Monitoring of Ralstonia eutropha KT1 in Groundwater in an

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Jun 18, 2001 - workshop: innovative potential of advanced biological systems for remedia- tion, 2 to 4 March 1998, Technical University Hamburg-Harburg, ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2002, p. 412–416 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.1.412–416.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 68, No. 1

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR Katsuji Tani,1 Masahiro Muneta,1 Kanji Nakamura,2 Katsutoshi Shibuya,3 and Masao Nasu1* Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871,1 Corporate Research and Development Center, Kurita Water Industries Ltd., Morinosato, Atsugi, Kanagawa 243-0124,2 and Institute of Technology, Simizu Corporation, Kohtoh, Tokyo 135-8530,3 Japan Received 18 June 2001/Accepted 10 October 2001

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 ⴛ 105 cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 ⴛ 107 cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR. (NH4)2SO4, 0.5 g; MgSO4 䡠 7H2O, 0.05 g; minor elements solution, 2.5 ml/liter (pH 7.2 ⫾ 0.2)] containing 2 mM toluene. The composition of the minor elements solution is follows: H3BO4, 0.232 g; ZnSO4 䡠 7H2O, 0.174 g; Fe2SO4(NH4)2SO4 䡠 6H2O, 0.116 g; CoSO4 䡠 7H2O, 0.096 g; (NH4)6Mo7O24 䡠 4H2O, 0.232 g; CuSO4 䡠 5H2O, 8.0 mg; MnSO4 䡠 4H2O, 8.0 mg/liter of distilled water. After the induction of the enzymes involved in TCE degradation, the cells were collected, washed, and suspended in dechlorinated tap water. Seven thousand liters of cell suspension (the optical density at 600 nm was approximately 1.0) was injected into the aquifer at the rate of 10 liter/min. After the injection of the cell suspension, dechlorinated water was injected into the aquifer at a rate of 10 liters/ min for 5 h in order to diffuse the activated cells of R. eutropha KT1 in the aquifer. It is theorized that the amount of activated R. eutropha KT1 required to degrade TCE in groundwater will decrease because the expression of enzymes involved in TCE degradation is inducible and there might not be inducers such as toluene. Thus, the groundwater was extracted continuously to remove R. eutropha KT1 at a rate of 3 liters/min in order to minimize the influence of the bacterial injection on the ecosystem at the test site. Field treatment for in situ bioaugmentation at the Kururi experimental site was started on 29 September 2000. Groundwater samples were collected from four sampling wells (W1, W2, W3, and the control well) at the middle of the aquifer 1 day before the start of the bioaugmentation treatment (28 September 2000) and then 0, 2, 4, 7, 12, 18, 25, 33, 46, 65, and 87 days after the start of the treatment. W1 is the injection and extraction well. W2 and W3 are located 0.7 and 2.05 m downstream from W1, respectively. Groundwater was also collected at the control well, which is located about 50 m upstream of the bioaugmentation experimental site and was not influenced by the treatment. Bacterial cells were collected by filtration and resuspended in freshly filtered 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C. After 16 h of incubation, the cells were washed twice in PBS

Bioremediation, the use of microbes to degrade environmental contaminants, has received increasing attention as an effective biotechnology to clean up polluted environments because it offers several advantages over the traditional chemical and physical treatments for diluted and widely dispersed contaminants. In situ treatment is one of the most attractive advantages of this technology, which enables us to remediate a contaminated site with minimum site disruption and without transportation of the contaminants (4, 12, 13). The establishment of methods to monitor microbes and their genes in the natural environment is desirable because it is necessary to understand the dynamics of bacteria that degrade pollutants in order to carry out bioaugmentation efficiently and safely. In particular, in Japan most contaminated sites are located close to residential areas. Thus, for public acceptance, it is very important to monitor microbes and to prove that this technology is safe (6, 14). In situ bioaugmentation treatment to remove trichloroethylene (TCE) in the groundwater was carried out in Japan. The remaining cells injected into the aquifer at the experimental bioaugmentation site were monitored by optimal in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. The experimental site is located at Kururi, Kimitsu City, Chiba, Japan, where the groundwater was contaminated with TCE at a concentration of approximately 200 ␮g/liter. The aquifer characteristics in this field have been described by Nakamura et al. (14). Cultured Ralstonia eutropha KT1 (5) in nutrient broth were harvested and resuspended in Pseudomonas Basel mineral medium [K2HPO4, 0.25 g; KH2PO4, 0.076 g;

* Corresponding author. Mailing address: Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8170. Fax: 81-6-6879-8174. E-mail: [email protected] -u.ac.jp. 412

