Wastewater Ecology - Environmentally Relevant Microorganisms

8/12/2019 Wastewater Ecology - Environmentally Relevant Microorganisms

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JOURNAL OF BIOSCIENCEAND BIOENGINEERINGVol. 8 9, No. 1 , l-11. 2000

REVIEWEnvironmentally Relevant Microorganisms

KAZUYA WATANABE* AND PAUL W. BAKERMarine Biotechnology Institute, Kamaishi Laboratories, 3-75-l Heita, Kamaishi, Iwate 026-0001, Japan

Received 19 November 1999/Accepted 3 December 1999The development of molecular microbial ecology in the 1990s has allowed sc ientists to realize that microbialpopulations in the natural environment are much more diverse than microorganisms so far isolated in thelaboratory. This finding has exerted a significant impact on environmental biotechnology, since knowledge inthis field has been large ly dependent on studies with pollutant-degrad ing bacteria isolated by conventionalculture methods. Researchers have thus started to use molecu lar ecological methods to analyze microbialpopulations relevant to pollutant degradation in the environment (called environmentally relevant microo r-ganisms, ERMs), although further effort is needed to gain prac tical benefits from these studies. This reviewhighlights the utili ty and limitations of molecular ecological methods for understanding and advancing en-vironmental biotechnology processes. The importance of the combined use of molecular ecological and phys-

iological methods for identifying ERMs is stressed.[Key words: environmental biotechnology , molecular microbial ecology , bioremediation, activate d sludge]

INTRODUCTION: CONSTRAINTS INENVIRONMENTAL BIOTECHNOLOGY

Environmental biotechnology is a technology that ap-plies biological systems to clean up environmental pol-lutants. This has been thought to be advantageous overphysical and chemical treatments due to its relatively lowcost and little d isturbance to the environment (1, 2). Inaddition, organic pollutants are biodegraded to inorgan-ic compounds (e.g., COZ, HzO, and Cl-), whereas phys-ical and chemical processes, e.g., vaporization, adsorp-tion and extraction, simp ly transfer the pollutants todifferent locations (1). The activated-sludge wastewater-treatment process has been a common practice for over80 years (3), while bioremediation of polluted soil, ground-water and marine environments has only recently beeninitiated (1, 4, 5).Environmental biotechnology relies on the pollutant-degrading capacities of naturally occurring m icrobial con-sortia (6), in which bacteria generally play central roles.Researchers are thus studying pollutant-degrading bac-teria which inhabit polluted environments. These studiesinclude the isolation of bacteria from the environment,their classification and physiological characterization,molecular analyses of their degradative enzymes andsometimes the construc tion of superbugs. Consequent-ly, extensive knowledge of bacterial physiology and themolecu lar features of degradative enzymes has been ob-tained (7-10). However, discrepancies between the phys-iology of isolated pollutant-degrading bacteria and thenature of in situ pollutant biodegradation have been re-ported (11). For example, the kinetics of the phenol-degrading ac tivity exhibited by total phenol-digesting ac-tivated sludge are significantly different from the kineticsof phenol-degrading bacteria isolated from activatedsludge and intensively studied in the laboratory (Fig. 1).This finding suggests that we cannot direc tly extrapolatethe data obtained using these laboratory -isolated phenol-

* Corresponding author.

degrading bacteria to assess the phenol-removal perfor-mance of activated sludge processes.A practical aspect of environmental biotechnology isthe use of microbial consortia as black boxes withoutanalyzing the constituent microbial populations. An ex-ample is bioremediation of oil-contaminated beaches (4,5, 12) in which inorganic nutrients, e.g., nitrogen andphosphorus, are supplemented to stimulate indigenoushydrocarbon-degrad ing bacteria. Although the biodegra-dation potentials expressed by total marine consortiahave been well characterized, the microbial populationsinvolved in the in situ hydrocarbon degradation have notbeen identified during the actual bioremediation trial s(4).There seems to exist two separate modes of study inenvironmental biotechnology: one biased toward labora-tory sc ience that focuses on isolated bacteria , and theother toward pure practice. Fusion of these modes ofstudy would be a promising way of advancing environ-mental biotechnology, although there are hurdles thathamper this. The first hurdle is the insufficient knowl -edge obtained from laboratory studies to interpret what ishappening in the real environment. The complexity ofmicrob ial consortia would be another hurdle that makesit difficult to analyze the structures of relevant microbialconsortia. Microbial consortia involved in environmentalbiotechnology, e.g., activated sludge and soi l consortia ,are genera lly very complex, enabling them to act on avariety of pollutants.A search for possible solutions to these constraints inenvironmental biotechnology began in the early 199Os,when good molecular biological techniques were devel-oped to study microbial ecology (called molecular micro-bial ecology). With this new discipline, we can under-stand natural microbial consortia in a more realisticmanner. This review describes how researchers have ap-plied this new discipline to environmental biotechnologyand also discusses how practical benefits can be drawnfrom such studies. A possible scheme to achieve thisgoal is then proposed, which inc lude the structure analy -


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2 WATANABEANDBAKER

FIG. 1 .

