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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: [email protected]

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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