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Microorganisms relevant to bioremediation


Kazuya Watanabe
Naturally occurring microbial consortia have been utilized in a
variety of bioremediation processes. Recent developments in
molecular microbial ecology offer new tools that facilitate
molecular analyses of microbial populations at contaminated
and bioremediated sites. Information provided by such
analyses aids in the evaluation of the effectiveness of
bioremediation and the formulation of strategies that might
accelerate bioremediation.
Addresses
Marine 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.
Abbreviations
AOB
ammonia-oxidizing bacteria
DGGE denaturing gradient gel electrophoresis
FISH
fluorescence in situ hybridization
PAH
polycyclic aromatic hydrocarbon
TCE
trichloroethylene
TGGE temperature-gradient gel electrophoresis

Introduction
Bioremediation is a technology that utilizes the metabolic
potential of microorganisms to clean up contaminated
environments. One important characteristic of bioremediation is that it is carried out in non-sterile open
environments that contain a variety of organisms. Of these,
bacteria, such as those capable of degrading pollutants,
usually have central roles in bioremediation, whereas other
organisms (e.g. fungi and grazing protozoa) also affect the
process. A deeper understanding of the microbial ecology
of contaminated sites is therefore necessary to further
improve bioremediation processes.
In the past two decades, molecular tools, exemplified by
rRNA approaches, have been introduced into microbial
ecology; these tools have facilitated the analysis of
natural microbial populations without cultivation.
Microbiologists have now realized that natural microbial
populations are much more diverse than those expected
from the catalog of isolated microorganisms. This is also
the case for pollutant-degrading microorganisms, implying that the natural environment harbors a wide range of
unidentified pollutant-degrading microorganisms that
have crucial roles in bioremediation. This article summarizes the results of recent studies of microbial populations
that are relevant to bioremediation.
Molecular ecological information is thought to be useful
for the development of strategies to improve bioremediation and for evaluating its consequences (including risk

assessment). Molecular tools are especially useful in


bioaugmentation, in which exogenous microorganisms that
are introduced to accelerate pollutant biodegradation need
to be monitored. This article discusses recent examples of
the successful application of molecular ecological tools to
the study of bioremediation.

Microorganisms relevant to methane oxidation


Traditionally, studies on pollutant biodegradation have
been initiated by the isolation of one or more microorganisms capable of degrading target pollutants; however,
conventional isolation methods have resulted in the isolation of only a fraction of the diverse pollutant-degrading
microorganisms in the environment. In addition, most isolated organisms have shown pollutant-degradation kinetics
that differ from those observed in the environment [1]. For
example, laboratory-cultivated methanotrophs exhibit
half-saturation constants for methane oxidation which are
one to three orders of magnitude higher than those
observed in soil. Using molecular phylogenetic analyses of
isotope-labeled DNA, Radajewski et al. [2] successfully
identified two novel methanotrophs that actively degrade
methane under environmental conditions. Molecular
approaches that target the 16S rRNA gene (16S rDNA)
and genes encoding enzymes involved in key metabolic
steps (e.g. those encoding particulate methane monooxygenase) have been applied to the analysis of
methanotrophs in rice field soil [3], lake sediments [4] and
forest soil [5]. Methanotrophs are considered to be important for reducing the emission of methane, a greenhouse
gas, from soil and sediment. In addition, methanotrophs
co-metabolize trichloroethylene (TCE); therefore, TCE
bioremediation often employs methane injection as a
means to stimulate the TCE-degrading activity of indigenous methanotrophs (i.e. methane biostimulation).
Methanotrophs which occurred at a methane biostimulation site were recently analyzed using denaturing gradient
gel electrophoresis (DGGE) of polymerase chain reaction
(PCR)-amplified 16S rDNA and soluble methane
monooxygenase gene fragments [6].

Marine petroleum hydrocarbon degradation


Molecular ecological approaches have also been used to
analyze bacterial populations that occur in petroleum-contaminated marine environments. Spilled-oil bioremediation
experiments conducted at a sandy beach found that phylotypes affiliated with the subclass of Proteobacteria
(-Proteobacteria) appeared in the DGGE fingerprints
obtained 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 the
Norwegian Arctic showed that 16S rDNA types affiliated
with the -Proteobacteria, especially those belonging to the
Pseudomonas and Cycloclasticus groups, were abundant in

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Environmental biotechnology

fertilized oil sands [8]. Microbial populations which occurred


in seawater after supplementation with petroleum and inorganic fertilizers have been analyzed using rRNA approaches;
it was reported that bacterial populations belonging to the
-Proteobacteria [9] and the genus Alcanivorax [9,10] showed
accelerated growth. These studies have indicated that some
groups of bacteria commonly occur in oil-contaminated
marine environments, although other populations change
under different environmental conditions.