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nmm. O8 0AX.An A.

-4b A:mAI , l *.,1 100 10000

Phenol concentration (FM)Comparison of specific phenol-oxygenating activi t iesexpresse d by representative phenol-degrading bacteria, Pseudo monassp. CF600 (A) and P. putida BH (0), and those by total phenol-digesting activate d sludge originating from a municipal wast ewat ertreatm ent plant (+) and an oil refinery (w ). Thes e activ ity cu rves

fol low Haldanes equation, v=( V,,. [s]) /( [s] +K,+ [s]~/&,), where[s] is the substrate concentration, K, is the half-saturation constant,KS1 is the inhibit ion constant, and V ,, is the theoretical maximumactivi tv. The K, values were estimated for s train CF600 (2.99 tiM),strain BH (3.2 0pM), the municipal sludge (0.70 ,cM ) and oil-refinerysludge (0.26 ,cM).sis of microbial consortia by molecular methods, func-tional analysis of in situ populations, isolation anddetailed physiological analyses of functionally importantpopulations (i.e., environmentally relevant organisms,ERMs), and the control of ERMs in the microbial con-sortia.

APPLICATION OF MOLECULAR MICROBIALECOLOGY TO ENVIRONMENTALBIOTECHNOLOGY

Molecular microbial ecology The advent of newmethods redefines our scope. This has been the case formicrobial ecology in the last two decades, when micro-biologists started to use molecular ecological methods,particularly that known as the 16s rRNA framework(13-15), for analyzing natural bacterial populations.Previously , microscopic observation and cultivation werethe methods w idely used for identifying bacteria in thenatural environment, even though these methods arethought to be insufficient for these purposes. Prob lemsassociated with m icroscopic observation are that ( i) themorphology of bacterial cells is generally too simple toserve as a basis for sound identification and to a llowreliable classification and (ii) microorganisms may adoptdifferent morphologies under different phys iolog icalconditions (16). Cultivation methods, e.g., viable platecount and most-probable-number (MPN) techniques,have been used for quantification of active ce lls in en-vironmenta l samples. However, because the mediumused in these methods always selects for certain organ-isms, the results are always biased toward these organ-isms (called cultivation bias). In addition, some bacterialcel ls may be viable but not be able to replicate understress conditions (16, 17). These problems have been real-ized by the observations that d irect microscopic countsof bacteria in aquatic and soil habitats exceed viableplate counts by several orders of magnitude (18-20). In

J. BIOSCI. BIOENG.,most cases, conventional cultivation methods can detect(and hence recover) only a small fraction of the micro-bial consortia.In contrast to traditional microbial ecology, molecularmicrobial ecology describes the consortium structurebased on DNA sequences recovered from the consortiumwithout cultivation. The DNA is extracted directly froma microbial consortium, so that the cultivation bias iseliminated. Certain gene fragments of different organ-isms are then cloned or amplified by PCR from the ex-tracted DNA in order to determine their sequences. Genecoding for the small subunit of ribosomal RNA (16srDNA for bacteria and archaea, and 18s rDNA foreukaryotes) is most commonly used for this purpose (13-15). The advantages of the use of this gene are: (i) allorganisms harbor this gene, and their evolutionary rela-tionships can be deduced (21), ( ii) a large number of se-quences of different organisms are stored in databases(22), (iii) un iversal PCR primers can be designed usingsequences in several highly conserved regions, and (iv)bacterial cells can be detected by in situ hybridizationtargeting abundant ribosomes in cell s. Using the 16s rDNAsequences, bacteria are classified into the phylogeneticgroups proposed by Weose (21), and the identifica tion ofnatural populations follows this phylogenetic classifica-tion.A typical scheme for analyzing bacterial populationsin the environment is illustra ted in Fig. 2 (refer to thelegend of this figure for the scheme). Among the me-thods, denaturing/temperature gradient gel electropho-resis (D/TGGE) is widely used in recent years for pro-filing microbial consortia (23, 24). The separation ofDNA fragments by D/TGGE is based on the decreasedelectrophoretic mobility of partially melted double-stranded DNA molecules in polyacrylamide gels contain-ing a linear gradient of DNA denaturants (DGGE) or atemperature gradient (TGGE). Molecules with differentsequences may have different melting behav iors and thusmigrate to different positions in a gel. This method isparticularly useful when temporal and spatial dynamicsof the population structure are analyzed, since it avoidsthe laborious aspects of cloning and sequencing.

