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Naturally occurring microbial consortia have been utilized in avariety of bioremediation processes. Recent developments inmolecular microbial ecology offer new tools that facilitatemolecular analyses of microbial populations at contaminatedand bioremediated sites. Information provided by suchanalyses aids in the evaluation of the effectiveness ofbioremediation and the formulation of strategies that mightaccelerate bioremediation.

AddressesMarine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan; e-mail: kazuya.watanabe@kamaishi.mbio.co.jp

Current Opinion in Biotechnology 2001, 12:237241

0958-1669/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsAOB ammonia-oxidizing bacteriaDGGE denaturing gradient gel electrophoresisFISH fluorescence in situ hybridizationPAH polycyclic aromatic hydrocarbonTCE trichloroethyleneTGGE temperature-gradient gel electrophoresis

IntroductionBioremediation is a technology that utilizes the metabolicpotential of microorganisms to clean up contaminatedenvironments. One important characteristic of bioremedi-ation is that it is carried out in non-sterile openenvironments that contain a variety of organisms. Of these,bacteria, such as those capable of degrading pollutants,usually have central roles in bioremediation, whereas otherorganisms (e.g. fungi and grazing protozoa) also affect theprocess. A deeper understanding of the microbial ecologyof contaminated sites is therefore necessary to furtherimprove bioremediation processes.

In the past two decades, molecular tools, exemplified byrRNA approaches, have been introduced into microbialecology; these tools have facilitated the analysis of natural microbial populations without cultivation.Microbiologists have now realized that natural microbialpopulations are much more diverse than those expectedfrom the catalog of isolated microorganisms. This is alsothe case for pollutant-degrading microorganisms, imply-ing that the natural environment harbors a wide range ofunidentified pollutant-degrading microorganisms thathave crucial roles in bioremediation. This article summa-rizes the results of recent studies of microbial populationsthat are relevant to bioremediation.

Molecular ecological information is thought to be usefulfor the development of strategies to improve bioremedia-tion and for evaluating its consequences (including risk

assessment). Molecular tools are especially useful inbioaugmentation, in which exogenous microorganisms thatare introduced to accelerate pollutant biodegradation needto be monitored. This article discusses recent examples ofthe successful application of molecular ecological tools tothe study of bioremediation.

Microorganisms relevant to methane oxidationTraditionally, studies on pollutant biodegradation havebeen initiated by the isolation of one or more microorgan-isms capable of degrading target pollutants; however,conventional isolation methods have resulted in the isola-tion of only a fraction of the diverse pollutant-degradingmicroorganisms in the environment. In addition, most iso-lated organisms have shown pollutant-degradation kineticsthat differ from those observed in the environment [1]. Forexample, laboratory-cultivated methanotrophs exhibithalf-saturation constants for methane oxidation which areone to three orders of magnitude higher than thoseobserved in soil. Using molecular phylogenetic analyses ofisotope-labeled DNA, Radajewski et al. [2] successfullyidentified two novel methanotrophs that actively degrademethane under environmental conditions. Molecularapproaches that target the 16S rRNA gene (16S rDNA)and genes encoding enzymes involved in key metabolicsteps (e.g. those encoding particulate methane mono-oxygenase) have been applied to the analysis of methanotrophs in rice field soil [3], lake sediments [4] andforest soil [5]. Methanotrophs are considered to be impor-tant for reducing the emission of methane, a greenhousegas, from soil and sediment. In addition, methanotrophsco-metabolize trichloroethylene (TCE); therefore, TCEbioremediation often employs methane injection as ameans to stimulate the TCE-degrading activity of indige-nous methanotrophs (i.e. methane biostimulation).Methanotrophs which occurred at a methane biostimula-tion site were recently analyzed using denaturing gradientgel electrophoresis (DGGE) of polymerase chain reaction(PCR)-amplified 16S rDNA and soluble methanemonooxygenase gene fragments [6].

Marine petroleum hydrocarbon degradationMolecular ecological approaches have also been used to analyze bacterial populations that occur in petroleum-conta-minated marine environments. Spilled-oil bioremediationexperiments conducted at a sandy beach found that phylo-types affiliated with the subclass of Proteobacteria(-Proteobacteria) appeared in the DGGE fingerprintsobtained for oiled plots but not in those for unoiled plots [7],suggesting their importance in spilled-oil bioremediation.Another oil-spill experiment conducted at a beach in theNorwegian Arctic showed that 16S rDNA types affiliatedwith the -Proteobacteria, especially those belonging to thePseudomonas and Cycloclasticus groups, were abundant in

Microorganisms relevant to bioremediationKazuya Watanabe

fertilized oil sands [8]. Microbial populations which occurredin seawater after supplementation with petroleum and inor-ganic fertilizers have been analyzed using rRNA approaches;it was reported that bacterial populations belonging to the-Proteobacteria [9] and the genus Alcanivorax [9,10] showedaccelerated growth. These studies have indicated that somegroups of bacteria commonly occur in oil-contaminatedmarine environments, although other populations changeunder different environmental conditions.

