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Development of an Amendment Recipe and Identification of Benzene Degraders for Anaerobic Benzene Bioremediation

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Abstract

This study aims to develop an optimal recipe of amendment (nutrients and electron acceptors) for anaerobic benzene bioremediation and identify the dominant indigenous benzene-degrading microorganisms in soil and groundwater collected from a fuel service station. Lessons learned in developing and optimizing the amendment recipe follow: (1) salinity and a high initial concentration of benzene were detrimental for benzene biodegradation, (2) a large dose of amendment can shorten the lag time for benzene biodegradation, (3) toluene was an essential co-substance for promoting benzene biodegradation. Stable isotope probing was used to identify microorganism incorporation of 13C from 13C– benzene. Under the experimental conditions, incorporation of 13C can be considered direct evidence of the occurrence of benzene biodegradation. Quantitative polymerase chain reaction showed the primary mechanism for benzene removal to be nitrate reduction. Microbial analyses (denaturing gradient gel electrophoresis and 16S ribosomal RNA) demonstrated that members of genus Dokdonella spp., Pusillimonas spp., and Advenella spp. were predominant in the microbial community and involved in anaerobic benzene bioremediation.

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References

  • Aburto, A., & Peimbert, M. (2011). Degradation of a benzene–toluene mixture by hydrocarbon-adapted bacterial communities. Annals of Microbiology, 61, 553–562.

    Article  CAS  Google Scholar 

  • American Public Health Association (APHA), American Water Works Association and Water Environmental Federation. (2012). Standard methods for the examination of water and wastewater (22nd ed.). Washington, D.C.: American Public Association.

    Google Scholar 

  • Baldwin, B. R., Peacock, A. D., Park, M., Ogles, D. M., Lstok, J. D., McKinley, J. P., Resch, C. T., & White, D. C. (2008). Multilevel samplers as microcosms to assess microbial response to biostimulation. Ground Water, 46, 295–304.

    Article  CAS  Google Scholar 

  • Barbaro, J., Barker, J., Lemon, L., & Mayfield, C. (1992). Biotransformation of BTEX under anaerobic, denitrifying conditions: field and laboratory observations. Journal of Contaminant Hydrology, 11, 245–272.

    Article  CAS  Google Scholar 

  • Barton, L. L., & Fauque, G. D. (2009). Biochemistry, physiology and biotechnology of sulfate-reducing bacteria. Advances in Applied Microbiology, 68, 41–98.

    Article  CAS  Google Scholar 

  • Beller, H. R., Kane, S. R., Legler, T. C., & Alvarez, P. J. (2002). A real-time polymerase chain reaction method for monitoring anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene. Environmental Science & Technology, 36, 3977–3984.

    Article  CAS  Google Scholar 

  • Braker, G., Fesefeldt, A., & Witzel, K. P. (1998). Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Applied and Environmental Microbiology, 64, 3769–3775.

    CAS  Google Scholar 

  • Cao, B., Ma, T., Ren, Y., Ren, Y., Li, G., Li, P., & Feng, L. (2011). Complete genome sequence of Pusillimonas sp. T7-7, a cold-tolerant diesel oil-degrading bacterium isolated from the Bohai Sea in China. Journal of Bacteriology, 193, 4021–4022.

    Article  CAS  Google Scholar 

  • Canadian Council of Ministers of the Environment (CCME). (2004). Canadian Soil Quality Guidelines for the Protection of Environmental and Human Health – Benzene.

  • Canadian Environmental Protection Act. (1999). First priority substances list (PSL1) assessments.

  • Choi, E. J., Jin, H. M., Lee, S. H., Math, R. L., Madsen, E. L., & Jeon, C. O. (2013). Comparative genomic analysis and benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) degradation pathways of Pseudoxanthomonas spadix BD-a59. Applied and Environmental Microbiology, 79, 663–671.

    Article  CAS  Google Scholar 

  • Cleveland, C. C., & Liptzin, D. (2007). C:N:P stoichiometry in soil: is there a “Redfield ratio” for the microbial biomass? Biogeochemistry, 85, 235–252.