In si tu hybridization with a fluorescence-labeled spe-cific probe is applicable for the quantitative analysis of amicrobial population (15). This method directly countslabeled bacterial ce lls under microscopic observation andcompares the count with the total cel l count determinedwith a DNA-binding fluorescent dye, such as 4,6-diami-dino-2-phenylindole (DAPI) or acridine orange (AO).Another useful method for consortium ana lysis (notincluded in Fig. 2) is terminal restriction fragment lengthpolymorphism (T-RFLP) analysis of PCR-amplified 16srDNA fragments (25, 26). This method identifies 16srDNA fragments based on the restrict ion endonucleasedigestion patterns rather than the sequences. It appearsto be advantageous over D/TGG E due to its resolutionand simplicity of identification.Since these molecu lar methods are capable of detect-ing microbial populations that are hardly detected byconventional culture-dependent methods (27-29), research-ers have started to apply them for analyzing microbialconsortia in environmental biotechnological processes.These applications are summarized below, and their util-ity and limitations are discussed.Activated sludge The activated sludge process iscurrently the mos t popular biological syste m for the


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VOL. 89, 2000 ENVIRONMENTALLY RELEVANT MICROORGAN ISMS 3

Microb ial consortium in the environment

(3)

band B

Consortium DNA (RNA)

PCR amplif ication with aset of universal primers

Denaturing/temperature gradient gelelectrophoresis (D/TGGE)

Determination of the

Excision of bands anddetermination of thesequences Database search

Design of aspecific probefor in-situdetection

Cc) 0.05 Woffnelfa succinogenesHelkwbacfer pylonSuffurospkillum defeyi8num

Arwbacter rWofi@lisThfomfcrospka denitrfffcans

DesutfovfMo des&icansRhodobacter capsulatusZoogfoea ramigeraRobrfvfvax geiatfnosus

FIG. 2. A typical scheme for analyzing a microbial consortium using molecular ecological methods. DNA is fi rst extracted from a microbialconsortium in the environment and used as a template for P CR to ampli fy 16s rDNA fragments with a set of universal pr imers. Thereafter, thePCR products (of the same length but with different sequences) are either separated by D/TGG E (see text for the separation mechanism) or eachproduct is cloned into E. col i . 16s rDNA fragments are then sequenced, and the determined sequences are compared with the sequences storedin nucleotide database s to phylogenetically identify the detected populations. Moreov er, the sequence information can be used to design anoligonucleotide probe for the detection and quantification of a spec ific bacterial population by fluorescenc e in situ hybridization (FIS H). (a) Cellsstained with DAPI showing the total population in a groundwater sam ple. (b) DGG E profiles showing the diversity and relative abundance ofPCR-ampli f ied 16s rDNA fragments; lanes 1 to 6 represent consortia in petroleum-contaminated groundwater, whi le lanes 7 to 10 represent thosein control groundwa ter. (c) A phylogenetic tree showing the position of the band-l sequenc e; band 1 was afhliated with the epsilon sub class ofthe class Proteobucteriu and most closely related to the genus Thiomicros pira. (d) Cells labeled with a DN A probe specific for the band-l popula-tion, an example of FISH .treatment of domestic and indus trial wastewater. Im-portant features of this process are: (i) heterotrophic bac-teria oxidatively digest organic pollutants in an aerationtank, and (ii ) microorganisms form floes , so that mostof the microorganisms are separated from the treatedwater in a settling tank (3, 30, 31). Bacterial populationsin the activated sludge of municipal sewage plants havebeen analyzed by in situ hybridization with fluorescence-labeled group-specific probes (26, 32). In these studies,bacteria affiliated with the beta subc lass of the class Pro-teobacteria were most abundantly detected, although cul -tivation on LB medium has indicated that the dominantgroup is the gamma subclass. Researchers have also ana-

lyzed bacterial populations in municipal sludge by clon-ing and sequencing of PCR-amplified 16s rDNA frag-ments (33-35). Again, the beta subclass was most abun-dantly detected, although the epsilon subc lass (i.e., anArcobacter population) was also detected at a high fre-quency (33). It is surpr ising that potentially pathogenicrepresentatives of the genus Arcobacter were observed insignificant numbers in activated sludge, although it hasnot yet been elucidated whether or not these Arcobacterpopulations are pathogenic. An interesting result fromthe study of Snaidr et al. (33) is that the total popula-tion structure evaluated from the frequencies of the 16srDNA clones of different phylogenetic groups was not so


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4 WATANABE AND BAKER .I. BIOSCI. BIOE~. .

