Recent technology on bio-remediation
of persistent organic pollutants (POPs)
and pesticides
Kazuhiro Takagi1),2), Ryota Kataoka1) and Kenichi Yamazaki2),1)
1)
Organochemicals Division,
National Institute for Agro-Environmental Sciences (NIAES),
Tsukuba, Japan
Graduate School of Agricultural Chemistry,
Tokyo Univ. of Agriculture, Tokyo, Japan
E-mail: [email protected]
2)
Abstract
The cleanup technology for contaminated soil and water with persistent organic pollutants (POPs) is
required but there are few reports for degradation of POPs. A novel aerobic pentachloronitrobenzene
(PCNB)-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in
a soil-charcoal perfusion system. Strain PD653 also degraded hexachlorobenzene (HCB) with liberation of
chloride ions to CO2 under aerobic conditions. It is the first aerobic bacteria capable of mineralizing HCB. As
well, aerobic dieldrin-degrading fungus, Mucor racemosus strain DDF, was isolated from annually applied
soil with endosulfan. DDF was able to degrade dieldrin more than 90% for 10 days incubation whereas
previously reports degraded dieldrin less than 50%. On the other hand, the application technology to the
contaminated sites is still inadequate to remediate s-triazine-contaminated site. Therefore, we developed a
new method to introduce the degrading-bacterial consortium into contaminated soil using a special charcoal
material which was enriched with a methylthio-s-triazine degrading bacterium and the chloro-s-triazine
degrading bacterial consortium CD7. The enriched charcoal was capable of degrading chloro-s-triazines
(simazine and atrazine) and methylthio-s-triazines (simetryn and dimethametryn) simultaneously in sulfurfree medium. Moreover, using enriched charcoal with CD7 in situ bioremediation study was conducted in a
contaminated site, where simazine is routinely applied for preservation of its turf. The material was effective
for preventing penetration of simazine into subsoils and aquatic environments nearby for approximately 2
years.
Key words: POPs, hexachlorobenzene (HCB), dieldrin, s-triazine, bioremediation.
Introduction
The organochlorine pesticides, e.g. hexachlorobenzene (HCB; C6Cl6) and dieldrin (C12H8Cl6O), are
persistent synthetic chemicals. HCB and dieldrin
have been extensively used for controlling the
fungal disease and insect pests, respectively. They
cause great damage to agricultural crops. However,
their use has been prohibited in many countries
since the 1970s because of their susceptibility to
biological magnification, high toxicity and long
persistence in the environment. Although the halflives of HCB and dieldrin in soil differ in extent
among reports, the average half-life of HCB and
dieldrin in soil are approximately more than 9 and
7 years, respectively (Barber et al., 2005; FAO,
2000). Therefore, HCB and dieldrin were listed
as the persistent organic pollutants (POPs) by the
Stockholm convention in 2001.
HCB and dieldrin are still found in the
environment such as agricultural or/and paddy field
even more than 30 years since their prohibition
(Seike et al., 2007; Hashimoto, 2005). Moreover,
in Japan, dieldrin at residual concentrations
exceeding the limit set by the Food Sanitation
Law of Japan (dieldrin: <0.02 ppm, fresh weight
basis) have been detected in cucumbers which are
produced in some agricultural area (Hashimoto,
2005). Thus, contamination with organochlorine
pesticides is still a serious environmental problem
which requires efficient method for remediation.
Meanwhile, the s-triazines are recognized as
a major class of herbicides and are widely used in
agriculture for controlling various weeds. They are
1
classified into three groups; chloro-, methylthioand methoxy-s-triazines. In the s-triazine family,
chloro-s-triazines such as atrazine and simazine
are the most popular. In particular, atrazine is used
globally to control annual grasses and broadleaf
weeds in fields of the major crops, such as corn,
sorghum, and sugar cane. They have been detected
in ground and surface water (Belluck et al., 1991;
Miles et al., 1996). Methylthio-s-triazines such as
simetryn and dimethametryn are used in paddy soil,
they have been detected in river water, (Miles et
al., 1996; Tanabe et al., 1996) a lake basin (Sudo
et al., 2002), and river sediments (Kawakami et
al., 2006). Chloro- and methylthio-s-triazines were
often detected in river water (Miles et al., 1996)
and river sediment (Kawakami et al., 2006).
Among the removal technologies for such
residual pesticides, bioremediation is considered
to be the most cost-effective because residues
of HCB, dieldrin or s-triazines are known to
have low concentration and has a wide range.
Microbial degradation is a promising effective
technique to remediate environmental pollutants.