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MONITORING OF R. EUTROPHA KT1 IN GROUNDWATER

FIG. 1. Changes in the number of bacteria at four sampling wells (n ⫽ 1). F, W1; Œ, W2; 䡺, W3; ⴛ, control well.

and suspended in 50% ethanol with deionized water. Fixed cells were stored at ⫺20°C. At all sampling wells, the concentration of bacteria was on the order of 105 cells/ml before injection of the cell suspension (Fig. 1). The concentration of bacteria reached 3 ⫻ 107 cells/ml at W1 and 8 ⫻ 106 cells/ml at W2 1 h after the start of the extraction of groundwater (day 0). At W2 and W3 the number of bacteria decreased and returned to the original level, which was similar to the value at the control well after 25 days. In contrast, the concentration at W1 then remained on the order of 106 cells/ml and reached the original level on day 65. An in situ PCR was carried out as described previously (16) with some modifications. Fixed cells were washed several times with PBS, and a 30-␮l aliquot (about 106 to 107 cells) was spotted onto amino alkylsilane-coated slides (Perkin-Elmer Cetus, Norwalk, Conn.) and dried under a vacuum. After dehydration in an ethanol series (50, 80, and 100% ethanol for 3 min each), the samples were incubated with lysozyme solution (0.5 mg of lysozyme per ml, 100 mM Tris-HCl [pH 8.2], 50 mM EDTA) for 15 min at room temperature, rinsed with deionized water, and dehydrated as described above. Permeabilization was furthered by treatment with proteinase K at a final concentration of 0.1 ␮g/ml for 20 min at room temperature. After permeabilization, RNA was removed from cells by DNase-free RNase (Sigma) at a final concentration of 10 mg/ml for more than 1 h at room temperature. Finally, samples were rinsed with a deionized water and ethanol series. They were sealed with 50 ␮l of PCR buffer (10 mM Tris-HCl [pH 9.0], 50 mM KCl, 1% Triton X-100) containing 2.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, 0.5 ␮M primers, and 0.5 U of Taq DNA polymerase. The PCR primers KT1pheC (5⬘-CGC CTGATACCGCAGCGGCATAG-3⬘) and Reuphe3r (5⬘-G CCATTCGCCAAAGCCGTGAAAG-3⬘) were designed and used for PCR amplification of the phenol hydroxylase gene of R. eutropha KT1. PCR cycles consisted of denaturing at 94°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 60 s. Amplification was repeated for 35 cycles with a thermal cycler (GenAmp in situ PCR system 1000; Perkin-

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Elmer Cetus). After thermal cycling, the samples were rinsed with a deionized water and ethanol series. They were sealed with 50 ␮l of hybridization buffer (0.9 M NaCl, 20 mM TrisHCl [pH 7.6], 5 mM EDTA, 0.01% sodium dodecyl sulfate, 50% formamide) containing 250 ng of Cy3-labeled KT1phe probe (5⬘-GCAGAACAACGGCCACCTGAACG-3⬘). After 10 min of heat treatment at 95°C, the samples were incubated at 42°C for more than 12 h, and then they were washed twice with washing buffer (75 mM NaCl, 20 mM Tris-HCl [pH 8.2], 5 mM EDTA, 0.01% sodium dodecyl sulfate) for 30 min at 45°C. Finally, samples were counterstained with 1 ppm of 4⬘,6diamidino-2-phenylindole (DAPI) for 10 min after being washed with deionized water, and they were mounted in immersion oil for observation by epifluorescence microscopy (BX-50; Olympus Corp.). Images were acquired by a cooled charge-coupled device camera (Cool Snap) and stored as digital files. In FISH, suspensions of fixed cells were spotted onto amino alkylsilane-coated slides and dried under a vacuum. After dehydration in an ethanol series (50, 80, and 100% ethanol for 3 min each), the samples were sealed with 50 ␮l of hybridization buffer containing 250 ng of Cy3-labeled R810 probe (5⬘-TAAGCTACGTTACTGAAGAAAT-3⬘) which was designed for this study, incubated at 42°C for 16 h, and then washed with washing buffer for 30 min at 45°C. Samples were counterstained with DAPI and observed as described above. In the in situ PCR and FISH analyses, at least 1,000 cells in different fields of view were counted for each sample. The microscopic enumeration results, in all cases, were obtained from three parallel samples. We confirmed the protocols of in situ PCR and FISH using cultivated R. eutropha KT1 and other standard strains (data not shown) and then analyzed the bioaugmentation samples. The remaining R. eutropha KT1 in the aquifer was monitored by in situ PCR and FISH (Fig. 2). Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 ⫻ 105 cells/ml, and the amount of bacteria carrying the phenol hydroxylase gene and Ralstonia as a percentage of the total bacterial cells was less than 0.1% at W1, W2, and the control well. At W1, the concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 ⫻ 107 cells/ml 1 h after injection, and the number of Ralstonia bacteria detected by FISH was similar. The number of bacteria detected by in situ PCR and FISH was similar to that detected by FISH 4 days after the start of the extraction of groundwater. The number of bacterial cells detected by FISH was less than that detected by in situ PCR on and after day 7. Figure 3 shows typical micrographs of bacterial cells detected by in situ PCR and FISH. In both methods, more than 90% of the total bacteria showed red fluorescence under green light excitation 1 h after the start of the extraction of groundwater. In contrast, the ratio of bacteria detected by FISH to total cells was less than that of bacteria detected by in situ PCR to total cells on day 33. In rRNAtargeted FISH, the fluorescence intensity was dependent on the copy number of rRNA in an individual cell. This was not the case for in situ PCR. R. eutropha KT1 injected into the groundwater probably did not have a ribosomal content high enough for detection by FISH because it did not synthesize proteins or grow actively in the oligotrophic conditions of the aquifer. The possibility of horizontal transfer of the phenol