different from the structure determined by in situ hybridi-zation with group-specific probes.Problem s frequently associated with the activated-sludge operation are bulking and foaming, which resultin poor sludge settling (36). Microscopic observationshave indicated that overgrowth of filamentous bacteriais involved in these problems (37, 38). Although manyfilamentous organisms have been isolated from activatedsludge (39-42), identification of organisms relevant tothese problems by conventional microbiological methodsis difficult due to their similar morphology. In situhybridization with group-specific probes (43) and probesdesigned from isolated bacteria (44, 45) and cloned 16srDNA fragments (45, 46) has thus been applied to detectand identify these organisms. de 10s Reyes et al. havedemonstrated that a vast majority of foaming-relatedMycobacterium in activated sludge comprised Gordonapopulations, although Gordona amarae, the most inten-sively studied Gordona bacterium in the laboratory,made up only a small percentage of the Gordona popula-tions (43).Removal of inorgan ic compounds Removal of nitro-gen (47) and phosphorus (48, 49) is also an importantrole of the activated sludge process. Scientists have at-tempted to identify bacteria l populations responsible fornitrogen (50-52) and phosphorus (53-55) removal by mol-ecular methods. Juretschko et al. have suggested Nitro-sococcus and Nitrospira-like populations to be the do-minant ammonia- and nitrite-oxidizers, respectively (52),although Nitrosomonas and Nitrobacter are the most fre-quently isolated counterparts. Bond and coworkers haveanalyzed the phylogenetic structures of efficient andnonefficient phosphate-removing activated-sludge consor-tia (54, 55), and found that populations affiliated withthe Rhodocyclus group in the beta subclass of Proteo-bacteria were abundantly detected only in the efficientsludge, suggesting that these populations are involved inphosphate removal.Anaerobic granules Anaerobic reactors are used totreat various organic wastes that are ultimately convertedto methane. The upflow anaerobic sludge blanket(UASB) is a popular anaerobic reactor that utilizesmicroorganisms to form a granular structure up to sev-eral millimeters in diameter (56). This type of reactor isthus able to maintain a high concentration of biomassand to treat an extremely high volumetric loading ratesof organic pollutants. Microscopic and immunologicalstudies have revealed the ultrastructure of layered organ-isms in a granule (57, 58), in which aceticlastic methano-genie archaea are surrounded by fermentative bacteria.This structure is considered important for the transfor-mation of organic pollutants first to acetate and then tomethane. Th is ultrastructure has been confirmed by insitu hybridization with group-specific probes (59-61).Cloning and sequencing of granule 16s rDNA fragmentshave revealed the existence of nove l bacterial popula-tions (62), and spec ific cloned sequences have been usedsuccessfu lly for in situ hybridization of sectionalizedgranules to investigate their spatial distribution (63).Groundwater bioremediation Contamination ofgroundwater and underground soi l with halogenated or -ganic compounds (64, 65) and petroleum hydrocarbons(66, 67) is a frequent occurrence. Organic compoundspersist in these environments due to the low oxygen con-centration, so that great effort is required for the remedi-ation of contaminated sites. Extensive studies have been

performed on the bioremediation of trichloroethylene(TCE) and related chlorinated compounds, and so farthese compounds are known to be cometabolica lly trans-formed by bacteria that contain nonspec ific oxygenases,e.g., methane-oxidizing bacteria (68-70), aromatic hydro-carbon-degrading bacteria (71-73) and ammonia-oxid iz-ing bacteria (74). Based on this knowledge, workers haveattempted in situ bioremediation by injecting methane(75), phenol (76) and toluene (77), although informationon bacteria l populations relevant to in situ transforma-tion of TCE has been quite lim ited (75). Bowman et al.examined the distribution of methanotrophs in TCE-con-taminated groundwater by culture-dependent techniquesand reported that most of the isolated methanotrophswere affiliated with type II methanotrophs (75). Since thetype II bacteria are known to possess soluble methanemonooxygenases (sMM0) capable of efficien tly trans-forming TCE, they have suggested that TCE can be effec-tive ly removed by stimulating these methanotrophs withmethane (75).We have analyzed bacterial populations in TCE-con-taminated soi l cores before and during the methane injec -tion for in situ biostimulation by using DGGE analysisof consortium 16s rDNA fragments (unpublished data).It was found that the population structure sign ificantlychanged due to the methane injection and bacteria l popu-lations closely related to some type I methanotrophsabundantly occurred after the methane injection. In thatstudy, we also analyzed methanotrophs by a MPN-cul-ture method and found that a population affiliated witha type II methanotroph was detected most abundantlyregardless of the methane injection. The discrepancy isconsidered to be ascribable to a bias associated with theMPN method in that only type II methanotrophs wouldhave grown well under the culture conditions employed.The resu lt thus suggests the inadequacy of conventionalculture-dependent methods for counting and identifyingmethanotrophic populations in the environment.Two groups of scientists have analyzed bacterial popu-lations in underground aquifers contaminated with JP-4jet fuel by 16s rRNA methods (78, 79). The aims ofsuch population analyses are to obtain a measure of thebioremediation effectiveness and data to determine theendpoint of the treatment (80). The population analysesalso provide information concerning the biodegradationpathways of pollutants. Dojka et al. cloned 16s rDNAsequences closely related to Syntrophus spp. and Methano-saeta spp. from a fuel-contaminated underground aqui-fer, and have hypothesized that aceticlast ic methanogene-sis is the terminal step of hydrocarbon biodegradation(78).Marine bioremediation Petroleum spills have had aserious impact on marine environments (4, 5). A famouscase is the Exxon Valdez oil sp ill in Alaska; in March1989, approximately 41 million liters of Alaskan NorthSlope crude oil was spilled from a tanker, the T/VExxon Va/a[dez, resulting in oil contamination of over2000 km of rocky intertidal shorelines. Another case isthe Nakhodka oil spill in Japan. In January 1997, morethan 5000 tons of heavy fuel oi l was spilled from theRussian tanker Nakhodka, which ran aground and sankin the sea of Japan close to Oki island; the oil contami-nated more than 500 km of the Japan sea coastline. Inthe case in Alaska, bioremediation was used to acceler-ate the natural degradation of residual oil. The effective-ness of bioremediation has been extensively evaluated,