Anaerobic petroleum hydrocarbon degradation


As petroleum hydrocarbons are persistent under anaerobic
conditions, their contamination of groundwater is a serious
environmental problem. The microbial diversity in a hydrocarbon- and chlorinated-solvent contaminated aquifer
undergoing intrinsic bioremediation was assessed by cloning
and sequencing bacterial and archaeal 16S rDNA fragments
[11]. This study detected phylotypes that were closely related to Syntrophus spp. (anaerobic oxidizers of organic acids
with the production of acetate and hydrogen) and
Methanosaeta spp. (aceticlastic methanogens), suggesting
their syntrophic association. Phylotypes affiliated with candidate divisions (that do not contain any isolated organisms)
were also obtained in abundance from the contaminated
aquifer [11], although their physiology is completely
unknown. A similar syntrophic association of bacteria and
archaea has also been reported in a methanogenic enrichment that slowly degrades hexadecane [12]. Likewise, a
toluene-degrading methanogenic consortium was characterized by rRNA approaches [13]. The consortium comprised
two archaeal species related to the genera Methanosaeta and
Methanospirillum, and two bacterial species, one related to the
genus Desulfotomaculum and the other unrelated to any previously described genus. Fluorescence in situ hybridization
(FISH) with group-specific rRNA probes was used to analyze a denitrifying microbial community degrading
alkylbenzenes and n-alkanes [14]; the Azoarcus/Thauera
group was found to be the major bacterial group. Bacteria
affiliated with the -Proteobacteria were found to grow in
petroleum-contaminated groundwater which accumulated at
the bottom of underground crude-oil storage cavities [15].
Microbial communities associated with anaerobic benzene
degradation under Fe(III)-reducing conditions in a petroleumcontaminated subsurface aquifer were also analyzed by
DGGE analysis [16], and it has been suggested that Fe(III)reducing Geobacter spp. have an important role in the
anaerobic oxidation of benzene. The available electron
acceptors are the principal determinants for the types of
microorganisms that occur in anaerobic environments, and
microbial populations identified in the above papers are considered important for petroleum hydrocarbon degradation in
subsurface environments under the respective conditions.
On the basis of these results, future developments in anaerobic hydrocarbon bioremediation are anticipated [17]. It is
noteworthy that phylotypes that are only distantly related to
known genera are often detected as major members of the
anaerobic communities, suggesting that parts of anaerobic
hydrocarbon biodegradation processes remain unidentified.

Polycyclic aromatic hydrocarbon degradation


Polycyclic aromatic hydrocarbons (PAHs) are compounds
of intense public concern owing to their persistence in the
environment and potentially deleterious effects on human
health [18]. A soil-derived microbial consortium capable of
rapidly mineralizing benzo[a]pyrene was analyzed by
DGGE profiling of PCR-amplified 16S rDNA fragments
[19]. The analysis detected 16S rDNA sequence types that
represented organisms closely related to known high molecular weight PAH-degrading bacteria (e.g. Burkholderias,
Sphingomonas and Mycobacterium), although the degradation
mechanisms have yet to be resolved. In soil environments,
the reduced bioavailability of PAHs due to sorption to natural organic matter is an important factor controlling their
biodegradation [20]. Friedrich et al. reported that different
phenanthrene-degrading bacteria occurred in soil enrichments when different sorptive matrices were present [21].
It has also been shown that the application of surfactants to
soil enrichments that degrade phenanthrene and hexadecane altered the microbial populations responsible for
the degradation [22]. These results have common implications for bioremediation; that is, nature harbors diverse
microbial populations capable of pollutant degradation
from which a few pollutant-degrading populations are
selected according to bioremediation strategies.

Metal bioremediation
Because of its toxicity, metal contamination of the environment is also a serious problem. Recent studies have applied
molecular tools to the analysis of bacterial [23,24] and
archaeal populations [25] that are capable of surviving in
metal-contaminated environments. Bacterial communities
in soil amended for many years with sewage sludge that
contained heavy metals were assessed using rRNA
approaches, including FISH and cloning and sequencing
[23]. The study found that two sequence groups affiliated
with the -Proteobacteria and Actinobacteria were frequently
obtained from clone libraries from the metal-contaminated
soil, although most Actinobacteria sequences showed low
similarity (<85%) to the sequences of any hitherto cultured
actinomycete. The detoxification machineries that some of
these organisms may have are considered useful for metal
bioremediation, and comparisons with the machineries of
previously isolated metal-resistant bacteria may yield interesting results. Recently, heavy-metal-tolerant Ralstonia
eutropha was genetically engineered to express mouse metallothionein on the cell surface [26]. It was demonstrated
that the inoculation of Cd2+-polluted soil with the genetically engineered Ralstonia significantly decreased the toxic
effects of the heavy metal on the growth of tobacco plants.

