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Microbial Community Analysis of Digested Liquids Exhibiting Different Methane Production Potential in Methane Fermentation of Swine Feces

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Abstract

Batch methane fermentation was conducted using seed sludge collected from six methane fermentation facilities. Swine feces were centrifuged and autoclaved, followed by its use as a substrate for methanogenesis. This “swine feces supernatant medium” facilitates the cultivation of the microbes of the seed sludge, sampling of the digested liquid using a syringe, and subculturing of the digested liquid in a subsequent medium using a syringe. Through 15 subcultures, digested liquids with high and low methane production potential were obtained, which were named “H-DS” and “L-DS,” respectively. On the day 10 of cultivation, chemical oxygen demand (COD) of H-DS significantly decreased by 31% and that of L-DS did not differ significantly compared with that on the day 0 of cultivation. Acetic acid concentration of H-DS (1009 mg/L) was significantly lower than that of L-DS (2686 mg/L). These chemical characteristics indicate that organics decomposition in L-DS was not successful and suggest that H-DS has high relative abundance of bacteria decomposing organic matter and methanogen utilizing acetic acid compared with those in L-DS. Microbial community analysis revealed that Shannon index of H-DS was significantly higher than that of L-DS, and the relative abundance of acetogenic bacteria (e.g., Syntrophomonas) and acetic acid-utilizing methanogen (Methanosarcina) in H-DS was significantly higher than that in L-DS. Thus, the high methane production potential of H-DS might be attributable to the smooth flow from acetogenesis to methanogenesis step in the methane fermentation, compared with the case of L-DS.

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References

  1. Batstone, D. J., Keller, J., Angelidaki, I., Kalyuzhnyi, S. V., Pavlostathis, S. G., Rozzi, A., Sanders, W. T. M., Siegrist, H., & Vavilin, V. A. (2002). The IWA anaerobic digestion model no 1 (ADM1). Water Science and Technology, 45(10), 65–73.

    CAS  PubMed  Google Scholar 

  2. Beaty, P. S., & McInerney, M. J. (1989). Effects of organic acid anions on the growth and metabolism of Syntrophomonas wolfei in pure culture and in defined consortia. Applied and Environmental Microbiology, 55(4), 977–983.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Boone, D. R., & Mah, R. A. (2015). Methanosarcina. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), In bergey’s manual of systematics of archaea and bacteria. https://doi.org/10.1002/9781118960608.gbm00519.

    Chapter  Google Scholar 

  4. Bosshard, P. P. (2015). Turicibacter. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), In bergey’s manual of systematics of archaea and bacteria. https://doi.org/10.1002/9781118960608.gbm00766.

    Chapter  Google Scholar 

  5. Cai, S., & Dong, X. (2010). Cellulosilyticum ruminicola gen. nov., sp. nov., isolated from the rumen of yak, and reclassification of Clostridium lentocellum as Cellulosilyticum lentocellum comb. nov. International Journal of Systematic and Evolutionary Microbiology, 60(4), 845–849.

    PubMed  Google Scholar 

  6. Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., Fierer, N., Peña, A. G., Goodrich, J. K., Gordon, J. I., Huttley, G. A., Kelley, S. T., Knights, D., Koenig, J. E., Ley, R. E., Lozupone, C. A., McDonald, D., Muegge, B. D., Pirrung, M., Reeder, J., Sevinsky, J. R., Turnbaugh, P. J., Walters, W. A., Widmann, J., Yatsunenko, T., Zaneveld, J., & Knight, R. (2010). QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5), 335–336.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Cerrillo, M., Viñas, M., & Bonmatí, A. (2016). Overcoming organic and nitrogen overload in thermophilic anaerobic digestion of pig slurry by coupling a microbial electrolysis cell. Bioresource Technology, 216, 362–372.

    CAS  PubMed  Google Scholar 

  8. Cibis, K. G., Gneipel, A., & König, H. (2016). Isolation of acetic, propionic and butyric acid-forming bacteria from biogas plants. Journal of Biotechnology, 220, 51–63.

    CAS  PubMed  Google Scholar 

  9. Edgar, R. C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26(19), 2460–2461.

    CAS  PubMed  Google Scholar 

  10. Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C., & Knight, R. (2011). UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27(16), 2194–2200.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Gerritsen, J., Fuentes, S., Grievink, W., Niftrik, L. V., Tindall, B. J., Timmerman, H. M., Rijkers, G. T., & Smidt, H. (2014). Characterization of Romboutsia ilealis gen. nov., sp. nov., isolated from the gastro-intestinal tract of a rat, and proposal for the reclassification of five closely related members of the genus Clostridium into the genera Romboutsia gen. nov., Intestinibacter gen. nov., Terrisporobacter gen. nov. and Asaccharospora gen. nov. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 5), 1600–1616.

