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JOURNALFBIOSCIENCENDBIOENGINEERING
Vol. 92 No. 1 1-8. 2001
REVIEW
Current Bioremediation Practice and Perspective
TOMOTADA IWAMOTO
AND
MASAO
NASu*
Department of Bacteriology, Kobe Institute of Health, 4-6 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046
and Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences,
Osaka University, J-6 Yamada-oku, Suita, Osaka 565-087J2, Japan
Received 8 March 2001IAccepted 7 May 2001
The use of microbes to clean up polluted environments, bioremediation, is a rapidly changing
and expanding area of environmental biotechnology. Although bioremediation is a promising ap-
proach to improve environmental conditions, our limited understanding of biological contribu-
tion to the effect of bioremediation and its impact on the ecosystem has been an obstacle to make
the technology more reliable and safer. Providing fundamental data to resolve these issues, ie.,
the behavior of the target bacteria directly related to the degradation of contaminants and the
changes in microbial communities during bioremediation, has been a challenge for microbio-
logists since many environmental bacteria cannot yet be cultivated by conventional laboratory
techniques. The application of culture-independent molecular biological techniques offers new
opportunities to better understand the dynamics of microbial communities. Fluorescence in situ
hybridization (FISH),
in situ
PCR, and quantitative PCR are expected to be powerful tools for
bioremediation to detect and enumerate the target bacteria that are directly related to the degra-
dation of contaminants. Nucleic acid based molecular techniques for fingerprinting the 16s ribo-
somal DNA (rDNA) of bacterial cells, ie., denaturing gradient gel electrophoresis (DGGE) and
terminal restriction fragment length polymorphism (T-RFLP), enable us to monitor the changes
in bacterial community in detail. Such advanced molecular microbiological techniques will pro-
vide new insights into bioremediation in terms of process optimization, validation, and the impact
on the ecosystem, which are indispensable data to make the technology reliable and safe.
[Key words:
bioremediation, 16s ribosomal RNA, fluorescence
in situ
hybridization,
in situ
PCR, quantitative
PCR, denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism]
The advances in technology have sustained our industri-
alized society. During the twentieth century, the explosive
development of chemical industries has produced a bewil-
dering variety of chemical compounds that have led to the
modernization of our lifestyles. The large-scale production
of a variety of chemical compounds, however, has caused
global deterioration of environmental quality. Among them,
xenobiotic compounds that greatly differ in chemical struc-
ture from natural organic compounds, such as polychlori-
nated biphenyls (PCBs), trichloroethylene (TCE), perchlo-
roethylene (PCE), trinitrotoluene (TNT), and so on, are the
chemical compounds of concern because of their toxicity,
resistance to biodegradation, and biomagnification via the
food web.
One of the worst environmental disasters caused by
chemical waste is the Love Canal case that happened in
Niagara Falls, N.Y., USA. The Love Canal area was origi-
nally the site of an abandoned canal that became a disposal
site for nearly 22,000 tons of chemical waste including
* Corresponding author. e-mail: nasu@phs.osaJca-u.ac.jp
phone: +81(0)6-6879-8170 fax: +81(0)6-6879-8174
PCBs, dioxin, and pesticides dumped by the Hooker Chem-
ical Company during the 1940s and early 1950s. Thereafter,
the site was filled with land and sold by the company to the
City of Niagara Falls, which allowed the construction of a
school and houses. In 1978, however, state officials detected
the leakage of toxic chemicals from the ground into the
basement of homes in that area. Abnormally high inci-
dences of miscarriages and birth abnormalities were re-
ported among the areas residents. Based on this disaster,
the Comprehensive Environmental Response Compensation
and Liability Act (CERCLA) of 1980 was enacted in the
United States. Along with subsequent amendments such as
the Superfund Amendments, the regulatory framework for
the disposal of hazardous waste and the cleaning up of sites
polluted by chemical compounds was established. This is-
sue created a new phase of environmental awareness,
i.e.,
special attention is now given to the remediation of contam-
inated soil and aquifers worldwide. In Japan, the Environ-
ment Agency amended the Water Pollution Control Law in
1996 and quality standards for groundwater were issued in
March 1997. With this amendment, the groundwater purifi-
cation order system that allows governors to take measures
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2
IWAMOTO AND NASU J. BIOSCI. BIOENG..
against polluters was created.
