Anaerobic digestion is an important technology for the bio-based economy. The stability of the process is crucial for its successful implementation and depends on the structure and functional stability of the microbial community. In this study, the total microbial community was analyzed during mesophilic fermentation of sewage sludge in full-scale digesters.
The digesters operated at 34–35°C, and a mixture of primary and excess sludge at a ratio of 2:1 was added to the digesters at 550 m3/d, for a sludge load of 0.054 m3/(m3·d). The amount and composition of biogas were determined. The microbial structure of the biomass from the digesters was investigated with use of next-generation sequencing.
The percentage of methanogens in the biomass reached 21%, resulting in high quality biogas (over 61% methane content). The abundance of syntrophic bacteria was 4.47%, and stable methane production occurred at a Methanomicrobia to Synergistia ratio of 4.6:1.0. The two most numerous genera of methanogens (about 11% total) were Methanosaeta and Methanolinea, indicating that, at the low substrate loading in the digester, the acetoclastic and hydrogenotrophic paths of methane production were equally important. The high abundance of the order Bacteroidetes, including the class Cytophagia (11.6% of all sequences), indicated the high potential of the biomass for efficient degradation of lignocellulitic substances, and for degradation of protein and amino acids to acetate and ammonia.
This study sheds light on the ecology of microbial groups that are involved in mesophilic fermentation in mature, stably-performing microbiota in full-scale reactors fed with sewage sludge under low substrate loading.
Amani, T., Nosrati, M. & Sreekrishnan, T.R. (2010). Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects – a review, Environmental Reviews, 18, pp. 255–278.
Amin, G.A. & Vriens, L. (2014), Optimization of up-flow anaerobic sludge blanket reactor for treatment of composite fermentation and distillation wastewater, African Journal of Biotechnology, 13, pp. 1136–1142.
de Bok, F.A.M., Plugge, C.M. & Stams, A.J.M. (2004). Interspecies electron transfer in methanogenic propionate degrading consortia, Water Research, 38, pp. 1368–1375.
Borrel, G., O’Toole, P.W., Harris, H.M.B., Peyret, P., Brugère, J.F. & Gribaldo, S. (2013). Phylogenomic data support a seventh order of methylotrophic methanogens and provide insights into the evolution of methanogenesis, Genome Biology and Evolution, 5, pp. 1769–1780.
Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S.M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J.A., Smith, G. & Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms, The ISME Journal, 6, pp. 1621–1624.
Cydzik-Kwiatkowska, A. (2015). Bacterial structure of aerobic granules is determined by aeration mode and nitrogen load in the reactor cycle, Bioresource Technolology, 181, pp. 312–320.
Cydzik-Kwiatkowska, A., Zieliński, M. & Jaranowska, P. (2012). Microwave radiation and reactor design influence microbial communities during methane fermentation, Journal of Industrial Microbiology and Biotechnology, 39(9), pp. 1397–1405.
Deng, D., Weidhaas, J.L. & Lin, L.S. (2016). Kinetics and microbial ecology of batch sulfidogenic bioreactors for co-treatment of municipal wastewater and acid mine drainage, Journal of Hazardous Materials, 305, pp. 200–208.
Díaz, C., Baena, S., Patel, B.K.C. & Fardeau, M.L. (2010). Peptidolytic microbial community of methanogenic reactors from two modified UASBS of brewery industries, Brazilian Journal of Microbiology, 41, pp. 707–717.
Edgar, R.C. (2010). Search and clustering orders of magnitude faster than BLAST, Bioinformatics, 12, pp. 1–3.
Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C. & Knight, R. (2011). UCHIIME improves sensitivity and speed of chimera detection, Bioinformatics, 27, 6, pp. 2194–2200.
Fernandez, A., Huang, S.Y., Seston, S., Xing, J., Hickey, R., Criddle, C. & Tiedje, J. (1999). How stable is stable? Function versus community composition, Applied and Environmental Microbiology, 65, pp. 3697–3704.
Forster, C.F. & Foot, R.J. (1997). The operation of a selector for the control of foam-foaming bacteria in activated sludge, Environmental Technolology, 18, pp. 237–241.
Guo, J., Peng, Y., Ni, B.-J., Han, X. & Yuan, L.F.N. (2015). Dissecting microbial community structure and methane-producing pathways of a full-scale anaerobic reactor digesting activated sludge from wastewater treatment by metagenomic sequencing, Microbial Cell Factories, 14, pp. 33.