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VOL. 89, 2000 ENVIRONMENTALLY RELEVANT MICROORGA NISMS 5

which includes chemical and microbiological studies (4,5, 12, 81), although researchers have not employedmolecular ecological methods.Recently, molecu lar methods in combination with fatty-acid analysis have been applied for evaluating bioremedia-tion of an experimental oil spi ll at a sandy beach (82). Inthat study, DGGE analysis indicated that the spilled oilpromoted the growth of bacteria with in the alpha-proteo-bacteria and Fkxibacter-Cytophaga-Bacteroides phyla. Acomparison of bacterial populations by DGGE betweenoil-contaminated and uncontaminated plots revealed sig-nificant differences even when the biodegradation hadceased. The data clea rly suggest the persistent influenceof spilled o il on marine biota.Utility and limitations of molecular ecological me-thods As described above, molecu lar methods haveenabled detection of numerous uncultured bacteria in en-vironmental biotechnology processes, some of which con-stitute major populations. It is natural to conside r thatmajor populations play some important roles in theseprocesses. The molecular methods can also provide in-sight into the functions of microbial populations,although only indirectly. If population dynamics cor-respond to fluctuations of a performance expressed bythe total consortium, the detected population may berelevant to that performance. An example is phosphate-removing activated sludge (54, 55). In addition, spatialpopulation dynamics may also provide useful informa-tion concerning the functions of the detected popula-tions; this principle has been applied to analyzing anaero-bic granules (59-61, 63).Another purpose of molecu lar methods is to providemeasures to assess the influences of pollu tion and en-forced remediation practices on the natural biota (80).Since a food web starts from bacteria l mineralization oforganic matter, analyses of the transition of bacteria lpopulations are quite important for such assessment.The above examples also suggest limitations of the 16srRNA approaches. First, we should consider biasesassociated with PCR amplification and DNA extraction(83-87), which can affect PCR-mediated quantificationof a microbial population. We should also bear in mindthat some populations are sti ll undetected. The abun-dance of a target population (a target sequence, ifaccurately described) can be more reliably estimated byusing specifically designed quantitative PCR (88-92)and/or in situ hybridization (15), although each methodwould include its own biases. Table 1 presents compari-son of ratios of a bacteria l population to the total popu-lation determined by three different methods (unpub-lished data). The ratios obtained by PCR-mediatedmethods, i.e. cloning and DGG E, are larger than theratios obtained by fluorescence in situ hybridization(FISH). It has been realized that PCR-mediated methodstend to overestimate the abundance of a detected popula-tion, since these methods fail to detect some populationswhose DNA fragments are difficu lt to amplify. In con-trast, FISH analysis sometimes underestimates the abun-dance of a microbial population, particularly of a slowlygrowing population, since metabolically inactive cells(not growing rapid ly) contain a small amount of ribo-somes and thus are hardly stained with a flouresentprobe targeting 16s rRNA. Cross checking of the resultsof several methods is thus recommended for quantitativepopulation analyses (83, 93). In addition, understandinginherent biases of each method is also important for the

TABLE 1. Com parison of the ratios of the band-l population(presented in Fig. 2) to the total population determinedby three different methodsGroundwater Band-l population/Total populations ( )Cloning DGGE FISHSample 1 54 18 11Sample 2 N D 35 24In the cloning meth od, the value represents the ratio of the numberof E. coli clones with the band-l sequence to the number of totalclones sequenced. In D GGE, the intensity of band 1 was comparedwith the total intensity as determined by densitometry. In FISH, cel lslabeled with the band-l DN A probe were counted under the micro-scope, and the number was compared with the number of cel ls stainedwith DAPI. ND . Not determined.

quantitative evaluation of the resul ts of molecu lar ap-proaches.The second limitation is related to the level of d ivers-ity of the 16s rRNA sequences. Relatively conserved 16srRNA sequences are very suitable for presenting thephylogenetic relationships of a wide range of bacteria,although the sequences often fail to discrim inate be-tween closely related bacteria l populations (92-94). Forexample, two phenol-degrading populations in activatedsludge harboring an identical 16s rDNA sequence (buttheir gyrB sequences were different) showed different be-haviors regarding floe formation, i.e., one is a floc-form-ing bacteria, and the other is nonfloccula ting bacteria(93). Quantitative PCR with specific gyrB-targetingprimers showed different population dynamics of thesetwo bacteria (93); gyrB was selected due to its highermolecular evolution rate (95).Third, the 16s rRNA approaches reveal only thephylogenetic trait of existing populations; the detectedpopulations are thus called phylogenetic strains. Wehence assume the function of a phylogenetic strain fromthe physiological traits of closely related isolates,although the phylogenetic feature is not necessarily cor-related with important physiological traits (93).In summary, we are now able to analyze the structuresof microbial consortia in environmental biotechnologyprocesses by molecular ecological methods. This knowl-edge should be related to the functional understandingof microbial populations in order to gain practical in-sight, and the molecular methods themselves are beingimproved in order to move toward this goal.

