Methanogenic community structure in simultaneous methanogenesis and denitrification granular sludge

Highlights

• UASB with SMD granules was operated with high removal efficiency of COD and NO₃^--N.

• Methanosaetaceae was absolute predominant methanogen in SMD granules.

• The methanogen quantity and activity decreased as C/N decreased from 20:1 to 5:1.

• Bacterial community succession happened with C/N decreasing.

Abstract

A laboratory scale up-flow anaerobic sludge bed (UASB) bioreactor fed with synthetic wastewater was operated with simultaneous methanogenesis and denitrification (SMD) granules for 235 days with a gradient decrease of C/N. Molecular cloning, qRT-PCR and T-RFLP were applied to study the methanogenic community structures in SMD granules and their changes in response to changing influent C/N. The results indicate that when C/N was 20:1, the methane production rate was fastest, and Methanosaetaceae and Methanobacteriaceae were the primary methanogens within the Archaea. The richness and evenness of methanogenic bacteria was best with the highest T-RFLP diversity index of 1.627 in the six granular sludge samples. When C/N was reduced from 20:1 to 5:1, the methanogenic activity of SMD granules decreased gradually, and the relative quantities of methanogens decreased from 36.5% to 10.9%. The abundance of Methanosaetaceae in Archaea increased from 64.5% to 84.2%, while that of Methanobacteriaceae decreased from 18.6% to 11.8%, and the richness and evenness of methanogens decreased along with the T-RFLP diversity index to 1.155, suggesting that the community structure reflected the succession to an unstable condition represented by high nitrate concentrations.

Graphical abstract

Keywords

Simultaneous methanogenesis and denitrification (SMD) / Methanogens / Community structure / Diversity index / Granular sludge

References

[1] Akizuki S, Izumi K, Nagao N, Shiotani T, Niwa C, Toda T (2013). Effect of COD/NO₃-N ratio and seed sludge on simultaneous methanogenesis and denitrification in intermittent organic solid waste treatment. International Biodeterioration & Biodegradation, 84(6): 8–13

[2] An Y, Yang F, Wong F S, Chua H C (2008). Simultaneous bioenergy (CH₄) production and nitrogen removal in a combined upflow anaerobic sludge blanket and aerobic membrane bioreactor. Energy & Fuels, 22(1): 103–107

[3] Angelidaki I, Alves M, Bolzonella D, Borzacconi L, Campos J L, Guwy A J, Kalyuzhnyi S, Jenicek P, van Lier J B (2009). Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Science and Technology, 59(5): 927–934

[4] Banihani Q, Sierra-Alvarez R, Field J A (2009). Nitrate and nitrite inhibition of methanogenesis during denitrification in granular biofilms and digested domestic sludges. Biodegradation, 20(6): 801–812

[5] Chen K C, Lin Y F (1993). The relationship between denitrifying bacteria and methanogenic bacteria in a mixed culture system of acclimated sludges. Water Research, 27(12): 1749–1759

[6] Chen S, Sun D, Chung J S (2009). Simultaneous methanogenesis and denitrification of aniline wastewater by using anaerobic-aerobic biofilm system with recirculation. Journal of Hazardous Materials, 169(1–3): 575–580

[7] Grosskopf R, Janssen P H, Liesack W (1998). Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Applied and Environmental Microbiology, 64(3): 960–969

[8] He Q M, Peng X Y, Li Z Y (2014). The treatment of animal manure wastewater by coupled simultaneous methanogenesis and denitrification (SMD) and shortcut nitrification-denitrification (SND). Journal of Chemical Technology and Biotechnology (Oxford, Oxfordshire), 89(11): 1697–1704

[9] Hendriksen H V, Ahring B K (1996). Integrated removal of nitrate and carbon in an upflow anaerobic sludge blanket (UASB) reactor: operating performance. Water Research, 30(6): 1451–1458