Bioremediation, which involves the use of microbes to
detoxify and degrade environmental contaminants, has re-
ceived increasing attention as an effective biotechnological
approach to clean up a polluted environment. In general, the
approaches to bioremediation are environmental modifica-
tion, such as through nutrient application and aeration, and
the addition of appropriate degraders by seeding. Bioreme-
diation offers several advantages over the conventional
chemical and physical treatment technologies, especially for
diluted and widely spread contaminants. In
situ
treatment is
one of the most attractive advantages of this technology.
The term
in situ
comes from Latin and means in its original
place.
In situ
bioremediation enables us to remediate a
contaminated site without transportation of contaminants
and with minimum site disruption. Manufacturing and in-
dustrial use of the site can continue while the bioremedia-
tion process is being implemented. Considering the situa-
tion in Japan, that is, in many instances contaminated sites
are located close to residential areas, this technology is ex-
tremely beneficial. To date, there have been several reports
stating that bioremediation has been successfully used to
treat petroleum-contaminated sites (1). Recently, the impor-
tance of bioremediation has been increasing in the field of
hazardous-waste management such as PCB, TCE, PCE,
BTEX (benzene, toluene, ethylene, and xylems).
However, we have to state that bioremediation is still an
immature technology. Although microbes play an essential
role in biogeochemical cycles (24) and they are the pri-
mary stimulant in the bioremediation of contaminated envi-
ronments, current knowledge of changes in microbial com-
munities during bioremediation is limited, and the microbial
community is still treated as a black box. The reason for
this is that many environmental bacteria cannot yet be cul-
tured by conventional laboratory techniques (5, 6). This has
led to two essential questions related to the implementation
of bioremediation in the field. These are (i) how to clarify
the biological contribution to the effectiveness of bioreme-
diation and (ii) how to assess the environmental impact
of bioremediation. Because of the technical limitations in
monitoring the target bacteria directly related to the degra-
dation of contaminants, bioremediation often faces the dif-
ficulty of identifying the cause and developing measures
in the case of failure remediation from a microbiological
standpoint. Moreover, our limited understanding of the
changes in microbial communities during bioremediation
makes it difficult to assess the impact of bioremediation on
the ecosystem.
The rapid advancement of molecular biological methods
has facilitated the study of microbial community structure
without bias introduced by cultivation. It is expected to pro-
vide new insights into process optimization, validation, and
the impact on the existing ecosystem. In this review, we de-
scribe (i) bioremediation systems and process, (ii) microbes
utilized for bioremediation, and (iii) potential of molecular
microbial ecological methods in bioremediation.
I. BIOREMEDIATION SYSTEMS
AND PROCESS
Bioremediation technologies can be broadly classified as
ex
situ
or
in situ. Ex situ
technologies are the treatments that
remove contaminants at a separate treatment facility.
In situ
bioremediation technologies involve the treatment of the
contaminants in the place itself. The
in situ
technologies of-
fer several advantages over physical and chemical remedia-
tion, as summarized in Table 1. Microbes have an extensive
capacity to degrade synthetic compounds; therefore, biore-
mediation can be applied to sites contaminated with a vari-
ety of chemical pollutants.
In situ
bioremediation processes
currently utilized in the field are classified into the follow-
ing three categories.
Bioattenuation
This is the method of monitoring the
natural progress of degradation to ensure that contaminant
concentration decreases with time at relevant sampling
points. Bioattenuation is widely used as a cleanup method
for underground storage tank sites with petroleum-contami-
nated soil and groundwater in the United States (7).
Biostimulation
If natural degradation does not occur
or if the degradation is too slow, the environment has to be
manipulated in such a way that biodegradation is stimulated
and the reaction rates are increased. The measures to be
taken, called biostimulation, include supplying the environ-
ment with nutrients such as nitrogen and phosphorus, with
electron acceptors such as oxygen, and with substrates such
as methane, phenol, and toluene. The chemical additives
used as substrates, phenol and toluene, are well-known
toxic chemicals. Thus, the concentrations of these chemi-
cals during biostimulation should be carefully monitored. In
Japan, the effectiveness of
in situ
biostimulation by methane
injection into TCE-contaminated groundwater was demon-
strated by small-scale field experiments funded separately
by the Environment Agency (8) and by the Ministry of In-
ternational Trade and Industry (9). By accumulating scien-
tific evidence through these kinds of field experiments,
in
situ
biostimulation is expected to become a reliable and safe
cleanup technology.
