JOURNAL

OFBIOSCIENCE
ANDBIOENGINEERING
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 approach to improve environmental
conditions, our limited understanding of biological contribution 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 microbiologists 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 degradation of contaminants. Nucleic acid based molecular techniques for fingerprinting the 16s ribosomal 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 provide 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 industrialized society. During the twentieth century, the explosive
development of chemical industries has produced a bewildering 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 structure from natural organic compounds, such as polychlorinated biphenyls (PCBs), trichloroethylene
(TCE), perchloroethylene (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 originally 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 Chemical 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 incidences of miscarriages and birth abnormalities
were reported 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 issue created a new phase of environmental
awareness, i.e.,
special attention is now given to the remediation of contaminated soil and aquifers worldwide. In Japan, the Environment Agency amended the Water Pollution Control Law in
1996 and quality standards for groundwater were issued in
March 1997. With this amendment, the groundwater purification order system that allows governors to take measures

IWAMOTO

AND NASU

against polluters was created.

Bioremediation,
which involves the use of microbes to
detoxify and degrade environmental
contaminants, has received increasing attention as an effective biotechnological
approach to clean up a polluted environment. In general, the
approaches to bioremediation
are environmental
modification, such as through nutrient application and aeration, and
the addition of appropriate degraders by seeding. Bioremediation 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 industrial use of the site can continue while the bioremediation process is being implemented. Considering the situation in Japan, that is, in many instances contaminated sites
are located close to residential areas, this technology is extremely beneficial. To date, there have been several reports
stating that bioremediation
has been successfully used to
treat petroleum-contaminated
sites (1). Recently, the importance 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 primary stimulant in the bioremediation of contaminated environments, current knowledge of changes in microbial communities 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 cultured 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 bioremediation and (ii) how to assess the environmental
impact
of bioremediation.
Because of the technical limitations in
monitoring the target bacteria directly related to the degradation of contaminants, bioremediation
often faces the difficulty 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 provide new insights into process optimization, validation, and
the impact on the existing ecosystem. In this review, we describe (i) bioremediation systems and process, (ii) microbes
utilized for bioremediation,
and (iii) potential of molecular
microbial ecological methods in bioremediation.

J. BIOSCI. BIOENG..

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 offer several advantages over physical and chemical remediation, as summarized in Table 1. Microbes have an extensive
capacity to degrade synthetic compounds; therefore, bioremediation can be applied to sites contaminated with a variety of chemical pollutants. In situ bioremediation processes
currently utilized in the field are classified into the following 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-contaminated 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 environment 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 chemicals during biostimulation should be carefully monitored. In
Japan, the effectiveness of in situ biostimulation by methane
injection into TCE-contaminated
groundwater was demonstrated by small-scale field experiments funded separately
by the Environment Agency (8) and by the Ministry of International Trade and Industry (9). By accumulating scientific 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 hierarchy is bioaugmentation,
which is a way to enhance the
biodegradative capacities of contaminated sites by inoculation 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 biostimulation does not work. However, we have to pay much attention to the application of bioaugmentation
because of its unknown effects on the ecosystem. Since large amounts of
degradative bacteria are added to contaminated sites, the effect 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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PERSPECTIVES OF BIOREMEDIATION

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 experiment, a phenol-utilizing
bacterium, Ralstonia eutropha
KT- 1, which was originally isolated from the same contaminated site, was injected without adding any substrates. One
challenging area of bioaugmentation
is the utilization of
genetically engineered microorganisms
(GEMS). Field bioaugmentation
study with a modified strain, Burkholderia
cepacia PRl,,,, was conducted at the Moffett Federal Airfield 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 collected 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 environment. The genes encoding enzymes related to toxic chemical degradation have been analyzed. Subsequently,
these
findings will expand the potential of bioremediation,
especially for recalcitrate chemical compounds. In the following, microbes capable of degrading toxic chemical compounds 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 magnitude of its pollution. So far, microbes capable of using
TCE as the sole energy source have not been isolated. However, it is well known that some microbes can degrade TCE
via a special type of metabolism, named cometabolism. In
cometabolism,
microbes gratuitously metabolize TCE utilizing the enzyme that are synthesized to degrade the primary substrate (13). Knowledge that TCE can be anaerobically 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 cometabolism of TCE by methanotrophs in 1985, many researchers
reported microbes capable of degrading TCE by cometabolism. Those are represented by methanotrophs (15) phenol
oxidizers (16) toluene oxidizers (17), ammonia oxidizers
(18), and propene utilizers (19). The low substrate speciticities of their enzymes (methane monooxygenase,
toluene dioxygenase, 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 electrical 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, PCBcontaminated
cooking oil, caused by a leaky heat exchanger, poisoned nearly a thousand people in Japan. Following 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 transformers 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 number 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 biphenyl 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 military 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 including 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. Adding an external carbon source to the soil such as acetate, soluble 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 bioremediation processes for the removal of dioxin-like compounds (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-