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FIG. 2. Changes in the number of bacteria carrying the phenol hydroxylase gene (F) detected by in situ PCR and Ralstonia (䡺) detected by FISH in W1 and W2. Data are averages of three replicates (n ⫽ 3), and bars indicate standard deviations.

hydroxylase gene into another genus of bacteria cannot be excluded, however. In W1, which was the injection and extraction well, R. eutropha KT1 could not be found on or after days 46 and 65 by FISH and in situ PCR, respectively. On the other hand, at W2, which is located 0.7 m downstream from W1, R. eutropha KT1 disappeared earlier than at W1. At the control well, the amount of Ralstonia bacteria detected by FISH or bacteria carrying the phenol hydroxylase gene detected by in situ PCR as a percentage of the total number of bacterial cells was less than 0.1%, which was under the limit of quantitative analysis during the field experiment. We have improved a direct in situ PCR method and applied it to the enumeration of bacteria carrying the Shiga-like toxin gene (sltII) in natural river water (9, 10). A direct in situ PCR in which the intracellular PCR products are labeled directly with compounds such as fluorescein isothiocyanate or digoxigenin makes it possible to detect specific cells simply and rapidly. It is difficult, however, to discriminate the target cells from cells containing false amplicons. Unavoidable background fluorescence also exists, and it is difficult to separate positive signals from this background fluorescence when environmental samples are analyzed (9). We employed FISH with a Cy3-labeled oligonucleotide probe hybridizing the region between the PCR primer positions to identify the target cells, and intracellular amplicons originating from the phenol hydroxylase gene of R. eutropha KT1 could be detected. Bioaugmentation, which is a way to enhance the biodegradative capacity of contaminated sites through inoculation with

bacteria possessing the desired catalytic capabilities, is considered an effective approach to cleaning up a polluted environment. The first bioaugmentation field experiment in Japan was conducted by the Ministry of International Trade and Industry (14). A field bioaugmentation study with genetically modified Burkholderia cepacia was also conducted at the Moffett Federal Airfield in the United States after a laboratory microcosm pilot study (12, 13). In order to use this technology efficiently and safely, the establishment of methods to monitor microbes in the natural environment is needed. Methods independent of culture are necessary in order to understand bacterial dynamics in the natural environment because most bacteria present in the natural environment cannot be cultured using traditional media (3). Quantitative PCR is a powerful technique to enumerate bacteria carrying a specific gene in natural environments because its sensitivity is high. The sensitivity of in situ PCR is inferior to quantitative PCR in the enumeration of specific bacteria (14). However, in situ PCR is recognized as a useful technique to detect bacteria carrying a specific gene because it can be used to identify the transcribed gene in an individual cell and reveal bacterial genetic diversity at the single-cell level (1, 2, 5, 7, 11, 15, 17, 18). In the near future, an analysis by in situ PCR with specific fluorochromes as a vital staining method (19) or a direct viable method (8) might be useful to reveal the physiologic characteristics of specific bacteria. Moreover, multiplex in situ PCR with primer sets for ribosomal DNA and the functional gene will make it possible to identify bacteria carrying a specific

FIG. 3. Typical micrographs of bacterial cells detected by in situ PCR and FISH. Only bacterial cells detected by the oligonucleotide probe showed the red fluorescence of Cy3 under green (G) excitation. The same microscopic fields are shown with UV excitation and green excitation.