For HCB, several studies have reported reductive
dechlorination (Fathepure et al., 1988; Chang
et al., 1998; Jayachandran et al., 2003) and
conversion of HCB to pentachlorophenol (PCP)
by using genetically engineered bacterial cells
(Jones et al., 2001). However, pure culture
capable of mineralizing HCB aerobically has
not been isolated yet. As well, the degradation
of dieldrin using bacteria (Matsumoto et al.,
2008) and fungi (Anderson et al., 1970) has been
reported. Matsumura & Boush (1967) found that
Trichoderma viride, isolated from soil that had been
heavily contaminated with various insecticides. It
could degrade, but not enough, the dieldrin and
there was little information about metabolites and
metabolic pathways of dieldrin. Moreover, no
further studies have since been reported. Therefore,
there is still an urgent need to isolate more effective
dieldrin-degrading microorganisms and develop an
effective bioremediation method for aerobic zones
polluted with organochlorine pesticides.
In contrast, Mandelbaum et al., (1995)
have isolated the triazine-degrading bacteria,
and the most famous degrader was Pseudomonas
sp. strain ADP. The catabolic pathways in the
degradation of chlorinated s-triazines have been
extensively studied in the strain ADP, and six
atrazine catabolic genes, atzABCDEF, have been
determined in this strain (Martinez et al. 2001).
Moreover, bioremediation in atrazine- or simazinecontaminated soil were performed by strain ADP
2
(Newcombe & Crowley 1999; Mora´n et al. 2006).
Here, we describe the aerobic biodegradation
of POPs (HCB and dieldrin) by isolated soil
microorganisms and bioremediation of persistent
pesticides (s-triazines). Keeping in mind, we
describe isolation and identification of aerobic
bacterium and fungus as HCB- and dieldrindegrader, respectively. For HCB, we report the
metabolic pathway caused by oxidative removal
of chlorine groups from HCB to CO2. Moreover,
we report about in situ bioremediation of simazinecontaminated site.
Aerobic biodegradation of POPs by
isolated soil microorganisms
Aerobic mineralization of
hexachlorobenzene by Nocardioides sp.
PD653
Enrichment culture by using the soil-charcoal
perfusion system
Enrichment of PCNB-degrading bacteria was
performed by the original soil-charcoal perfusion
method (Takagi & Yoshioka, 2000; Iwasaki et al.,
2007; Takagi et al., 2009). A soil sample, to which
PCNB had been annually applied for more than
5 years, was taken from a cabbage field (Ibaraki,
Japan) at a depth of 0-20 cm. The soil sample (40
g, dry weight) was mixed with Charcoal A100 (2
g, grain size 5 to 10 mm, BET specific surface area
of 95-110 m2/ g, pH 7.8) as a microhabitat and an
adsorbent of PCNB. Enrichment culture was carried
out under dark conditions at 25°C by circulating
300 ml mineral salt medium (MM) containing 5
mg/L of PCNB through the soil-charcoal mixture
in a perfusion apparatus. The perfusion rate of the
medium was adequately controlled by a portable
air pump and smooth leaching was maintained.
The medium was replaced every week. Aliquots of
culture fluids were centrifuged at 12,000 rpm for 10
min. Concentration of chloride ion in the supernatant
was measured by an ion chromatography.
For determining the PCNB concentration, the
supernatant (5 ml) was passed through a SepPak C18 cartridge, which was pre-conditioned by
washing with acetone, methanol and distilled water,
with a Waters vacuum manifold. The “concentrated”
cartridge was then dried over the vacuum manifold
and subsequently eluted with acetone (5 ml). The
eluate was dried and re-dissolved with 1 ml acetone
for GC/ECD. Following PCNB degradation in the
first enrichment culture, 0.25 g of the charcoal was
transferred to another apparatus with 7.5 g of new
autoclaved Charcoal A100. Further enrichment and
purification were performed by circulating 300 ml
of MM containing 10 mg/L of PCNB.
At the beginning of enrichment, the
generation of chloride ions in the perfusion
apparatus was detected after 1 day of circulation.
PCNB was not detected in the culture fluid.
During 3 weeks of circulation and 2 exchanges of
the medium, the generation rate of chloride ions
increased (Fig. 1-A). After 23 days of circulation,
the charcoal was transferred to the secondary
enrichment culture. The PCNB-degrading bacteria
were highly enriched in the charcoal during the
second enrichment. After 45 days of circulation and
4 exchanges of the medium, the enriched charcoal
was harvested to carry out subsequent colony
isolation (Fig. 1-B).