    Article  Google Scholar 

  • Coates, J. D., Chakraborty, R., Lack, J. G., O'Connor, S. M., Cole, K. A., Bender, K. S., & Achenbach, L. A. (2001). Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas. Nature, 411, 1039–1043.

    Article  CAS  Google Scholar 

  • Cunningham, J. A., Hopkins, G. D., Lebron, C. A., & Reinhard, M. (2000). Enhanced anaerobic bioremediation of groundwater contaminated by fuel hydrocarbons at Seal Beach, California. Biodegradation, 11, 159–170.

    Article  CAS  Google Scholar 

  • Ding, G.-C., Heuer, H., & Smalla, K. (2012). Dynamics of bacterial communities in two unpolluted soils after spiking with phenanthrene: soil type specific and common responders. Frontiers in Microbiology, 3, 1–16.

    Article  Google Scholar 

  • Dong, H., Zhang, G., Jiang, H., Yu, B., Chapman, L. R., Lucas, C. R., & Fields, M. W. (2006). Microbial diversity in sediments of saline Qinghai Lake, China: linking geochemical controls to microbial ecology. Microbial Ecology, 51, 65–82.

    Article  CAS  Google Scholar 

  • Edwards, E. A., & Grbic-Galic, D. (1992). Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions. Applied and Environmental Microbiology, 58, 2663–2666.

    CAS  Google Scholar 

  • Farhadian, M., Vachelard, C., Duchez, D., Larroche, C. (2008). In situ bioremediation of monoaromatic pollutants in groundwater: a review. Bioresource Technology, 99, 5296–5308.

  • Fingas, M. (2013). The basics of oil spill cleanup (3rd ed.). Boca Raton: CRC Press.

    Google Scholar 

  • Fritz, P., Reardon, E. J., Barker, J., Brown, R. M., Cherry, J. A., Killey, R. W. D., & McNaughton, D. (1978). The carbon isotope geochemistry of a small groundwater system in northeastern Ontario. Water Resources Research, 14, 1059–1067.

    Article  CAS  Google Scholar 

  • Geyer, R., Peacock, A., Miltner, A., Richnow, H., White, D., Sublette, K., & Kästner, M. (2005). In situ assessment of biodegradation potential using BioTraps amended with 13C-labelled benzene or toluene. Environmental Science & Technology, 39, 4983–4989.

    Article  CAS  Google Scholar 

  • Guckert, J. B., Antworth, C. P., Nichols, P. D., & White, D. C. (1985). Phospholipid, ester-linked fatty acid profiles as reproducible assays for changes in prokaryotic community structure of estuarine sediments. FEMS Microbiology Letters, 31, 147–158.

    Article  CAS  Google Scholar 

  • Harwood, C. S., Parales, R. E. (1996). The β-ketoadipate pathway and the biology of self-identity. Annual Reviews in Microbiology, 50, 553–590.

  • Harvey, R. W., Smith, R. L., & George, L. (1984). Effect of organic contamination upon microbial distributions and heterotrophic uptake in a Cape Cod, Mass., aquifer. Applied and Environmental Microbiology, 48, 1197–1202.

    CAS  Google Scholar 

  • Holmes, D. E., Risso, C., Smith, J. A., & Lovley, D. R. (2011). Anaerobic oxidation of benzene by the hyperthermophilic archaeon Ferroglobus placidus. Applied and Environmental Microbiology, 77, 5926–5933.

    Article  CAS  Google Scholar 

  • Hug, L. A., Edwards, E. A., Vrionis, H., & Major, D. W. (2013). Research needs for bioaugmentation. In H. F. Stroo, A. Leeson, & C. H. Ward (Eds.), Bioaugmentation for Groundwater Remediation. Springer science. New York.

  • Johnsen, A. R., Winding, A., Karlson, U., & Roslev, P. (2002). Linking of microorganisms to phenanthrene metabolism in soil by analysis of 13C-labelled cell lipids. Applied and Environmental Microbiology, 68, 6106–6113.