Waste treatment
Microbial consortia involved in wastewater treatment have
been a major subject of microbial ecology, and many
papers have been published in which molecular tools were
used for community analyses. Bacterial community structures and physiological states within an industrial phenol
bioremediation system were recently analyzed [27].

Microorganisms relevant to bioremediation Watanabe

Comparisons made between the amounts of group-specific


rRNAs and the process chemistry enabled the authors to
identify some phylogenetic groups of bacteria important
for the process performance. The phylogenetic diversity of
bacterial communities supported by a seven-stage, fullscale bioreactor used to treat pharmaceutical wastewater
was studied using PCR-based techniques (i.e. DGGE fingerprinting and cloning of 16S rDNA fragments [28]).
These two techniques detected similar phylotypes,
although they failed to concede on their relative distribution, suggesting difficulties in quantitative interpretation
based on these methods. A combination of 16S rDNA
cloning, hybridization with oligonucleotide probes for
ammonia-oxidizing bacteria (AOB) and sequencing of the
hybridization-positive clones suggested that novel
Nitrosospira-like populations were the major AOB in a rhizosphere zone used to treat wastewater (rhizoremediation)
[29]. To identify microbial populations responsible for
phosphorus removal in activated-sludge, the structure of
the bacterial population was analyzed by FISH during the
operation of a laboratory-scale reactor with various phosphorus removal rates [30,31]. FISH has also been used to
analyze microbial populations in mesophilic and thermophilic sludge granules [32], foaming activated-sludge [33]
and bulking activated-sludge [34].
Temperature-gradient gel electrophoresis (TGGE) of
PCR-amplified 16S rDNA fragments was used to identify
the major phylotypes in phenol-digesting activated-sludge.
Physiological characterization of isolated bacteria corresponding to these phylotypes identified microbial
transition that caused a failure in the phenol treatment
[35]. The ecological information obtained in this study
was successfully used to develop a countermeasure against
the 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.

Bioaugmentation
The introduction of exogenous microorganisms into environments (bioaugmentation) has been used in an attempt
to accelerate bioremediation. It is desirable that the fate of
an introduced organism be monitored in order to prove its
contribution to pollutant degradation and to assess its
influence on the ecosystem. Molecular tools have been
used for this purpose. DGGE/TGGE fingerprinting of 16S
rDNA fragments has been used to examine the effects of
bioaugmentation on indigenous bacterial community
structures in a range of situations: a laboratory-scale
semicontinuous activated-sludge system loaded with
3-chloroaniline [37]; experimental model sewage plants
subjected to shock loads of chlorinated and methylated
phenols [38]; and in 2,4-dichlorophenoxyacetic-acidcontaminated soil horizons [39]. Quantitative PCR assays
targeting catabolic genes [40,41] and gyrB (the gene coding
for the subunit B protein of DNA gyrase) [42] have
successfully been used to monitor the fates of introduced

239

bacteria in complex microbial communities (e.g. those in


activated-sludge and in soil). In some cases, where genetically modified organisms were utilized, bioaugmentation
improved pollutant-biodegradation rates in the environment due to the establishment of transconjugants capable
of degrading the pollutants rather than the direct contribution of the inoculated organisms [39,43].

Conclusions
Bioremediation is still considered to be a developing technology. One difficulty is that bioremediation is carried out in
the natural environment, which contains diverse uncharacterized organisms. Most pollutant-degrading microorganisms
isolated and characterized in the laboratory are now thought
to make a minor contribution to bioremediation. Another
difficulty is that no two environmental problems occur under
completely identical conditions; for example, variations
occur in the types and amounts of pollutants, climate conditions and hydrogeodynamics. These difficulties have caused
the bioremediation field to lag behind knowledge-based
technologies that are governed by common rationales.
As summarized in this review, information on microbial
populations relevant to bioremediation is accumulating
rapidly with the aid of molecular ecological approaches.
Although our knowledge is not yet complete, it is time to
initiate more comprehensive approaches to find common
rationales in bioremediation. In some cases, for example,
marine petroleum bioremediation, we have already found
that similar bacterial populations occur even at geographically distant sites. Understanding the physiology and
genetics of such populations may prove very useful to
assess and improve bioremediation. Most importantly, we
need to identify general aspects in certain types of
bioremediation. For this purpose, I wish to propose the
construction of a database that collects the results of
molecular ecological assessments of contaminated and
bioremediated sites. The database would provide bioremediation with ecological backgrounds and, in concert
with currently available databases relevant to bioremediation, would facilitate the development of commonly
applicable schemes for certain types of bioremediation.

Acknowledgements
The author wishes to acknowledge the support provided by the New
Energy and Industrial Technology Development Organization (NEDO).
Thanks are also given to Shigeaki Harayama and Robert Kanaly for critical
reading of this manuscript.

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