Isolation of PCNB-degrading bacteria
The enriched charcoal (1g) was crushed and
suspended in 50 mM phosphate buffer (pH 7.0). The
suspension was diluted with the same buffer and
was inoculated on the MM agar plate containing 50
mg/L of PCNB. Following successive incubation at
25°C for 3 weeks, colonies showing a clear zone on
the plate were isolated and subcultured on the same
agar medium. Further purification of a PCNBdegrading bacterium was conducted on R2Aagar
plates at 30°C.
Some bacterial colonies showing clear
zones on MM agar containing 50 mg/L of PCNB
were isolated. Further colony purification of
PCNB-degrading bacteria was performed on R2A
agar. Several types of colonies having different
morphology were observed on R2A agar. The
PCNB-degrading abilities of the individual
secondary isolates were examined in the tube
cultures and subsequent flask cultures. An isolate
that showed distinctive formation of chloride ions
(6.6 mg/L) and nitrite ions (1.7 mg/L) from PCNB
(total 10 mg/L) after a 9-day cultivation in a flask
culture was obtained, and named strain PD653.
Identification of the isolated PCNB-degrading
bacteria
The isolated strain, PD653, was characterized on
the basis of comparative morphology, physiology
and comparison of the 16S rRNA sequences. The
known primers named fD1, fD2, rP1, rP2 and rD1
was used for 16S rRNA gene amplification, and the
following 5’-Texas Red-labeled primer was used
for the cycle sequencing reaction: fD1, rD1, 341f,
534r, 799f (5’-CAAACAGGATTAGATACCC-3’),
907f, 907r and 1223r.
The isolated strain PD653 belongs to a species
of gram-negative, catalase- positive, oxidasenegative, non-spore-forming and non-motile rods
(0.7-0.8 x 1.0-1.2µm in size), which form pale
yellow circular colonies. The GC content of the
Fig. 1. (A) First enrichment culture of PCNB-degrading bacteria in Charcoal A100
using a soil-charcoal perfusion method. (B) Secondary enrichment culture using
a charcoal perfusion method. Changes in concentration of PCNB (o) and chloride
ions (n) in culture fluid are indicated. Downward arrows indicate the time points
of replacement of perfusion fluid. Concentrations of PCNB at the initial point and
replacement points are calculated values of the added PCNB (Takagi et al., 2009).
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strain was 70.8%. The 16S rRNA sequence of strain
PD653 (1,487 nucleotides) was compared with
those of the bacterial sequences in the GenBank.
Strain PD653 exhibited a high sequence similarity
with those of bacteria classified as Nocardioides.
The highest sequence similarity (97.1%) of the
16S rRNA gene was found in Nocardioides sp.
OS4 (Lee et al., 1997). The 16S rRNA of strain
PD653 was aligned with those of the representative
strains of the Actinomycetales, and a phylogenetic
dendrogram was constructed (Fig. 2). Based on
these results, strain PD653 is assigned to a novel
species in the genus Nocardiodes.
HCB degradation
HCB was dissolved prior to experiments in an
acetone solution at a concentration of 500 mg/L.
An appropriate aliquot of this stock solution
was then added to a sterilized Erlenmeyer flask,
and the solvent mixture was evaporated in the
ambient atmosphere. HCB (7.2 mM) in 50 ml
Erlenmeyer flasks was dissolved by adding 20
ml of MM with subsequent sonication, and then
a single colony of PD653 was inoculated. During
the shaking incubation under dark conditions at
25°C, the increase of OD600 was monitored as cell
growth. Decrease of HCB and release of Cl- were
monitored by HPLC and by an ion chromatography,
respectively.
On aerobically culturing strain PD653 in
MM containing HCB, the initial concentration of
8.0 mM of HCB decreased to 1.5 mM during 9 days
of cultivation and accumulation of chloride ions
up to 34.0 mM was observed (Fig. 3). Apparent
increase of OD600 was not obtained after 9 days of
cultivation (Fig. 3).
Mineralization of HCB
The PCNB-degrading bacterium grown on R2A
agar plate was inoculated into glass jars containing
20 ml of MM supplemented with 3.6 mM 14C-HCB.
The jars were shaken in a thermostatic chamber
(30OC, 100 rpm) with a continuous supply of filtersterile air. The exhaust was passed through a PUF
column and a pair of 1-M NaOH traps. Remaining
HCB concentration was determined by HPLC,
and the collected NaOH solution was analyzed by
liquid scintillation counting (LSC). As a control
experiment, duplicate jars were prepared in the
same manner without inoculation of the bacterium
and periodically analyzed.
A study on mineralization of HCB was then
performed using 3.6 mmM 14C-HCB (Fig. 4).