[10] Klüber H D, Conrad R (1998). Inhibitory effects of nitrate, nitrite, NO and N₂O on methanogenesis by Methanosarcina barkeri and Methanobacterium bryantii. FEMS Microbiology Ecology, 25(4): 331–339

[11] Kodera T, Akizuki S, Toda T (2017). Formation of simultaneous denitrification and methanogenesis granules in biological wastewater treatment. Process Biochemistry, 58: 252–257

[12] Liang L H, Zuo J E (2008). Review of modern culture-independent methods used to study community structure and function of denitrifying microorganisms. Acta Scientiae Circumstantiae, 28(4): 599–605 (in Chinese)

[13] Lin Y F, Chen K C (1995). Denitrification and methanogenesis in a co-immobilized mixed culture system. Water Research, 29(1): 35–43

[14] Liu W T, Chan O C, Fang H H P (2002). Characterization of microbial community in granular sludge treating brewery wastewater. Water Research, 36(7): 1767–1775

[15] Lueders T, Chin K J, Conrad R, Friedrich M (2001). Molecular analyses of methyl-coenzyme M reductase a-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage. Environmental Microbiology, 3(3): 194–204

[16] Mosquera-Corral A, Sánchez M, Campos J L, Méndez R, Lema J M (2001). Simultaneous methanogenesis and denitrification of pretreated effluents from a fish canning industry. Water Research, 35(2): 411–418

[17] Osborn A M, Moore E R B, Timmis K N (2000). An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environmental Microbiology, 2(1): 39–50

[18] Ovreås L, Forney L, Daae F L, Torsvik V (1997). Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Applied and Environmental Microbiology, 63(9): 3367–3373

[19] Song M, Shin S G, Hwang S (2010). Methanogenic population dynamics assessed by real-time quantitative PCR in sludge granule in upflow anaerobic sludge blanket treating swine wastewater. Bioresource Technology, 101(Suppl 1): S23–S28

[20] Stahl D A, Amann R (1998). Development and application of nucleic acid probes. In: Nucleic Acid Techniques in Bacterial Systematics. Chichester: John Wiley Sons Ltd., 205–248

[21] 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(8): 1214–1225

[22] Tugtas A E, Pavlostathis S G (2007). Inhibitory effects of nitrogen oxides on a mixed methanogenic culture. Biotechnology and Bioengineering, 96(3): 444–455

[23] Tugtas A E, Pavlostathis S G (2008). Inhibitory effects of nitrate reduction on methanogenesis in the presence of different electron donors. Water Science and Technology, 57(5): 693–698

[24] Wagman D D, Evans W H, Parker V B, Schumm R H, Halow I, Bailey S M, Churney K L, Nuttall R L (1982). The NBS tables of chemical thermodynamic properties. Journal of Physical and Chemical Reference Data, 11(2): 7–38

[25] Wang B, Peng D, Zhang X, Wang X (2016). Structure and formation of anoxic granular sludge—A string-bag hypothesis. Frontiers of Environmental Science and Engineering, 10(2): 311–318

[26] Xie L, Chen J, Wang R, Zhou Q (2012). Effect of carbon source and COD/NO₃-N ratio on anaerobic simultaneous denitrification and methanogenesis for high-strength wastewater treatment. Journal of Bioscience and Bioengineering, 113(6): 759–764

[27] Yi X H, Wan J, Ma Y, Wang Y, Guan Z, Jing D D (2017). Structure and succession of bacterial communities of the granular sludge during the initial stage of the simultaneous denitrification and methanogenesis process. Water, Air, and Soil Pollution, 228(3): 121–132

[28] Yin Z, Xie L, Zhou Q, Bi X (2018). Simultaneous carbon and nitrogen removal from anaerobic effluent of the cassava ethanol industry. Journal of Bioscience and Bioengineering, 125(3): 346–352

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 51678054 and 51378064).

RIGHTS & PERMISSIONS

2018 Higher Education Press and Springer–Verlag GmbH Germany, part of Springer Nature