ENVIRONMENTALLY RELEVANTMICROORGANISMS

Functional analyses of microbial populations in the en-vironment One important current issue in microbialecology is to analyze the functions (physiological fea-tures) of microbial populations that are detected bymolecular ecological methods. Several culture-indepen-dent methods for analyzing the in situ functions andphysiology of microbial populations have been devel-oped.Metabolically active members of microbial consortiacan be identified by quantifying rRNA molecules ofdifferent species, since the ribosome content of bacterialcells is linearly related to their growth rate (96-98).Nogales et al. have identified presumptive metabolicallyactive populations in moorland soil highly pollutedwith polychlorinated biphenyls by sequencing of cloned


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6 WATANABE AND BAKER .I. Brom. BIOENG.,reverse transcription-PCR (RT-PCR) products of 16srRNA generated from total RNA extracts (99). Consorti-um 16s rDNA fragments have been amplified by PCRfrom extracted DNA and by RT-PCR from extractedRNA, and their D/TGGE profiles have been comparedin order to detect metabolically active populations (loo-102). In this way, Felske et al. demonstrated that uncul-tured Actinobacteria were active in soil (100). Thefluorescence intensity of individual cells labeled by FISHis correlated with the ribosome content of a bacterial cell(15, 98), which is thus another possib le indicator of thein situ activity (the growth rate).Microautoradiography of individual cells after incuba-tion w ith a radioac tive substrate has been used in combi-nation with fluorescence in situ hybridization to identifymetabolically active cells (103). Urbach et al. demonstrat-ed another usage of substrate uptake to identify metabol-ically active cells without cultivation (104); DNA labeledwith exogenously supplied bromodeoxyuridine (BrdU)was immunologically purified from total DNA extractedfrom a lakewater consortium and subjected to lengthheterogeneity PCR (85) to identify metabolically activeribotypes.It is very helpful for analyzing functions of localizedpopulations if one can know the chemical gradients cou-pled with spatial population dynamics, and microsensorshave been applied for this purpose (105). This techniquehas been applied in combination with molecu lar eco-logical methods to analyze sulfidogenic biofilms (106),nitrifying biofilm s (107, 108) and activated sludge (109).These studies have shown that some of the 16s rRNA-probe labeled cells are metabolically active.Catabo lic gene fragments are amplified by PCR fromenvironmental DNA samples and sequenced to analyzethe composition and diversity of catabolic populations.Genes used for th is purpose include ammonia mono-oxygenase (1 IO), methane monooxygenase (11 l-l 13),catechol dioxygenase (114, 115), phenol hydroxylase(116) and hydrogenase of Desul fovibrio spp. (117, 118).Rotthauwe et al. (110) have suggested that the ammoniamonooxygenase gene (amoA) represents a powerfulmolecular tool for analyzing indigenous ammonia-oxidiz-ing communities due to (i) its specificity, (ii) its fine-scaleresolution of closely related populations and (iii) the factthat a functional trait rather than a phylogenetic trait i sdetected. However, because PCR primers used in thesestudies were designed by comparing limited numbers ofpreviously cloned genes, it is likely that large parts oftotal enzyme populations remained undetected. Simu l-taneous use of multip le genes, e.g., catabo lic genes andthe 16s rRNA gene, is thus necessary for comprehensiveanalysis of catabo lic populations (110, 116).Isolation of microorganisms Although the above-mentioned techniques are quite useful for studying func-tional and physiological traits of microbial populationsin the environment, pure culture experiments are in-dispensable for detailed analyses of functions of eachpopulation (27, 93), particularly for manifesting con-cealed physiological traits like ly to be important for theestablishment of the consortium. To date, many pol-lutant-degrading bacteria have been isolated from natu-ral mixed populations after batch-culture enrichment inmedia containing relat ively high concentrations of thepollutants. However, th is batch-culture enrichment isnot considered suitable for ecological studies, becausesuch methods isolate a very limited number of bacteria