Presence or absence of 14C-HCB in the specimen
was assigned using HPLC equipped with a
radioactivity flow-through detector. In the14day culture fluid, the radioactivity decreased by
39.5% of its initial theoretical value. Radioactive
Fig. 2. Phylogenetic affiliation based on the 16S rRNA sequence data,
showing the relationship of strain PD653 to the most closely
related genera (Takagi et al., 2009).
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Fig. 3. Aerobic degradation of HCB by Nocardioides sp. strain PD653 in the
mineral salt medium (MM). The changes in OD600 of strain PD653 (s) and
evolution of chloride ions (p) in accordance with the HCB degradation (l) are
demonstrated. OD600 and concentrations of the materials are mean values of
the duplicate experiments. Error bars indicate S.D. (Takagi et al., 2009).
Fig. 4. Mineralization of [U-ring-14C] HCB by Nocardioides sp. strain PD653.
Disappearance of 14C-HCB (l) and evolution of 14C-labelled unidentified
water soluble metabolites (p) and 14CO2 (s) are demonstrated. Volatile
14
C-HCB (m) captured on the PUF column is also shown. Radioactivity of
the materials is mean value of the duplicate experiments, and is expressed
as the percentage of that of the initial applied 14C. Error bars indicate S.D.
(Takagi et al., 2009).
HCB was not detected in the culture fluid, and the
radioactivity was predominantly found in unknown
water-soluble metabolites after one day of
cultivation. The adsorbed 14C-HCB residue onto the
jar walls was only 2.2%. The PUF column captured
13.1% of the residue, which was ascertained as
HCB volatilized during cultivation. The NaOH
traps recovered 36.8%, and 84.7% of the trapped
radioactivity was precipitated as Ba14CO3 by
adding BaCl2, indicating that the radioactivity was
attributed to 14CO2.
5
Biotransformation experiment by using the
resting cells
Cells of the PCNB-degrading bacterium were
grown on R2A medium until they reached an
optical density (OD600) of approximately 1.2. After
harvesting the cells by centrifugation (4,000 rpm,
20 min), the cell pellets were washed twice with a
20 mM phosphate buffer (pH 7.0) and suspended
in the same buffer. For turnover experiments with
resting cells, 10 ml portions of a cell suspension
(OD600=1.0) were added to 50 ml Erlenmeyer flasks
containing HCB and pentachlorophenol (PCP)
(21.6 mM each). After shaking for several times
in a rotary shaker at 22oC, 1 ml of 1 N HCl and
10 ml of CH3CN were added to stop the reaction.
Aliquots were withdrawn (1.0 ml), then the sample
was centrifuged (12,000 rpm, 10 min), and 20 ml
of the supernatant was then subjected to HPLC
analysis. For the detection of chlorohydroquinones,
the culture was acetylated by adding 3 ml of 1 M
K2CO3 and 1 ml of acetanhydride, subsequently
extracted by ethyl acetate. The acetylated sample
was analyzed by GC/MS.
In order to identify the intermediate of HCB
catabolism, resting cells of strain PD653 were used
in a degradation experiment. By incubating HCB
with the resting cells, decreased HCB levels and
increased levels of a metabolite were observed
(Fig. 5).
This metabolite was identified as PCP
by its HPLC retention time and UV spectrum;
they were found to be identical to those of an
authentic sample. A stoichiometrically equal
amount of chloride ions corresponding to the
generated PCP was detected in the incubation fluid
(data not shown). Although the only metabolite
detectable by HPLC analysis was PCP, GC/MS
analysis made it possible to detect other minor
metabolites. To detect such minor metabolites,
PCP was incubated with the culture of the resting
cells for a short period (2 h). Though the decrease
of PCP was very little, tetrachlorohydroquinone
and 2,6-dichlorohydroquinone were detected as
acetylated derivatives (Fig. 6).
These intermediates were identified by the
comparison of their GC retention times and MS
spectra with authentic samples. Degradation of
HCB and appearance of these metabolites were
not observed in the control culture with autoclaved
resting cells (Fig. 5). According to the results of
resting-cell study, the putative metabolic pathway
of HCB in strain PD653 is proposed in Fig. 7.
Fig. 5. Generation of PCP accompanied by degradation of HCB by
resting cells of Nocardioides sp. strain PD653. The time courses of
disappearance of HCB (l) and generation of PCP (s) are demonstrated.
The concentrations of HCB (m) and PCP (r) in heat-killed control
cultures are also demonstrated. Mean values (n = 3) and S.D. of
concentrations of the materials are shown (Takagi et al., 2009).