    CAS  PubMed  Google Scholar 

  12. Grabowski, A., Tindall, B. J., Bardin, V., Blanchet, D., & Jeanthon, C. (2005). Petrimonas sulfuriphila gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir. International Journal of Systematic and Evolutionary Microbiology, 55(3), 1113–1121.

    CAS  PubMed  Google Scholar 

  13. Guo, L., Li, X. M., Bo, X., Yang, Q., Zeng, G. M., Liao, D. X., & Liu, J. J. (2008). Impacts of sterilization, microwave and ultrasonication pretreatment on hydrogen producing using waste sludge. Bioresource Technology, 99(9), 3651–3658.

    CAS  PubMed  Google Scholar 

  14. Hahnke, S., Langer, T., Koeck, D. E., & Klocke, M. (2016). Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov., isolated from mesophilic laboratory-scale biogas reactors, and emended description of the genus Proteiniphilum. International Journal of Systematic and Evolutionary Microbiology, 66, 1466–1475.

    CAS  PubMed  Google Scholar 

  15. Horino, H., Fujita, T., & Tonouchi, A. (2014). Description of Anaerobacterium chartisolvens gen. nov., sp. nov., an obligately anaerobic bacterium from Clostridium rRNA cluster III isolated from soil of a Japanese rice field, and reclassification of Bacteroides cellulosolvens Murray et al. 1984 as Pseudobacteroides cellulosolvens gen. nov., comb. nov. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 4), 1296–1303.

    CAS  PubMed  Google Scholar 

  16. Ikeda, S., Watanabe, K. N., Minamisawa, K., & Ytow, N. (2004). Evaluation of soil DNA from arable land in Japan using a modified direct-extraction method. Microbes and Environments, 19, 301–309.

    Google Scholar 

  17. Japan Sewage Works Association. (1997). Japanese standard testing methods for sewage. Japan Sewage Works Association (in Japanese).

  18. Jarrell, K. F., Saulnier, M., & Ley, A. (1987). Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge. Canadian Journal of Microbiology, 33(6), 551–554.

    CAS  PubMed  Google Scholar 

  19. Jiménez, J., Guardia-Puebla, Y., Romero-Romero, O., Cisneros-Ortiz, M. E., Guerra, G., Morgan-Sagastume, J. M., & Noyola, A. (2014). Methanogenic activity optimization using the response surface methodology, during the anaerobic co-digestion of agriculture and industrial wastes. Microbial community diversity. Biomass Bioenergy., 71, 84–97.

    Google Scholar 

  20. Kim, E., Lee, J., Han, G., & Hwang, S. (2018). Comprehensive analysis of microbial communities in full-scale mesophilic and thermophilic anaerobic digesters treating food waste-recycling wastewater. Bioresource Technology, 259, 442–450.

    CAS  PubMed  Google Scholar 

  21. Lechner, U. (2015). Sedimentibacter. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), In bergey’s manual of systematics of archaea and bacteria. https://doi.org/10.1002/9781118960608.gbm00718.

    Chapter  Google Scholar 

  22. Li, J., Jha, A. K., & Bajracharya, T. R. (2014). Dry anaerobic co-digestion of cow dung with pig manure for methane production. Applied Biochemistry and Biotechnology, 173(6), 1537–1552.

    CAS  PubMed  Google Scholar 

  23. Li, D., Liu, S., Mi, L., Li, Z., Yuan, Y., Yan, Z., & Liu, X. (2015). Effects of feedstock ratio and organic loading rate on the anaerobic mesophilic co-digestion of rice straw and pig manure. Bioresource Technology, 187, 120–127.

    CAS  PubMed  Google Scholar 

  24. Magoc, T., & Salzberg, S. L. (2011). FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 27(21), 2957–2963.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Meng, X., Zhang, Y., Sui, Q., Zhang, J., Wang, R., Yu, D., Wang, Y., & Wei, Y. (2018). Biochemical conversion and microbial community in response to ternary pH buffer system during anaerobic digestion of swine manure. Energies, 11(11), 2991.