Anaerobic petroleum hydrocarbon degradationAs petroleum hydrocarbons are persistent under anaerobicconditions, their contamination of groundwater is a seriousenvironmental problem. The microbial diversity in a hydro-carbon- and chlorinated-solvent contaminated aquiferundergoing intrinsic bioremediation was assessed by cloningand sequencing bacterial and archaeal 16S rDNA fragments[11]. This study detected phylotypes that were closely relat-ed to Syntrophus spp. (anaerobic oxidizers of organic acidswith the production of acetate and hydrogen) andMethanosaeta spp. (aceticlastic methanogens), suggestingtheir syntrophic association. Phylotypes affiliated with candi-date divisions (that do not contain any isolated organisms)were also obtained in abundance from the contaminatedaquifer [11], although their physiology is completelyunknown. A similar syntrophic association of bacteria andarchaea has also been reported in a methanogenic enrich-ment that slowly degrades hexadecane [12]. Likewise, atoluene-degrading methanogenic consortium was character-ized by rRNA approaches [13]. The consortium comprisedtwo archaeal species related to the genera Methanosaeta andMethanospirillum, and two bacterial species, one related to thegenus Desulfotomaculum and the other unrelated to any pre-viously described genus. Fluorescence in situ hybridization(FISH) with group-specific rRNA probes was used to ana-lyze a denitrifying microbial community degradingalkylbenzenes and n-alkanes [14]; the Azoarcus/Thaueragroup was found to be the major bacterial group. Bacteriaaffiliated with the -Proteobacteria were found to grow inpetroleum-contaminated groundwater which accumulated atthe bottom of underground crude-oil storage cavities [15].Microbial communities associated with anaerobic benzenedegradation under Fe(III)-reducing conditions in a petroleum-contaminated subsurface aquifer were also analyzed byDGGE analysis [16], and it has been suggested that Fe(III)-reducing Geobacter spp. have an important role in theanaerobic oxidation of benzene. The available electronacceptors are the principal determinants for the types ofmicroorganisms that occur in anaerobic environments, andmicrobial populations identified in the above papers are con-sidered important for petroleum hydrocarbon degradation insubsurface environments under the respective conditions.On the basis of these results, future developments in anaer-obic hydrocarbon bioremediation are anticipated [17]. It isnoteworthy that phylotypes that are only distantly related toknown genera are often detected as major members of theanaerobic communities, suggesting that parts of anaerobichydrocarbon biodegradation processes remain unidentified.

Polycyclic aromatic hydrocarbon degradationPolycyclic aromatic hydrocarbons (PAHs) are compoundsof intense public concern owing to their persistence in theenvironment and potentially deleterious effects on humanhealth [18]. A soil-derived microbial consortium capable ofrapidly mineralizing benzo[a]pyrene was analyzed byDGGE profiling of PCR-amplified 16S rDNA fragments[19]. The analysis detected 16S rDNA sequence types thatrepresented organisms closely related to known high mol-ecular weight PAH-degrading bacteria (e.g. Burkholderias,Sphingomonas and Mycobacterium), although the degradationmechanisms have yet to be resolved. In soil environments,the reduced bioavailability of PAHs due to sorption to nat-ural organic matter is an important factor controlling theirbiodegradation [20]. Friedrich et al. reported that differentphenanthrene-degrading bacteria occurred in soil enrich-ments when different sorptive matrices were present [21].It has also been shown that the application of surfactants tosoil enrichments that degrade phenanthrene and hexa-decane altered the microbial populations responsible forthe degradation [22]. These results have common implica-tions for bioremediation; that is, nature harbors diversemicrobial populations capable of pollutant degradationfrom which a few pollutant-degrading populations areselected according to bioremediation strategies.