6
Fig. 6. GC/MS analysis of metabolites obtained from degradation of PCP by
resting cells of Nocardioides sp. strain PD653. Acetylated derivatives of
the metabolites were analyzed. The scanning was carried out at mass
range of 50–600 (m/z) (Takagi et al., 2009).
Fig. 7.Possible metabolic pathway of HCB by Nocardioides sp. strain
PD653 under strict aerobic conditions (Takagi et al., 2009).
Degradation of dieldrin using fungi
isolated from endosulfan contaminated soil
Microorganisms and isolation of soil fungi from
contaminated soil with endosulfan
Thirty-five of Trichoderma spp. were provided
from Dr. Yokota, Tokyo Univ. of Agriculture, which
isolated from raw wood cultivated by Lentinula
edodes (Berk.) Pegler.
Six soil samples collected from different
sites having a history of repeated endosulfan
applications were used in this enrichment study
for the isolation of dieldrin-degrading fungi. One
gram of each soil was mixed with 20 ml of sterile
distilled water with a Universal Homogenizer, and
then diluted to 10-2, 10-3 and 10-4; 100 µl of the
resulting soil solutions were placed onto a plate of
7
Martin agar medium with chloramphenicol (0.25
g/L). The plates were then incubated at 25°C for
5 days. The fungal colonies were classified into
several groups according to their morphology,
growth rate and color; and representatives of each
group were picked up with a sterile needle and
subcultured on potato dextrose agar (PDA) plates.
Sixty three fungal strains were isolated from soil
contaminated with endosulfan.
Degradation experiment
All fungi isolated were grown on PDA agar
medium in Petri dishes at 25oC. Fungal disks (6
mm diameter) were taken from the margin of the
fungal colonies grown for 14 days. A fungal disk of
each fungus was transfered into a modified czapek
dox (MCD) liquid medium for the degradation
experiment. After pre-incubation for 7 days,
50 µl of dieldrin in acetone was added to each
inoculated flask (final concentration: 13.2 µM/
flask). To prevent the volatilization of dieldrin, the
flask was sealed with a glass stopper. The cultures
were incubated statically for 14 days at 25oC.
As a control, the cultures were killed by heating
treatment using autoclave after an initial 7 days of
incubation. The cultures were homogenized with
15 ml of acetone, and the residual biomass was
removed by centrifugation at 3,000 x g for 10 min.
One ml of supernatant was transferred into the test
tube, and extracted by 5 ml of hexane. Dieldrin
was analyzed using GC/ECD. Moreover, the time
course of degradation test was performed using
superior fungus for degrading dieldrin.
At first, the degradation experiment was
performed using 35 strains of Trichoderma spp.
Almost all fungi could not degrade dieldrin
compared with control. However, we found a
Trichoderma sp. strain 93155 which was capable of
degrading dieldrin. The degradation of dieldrin was
19.7 %. The previous reports used Trichoderma
spp. also degraded approximately 20%. We think
Trichoderma spp. can degrade 20% at best. But
it should be difficult if Trichoderma spp. are used
in situ remediation. Therefore, further screening to
find superior fungus compared with Trichoderma
spp. was performed. As a result, we found two fungi
isolated from soil contaminated by endosulfan
and named ACM and DDF. Their degradation of
dieldrin in strain ACM and DDF was 43.3 % and
95.8 %, respectively. Especially, DDF degraded 96
% of dieldrin in MCD culture media. Moreover,
time coarse changing of degrading dieldrin for
strain DDF was performed for 3 weeks. The result
showed that DDF degraded dieldrin rapidly for 10
days with the growth of the fungus, and the mean
values were more than 90% (Fig. 8). This result
created a strong impact because the degradation
was less than 50% in almost all of previous reports.
Identification of dieldrin-degrading fungus
To identify the fungal species of ACM and DDF, DNA
was extracted from pure cultures of fungi by using
a FastDNA Kit as described by the manufacturer
(Q-BIOgene). The internal transcribed spacer (ITS)
Fig. 8. The time courses of disappearance of dieldrin (circles)
and growth of strain DDF (squares) are demonstrated. Three
replicates was performed in this study, and error bar means S.D.
8
region of ribosomal DNA was amplified using
primers ITS1 and ITS4. DNA sequences were
determined using the ABI 3130 Genetic Analyzer
with a reaction kit (Big Dye Terminator v.1.1 Cycle
Sequencing Kit) following the manufacturer’s
manual. The DNA sequence was compared with
the sequences of known species in the GenBank
database. In the case of identities above 97% in
both ITS1 and ITS2 regions, the species name was
assigned to the morphotype. All sequence data,
including newly obtained and retrieved sequences,
were aligned with the computer program ClustalX.