    CAS  Google Scholar 

  26. Morotomi, M., Nagai, F., & Watanabe, Y. (2012). Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposal of Christensenellaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 62(1), 144–149.

    CAS  PubMed  Google Scholar 

  27. Mumtaz, T., Abd-Aziz, S., Yee, P. L., Yunus, W. M. Z. W., Shirai, Y., & Hassan, M. A. (2010). Synthesis, characterization and structural properties of intracellular copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Comamonas sp. EB172 from renewable resource. International Journal of Polymer Analysis and Characterization, 15(6), 329–340.

    Google Scholar 

  28. Niu, Q., Kubota, K., Qiao, W., Jing, Z., Zhang, Y., & Yu-You, L. (2015). Effect of ammonia inhibition on microbial community dynamic and process functional resilience in mesophilic methane fermentation of chicken manure. Journal of Chemical Technology and Biotechnology, 90, 2161–2169.

    CAS  Google Scholar 

  29. Oz, N. A., Ince, O., Turker, G., & Ince, B. K. (2012). Effect of seed sludge microbial community and activity on the performance of anaerobic reactors during the start-up period. World Journal of Microbiology and Biotechnology, 28(2), 637–647.

    CAS  PubMed  Google Scholar 

  30. Peng, X., Zhang, Z., Luo, W., & Jia, X. (2013). Biodegradation of tetrabromobisphenol a by a novel Comamonas sp. strain, JXS-2-02, isolated from anaerobic sludge. Bioresource Technology, 128, 173–179.

    CAS  PubMed  Google Scholar 

  31. Rainey, F. A., Hollen, B. J., & Small, A. M. (2015). Clostridium. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), In bergey’s manual of systematics of Archaea and Bacteria. https://doi.org/10.1002/9781118960608.gbm00619.

    Chapter  Google Scholar 

  32. Ren, J., Yuan, X., Li, J., Ma, X., Zhao, Y., Zhu, W., Wang, X., & Cui, Z. (2014). Performance and microbial community dynamics in a two-phase anaerobic co-digestion system using cassava dregs and pig manure. Bioresource Technology, 155, 342–351.

    CAS  PubMed  Google Scholar 

  33. Schulz, H., & Eder, B. (2002). Biogas Praxis (Japanese translation edition by Y. Ukita). Ohmsha: Tokyo (in Japanese).

    Google Scholar 

  34. Sekiguchi, Y. (2015). Syntrophomonas. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), bergey’s manual of systematics of archaea and bacteria. https://doi.org/10.1002/9781118960608.gbm00682.

    Chapter  Google Scholar 

  35. Shah, H. N., & Hookey, J. V. (2015). Tissierella. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), In bergey’s manual of systematics of archaea and bacteria. https://doi.org/10.1002/9781118960608.gbm00721.

    Chapter  Google Scholar 

  36. Shen, P., Fei, H., Shuquan, S., Junya, Z., Zhineng, C., Junfang, L., Jiayi, G., Bin, F., & Bo, W. (2014). Using pig manure to promote fermentation of sugarcane molasses alcohol wastewater and its effects on microbial community structure. Bioresource Technology, 155, 323–329.

    CAS  PubMed  Google Scholar 

  37. Shiratori-Takano, H., & Ueda, K. (2017). Lutispora. In W. B. Whitman, F. Rainey, P. Kämpfer, M. Trujillo, J. Chun, P. DeVos, B. Hedlund, & S. Dedysh (Eds.), In bergey’s manual of systematics of Archaea and Bacteria. https://doi.org/10.1002/9781118960608.gbm01393.

    Chapter  Google Scholar 

  38. Takahashi, S., Tomita, J., Nishioka, K., Hisada, T., & Nishijima, M. (2014). Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing. PLoS One, 9(8), e105592.

    PubMed  PubMed Central  Google Scholar 

  39. Tang, Y.-Q., Shigematsu, T., Morimura, S., & Kida, K. (2015). Dynamics of the microbial community during continuous methane fermentation in continuously stirred tank reactors. Journal of Bioscience and Bioengineering, 119, 375–383.

    CAS  PubMed  Google Scholar 

  40. Ueki, A., Ohtaki, Y., Kaku, N., & Ueki, K. (2016). Descriptions of Anaerotaenia torta gen. nov., sp. nov. and Anaerocolumna cellulosilytica gen. nov., sp. nov. isolated from a methanogenic reactor of cattle waste and reclassification of Clostridium aminovalericum, Clostridium jejuense and Clostridium xylanovorans as Anaerocolumna species. International Journal of Systematic and Evolutionary Microbiology, 66(8), 2936–2943.