that always grow most rapid ly in laboratory media (119).Dunber and coworkers have thus proposed an alterna-tive method, namely autoradiographic direct plating, forisolating diverse microbial species with unique catabolictraits (119, 120). It has also been demonstrated that con-tinuous culture enrichment is useful for isolating diversephenol-degrading bacteria with high affinities for the sub-strate (121, 122). Marine oligotrophic bacteria have beensuccessfully isolated by the dilution culture techniqueproposed by Button et al. (123). This technique simplydilutes raw seawater with sterile seawater (low concentra-tions of organic nutrients may be added), and pure cul-tures may be obtained by the MPN manner. These alter-native isolation techniques have facilitated the isolationof diverse bacteria, most of which exhib it genotyp ic andphenotypic traits different from those of the batch-enrich-ment isolates.Molecula r ecological methods are also useful for themonitoring of enrichment and isolation processes. Teskeet al. used DGGE phylogenetic data from sulfate-reduc-ing enrichment to select methods (e.g., culture media)for isolating the component m icroorganisms from theenrichment (124). Heuer et al. have reported a method forsearching for bacterial isolates that correspond to popula-tions detected by a molecula r ecological method (125).They generated digoxygenin-labeled polynucleotide probesfrom major bands in TGG E community fingerprints,and the probes were used to screen bacteria l isolates bydot blot hybrid ization (125). In situ hybridization hassuccessfully been applied to check the purity of a bacteri-al phylotype in an enrichment culture and in purificationsteps by density gradient centrifugation (126). In thisway, Strous et al. obtained an almost pure cell suspen-sion of planctomycetes (99.6 ) that could not be cult i-vated by conventional microbiological techniques andshowed anaerobic ammonia oxidation (126).Environmentally relevant microorganisms (ERMs)A microorganism that plays a major role in the environ-ment (or in an environmental biotechnology process) istermed an environm entally relevant microorganism(ERM), while, in this review, this term is specificall yused for an isolated bacterium having such ecologicalcharacteristics (hence, its physiology and genetics shouldbe characterized in the laboratory). To date, only a fewmicroorganisms can be considered as ERMs, and thesefew examples are presented below in order to depict howthey were isolated and how they are different from typi-cal laboratory strains.The first example is high-affin ity methanotrophs (127).It has been recognized that the apparent half saturationconstant [Km(app)] or methane oxidation in soil is 1 to 3orders of magnitude lower than those observed withmethanotrophs previously isolated in the laboratory.Dunfield et al. applied low gaseous mixing ratios (< 275parts per million of volume) for the enrichment ofmethanotrophs from soil , and a stable mixed culture wasobtained which exh ibited a Km(app) ompatible with thatof the organisms in soil. DGGE with the methanemonooxygenase gene and 16s rDNA fragments showedthat a population affiliated with the type II methano-troph dominated the enrichment. This population wasthen isolated by direct plating under a low gaseous mix-ing ratio of methane. However, when the enrichmentwas transferred to a higher mixing ratio of methane(over I ), the Qapp) value increased without a changein the DGGE pattern, suggesting that the Km(app) alue is


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VOL. 89, 2000dependent on the growth condition (the phys iolog icalstate) rather than different K,,,(app) alues being expressedby different methanotrophs. This paper emphas izes theimportance of choosing appropriate conditions forlaboratory experiments in ecological studies.It has been well known that the majori ty of marinebacteria resist cultivation in standard laboratory mediaand the culturabil ity seldom exceeds 0.01 (123). Theuncultured bacteria are believed to be oligotrophs, be-cause the total o rganic concentration in seawater is gener-ally very low. Oligotrophs only are able to utilize dilutedorganic compounds and exis t in the form of very smallcells. They cannot attain high cell concentrations. An iso-tope incorporation experiment has suggested that oligo-trophs predominantly incorporate ambient organic com-pounds in seawater (128). This is also likel y to be thecase for marine oil spills, which has been suggested by atoluene turnover study (129). A marine oligotroph ic bac-terium capable of growing on toluene, xylenes and somepolynuclear aromatics was isolated and identified asCycloclasticus oligotrophus (130). It was found that thisbacterium harbors genes for aromatic hydrocarbon degra-dation, sim ilar to those of soil Pseudomonads (130), andthat the bacterium exhib its an affinity constant for tol-uene incorporation (e.g., 1.3 /*g per liter ) similar to thatmeasured in seawater (131). These data suggest that th isorganism is involved in toluene degradation in the ma-rine environment, although molecu lar ecological me-thods have not yet been applied to its detection in themarine environment.A functionally dominant phenol-degrading populationin phenol-digesting activated sludge has been identifiedby the combined use of molecu lar ecological detectionand physiolog ical comparison (116). In that study,TGG E analysis of PCR products of 16s rDNA and ofthe gene encoding phenol hydroxylase (LmPH) revealeda few dominant populations in activated sludge after20d of incubation with phenol. Bacteria closely relatedto Comamonas spp. were isolated by direct plating andcontinuous enrichment methods and were found to pos-sess 16s rDNA and LmPH sequencese identical to thosepredominantly found by the TGGE analyses. Although thesebacteria are capable of growing on phenol as the solecarbon source, their growth on phenol at concentrationsof over 100 mg per liter was very poor. A kinetic analysisof the phenol-oxygenating activity indicated that theComamonas bacteria exhibit the affinity and inhibitionconstants in Haldane s equation almost identical tothose of the phenol-digesting activated sludge (Fig. 3).Since the three approaches (i.e., TGGE using 16srDNA, that using LmPH and kinetic analysis) were con-sistent, the Comamonas bacteria have been identified tobe the dominant phenol-degrading population in activa t-ed sludge. This study has thus demonstrated an eco-logical theory (132) that although naturally occurringmicrobial consortia harbor diverse bacteria possessing aspecific function, only limited species dominate the nicheunder stable environmental conditions.Concentrations of organic compounds in the naturalenvironment are genera lly much lower than those inlaboratory media. Hence, ERMs listed above show highaffinities for organic substrates, suggesting the impor-tance of kinetic studies for physiological identification ofERMs. If a microorganism expresses pollutant-degrada-tion kinetics compatible with those observed in theenvironment and its population dynamics as analyzed by

ENVIRONMENTALLY RELEVANT MICROORGAN ISMS 7

oooorWAS2,----,

Group 2 (0,--\ CF6k2 O?

to 0:AH- - -rP6

\\ o ,I;&y --_*1000 ;Bl

Group 1-.