Distance- based phylogenetic trees were generated
by the model of Jukes and Cantor (1969) and a
neighbor-joining algorithm (Saitou and Nei 1987).
The topology of phylogenetic trees was evaluated
by bootstrap resampling (1000 replicates). Clustal
W provided by DNA Data Bank of Japan was used
for the analyses.
The rRNA gene sequences have been shown
to provide taxonomically useful information. The
fungi represented 43.3 % and 95.8 % of degradation
were Actinomucor sp. and Mucor sp., respectively.
Individual homology was over 95 %. Moreover,
to determine whether Mucor sp. could accurately
identify relationships among diverse mold taxa,
we compared phylogenetic trees constructed with
ITS sequences. In addition, high bootstrap values
are observed at the deeply branching nodes, and
similarly high values are observed at branches
separating more closely related genera. Thus,
Mucor sp. closely related to Mucor racemosus f.
racemosus (Fig. 9). To date, Mucor alternans was
previously reported to degrade dieldrin and DDT
by Anderson et al. (1970). However, M. alternans
isolated by them degraded only 20% of dieldrin.
The degradation of strain DDF is superior to M.
alternans, therefore, strain DDF was expected to
use bioremediation of dieldrin.
Fig. 9. Phylogenetic affiliation based on the 16S rRNA sequence data,
showing the relationship of strain DDF to the most closely related genera.
9
Bioremediation of persistent
pesticides(s-triazines)
Simultaneous biodegradation of chloro
and methylthio-s-triazines with a newly
bacterial consortium
Bacterial consortium CD7 was obtained which
can mineralize simazine by using the soilcharcoal method (Takagi & Yoshioka, 2000), and
a degrading bacterium b-Proteobacteria CDB21
was isolated from CD7 by Iwasaki et al. (2007).
It was considered that CD7 was more effective
for bioremediation than strain CDB21 because
CD7 could utilize simazine as sole carbon and
nitrogen sources in mineral salt medium (Takagi &
Yoshioka, 2000; Iwasaki et al., 2007). In addition,
it was considered that a special type of charcoal
A100 was effective as a bacterial carrier because
Charcoal A100 has the following benefits. Charcoal
A100 is effective as a high absorbent material
of organic compounds because surface specific
area of Charcoal A100 (100 m2/g) is higher than
surface specific area of a soil. The second benefit is
useful as a microhabitat of degrading bacteria and
degrader can keep their degrading activity in long
term, because a micropore of Charcoal A100 was
optimized as a microhabitat. The effectiveness of
Charcoal A100 enriched with CD7 was described
previously (Takagi & Yoshioka, 2000; Takagi et
al., 2004). Thus, Charcoal A100 enriched with
CD7 is considered to be an effective material for
bioremediation. However, CD7 and strain CDB21
could not degrade methylthio-s-triazines, while
Rhodococcus sp. FJ1117YT (Fujii et al., 2007) can
transform methylthio-s-triazines to their hydroxyl
analogues via sulfur oxidation, and accumulate
hydroxy-triazines. The expected metabolic
pathways of chloro- and methylthio-s-triazines in
the mixed culture are shown in Fig. 10.
To take advantage of these useful properties
of CD7 and Charcoal A100, this topic describes
the development of Charcoal A100 enriched with
strain FJ1117YT and CD7, and demonstration
of the simultaneous degradation of chloro- and
methylthio-s-triazines using this novel material.
Enrichment of Charcoal A100 with strain
FJ1117YT and CD7
At first, washed Charcoal A100 was packed in a
perfusion apparatus and autoclaved. Subsequently,
two pieces of stab cultures of CD7 on an Mineral salt
medium (MM) agar plate containing simazine were
Fig. 10. The expected metabolic pathways of chloro- and methylthio-s-triazines
degraded by CD7 (strain CDB21) and strain FJ1117YT. Simazine (R1, R2= C2H5)
and atrazine [R1= C2H5, R2=CH(CH3)2] were selected as chloro-s-triazines, and
simetryn (R1, R2= C2H5) and dimethametryn [R1= C2H5, R2= CH(CH3)CH(CH3)2]
were used as methylthio-s-triazines (Yamazaki et al., 2008).
10
placed on the charcoal. The surface of charcoal
layer was covered with a glass microfiber filter.
Enrichment was performed in the dark conditions
at 25˚C. MM containing 5 mg/l simazine was
perfused with air lift using an air pump for 14 days.