Metal bioremediationBecause of its toxicity, metal contamination of the environ-ment is also a serious problem. Recent studies have appliedmolecular tools to the analysis of bacterial [23,24] andarchaeal populations [25] that are capable of surviving inmetal-contaminated environments. Bacterial communitiesin soil amended for many years with sewage sludge thatcontained heavy metals were assessed using rRNAapproaches, including FISH and cloning and sequencing[23]. The study found that two sequence groups affiliatedwith the -Proteobacteria and Actinobacteria were frequentlyobtained from clone libraries from the metal-contaminatedsoil, although most Actinobacteria sequences showed lowsimilarity (

Comparisons made between the amounts of group-specificrRNAs and the process chemistry enabled the authors toidentify some phylogenetic groups of bacteria importantfor the process performance. The phylogenetic diversity ofbacterial communities supported by a seven-stage, full-scale bioreactor used to treat pharmaceutical wastewaterwas studied using PCR-based techniques (i.e. DGGE fin-gerprinting and cloning of 16S rDNA fragments [28]).These two techniques detected similar phylotypes,although they failed to concede on their relative distribu-tion, suggesting difficulties in quantitative interpretationbased on these methods. A combination of 16S rDNAcloning, hybridization with oligonucleotide probes forammonia-oxidizing bacteria (AOB) and sequencing of thehybridization-positive clones suggested that novelNitrosospira-like populations were the major AOB in a rhi-zosphere zone used to treat wastewater (rhizoremediation)[29]. To identify microbial populations responsible forphosphorus removal in activated-sludge, the structure ofthe bacterial population was analyzed by FISH during theoperation of a laboratory-scale reactor with various phos-phorus removal rates [30,31]. FISH has also been used toanalyze microbial populations in mesophilic and thermo-philic sludge granules [32], foaming activated-sludge [33]and bulking activated-sludge [34].

Temperature-gradient gel electrophoresis (TGGE) ofPCR-amplified 16S rDNA fragments was used to identifythe major phylotypes in phenol-digesting activated-sludge.Physiological characterization of isolated bacteria corre-sponding to these phylotypes identified microbialtransition that caused a failure in the phenol treatment[35]. The ecological information obtained in this studywas successfully used to develop a countermeasure againstthe failure in the phenol treatment [36]. These papers present successful examples which showed the utility of molecular ecological approaches for manipulating microbial consortia for bioremediation.

BioaugmentationThe introduction of exogenous microorganisms into envi-ronments (bioaugmentation) has been used in an attemptto accelerate bioremediation. It is desirable that the fate ofan introduced organism be monitored in order to prove itscontribution to pollutant degradation and to assess itsinfluence on the ecosystem. Molecular tools have beenused for this purpose. DGGE/TGGE fingerprinting of 16SrDNA fragments has been used to examine the effects ofbioaugmentation on indigenous bacterial communitystructures in a range of situations: a laboratory-scale semicontinuous activated-sludge system loaded with3-chloroaniline [37]; experimental model sewage plantssubjected to shock loads of chlorinated and methylatedphenols [38]; and in 2,4-dichlorophenoxyacetic-acid-contaminated soil horizons [39]. Quantitative PCR assaystargeting catabolic genes [40,41] and gyrB (the gene codingfor the subunit B protein of DNA gyrase) [42] have successfully been used to monitor the fates of introduced

bacteria in complex microbial communities (e.g. those inactivated-sludge and in soil). In some cases, where geneti-cally modified organisms were utilized, bioaugmentationimproved pollutant-biodegradation rates in the environ-ment due to the establishment of transconjugants capableof degrading the pollutants rather than the direct contribu-tion of the inoculated organisms [39,43].

ConclusionsBioremediation is still considered to be a developing tech-nology. One difficulty is that bioremediation is carried out inthe natural environment, which contains diverse uncharac-terized organisms. Most pollutant-degrading microorganismsisolated and characterized in the laboratory are now thoughtto make a minor contribution to bioremediation. Another difficulty is that no two environmental problems occur undercompletely identical conditions; for example, variationsoccur in the types and amounts of pollutants, climate condi-tions and hydrogeodynamics. These difficulties have causedthe bioremediation field to lag behind knowledge-basedtechnologies that are governed by common rationales.

As summarized in this review, information on microbialpopulations relevant to bioremediation is accumulatingrapidly with the aid of molecular ecological approaches.Although our knowledge is not yet complete, it is time toinitiate more comprehensive approaches to find commonrationales in bioremediation. In some cases, for example,marine petroleum bioremediation, we have already foundthat similar bacterial populations occur even at geographi-cally distant sites. Understanding the physiology andgenetics of such populations may prove very useful toassess and improve bioremediation. Most importantly, weneed to identify general aspects in certain types of bioremediation. For this purpose, I wish to propose theconstruction of a database that collects the results of molecular ecological assessments of contaminated andbioremediated sites. The database would provide biore-mediation with ecological backgrounds and, in concertwith currently available databases relevant to bioremedia-tion, would facilitate the development of commonlyapplicable schemes for certain types of bioremediation.

AcknowledgementsThe author wishes to acknowledge the support provided by the NewEnergy and Industrial Technology Development Organization (NEDO).Thanks are also given to Shigeaki Harayama and Robert Kanaly for criticalreading of this manuscript.

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