.Lynch J M. 2004. Plant growth-promoting agents. In MicrobialDiversity and Bioprospecting, pp. 391–396. Ed. A T Bull.Washington DC: ASM Press.Lynch J M, Whipps J M. 1990. Substrate flow in therhizosphere. Plant and Soil 129:1–10.Lynch J M, Wilson K L, Ousley M A, Whipps J M. 1991.Response of lettuce to Trichoderma treatment. Letters inApplied Microbiology 12:59–61.Moffat A J. 2001. Increasing woodland in urban areas inthe UK – meeting ecological and environmental standards.International Forestry Review 3:198–205.NathanailCP,BardosRP.2004. ReclamationofContaminatedLand. Chichester: J Wiley.Oliveira R S, Dodd J C, Castro PM L. 2001. The mycorrrhizalstatus of Phragmites australis in several polluted soils andsediments of an industrialised region of Northern Portugal.Mycorrhiza 10:241–247.Senior E, Bull A T, Slater J H. 1976. Enzyme evolution ina microbial community growing on the herbicide Dalapon.Nature, London 263:476–479.Valdes J J. (Ed.) 2000. Bioremediation. Dordrecht: Kluwer.(Revised version accepted 3 February 2005;Received 10 September 2004)

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