The perfusion fluid was replaced twice during the
enrichment. A phosphate buffer suspension (pH 6.9)
of strain FJ1117YT was, then, placed on the filter
paper. MM containing 5 mg/l each of simazine and
simetryn was perfused under the similar conditions
for 43 days. The perfusion fluid was replaced four
times during the second enrichment Charcoal A100
enriched with strain FJ1117YT and CD7 showed
faster decrease in the concentrations of simazine
and simetryn in MM than the non-inoculated
control. This result indicated the enrichment of
strain FJ11177YT and CD7 in Charcoal A100.
Detection of bacterial community in
Charcoal A100 enriched with strain
FJ1117YT and CD7
After enrichment of strain FJ1117YT and CD7 in
Charcoal A100, the establishment of the bacterial
community in Charcoal A100 was monitored by
PCR-DGGE. Primers designed from a sequence of
the variable V3 region of 16S rRNA, were used.
PCR-DGGE was performed and strain FJ1117YT
and all the species consisting of CD7 were detected.
Simultaneous degradation of chloro- and
methylthio-s-triazines using Charcoal A100
enriched with strain FJ1117YT and CD7
Charcoal A100 enriched with strain FJ1117YT and
CD7 (0.4 g dry weight) was inoculated in sulfur-free
mineral salt medium (MM-S) (30 ml) containing
5 mg/l each of simazine, atrazine, simetryn, and
dimethametryn or in MM-S containing 5 mg/l of
each herbicide. The flasks were shaken at 120 rpm
at 25˚C for 15 days. As controls, sterile Charcoal
A100 and/or Charcoal A100 enriched with only
CD7 were inoculated and shaken under the same
conditions. The concentration of s-triazines in the
medium was determined periodically by HPLC.
Charcoal A100 enriched with strain FJ1117YT
and CD7 was applied to simultaneous degradation
of chloro- and methylthio-s-triazines. Simazine
and atrazine were degraded to by 80−100% with
Charcoal A100 enriched with both strain FJ1117YT
and CD7 or CD7 alone after 9 days, and were
completely degraded after 15 days (Fig. 11-A, B).
On the other hand, simetryn and dimethametryn
were degraded by over 80% with strain FJ1117YT
and CD7 enriched charcoal in sulfur-free medium,
but they were not degraded in the presence of sulfate
(in MM-S) (Fig. 11-C, D). However, Charcoal A100
enriched with strain FJ1117YT and CD7 could be
a promising model to construct a multifunctional
material enriched with bacterial consortium for
in situ bioremediation. On the basis of this study,
we constructed another charcoal material, which
include methylthio-s-triazines-degrading bacteria
(Nocardioides sp. strain MTD22; Yamazaki et al.,
2008) that are not suppressed by external sulfur
sources (Takagi et al., 2008). And degradation of
muliti-triazines in soil was successful by this new
material (Charcoal A100 enrich with CD7+strain
MTD22; Yamazaki et al., 2009).
In situ bioremediation of simazinecontaminated site by using Charcoal A100
enriched with bacterial consortium
It has been obscure whether the charcoal enriched
with degrading bacteria has been effective or not
under field conditions. To demonstrate this, we
conducted a field experiment by laying charcoal
enriched with simazine-degrading bacterial
consortium CD7 under the subsoil of a golf course
to prevent the contamination of subsoils, rivers, and
groundwater with the herbicide simazine, which is
widely used on golf courses throughout Japan and
frequently detected in river water.
In situ bioremediation of simazine-contaminated
site
We placed a 1-cm-thick layer of the charcoal A
enriched with CD7 under the subsoil at 15 cm deep
of a treatment plot in a golf course. In the control
plot, charcoal without CD7 was laid in the same
manner as in the treated plot. Porous glass cups
were inserted at 4 locations in each plot to collect
the soil solution directly beneath the charcoal layer.
After the simazine application, we periodically
examined changes in the simazine concentration in
the soil solution and the soil and charcoal layers.
The simazine was applied twice a year for 2 years.
The results were as follows.
Simazine conc. in soil solution
In the control plot, simazine at a conc. of 0.01
mg/L or more was detected at all locations until
the seventh week after the first application. In
the treated plot, the simazine conc. was 0.005
11
Fig. 11. Time course of simultaneous degradation of chloro- and methylthio-striazines with Charcoal A100 enriched with strain FJ1117YT and CD7 (Yamazaki
et al., 2008). Degradation of simazine (A), atrazine (B), simetryn (C), and
dimethametryn (D) by strain FJ1117YT and CD7 with (s)or without (o) sulfate,
with CD7 alone (m), and using non-enriched Charcoal A100 as a control (n) are
shown (Yamazaki et al., 2008).
mg/L or less at all the sampling locations until
the sixth week after the first application; it was
not detected from the seventh week onward. The
simazine-degradation rate in the soil water of the
treatment plot reached 92% in 6 months after the
first application, compared with the control plot.