Bioaugmentation
The third choice in the treatment hi-
erarchy is bioaugmentation, which is a way to enhance the
biodegradative capacities of contaminated sites by inocula-
tion of bacteria with the desired catalytic capabilities. This
is considered to be an effective approach in the case of very
recalcitrant chemicals where bioattenuation or biostimula-
tion does not work. However, we have to pay much atten-
tion to the application of bioaugmentation because of its un-
known effects on the ecosystem. Since large amounts of
degradative bacteria are added to contaminated sites, the ef-
fect of the bacteria on both human and environment must be
clarified in advance. Moreover, it needs to be confirmed that
TABLE 1. Advantages of
in situ
bioremediation
Can be done on site
Eliminates transportation cost
Eliminates waste permanently
- Site disruption can be minimized
Applicable to diluted and widely diffused contaminants
Affordable
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VOL 92 2001
PERSPECTIVES OF BIOREMEDIATION
3
the injected bacteria have perished after the remediation and
thus do not affect the indigenous microbial community for a
long period. The first field experiment on bioaugmentation
in Japan was conducted in 2000 under strict control by the
Ministry of International Trade and Industry (10). In the ex-
periment, a phenol-utilizing bacterium,
Ralstonia eutropha
KT- 1, which was originally isolated from the same contami-
nated site, was injected without adding any substrates. One
challenging area of bioaugmentation is the utilization of
genetically engineered microorganisms (GEMS). Field bio-
augmentation study with a modified strain, Burkholderia
cepacia PRl,,,,
was conducted at the Moffett Federal Air-
field in the U.S. after laboratory microcosm studies (11).
The modified strain,
B. cepacia
PRl,,,, can degrade TCE
effectively while growing on lactate. This avoids the use of
toxic chemicals such as toluene or phenol as a substrate
(12). The field experiment was carried out to evaluate the
effectiveness of B. cepacia PRl,,, in removing TCE along
with lactate. While bioaugmentation showed the potential to
remove recalcitrant chemicals, comprehensive scientific
data to ensure the safety of this technology must be col-
lected before commercializing this technology.
II. MICROBES UTILIZED FOR
BIOREMEDIATION
The rapid advancement of molecular microbiological
methods has facilitated research activities to understand the
fundamental mechanism of biodegradation. A number of
bacterial strains capable of metabolizing environmental
contaminants have been isolated from the natural environ-
ment. The genes encoding enzymes related to toxic chemi-
cal degradation have been analyzed. Subsequently, these
findings will expand the potential of bioremediation, espe-
cially for recalcitrate chemical compounds. In the follow-
ing, microbes capable of degrading toxic chemical com-
pounds are summarized.
Trichloroethylene (TCE) Chlorinated ethanes and
ethenes are commonly used as cleaning solvents and in dry
cleaning operations. TCE has received the most attention
among these chemicals because of its toxicity and the mag-
nitude of its pollution. So far, microbes capable of using
TCE as the sole energy source have not been isolated. How-
ever, it is well known that some microbes can degrade TCE
via a special type of metabolism, named cometabolism. In
cometabolism, microbes gratuitously metabolize TCE uti-
lizing the enzyme that are synthesized to degrade the pri-
mary substrate (13). Knowledge that TCE can be anaero-
bically dechlorinated to a carcinogenic intermediate, vinyl
chloride, has prompted many intensive investigations into
the aerobic, oxygenase-mediated cometabolism of TCE.
After Wilson and Wilson (14) have shown the cometabo-
lism of TCE by methanotrophs in 1985, many researchers
reported microbes capable of degrading TCE by cometabo-
lism. Those are represented by methanotrophs (15) phenol
oxidizers (16) toluene oxidizers (17), ammonia oxidizers
(18), and propene utilizers (19). The low substrate specitici-
ties of their enzymes (methane monooxygenase, toluene di-
oxygenase, phenol hydroxylase, ammonia monooxygenase,
or propene monooxygenase) allow the conversion of TCE
to TCE epoxide, which subsequently hydrolyzes to polar
products (e.g., formic, glyoxylic, and dichloroacetic acids)
utilizable by microorganisms (20).