    CAS  PubMed  Google Scholar 

  41. Vrieze, J. D., Hennebel, T., Boon, N., & Verstraete, W. (2012). Methanosarcina: The rediscovered methanogen for heavy duty biomethanation. Bioresource Technology, 112, 1–9.

    PubMed  Google Scholar 

  42. Wang, M., Zhou, J., Yuan, Y. X., Dai, Y. M., Li, D., Li, Z. D., Liu, X. F., Zhang, X. Y., & Yan, Z. Y. (2017). Methane production characteristics and microbial community dynamics of mono-digestion and co-digestion using corn stalk and pig manure. International Journal of Hydrogen Energy, 42(8), 4893–4901.

    CAS  Google Scholar 

  43. Yabe, S., Sakai, Y., Abe, K., & Yokota, A. (2017). Diversity of Ktedonobacteria with Actinomycetes-like morphology in terrestrial environments. Microbes and Environments, 32, 61–70.

    PubMed  PubMed Central  Google Scholar 

  44. Yang, Z., Wang, W., He, Y., Zhang, R., & Liu, G. (2018). Effect of ammonia on methane production, methanogenesis pathway, microbial community and reactor performance under mesophilic and thermophilic conditions. Renewable Energy, 125, 915–925.

    CAS  Google Scholar 

  45. Yokoyama, M. T., Spence, C., Hengemuehle, S. M., Whitehead, T. R., von Bernuth, R., & Cotta, M. (2016). Sodium tetraborate decahydrate treatment reduces hydrogen sulfide and the sulfate-reducing Bacteria population of swine manure. Journal of Environmental Quality, 45(6), 1838–1846.

    CAS  PubMed  Google Scholar 

  46. Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International Journal of Systematic and Evolutionary Microbiology, 67(5), 1613–1617.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Yutin, N., & Galperin, M. Y. (2013). A genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia. Environmental Microbiology, 15(10), 2631–2641.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zakaria, M. R., Abd-Aziz, S., Ariffin, H., Rahman, N. A. A., & Lai, P. (2008). Comamonas sp. EB172 isolated from digester treating palm oil mill effluent as potential polyhydroxyalkanoate (PHA) producer. African Journal of Biotechnology, 7, 4118–4121.

    CAS  Google Scholar 

  49. Zhao, H., Yang, D., Woese, C. R., & Bryant, M. P. (1993). Assignment of fatty acid-β-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses. International Journal of Systematic and Evolutionary Microbiology, 43(2), 278–286.

    CAS  Google Scholar 

  50. Zhao, Y., Yuan, X., Wen, B., Wang, X., Zhu, W., & Cui, Z. (2017). Methane potential and microbial community dynamics in anaerobic digestion of silage and dry cornstalks: a substrate exchange study. Applied Biochemistry and Biotechnology, 181(1), 91–111.

    CAS  PubMed  Google Scholar 

  51. Zhou, J., Zhang, R., Liu, F., Yong, X., Wu, X., Zheng, T., Jiang, M., & Jia, H. (2016). Biogas production and microbial community shift through neutral pH control during the anaerobic digestion of pig manure. Bioresource Technology, 217, 44–49.

    CAS  PubMed  Google Scholar 

  52. Zhou, S., Nikolausz, M., Zhang, J., Riya, S., Terada, A., & Hosomi, M. (2016). Variation of the microbial community in thermophilic anaerobic digestion of pig manure mixed with different ratios of rice straw. Journal of Bioscience and Bioengineering, 122(3), 334–340.

    CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Mitsui E&S Environment Engineering Co., Ltd., and Misaki Youton Corp. for providing the digested liquids, and Shoji Ookutsu (Kagoshima University) for providing the swine feces.

Funding

This research was supported by Research grant for advanced research, United Graduate School of Agricultural Sciences, Kagoshima University.

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Correspondence to Hirohito Tsurumaru.

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Nakamura, Y., Ishibashi, M., Kamitani, Y. et al. Microbial Community Analysis of Digested Liquids Exhibiting Different Methane Production Potential in Methane Fermentation of Swine Feces. Appl Biochem Biotechnol 191, 1140–1154 (2020). https://doi.org/10.1007/s12010-020-03228-7

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