Download 24F07-07 (1238) Dynamic Behavior of Bacteria in PCE

Proceedings of International Symposium on EcoTopia Science 2007, ISETS07 (2007)
Dynamic Behavior of Bacteria in PCE Contaminated Surface
During Bioremediation
Arpita Bhowmik1, Akane Asahino 2, Takanori Shiraki 2, Kohei Nakamura2
and Kazuhiro Takamizawa1, 2
1. United Graduate School of Agricultural Science, Gifu University, Gifu, Japan.
2. Faculty of Applied Biological Science, Gifu University, Gifu , Japan
Abstract: Among the most ubiquitous chlorinated compounds as major environmental pollutants, tetrachloroethylene (PCE) is a common groundwater contaminant due to its widespread use as solvent. It is
used in dry cleaning and also used to degrease metal parts in the automotive and other metalworking industries. PCE is toxic at low levels, and its density causes it to sink below the water table. A potential
method for managing PCE contaminated sites is to evaluate the intrinsic and enhanced bioremediation. In
this study we characterized the microbial community in the PCE contaminated soil. DNA has been extracted in soil samples from PCE-contaminated sites. Variable behavior of microbes has been observed
between natural attenuation experiment and biostimulation experiment that has been mediated by the addition of nutrients. Results of Denaturing Gradient Gel electrophoresis (DGGE) of amplified bacterial
16S rDNA in case of biostimulation show that the microbial community was dominated by species phylogenetically related to the βproteobacteria. In case of natural attenuation, sequences have been found
belonging to the multiple species of different phyla. Interestingly, we have found sequences matched to
the species belonging to the Firmicutes which contains bacteria capable of reductive dehalogenation. According to this finding we are assuming the possibility of the presence of some Clostridium like PCE degraders within the microbial community in either bioremediation or biostimulation experiments. We are
continuing our study for the finding of microbial species with more specific and significant characteristics
in this regard.
Keywords: Dynamic behavior of bacteria, DGGE, PCE, Bioremediation and Biostimulation.
(DGGE) since it is an electrophoretic separation method
based on differences in melting behavior of double
stranded DNA fragments for even a singe base pair
change and DGGE can detect the presence of particular
bacterial genera that is present in large portion of the
population [4].
1. INTRODUCTION
Anthropogenic chlorinated organic compounds such as
tetrachloroethylene (PCE) and trichloroethylene (TCE)
are common groundwater contaminants due to their
widespread use as solvents which can cause even liver
cancer in human. PCE is not soluble in water and resistant to biodegradation in aerobic subsurface environment.
Several studies showed that under anaerobic conditions,
these compounds can be reductively dehalogenated to
less chlorinated ethenes or innocuous ethane by microorganisms through dehalorespiration [1]. Because this
process has the potential of completely detoxifying chlorinated ethenes, it is recognized as a promising approach
for the cleanup of contaminated sites and has been extensively studied for in situ bioremediation.
The factors those affect reductive dehalogenation are
the metabolic capabilities of microorganisms and interactions among the microorganisms involved. To date a
number of bacteria have been cultivated that are capable
of reductive dehalogenation of chlorinated ethenes [2] In
this study we report on phylogenetic identification of organisms in a microbial community pursuing both biostimulation and natural attenuation experiment with the
samples collected from PCE contaminated sites and we
have found some dominant phyla those contain PCE degraders. In this connection, for extracted DNA from soil
samples, we have carried out experiments by amplifying
16S rRNA genes (16S rDNA) using universal primers [3]
pursuing Denaturing Gradient Gel Electrophoresis
2. MATERIALS AND METHODS
2.1. Soil samples
Ground water polluted soils have been collected from
PCE contaminated sites belonging to a company.
2.2. DNA Extraction
Mo Bio ULTRACLEANTM soil DNA kit has been used
for DNA extraction according to the directions of the
manufacturer.
.
2.3. Analytical method
PCE, TCE, cis-dichloroethylene (cis-DCE), vinyl chloride (VC) were quantified using 250μL head space sample in gas chromatography system equipped with a flame
ionization detector according to the Japanese Standard
Method JIS K0102.
2.4. 16S rDNA amplification and DGGE analysis
For Bacteria-specific PCR of 16S rDNA fragments, the
primers GC 341F (E-coli position 341-358) (5′-CGC
Corresponding author: K.Takamizawa, [email protected]
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Proceedings of International Symposium on EcoTopia Science 2007, ISETS07 (2007)
CCG CCG CGC GCG GCG GGC GGG GCG GGG
GCA CGG GGG G CCT ACG GGA GGC AGC
AG-3′) and 517 R (E-coli position 517-534) ( 5′-ATT
ACC GCG GCT GCT GG -3′) were used.