/ i&q Ph:nol-dlgesting actwated sludge~3 02

0) R2,oo '. 0 p' I I

0 -rc4 , 2 3

FIG. 3. A kinetic comparisonof the phenol-oxygenating ctivi-ties of phenol-digesting activate d sludge and isolated phenol-degrad-ing bacteria (116). E2, Ralsronia eutropha E2; E6, Comamonas sp.E6; RS, C. restosteroni RS; W, C. restosteroni R2; rC4, Nevskia sp.rC4; rN7, Comam onas sp. rN7; AH, Acinetobacter calcoaceticus AH;rP6, Bacteroids sp. rP6; WAS2, Pseudomonas sp. WASZ; CF600,Pseudomonas sp. CF600; BH, Pseudomonas putida BH; rB1, P.putida rB1. All these strains were isolated from activate d sludge.Kinetic cons tants are mentioned in the legend for Fig. 1. Based on theirK, and KS, values , these bacteria are classified into three groups(groups 1 to 3). Strain rN7, identi f ied as the ERM, showed alm ostidentical kine tic properties to those of phenol-digesting activate dsludge.molecular ecological methods (hopefully by severalmethods) correspond to changes in a spec ific function,one is allowed to declare that an ERM has been iden-tified. One important aspect is that all these ERMs havebeen enriched and isolated under low substrate concen-trations.Control of ERMs in microbial consortia An exam-ple is presented in which the growth of ERMs was con-trolled to improve an environmental biotechnologyprocess . When the phenol-loading rate of an activatedsludge process was increased stepwise from 0.5 to 1.0,and then to 1.5g.Zp1.dp1, the process broke downwithin one week after the loading rate was increased to1.5 g .I-. d-l (93). Trans itions of major bacteria l popula-tions were analyzed using molecular tools (93), and wefound that phenol-degrading populations changed fromfloe-forming bacteria to nonfloccula ting bacteria at theloading rate of 1.5 g. 1-l. d-l; this population changewas observed concomitantly with sludge washout, sug-gesting that it was probably respons ible for the processbreakdown. To circumvent this problem, a preferentialgrowth substrate for the floe-forming phenol-degradingpopulation was additionally supplied at high phenol-load-ing rates; this measure was found to be effective forenhancing the floe-forming population and suppressing thenonfloccula ting populations in activated sludge (unpub-lished data). Consequently, the activated-sludge processwas capable of degrading phenol at phenol-loading rateof 1.5 g.I-.d-. Although the supply of galactose isimpractically expensive, this study has demonstrated thatcontrol of ERMs enables the improvement of environ-mental biotechnology processes.Figure 4 illustrates factors governing the size of bac-terial populations in a natural microbial consortium.The four factors indicated by arrows in the figure can be


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8 WATANABE AND BAKER .i. BI~SCI. BIOEN~..

Invasion ICo

FIG . 4. Fact ors governing the sizes of bacterial populations in anatural habitat. A rrows po inting tow ard bacteria work positively forbacterial populations, while those pointing away from bac teria worknegatively.used to control bacterial populations. Biostimulat ion sup-plements nutrients to activate pollutant-degrading micro-organisms (related to arrow 1 in Fig. 4), while bioaug-mentation introduces exogenous pollutant-degradingmicroorganisms (shown by arrow 3). In the above exam-ple, we selectively stimulated the growth of the ERMpopulation, which is hence termed selective biostimula-tion. The other two factors (indicated by arrows 2 and4) are also known to exert large effects on the bacterialpopulations in the environment (133), so that these fac-tors may also be applicable in the field of environmentalbiotechnology in order to improve microbial consortia.

CONCLUSIONManipulation of ERMs is a possible way to microbio-

logically improve environmental biotechnology proces-ses. To achieve this, we should understand the physiologi-cal traits of ERMs in detail and compare these with thetraits of other major members of the microbial consort i-um. However, identification of an ERM is sti ll labori-ous, even if we can effectively use molecular ecologicaltools. Furthermore, compared with typical laboratorystrains, ERMs often grow very slowly in laboratory me-dia, and the cell concentrations achieved in their culturesare generally very low, which hampers their physiologi-cal characterization. We therefore propose that an im-portant subject that should be tackled in the next decadeis the development of physiological means that facilitatethe study of ERMs in the laboratory.ACKNOWLEDGMENTS

This work was supported, in part, by New Energy and Industr ialTechnology Development Organization (NEDO) and Research In-sti tute of Innovative Technology for the Earth (RITE). We thankShigeaki Harayam a for his continuous encouragem ent and helpfuldiscussion, and Ikuko Hiramatsu and Sachiko Kawasaki for theirtechnical assistance.REFERENCES

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