Irgens, R.L. (2015). Meniscus, Bergey’s Manual of Systematics of Archaea and Bacteria, Wiley Online Library, pp. 1–3.
Kampmann, K., Ratering, S., Kramer, I., Schmidt, M. & Zerr, W.S. (2012). Schnell unexpected stability of Bacteroidetes and Firmicutes communities in laboratory biogas reactors fed with different defined substrates, Applied and Environmental Microbiology, 78, 7, pp. 2106–2119.
Kim, W., Hwang, K., Shin, S.G., Lee, S. & Hwang, S. (2010). Effect of high temperature on bacterial community dynamics in anaerobic acidogenesis using mesophilic sludge inoculums, Bioresource Technol, 101, pp. 17–22.
Klimiuk, E. & Łebkowska, M. (2003). Biotechnology in environmental protection, Wyd. Naukowe PWN, Warszawa 2003. (in Polish)
Lee, Y.J., Romanek, C.S., Mills, G.L., Davis, R.C., Whitman, W.B. & Wiegel, J. (2006). Gracilibacter thermotolerans gen. nov., sp. nov., an anaerobic, thermotolerant bacterium from a constructed wetland receiving acid sulfate water, International Journal of Systematic and Evolutionary Microbiology, 56, 9, pp. 2089–2093.
Li, Y.-F., Abraham, Ch., Nelson, M.C., Chen, P.-H., Graf, J. & Yu, Z. (2015). Effect of organic loading on the microbiota in a temperature-phased anaerobic digestion (TPAD) system co-digesting dairy manure and waste whey, Applied Microbiology and Biotechnology, 99, pp. 8777–8792.
Liu, Y. & Whitman, W.B. (2008). Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea, Annals of the New York Academy of Sciences, 1125, pp. 171–189.
Lu, Q., Yi, J. & Yang, D. (2016). Comparative analysis of performance and microbial characteristics between high solid and low solid anaerobic digestion of sewage sludge under mesophilic conditions, Journal of Microbiology and Biotechnology, 26, 1, pp. 110–119.
Lv, W., Zhang, W. & Yu, Z. (2013). Evaluation of system performance and microbial communities of a temperature-phased anaerobic digestion system treating dairy manure: Thermophilic digester operated at acidic pH, Bioresource Technology, 142, pp. 625–632.
Lykidis, A., Chen, C.-L., Tringe, S.G., McHardy, A.C., Copeland, A., Kyrpides, N.C., Hugenholtz, P., Macarie, H., Olmos, A., Monroy, O. & Liu, W.-T. (2011). Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium, The ISME Journal, 5, pp. 122–130.
Montero, B., Garcia-Morales, J.L., Sales, D. & Solera, R. (2008). Evolution of microorganisms in thermophilic-dry anaerobic digestion, Bioresource Technology, 99, pp. 3233–3243.
Narihiro, T., Terada, T., Kikuchi, K., Iguchi, A., Ikeda, M., Yamauchi, T., Shiraishi, K., Kamagata, Y., Nakamura, K. & Sekiguchi, Y. (2009). Comparative analysis of bacterial and archaeal communities in Methanogenic sludge granules from upflow anaerobic sludge blanket reactors treating various food-processing, high-strength organic wastewaters, Microbes and Environments, 24, pp. 88–96.
Pokój, T., Gusiatin, Z.M., Bułkowska, K. & Dubis, B. (2014). Production of biogas using maize silage supplemented with residual glycerine from biodiesel manufacturing, Archives of Environmental Protection, 40, 4, pp. 17–29.
Ragsdale, S.W. & Pierce, E. (2008). Acetogenesis and the Wood-Ljungdahl pathway of CO(2) fixation, Biochimica et Biophysica Acta, 1784, 12, pp. 1873–1898.
Rincón, B., Borja, R., González, J.M., Portillo, M.C. & Sáiz-Jiménez, C. (2008). Influence of organic loading rate and hydraulic retention time on the performance, stability and microbial communities of one-stage anaerobic digestion of two-phase olive mill solid residue, Biochemical Engineering Journal, 40, pp. 253–261.
Salunkhe, D.B. (2012). Biogas technology, International Journal of Engineering Science and Technology, 4, 12, pp. 4934–1940.