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gene phylogenetically. This technique may give us valuable information about horizontal gene transfer in the natural environment. We anticipate that this technique will contribute to our knowledge of bacteria in the natural environment and help to establish bioaugmentation as a reliable and safe technology. This work was conducted as part of research handled by the Research Institute of Innovative Technology for the Earth (RITE), Japan, and funded by the Ministry of International Trade and Industry (MITI) through the New Energy and Industrial Technology Development Organization (NEDO). We thank Kimitsu City Hall in Chiba Prefecture for kind cooperation. REFERENCES 1. Chen, F., J. M. Gonzalez, W. A. Dustman, M. A. Moran, and R. E. Hodson. 1997. In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes. Appl. Environ. Microbiol. 63:4907– 4913. 2. Chen, F., B. Binder, and R. E. Hodson. 2000. Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR. FEMS Microbiol. Lett. 184:291–295. 3. Colwell, R. R., and D. J. Grimes. 2000. Nonculturable microorganisms in the environment. ASM Press, Washington, D.C. 4. Hanada, S., T. Shigematsu, K. Shibuya, M. Eguchi, T. Hasegawa, Y. Suda, Y. Kamagata, T. Kanagawa, and R. Kurane. 1998. Phylogenetic analysis of trichloroethylene-degrading strain newly isolated from the polluted soil with this contaminant. J. Ferment. Bioeng. 86:539–544. 5. Hodson, R. E., W. A. Dustman, R. P. Garg, and M. A. Moran. 1995. In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities. Appl. Environ. Microbiol. 61:4074– 4082. 6. Iwamoto, T., K. Tani, K. Nakamura, Y. Suzuki, M. Kitagawa, M. Eguchi, and M. Nasu. 2000. Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE. FEMS Microbiol. Ecol. 32: 129–141. 7. Jacobs, D., M. L. Angels, A. E. Goodman, and B. A. Neilan. 1997. Improved

8. 9. 10. 11. 12.

13.

14.

15. 16. 17. 18. 19.

methods for in situ enzymatic amplification and detection of low copy number genes in bacteria. FEMS Microbiol. Lett. 152:65–73. Kogure, K., U. Shimizu, and N. Taga. 1979. A tentative direct microscopic method for counting living marine bacteria. Can. J. Bacteriol. 25:415–420. Kurokawa, K., K. Tani, and M. Nasu. 1997. Direct in situ PCR method for the detection of verotoxin-producing Escherichia coli. Jpn. J. Bacteriol. 52: 513–518. Kurokawa, K., K. Tani, M. Ogawa, and M. Nasu. 1999. Abundance and distribution of bacteria carrying sltII gene in natural river water. Lett. Appl. Microbiol. 28:405–410. Lange, M., T. Tolker-Nielsen, S. Molin, and B. K. Ahring. 2000. In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells. Appl. Environ. Microbiol. 66:1796–1800. McCarty, P. L. 1998. Field evaluations of trichloroethylene cometabolism in groundwater with and without bioaugmentation, p. 15–21. In International workshop: innovative potential of advanced biological systems for remediation, 2 to 4 March 1998, Technical University Hamburg-Harburg, Germany. Munakata-Marr, J. 1996. Enhancement of trichloroethylene degradation in aquifer microcosms bioaugmentation with wild type and genetically altered Burkholderia (Pseudomonas) cepacia G4 and PR1. Environ. Sci. Technol. 30:2045–2052. Nakamura, K., H. Ishida, T. Iizumi, K. Shibuya, and K. Okamura. 2000. Quantitative PCR-detection of phenol-utilizing bacterium, Ralstonia eutropha KT1, injected to a trichloroethylene-contaminated site. Environ. Eng. Res. 37:267–278. Porter, J., R. Pickup, and C. Edwards. 1995. Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction. FEMS Microbiol. Lett. 134:51–56. Tani, K., K. Kurokawa, and M. Nasu. 1998. Development of a direct in situ PCR method for detection of specific bacteria in natural environments. Appl. Environ. Microbiol. 64:1536–1540. Tolker-Nielsen, T., K. Holmstrom, and S. Molin. 1997. Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR. Appl. Environ. Microbiol. 63:4196–4203. Tolker-Nielsen, T., K. Holmstrom, L. Boe, and S. Molin. 1998. Non-genetic population heterogeneity studied by in situ polymerase chain reaction. Mol. Microbiol. 27:1099–1105. Yamaguchi, N., T. Kenzaka, and M. Nasu. 1997. Rapid in situ enumeration of physiologically active bacteria in river waters using fluorescent probes. Microb. Environ. 12:1–8.