The PCR cocktail contained 3μl of 4μM of each of
primers, 5μl of 10xPCR buffer (Applied Bio system), 5μl
of 2mM dNTP (TAKARA), 1μl of Bovine serum albumin
(20mg/ml) (TAKARA), 1μl of DNA extract and 0.25μl of
Ampli Taq goldTM (5U/μl) (Applied Biosystem) in a final
volume of 50μl. PCR amplification was carried out with
TAKARA PCR Thermo Cycler. After initial denaturation
at 950C for 15 min, 35 cycles of denaturation at 94ºC for
1 min, primer annealing at 53ºC for 1 min and extension
at 72ºC for 2 min was performed followed by a final extension step at 72ºC for 120 min.
DGGE analysis of 16S rDNA fragments was performed using DCodeTM universal mutation detection system (BIO-RAD). Gels (16cm x 16cm) consisted of 40%
bis-: acrylamide (37:5:1) and a denaturant gradient of
30%-60%. Electrophoresis was performed in 1xTAE
buffer at 600C and 130V for 5h. Gels were attained for 30
min with a 1:10000 dilution of SYBR gold (Invitrogen)
and analyzed using TOYOBO Electronic U, V, Transilluminator and Dark Reader Transilluminator (Clare
Chemical Research).
changes in the bacterial community throughout the experiment. Because the main purpose of this study was to
identify the dominant microorganisms involved dehalogenation. (Fig.2).
2.5. Sequence analysis
Sequence analysis has been performed through a
BLAST [5] search against the Gene Bank database.
3. RESULT
3.1. PCE dechlorination activities of soil samples:
The concentration of the intermediate products of PCE
degradation has been measured with four samples (Fig .1).
Two samples were subject to natural attenuation and the
other two were subject to biostimulation. In each case it
has been observed that dechlorination of PCE has been
occurred.
Fig.2. DGGE patterns of 16S rDNA fragments from
the corresponding samples. Strain KYT-1 has been used
as a control, since its sequence is known previously. Four
samples have been taken for experiment of which two for
biostimulation (Bios.1 and Bios. 2) and two for natural
attenuation (N.ATT.1 and N.ATT.2) experiment. Bands
subject to sequence analysis are labeled with numbers
(Refer to Table 1).
50
40
30
20
10
BIOS. 1
N. ATT. 1
N. ATT. 2
VC
cisDCE
TCE
PCE
VC
cisDCE
TCE
PCE
VC
cisDCE
TCE
PCE
VC
cisDCE
TCE
0
PCE
Concentration (mg/l)
3.2. DGGE and Sequence analysis
Samples were subject to DGGE analysis to assess
BIOS. 2
Soil samples
Fig.1. PCE-dechlorinating activities in soil samples under biostimulation (BIOS.1 and Bios. 2) and natural
attenuation (N. ATT. 1 and N.ATT. 2) conditions.
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Proceedings of International Symposium on EcoTopia Science 2007, ISETS07 (2007)
Table 1. Phylogenetic affiliation of 16SrDNA fragments retrieved in this study
(Band numbering refers to the DGGE gel in Fig. 2)
Band
number
Sample
Microorganism
Homology (%)
Phylum
１
KYT-1
Clostridium sp.KYT-1
95
Firmicutes
Massilia timonae
91
Proteobacteria(β）
Petrobacter succinimandens
85
Proteobacteria(β）
4
Dyganella sp. clone
92
Proteobacteria(β）
5
Burkholderia sp.clone
92
Proteobacteria(β）
6
Dysgonomonas gradei
93
Bacteroidetes
Bacteroides sp.
95
Bacteroidetes
8
Prevotera sp.
91
Bacteroidetes
9
Clostridium sp.clone
97
Firmicutes
10
Clostridium sp.
96
Firmicutes
Clostridium sp.
87
Firmicutes
12
Clostridium sp.clone
97
Firmicutes
13
Clostridium sp.
96
Firmicutes
14
Clostridium sp.
96
Firmicutes
15
Nitrosomonas sp.
96
Proteobacteria(β）
Nitrosomonas sp.
95
Proteobacteria(β）
17
Zoogloea sp.
92
Proteobacteria(β）
18
Azoarcus denitrificans
92
Proteobacteria(β）
2
3
BIOS- 1
7
N.ATT-1
11
N.ATT-2
16
BIOS-2
4. DISCUSSION
In one case, as shown in Table 1, we have found sequences matched to the species belonging to the phylum
Firmicutes which contains bacteria capable of reductive
dehalogenation. According to this finding we are assuming the possibility of the presence of some Clostridium
and Dehalobacter-like PCE degraders within the microbial community in the experimented site.
In case of some samples it has been found that β
Proteobacteria tend to be dominant. Since they are facultative bacteria those can exist in anaerobic condition, we
are hypothesizing that some microorganisms of this phylum capable of dehalogenation of PCE could be present
in the site.
We are continuing our study in this regard to find out
and characterize these PCE degraders more specifically.
In this connection we have taken three points in consid-
eration that which PCE degraders exist in the contaminated site and whether in case of biostimulation the nutrients also stimulate some pathogens and as a result of
that whether any secondary pollution occurs.
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