Polychlorinated biphenyls (PCB)
PCBs are a group
of manmade compounds composed of biphenyl molecules
containing from one to ten chlorines. They are oily fluids
with high boiling point, high chemical resistance, low elec-
trical conductivity, and high refractive index. Because of
these properties, they have been used mainly as insulators
in electrical transformers and capacitors, as heat exchange
fluids, and as plasticizers. Their toxicity, bioconcentration,
and persistence have been well documented. In 1968, PCB-
contaminated cooking oil, caused by a leaky heat ex-
changer, poisoned nearly a thousand people in Japan. Fol-
lowing this experience, the manufacture of PCBs was
stopped and usage was limited in 1972 in Japan. The use
and discharge of PCBs in the United States came under a
complete government ban in 1978. However, PCBs are still
serious environmental pollutants globally since previously
contaminated sediments, landfills, and older electric trans-
formers are still exist as sources of PCB pollution. Although
PCBs are relatively resistant to biodegradation, it has been
shown that a number of bacteria can cometabolize various
PCB components (21, 22). Biphenyl dioxygenase is known
to play a critical role in PCB degradation. Bioremediation,
therefore, is expected to be an effective approach to remove
PCBs from the contaminated sites. Since Furukawa and
Miyazaki (23) had cloned biphenyl and PCB catabolism
genes bphA, bphB, and bphC) from the chromosomal DNA
of Pseudomonas pseudoalcaligenes IW707 in 1986, a num-
ber of PCBs degrading genes have been cloned (24-26) and
sequenced (27,28). Erickson and Mondello (28) determined
the nucleotide sequence of the DNA region encoding the bi-
phenyl dioxygenase of
Pseudomonas
species strain LB400,
which is a potentially valuable organism for bioremediation
of PCBs as it is able to oxidize a wide variety of PCBs.
2,4,6-Trinitrotoluene (TNT) TNT is a common mili-
tary explosive that is found wherever munition is produced,
loaded, handled or packed. Its manufacturing and disposal
left many sites polluted. Although many aerobic bacteria
have the potential to degrade nitroaromatic compounds in-
cluding TNT (29), no successful bioremediation has been
reported with an aerobic treatment. Anaerobic bacteria such
as chlostridia (30), sulfate reducers (31, 32), methanogens
(33),
Desulfovibrio
species (31, 32), and Fe (III)-reducing
bacteria (34,35) can reduce nitroaromatic compounds. Add-
ing an external carbon source to the soil such as acetate, sol-
uble starch, and glucose, favors the formation of anaerobic
conditions that promote the initial metabolic steps in the
biodegradation of TNT (36). So far, the best approach to
treat TNT-contaminated sites seems to be a sequence of
anaerobic and aerobic processes (37,38).
Dioxin-like compounds The implementation of bio-
remediation processes for the removal of dioxin-like com-
pounds (e.g., polychlorinated dibenzo-p-dioxins (PCDD)
and polychlorinated dibenzofurans (PCDF)) remains to be a
challenge for microbiologists and environmental engineers.
Sphingomonas
sp. RWl has the dioxin dioxygenase system
but it can degrade only low chlorinated dibenzo-p-dioxin
(DD) and dibenzofuran (DF) (39). So far, no bacteria capa-
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4 IWAMOTO AND NASU
J. BIOSCL
BIOENG.
ble of degrading PCDD and PCDF have been found. Exten-
sion of the substrate range of DD- and DF-degrading bacte-
ria is expected to be achieved by mutagenesis of the catalyt-
ically active a subunit of the dioxygenase (40). Bumpus
et
al. (41) reported that the white-rot fungus
Phanerochaete
chrysosporium can degrade 2,3,7&tetrachlorodibenzodi-
oxin (TCDD). Takada et al. 42) studied the degradation of
2,3,7,8-TCDD/F by the peroxidases produced by the myce-
lium of Phunerochuete sordidu strain. Their results showed
significant degradation rates and metabolite formation. Uti-
lization of white-rot fungi may be another approach for
treating dioxin-like compounds.
Toxic metals
Besides its use in attacking organic com-
pounds, bioremediation can be used to treat sites contami-
nated with heavy metals. Some bacteria have been reported
to reduce anaerobically hexavalent chromium that is toxic
and mutagenic, to its trivalent form that is less toxic (43).
Bioprecipitation by sulfate-reducing bacteria has been well
studied. They convert sulfate in the groundwater to hydro-
gen sulfide which, in turn, reacts with heavy metals to form
insoluble metal sulfides such as zinc sulfide and cadmium
sulfide. Biomethylation to yield volatile derivatives such as
dimethylselenide or trimethylarsine is a well-known phe-
nomenon catalyzed by a variety of bacteria, algae, and fungi
(44). These mechanisms show a high potential for bioreme-
diation on heavy metal contaminated sites.
