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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
2)Graduate School of Agricultural Chemistry,
Tokyo Univ. of Agriculture, Tokyo, Japan
E-mail: ktakagi@affrc.go.jp, 2011-07-13

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 sulfur-free 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.

Key words: POPs, hexachlorobenzene (HCB), dieldrin, s-triazine, bioremediation.

Introduction

The organochlorine pesticides, e.g. hexachloro-benzene (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 half-lives 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 classified into three groups; chloro-, methylthio- and 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 simazine-contaminated soil were performed by strain ADP (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 dieldrin-degrader, 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 simazine-contaminated 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 Sep-Pak 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(1081)-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(1081)-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 PCNB-degrading 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 strainPD653.

  • 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, oxidase-negative, 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 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(1266)). 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 ?M) 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 ??M of HCB decreased to 1.5 ?M during 9 days of cultivation and accumulation of chloride ions up to 34.0 ?M was observed ( Fig. 3(1138)). Apparent increase of OD600 was not obtained after 9 days of cultivation ( Fig. 3(1138)).

  • 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 ?M 14C-HCB. The jars were shaken in a thermostatic chamber (30OC, 100 rpm) with a continuous supply of filter-sterile 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 ?mM 14C-HCB ( Fig. 4(1329)). Presence or absence of 14C-HCB in the specimen was assigned using HPLC equipped with a radioactivity flow-through detector. In the14-day culture fluid, the radioactivity decreased by 39.5% of its initial theoretical value. Radioactive 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.

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 ?M 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 ?l 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(1016)).

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(1159)).

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(1016)). According to the results of resting-cell study, the putative metabolic pathway of HCB in strain PD653 is proposed in Fig. 7(1081).

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 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(1245)). 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) 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(1109)). 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.

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 soil-charcoal method (Takagi & Yoshioka, 2000), and a degrading bacterium ?-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(1145).

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 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(1069)-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(1069)-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 Simazine-Contaminated 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 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 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(1097)).

Long-Term Monitoring of Simazine-Degrading 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, PCR-DGGE 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 2 years of the bioremediation study ( Fig. 13(1100)). 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 degradedby 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 POPs-degrading 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.

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Index of Images

Figure 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 (?) 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).

Figure 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 (?) 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).

Figure 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).

Figure 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).

Figure 3 Aerobic Degradation of HCB by Nocardioides SP. Strain PD653 in the Mineral Salt Medium (MM). the Changes in Od600 of Strain PD653 (?) and Evolution of Chloride Ions (?) in Accordance with the HCB Degradation (?) Are Demonstrated. Od600 and Concentrations of the Materials Are Mean Values of the Duplicate Experiments. Error Bars Indicate S.D. (Takagi Et Al., 2009).

Figure 3 Aerobic Degradation of HCB by Nocardioides SP. Strain PD653 in the Mineral Salt Medium (MM). the Changes in Od600 of Strain PD653 (?) and Evolution of Chloride Ions (?) in Accordance with the HCB Degradation (?) Are Demonstrated. Od600 and Concentrations of the Materials Are Mean Values of the Duplicate Experiments. Error Bars Indicate S.D. (Takagi Et Al., 2009).

Figure 4 Mineralization of [U-Ring-14C] HCB by Nocardioides SP. Strain PD653. Disappearance of 14C-HCB (?) and Evolution of 14C-Labelled Unidentified Water Soluble Metabolites (?) and 14co2 (?) Are Demonstrated. Volatile 14C-HCB (?) 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).

Figure 4 Mineralization of [U-Ring-14C] HCB by Nocardioides SP. Strain PD653. Disappearance of 14C-HCB (?) and Evolution of 14C-Labelled Unidentified Water Soluble Metabolites (?) and 14co2 (?) Are Demonstrated. Volatile 14C-HCB (?) 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).

Figure 5 Generation of PCP Accompanied by Degradation of HCB by Resting Cells of Nocardioides SP. Strain PD653. the Time Courses of Disappearance of HCB (?) and Generation of PCP (?) Are Demonstrated. the Concentrations of HCB (?) and PCP (?) 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).

Figure 5 Generation of PCP Accompanied by Degradation of HCB by Resting Cells of Nocardioides SP. Strain PD653. the Time Courses of Disappearance of HCB (?) and Generation of PCP (?) Are Demonstrated. the Concentrations of HCB (?) and PCP (?) 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).

Figure 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).

Figure 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).

Figure 7 Possible Metabolic Pathway of HCB by Nocardioides SP. Strain PD653 under Strict Aerobic Conditions (Takagi Et Al., 2009).

Figure 7 Possible Metabolic Pathway of HCB by Nocardioides SP. Strain PD653 under Strict Aerobic Conditions (Takagi Et Al., 2009).

Figure 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.

Figure 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.

Figure 9 Phylogenetic Affiliation Based on the 16S Rrna Sequence Data, Showing the Relationship of Strain DDF to the Most Closely Related Genera.

Figure 9 Phylogenetic Affiliation Based on the 16S Rrna Sequence Data, Showing the Relationship of Strain DDF to the Most Closely Related Genera.

Figure 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).

Figure 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).

Figure 11 Time Course of Simultaneous Degradation of Chloro- and Methylthio-S-Triazines 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 (?)or without (?) Sulfate, with CD7 Alone (?), and Using Non-Enriched Charcoal A100 As a Control (?) Are Shown (Yamazaki Et Al., 2008).

Figure 11 Time Course of Simultaneous Degradation of Chloro- and Methylthio-S-Triazines 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 (?)or without (?) Sulfate, with CD7 Alone (?), and Using Non-Enriched Charcoal A100 As a Control (?) Are Shown (Yamazaki Et Al., 2008).

Figure 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).

Figure 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).

Figure 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).

Figure 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).

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