IWAMOTO

J. BIOSCL BIOENG.,

AND NASU

ble of degrading PCDD and PCDF have been found. Extension of the substrate range of DD- and DF-degrading bacteria is expected to be achieved by mutagenesis of the catalytically active a subunit of the dioxygenase (40). Bumpus et
al. (41) reported that the white-rot fungus Phanerochaete
chrysosporium can degrade 2,3,7&tetrachlorodibenzodioxin (TCDD). Takada et al. (42) studied the degradation of
2,3,7,8-TCDD/F by the peroxidases produced by the mycelium of Phunerochuete sordidu strain. Their results showed
significant degradation rates and metabolite formation. Utilization of white-rot fungi may be another approach for
treating dioxin-like compounds.
Toxic metals
Besides its use in attacking organic compounds, bioremediation
can be used to treat sites contaminated 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 hydrogen 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 phenomenon catalyzed by a variety of bacteria, algae, and fungi
(44). These mechanisms show a high potential for bioremediation on heavy metal contaminated sites.
III. POTENTIAL OF USING MOLECULAR
MICROBIAL ECOLOGICAL METHODS
IN BIOREMEDIATION
To implement bioremediation in the field, biological contribution 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 bioremediation is indispensable. It has been a challenge for microbiologists to analyze microbial communities in natural environments since most environmental
bacteria cannot be cultivated 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 microbial ecological studies. In the following, we describe molecular microbial ecological methods that can be utilized in in
situ bioremediation.
Detection and monitoring of target bacteria
The detection 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 recognized as efficient techniques to detect and enumerate certain bacteria in complex communities (4547). Most notably, fluorescence in situ hybridization
(FISH) with ribosomal RNA (rRNA) targeted oligonucleotide
probes has
been used successfully in microbial ecological studies. The
rRNA molecules comprise highly conserved domains interspersed 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 efficiently, 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 sensitivity. In general, the use of standard FISH with monoFITC-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) reported a new fluorescence in situ hybridization technique,
HNPP-FISH, using 2-hydroxy-3-naphthoic
acid 2-phenylanilide 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 detection of target genes are carried out inside individual bacterial cells (Fig. 2). This technique enables us to detect individual 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 distribution 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

FIG

1.

Principles of Cy-3 FISH and HNPP-FISH.

Cell wall and

PERSPECTIVESOF BIOREMEDIATION

VOL. 92,200l

:
Permeabilization
(Lysozyme, Proteinase K) :

DNA polymerase
Primers
dNTP
Labeled dUTP

Labeled PCR product

Labeled dUTP

Taq DNA polymerase

FIG 2. Principle of in situ PCR.

ditions. Chen et al. (63) have used the technique for the detection 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.
Microbial
Monitoring changes in bacterial diversity
communities play an essential role in biogeochemical
cycles (2-4) and contribute to the maintenance of the ecosystem. Therefore, investigating the influence of bioremediation on the microbial community is indispensable to prove
the safety of in situ bioremediation.
Denaturing gradient gel electrophoresis (DGGE) of PCRamplified 16s rDNA fragments has emerged as a powerful 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 separated into discrete bands during electrophoresis in a polyacrylamide 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 denature according to their sequences. Partial denaturation
causes their migration to stop at a unique position, thereby forming discrete bands in the gel. Consequently, the diversity 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
DNA fragment

Gc clamp
Mobility: High

\
w
Denature

Low

I
I
Denam Wm.

Gc clamp
Mobility: Low

Fmnamidc + Urea;

Denature

Mobility: Stop

High

L-

\
Polyaclylamide gel

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.

.I. BIOSCI.BIOENG.,

IWAMOTO AND NASU

@
c*--

ation of contaminated
environments.
However, current
knowledge of biological contribution to the effect of bioremediation 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 obtained by the advanced molecular microbiological
techniques will make bioremediation
a more reliable and safer
technology.

z.,

c;g

DNAextraction

Bacterial community

PCR with
labeled primer

I
*

0
0
0
0

REFERENCES

---I
Restriction
enzyme digestion
I

-=
-

I
:~

L-

L--/

digested fkgment
with labeledprimer
Fluorescence-basedsequencer

-3

digested fragment

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 behavior 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-labeled 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 research tool that provides an investigator a rapid way to determine optimal primer and restriction enzyme combinations for bacterial community analysis by T-RFLP. It is located at the Ribosomal Database Project website. This will
facilitate microbial community analysis by T-RFLP.
CONCLUSION
Bioremediation is an interdisciplinary
technology involving microbiology, engineering, ecology, geology, and chemistry. Microbes are the primary stimulant in the bioremedi-

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