III. POTENTIAL OF USING MOLECULAR
MICROBIAL ECOLOGICAL METHODS
IN BIOREMEDIATION
To implement bioremediation in the field, biological con-
tribution to the effect of bioremediation and the impact on
the ecosystem need to be clarified. To this end, the analysis
of microbial communities that take part in
in situ
bioremedi-
ation is indispensable. It has been a challenge for microbiol-
ogists to analyze microbial communities in natural environ-
ments since most environmental bacteria cannot be culti-
vated by conventional laboratory techniques so far (5,6). To
obtain a better understanding of the structure and dynamics
of natural microbial communities, other approaches that
complement conventional culture-dependent techniques are
needed. The application of molecular biological techniques
to detect and identify microorganisms by certain molecular
markers has been more and more frequently used in micro-
bial ecological studies. In the following, we describe molec-
ular microbial ecological methods that can be utilized in
in
situ
bioremediation.
Detection and monitoring of target bacteria
The de-
tection and monitoring of target bacteria that are directly
related to the degradation of contaminants are needed for
process monitoring and optimization of bioremediation.
Single-cell level detections of specific bacteria are well rec-
ognized as efficient techniques to detect and enumerate cer-
tain bacteria in complex communities (4547). Most nota-
bly, fluorescence in situ hybridization (FISH) with ribo-
somal RNA (rRNA) targeted oligonucleotide probes has
been used successfully in microbial ecological studies. The
rRNA molecules comprise highly conserved domains inter-
spersed with more variable regions (48, 49). Thus, rRNA
sequences are commonly used to construct phylogenetic
trees. The specific sequences for a number of the certain
bacterial groups and species (50-52) have been identified.
FISH involves hybridization of fluorescence-labeled
oligonucleotide probes to intracellular rRNA. Cells showing
specific hybridization with the probe can be identified and
enumerated by epifluorescence microscopy. More effi-
ciently, analysis by flow cytometry enables us to identify
and enumerate a large number of cells in a short time (one
thousand cells per second) (45). The problem in utilizing
FISH in studies of natural bacterial communities is its sen-
sitivity. In general, the use of standard FISH with mono-
FITC-labeled probes gives a strong signal only if cells are
metabolically active, and, hence, contain large number of
rRNAs (53-55). Various approaches have been taken to
improve the sensitivity (56, 57). Yamaguchi et al. (58) re-
ported a new fluorescence in situ hybridization technique,
HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2-phenyla-
nilide phosphate (HNPP) and Fast Red TR, which enhances
the fluorescence signals eightfold compared to FITC-FISH.
The use of a Cy3 labeled oligonucleotide probe is also
known as an effective approach to improve sensitivity (59,
60). The principles of these methods are shown in Fig. 1.
Another single-cell level detection that has been used in
microbial ecological studies is
in situ
PCR (61). This is a
unique modification of PCR in which amplification and de-
tection of target genes are carried out inside individual bac-
terial cells (Fig. 2). This technique enables us to detect indi-
vidual functional genes present in single copy or low copy
numbers in intact bacterial cells that cannot detected by
FISH. Kurokawa et al. (62) reported the abundance and dis-
tribution of bacteria carrying the
skII
gene in natural river
water by in situ PCR. Using a combination of in situ reverse
transcription and
in situ
PCR, we can investigate how gene
expression in bacterial cells responds to environmental con-
Cy3-FISH
Cell wall and
Hybridization
HNPP-FISH
Cell wall and
FIG 1. Principles of Cy-3 FISH and HNPP-FISH.
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VOL. 92,200l
PERSPECTIVESOF BIOREMEDIATION
FIG 2. Principle
of in
situ PCR.
Labeled PCR product
Taq
DNA polymerase
Labeled dUTP
- :
Permeabilization
(Lysozyme, Proteinase K) :
DNA
polymerase
Primers
dNTP
Labeled dUTP
ditions. Chen
et al. 63)
have used the technique for the de-
tection of
Pseudomonas putida
Fl expressing the
tod Cl
gene in seawater exposed to toluene vapor.
The recent development of real-time PCR devices has
made quantitative PCR much easier. Besides single-cell
level detection, the quantitative PCR approach utilizing
bulk DNA from natural bacterial communities may be an
effective approach to monitor target bacteria. Nakamura et
al.
(10) successfully monitored the number of
Ralstonia
eutropha
KT-1 during field experiments of bioaugmentation
in TCE-contaminated groundwater by quantitative PCR
with LightCyclerTM (Roche) targeting repetitive extragenic
palindromic (REP) sequence.