However, this rate was slightly retarded to 70%
after the second application, compared with the
control plot. After the third and fourth application,
the degradation rate in the soil water of the treated
plot was still more than 60%.
Simazine conc. in the charcoal layer
The conc. of simazine in the charcoal layer was
maintained at 5 to 8 mg/kg dry matter until 6
months after the first application in the control
plot, owing to the adsorption of simazine. In the
treated plot, the conc. reached a maximum (3 mg/
kg dry mater) 1 month after the first application,
and afterward decreased to 1/20 of the residual
amount in the control plot at 5.5 months after the
12
first application. After the 2nd application a similar
trend was observed. In the treatment plot, because
the bacteria living within the charcoal degraded
the simazine adsorbed on it, the residual amount
of simazine was much less than in the control plot
(Fig. 12).
Long-term monitoring of simazinedegrading bacteria in charcoal by DGGE
methods
In long-term bioremediation studies, changes
of bacterial community and resulting changes
of initially inoculated bacterial consortia are
common. To elucidate whether composition of
CD7 has been changed or not during the study,
PCR-DGGE study of the bacterial DNA extracted
from the charcoal was examined. As a result, PCRDGGE analysis showed that composition of CD7
has been unchanged and three bacteria, which have
constituted the consortium CD7, have survived
stably in the charcoal placed under subsoil during
Fig. 12. Change in concentration of simazine in soil water (15-20 cm)
at both plots and number of simazine-degrading bacteria in charcoal
after introducing into subsoil at treatment plot. In the control plot, a
charcoal material without bacterial enrichment was laid in the same
manner as in the treatment plot. Simazine application was performed
4 times (see arrows) during the experiment (Takagi et al., 2004).
Fig. 13. Long-term monitoring of CD7 in the charcoal A by PCR-DGGE methods (B).
Sampling was performed on Nov. 22, 2000 (1), Dec. 26, 2000 (2), Feb. 11, 2001
(3), Mar. 31, 2001 (4), May. 2, 2001 (5), Oct. 13, 2001 (6), Mar. 31, 2002 (7)
and May. 25, 2002 (8). Lane C is a result in the simazine-degrading bacterial
consortium, consisting of strain CDB21, Bradyrhizobium japonicum CSB1 and
Arthrobacter sp. strain CD7w (Takagi et al., 2004).
13
2 years of the bioremediation study (Fig. 13). More
precise studies using specific DNA probes for the
simazine-degrading bacterium are being carried
out.
Therefore, by laying charcoal enriched with
simazine-degrading bacterial consortium under
the subsoil in contaminated fields, we are able to
minimize simazine pollution of the subsoil, river
water, and groundwater for at least several years.
Conclusion
The soil pollution with organic chemicals (e.g.
POPs, persistent pesticides) is a worldwide
environmental problem affecting the ecosystems
and human health. Therefore, soil remediation is
necessary for polluted arable land in most countries
including Asian countries to produce safe crops
and reduce environment risks. Soil microbes
characterized by wide catabolic capabilities,
are known to degrade contaminants. Their
biotechnological use might lead to the development
of bioremediation processes for cleaning
contaminated soils. Nevertheless, the isolation of
degraders against POPs is inadequate to remediate
the contaminated environment. In order to rapidly
enrich and isolate POPs-degrading bacteria, we
have developed a soil-charcoal perfusion method
using special charcoal (Charcoal A100) as a
microhabitat and adsorbent of organic chemicals.
Using this method, we have already isolated several
aerobic bacteria capable of degrading persistent
pesticides (e.g. s-tirazines, PCNB, PCP, HCB)
Furthermore, we have developed charcoal enriched
with degrading bacterial consortium as a material
for bioremediation. Indeed, by mixing the material
with contaminated soils, persistent pesticides were
adsorbed onto the charcoal and rapidly degraded
by bacteria living in it. By laying charcoal enriched
with simazine-degrading bacterial consortium
under the subsoil in contaminated fields, we have
demonstrated to minimize simazine pollution
of the subsoil and aquatic environments for 2
years. The Charcoal A100 enriched with POPsdegrading bacterial consortium could be applicable
to POPs-contaminated sites. As well, Mucor
racemosus DDF as a dieldrin-degrader was
isolated from contaminated soil with endosulfan
using a conventional method. However, this strain
degraded dieldrin the most effective compared with
previous reports. This strain is expected to apply
to in situ bioremediation. We have to develop a
suitable carrier and substrate for this soil fungus
before introducing into contaminated site.
14
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