Schlüter, A., Bekel, T., Diaz, N.N., Dondrup, M., Eichenlaub, R., Gartemann, K.-H., Krahn, I., Krause, L., Krömeke, H., Kruse, O., Mussgnug, J.H., Neuweger, H., Niehaus, K., Pühler, A., Runte, K.J., Szczepanowski, R., Tauch, A., Tilker, A., Viehöver, P. & Goesmann, A. (2008). The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analyzed by the 454-pyrosequencing technology, Journal of Biotechnology, 136, 1–2, pp. 77–90.
Shah, F.A., Mahmood, Q., Shah, M.M., Pervez, A. & Asad, S.A. (2014). Microbial ecology of anaerobic digesters: the key players of anaerobiosis, The Scientific World Journal, pp. 1–21.
Sun, L., Pope, B.P., Eijsink, V.G.H. & Schnürer, A. (2015). Characterization of microbial community structure during continuous anaerobic digestion of straw and cow manure, Microbial Biotechnology, 8, 5, pp. 815–827.
Sundberg, C., Al-Soud, W.A., Larsson, M., Alm, E., Yekta, S.S., Svensson, B.H., Sørensen, S.J. & Karlsson, A. (2013). 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters, FEMS Microbioogyl Ecology, 85, pp. 612–626.
Tabatabaei, M., Rahim, R.A., Abdullah, N., Wright, A.-D.G., Shirai, Y., Sakai, K., Sulaiman, A. & Hassan, M.A. (2010). Importance of the methanogenic archaea populations in anaerobic wastewater treatments, Process Biochemistry, 45, pp. 1214–1225.
Tang, Y.Q., Shigematsu, T., Morimura, S. & Kida, K. (2005). Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation, Journal of Bioscience and Bioengineering, 99, pp. 150–164.
Wang, X., Duan, X., Chen, J., Fang, K., Feng, L., Yan, Y. & Zhou, Q. (2016). Enhancing anaerobic digestion of waste activated sludge by pretreatment: effect of volatile to total solids, Environmental Technology, DOI:10.1080/09593330.2015.1120783.
Ward, L.M., Hemp, J., Pace, L.A. & Fischera, W.W. (2015). Draft genome sequence of Leptolinea tardivitalis YMTK-2, a mesophilic anaerobe from the Chloroflexi class Anaerolineae, Genome Announcements, 3, 6.
Whitman, W.B., Bowen, T.L. & Boone, D.R. (2006). The methanogenic bacteria, Prokaryotes, 3, pp. 165–207.
Wu, J.-H., Chuang, H.-P., Hsu, M.-H. & Chen, W.-Y. (2013). Use of a hierarchical oligonucleotide primer extension approach for multiplexed relative abundance analysis of methanogens in anaerobic digestion systems, Applied and Environmental Microbiology, 79, 24, pp. 7598–7609.
Yamada, T., Sekiguchi, Y., Hanada, S., Imachi, H., Ohashi, A., Harada, H. & Kamagata, Y. (2006). Anaerolinea thermolimosa sp. nov., Levilinea saccharolytica gen. nov., sp. nov. and Leptolinea tardivitalis gen. nov., sp. nov., novel filamentous anaerobes, and description of the new classes Anaerolineae classis nov. and Caldilineae classis nov. in the bacterial phylum Chloroflexi, International Journal of Systematic and Evolutionary Microbiology, 56, 6, pp. 1331–1340.
Yuan, Y., Wanga, S., Liu, Y., Li, B., Wang, B. & Peng, Y. (2015). Long-term effect of pH on short-chain fatty acids accumulation and microbial community in sludge fermentation systems, Bioresource Technology, 197, pp. 56–63.
Zhang, X., Qu, Y., Ma, Q., Zhang, Z., Li, D., Wang, J., Shen, W., Shen, E. & Zhou, J. (2015). Illumina MiSeq sequencing reveals diverse microbial communities of activated sludge systems stimulated by different aromatics for indigo biosynthesis from indole, PLoS ONE, 10, 4, e0125732. doi:10.1371/journal.pone.0125732.
PN-81/G-04516 – Polish standard of determination of combustion heat and calculation of calorific value.
PN-EN 872:2007 – Polish standard of determination of total solids – Method by filtration through glass fiber filters
PN-EN ISO 1716 – Polish standard of determination of heat of combustion (calorific value)
PN-93/M-53950/01 – Polish standard of determination of density of gases