    Article  CAS  Google Scholar 

  • Kandeler, E., Deiglmayr, K., Tscherko, D., Bru, D., & Philippot, L. (2006). Abundance of narG, nirS, nirK, and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland. Applied and Environmental Microbiology, 72, 5957–5962.

    Article  CAS  Google Scholar 

  • Kazumi, J., Caldwell, M., Suflita, J., Lovley, D., & Young, L. (1997). Anaerobic degradation of benzene in diverse anoxic environments. Environmental Science & Technology, 31, 813–818.

    Article  CAS  Google Scholar 

  • Kostka, J. E., Parakash, O., Overholt, W. A., Green, S. J., Freyer, G., Canion, A., Delgardio, J., Norton, N., Hazen, T. C., & Huettel, M. (2011). Hydrocarbon-degrading bacteria and the bacterial community response in Gulf of Mexico Beach sands impacted by the deepwater horizon oil spill. Applied and Environmental Microbiology, 77, 7962–7974.

    Article  CAS  Google Scholar 

  • Lee, G., Ro, H., Lee, S., & Lee, S. (2006). Electrokinetically enhanced transport of organic and inorganic phosphorus in a low permeability soil. Geosciences Journal, 10, 85–89.

    Article  CAS  Google Scholar 

  • Li, W., Wang, L. Y., Duan, R. Y., Liu, J. F., Gu, J. D., & Mu, B. Z. (2012). Microbial community characteristics of petroleum reservoir production water amended with alkanes and incubated under nitrate-, sulfate-reducing and methanogenic conditions. International Biodeterioration & Biodegradation, 69, 87–96.

    Article  CAS  Google Scholar 

  • Li, X., Wang, X., Zhang, Y., Zhao, Q., Yu, B., Li, Y., & Zhou, Q. (2016). Salinity and conductivity amendment of soil enhanced the bioelectrochemical degradation of petroleum hydrocarbons. Nature, 6(32861), 1–11.

    Article  Google Scholar 

  • Nales, M., Butler, B. J., & Edwards, E. A. (1998). Anaerobic benzene biodegradation: a microcosm survey. Bioremediation Journal, 2, 125–144.

    Article  CAS  Google Scholar 

  • National Research Council. (2003). Oil in the sea III, inputs, fate, and effects. Washington: The National Academies Press.

    Google Scholar 

  • Nielsen, D. M. (2005). Practical Handbook of Environmental Site Characterization and Ground-Water Monitoring (2nd edition). Boca Raton, Florida: Taylor & Francis Group.

    Book  Google Scholar 

  • Painchaud, J., Therriault, J., & Legendre, L. (1995). Assessment of salinity-related mortality of freshwater bacteria in the Saint Lawrence estuary. Applied and Environmental Microbiology, 61, 205–208.

    CAS  Google Scholar 

  • Pelz, O., Chatzinotas, A., Zarda-Hess, A., Abraham, W. R., & Zeyer, J. (2001). Tracing toluene-assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell hybridization. FEMS Microbiology Ecology, 38, 123–131.

    Article  CAS  Google Scholar 

  • Phelps, C. D., & Young, L. (1999). Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments. Biodegradation, 10, 15–25.

    Article  CAS  Google Scholar 

  • Roychoudhury, A. N., & Merrett, G. L. (2006). Redox pathways in a petroleum contaminated shallow sandy aquifer: iron and sulfate reductions. Science of the Total Environment, 366, 262–274.

    Article  CAS  Google Scholar 

  • Shultz, D. J., & Calder, J. A. (1976). Organic carbon 13C12C variations in estuarine sediments. Geochimica et Cosmochimica Acta, 40, 381–385.

    Article  CAS  Google Scholar 

  • Smith, M. R. (1990). The biodegradation of aromatic hydrocarbons by bacteria. Biodegradation, 1, 191–206.

    Article  CAS  Google Scholar 

  • Stults, J. R., Snoeyenbos-West, O., Methe, B., Lovley, D. R., & Chandler, D. P. (2001). Application of the 5’ fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA. Applied and Environmental Microbiology, 67, 2781–2789.