Monitoring changes in bacterial diversity
Microbial
communities play an essential role in biogeochemical cy-
cles (2-4) and contribute to the maintenance of the ecosys-
tem. Therefore, investigating the influence of bioremedia-
tion on the microbial community is indispensable to prove
the safety of in situ bioremediation.
Denaturing gradient gel electrophoresis (DGGE) of PCR-
amplified 16s rDNA fragments has emerged as a power-
ful and convenient tool for determining temporal or spatial
differences in bacterial populations and for monitoring
changes in the diversity of bacterial communities (64-71).
In this method, PCR-amplified 16s rDNA fragments from a
bacterial community, essentially the same size, can be sepa-
rated into discrete bands during electrophoresis in a poly-
acrylamide gel containing a linearly increasing gradient of
DNA denaturant,
i.e.,
a mixture of urea and formamide.
This separation is based on the decreased electrophoretic
mobility of partially denatured DNA molecule in the gel.
In DGGE, individual double-stranded DNA molecules de-
nature according to their sequences. Partial denaturation
causes their migration to stop at a unique position, there-
by forming discrete bands in the gel. Consequently, the di-
versity of a bacterial community can be visualized in terms
of their banding patterns in DGGE. By the attachment of a
GC clamp, which is GC-rich sequence, to the DNA fiag-
ment, all sequence variants can be detected (72). Figure 3
illustrates the principle of DGGE. Individual bands can be
excised, re-amplified and sequenced or hybridized with
oligonucleotide probes to determine the composition of the
v
0
Low
I
Denam Wm.
Fmnamidc Urea;
I
0
High
DNA fragment
\
Gc clamp
w Mobility: High
Denature
I
Gc clamp
*
Mobility: Low
Denature
\
Polyaclylamide gel
L-
Mobility: Stop
1. Mobility: double stranded DNA > partially denatured DNA
2. Conditions (concentration of denahuant, temperature) for denahtring DNA
depend on the sequence
Bacterial species
--
m---
Neutral polyacryhmide
Separation by DGGE based on sequece
A, B, C have the same length but different sequences
FIG 3. Principle of DGGE.
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6
IWAMOTO AND NASU
@-- z.,
c;g
DNA
extraction
Bacterial community
PCR with
labeled primer
I
0
0
0
0
0
---I
Restriction
enzyme digestion
I
-=
-
I
:~
L-
L---
/
\
-3
digested fkgment
digested fragment
with labeled primer
Fluorescence-based equencer
Fragment length after restriction enzyme
digestion depends on the DNA sequence
(The difference in restriction enzyme site must be reflected by the difference in sequence)
FIG 4. Principle of T-RFLP
bacterial community. Besides, quantitative banding pattern
analysis makes DGGE more powerful to monitor the behav-
ior of the bacterial community over a long period (73-77).
Some researchers have successfully monitored the changes
in bacterial diversity during in situ bioremediation by
DGGE (78-80).
Another efficient method for the analysis of microbial
community diversity in various environments is terminal
restriction fragment length polymorphism (T-RFLP) (81).
In this method, a fluorescence-labeled primer is used to
amplify a selected region of bacterial genes encoding 16s
rRNA from a bacterial community. The PCR products are
digested with restriction enzymes, and the fluorescence-la-
beled terminal restriction fragment is precisely measured
by an automated DNA sequencer (Fig. 4). Moesender et al.
(82) compared the results of T-RFLP and DGGE analyses of
complex marine bacterial communities. The result showed
that T-RFLP fingerprinting had a slightly higher resolution
than DGGE. Marshi
et al.
(83) developed a web-based re-
search tool that provides an investigator a rapid way to de-
termine optimal primer and restriction enzyme combina-
tions for bacterial community analysis by T-RFLP. It is lo-
cated at the Ribosomal Database Project website. This will
facilitate microbial community analysis by T-RFLP.
CONCLUSION
Bioremediation is an interdisciplinary technology involv-
ing microbiology, engineering, ecology, geology, and chem-
istry. Microbes are the primary stimulant in the bioremedi-
.I.
BIOSCI.
BIOENG.,
ation of contaminated environments. However, current
knowledge of biological contribution to the effect of biore-
mediation and its impact on the ecosystem is limited, and
the microbial community is still treated as a black box.
The molecular microbiological techniques described in this
review are expected to catalyze research activities to clarify
these issues. We anticipate that new insights into process
optimization, validation, and impact on the ecosystem ob-
tained by the advanced molecular microbiological tech-
niques will make bioremediation a more reliable and safer
technology.
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