    Article  CAS  Google Scholar 

  • Sublette, K., Peacock, A., White, D., Davis, G., Ogles, D., Cook, D., Kolhatkar, R., Beckmann, D., & Yang, X. (2006). Monitoring subsurface microbial ecology in a sulfate-amended, gasoline-contaminated aquifer. Ground Water Monitoring & Remediation, 26, 70–78.

    Article  CAS  Google Scholar 

  • Sun, D., Li, J., Xu, M., An, T., Sun, G., & Guo, J. (2013). Toluene removal efficiency, process robustness, and bacterial diversity of a biotrickling filter inoculated with Burkholderia sp. strain T3. Biotechnology and Bioprocess Engineering, 18, 125–134.

    Article  CAS  Google Scholar 

  • Tao, Y., Fishman, A., Bentley, W. E., & Wood, T. K. (2004). Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1. Applied and Environmental Microbiology, 70, 3814–3820.

    Article  CAS  Google Scholar 

  • Ten, L. N., Jung, H. M., Im, W. T., Oh, H. W., Yang, D. C., Yoo, S. A., & Lee, S. T. (2009). Dokdonella ginsengisoli sp. nov., isolated from soil from a ginseng field, and emended description of the genus Dokdonella. International Journal of Systematic and Evolutionary Microbiology, 59, 1947–1952.

    Article  Google Scholar 

  • United States Environmental Protection Agency (USEPA). (1998). Carcinogenic effects of benzene: an update. DC: Washington.

    Google Scholar 

  • Vogt, C., Kleinsteuber, S., & Richnow, H. (2011). Anaerobic benzene degradation by bacteria. Microbial Biotechnology, 4, 710–724.

    Article  Google Scholar 

  • Wagner, M., Loy, A., Klein, M., Lee, N., Ramsing, N. B., Stahl, D. A., & Friedrich, M. W. (2005). Functional marker genes for identification of sulfate-reducing prokaryotes. Methods in Enzymology, 397, 469–489.

    Article  CAS  Google Scholar 

  • Wenderoth, D. F., Rosenbrock, P., Abraham, W.-R., Pieper, D. H., & Höfle, M. G. (2003). Bacterial community dynamics during biostimulation and bioaugmentation experiments aiming at chlorobenzene degradation in groundwater. Microbial Ecology, 46, 161–176.

    Article  CAS  Google Scholar 

  • Winderl, C., Schaefer, S., & Lueders, T. (2007). Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker. Environmental Microbiology, 9, 1035–1046.

    Article  CAS  Google Scholar 

  • Xiong, W., Mathies, C., Bradshaw, K., Carlson, T., Tang, K., & Wang, Y. (2012). Benzene removal by a novel modification of enhanced anaerobic biostimulation. Water Research, 46, 4721–4731.

    Article  CAS  Google Scholar 

  • Yan, S., Subramanian, B., Surampalli, R. Y., Narasiah, S., Tyagi, R. D. (2007). Isolation, characterization, and identification of bacteria from activated sludge and soluble microbial products in wastewater treatment systems. Practice Periodical of Hazardous, Toxic, and Radioactive Waste Management, 11, 240–258.

  • Yoon, J. H., Kang, S. J., & Oh, T. K. (2006). Dokdonella koreensis gen. nov., sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology, 56, 145–150.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are grateful for financial support provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant, the Stantec Consulting Inc. Research and Development Fund, and the Mitacs-Accelerate Graduate Research Internship Program. We also would like to express our thanks to Mr. D. Fisher, technologist at the University of Saskatchewan, for his assistance in the environmental laboratory and Ms. A. Strilchuk, senior technical editor at Stantec Consulting Ltd., for her editorial QA/QC review.

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Xiong, W., Lu, Z. & Peng, J. Development of an Amendment Recipe and Identification of Benzene Degraders for Anaerobic Benzene Bioremediation. Water Air Soil Pollut 229, 7 (2018). https://doi.org/10.1007/s11270-017-3663-3

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