THE MICROBIAL COMMUNITY COMPOSITION OF AN ANAEROBIC REACTOR IN A SUGAR INDUSTRY WASTEWATER TREATMENT PLANT-FROM CLASSICAL TO NEW APPROACHES

Year 2021, Volume: 22 Issue: 1, 49 - 58, 15.04.2021

Nilgün Poyraz

https://doi.org/10.23902/trkjnat.835403

Abstract

In this study, the microbial characteristics of the anaerobic reactor of a sugar industry wastewater treatment plant were analyzed using cloning, FISH (Fluoresan in situ hybridization) and metagenomic analysis. Samples were obtained from seven different ports of the reactor on the 148th day of operation. The temperature was maintained at mesophilic conditions. The system’s pH range was operated at 6.8. The cloning results showed that most of the bacterial clones belonged to uncultured members of the Bacteria domain. Many archaeal clones were related to uncultured Archaea and Methanosarcina. The FISH method was applied to determine the microbial composition of the samples, which showed that bacterial and archaeal species had nearly equal rates. Rod-shaped cells, long bacilli, coccus and long chains were detected in the samples.
After metagenomic analysis, in all samples, Archaea domain members ranged between 60-36% and Bacteria domain members ranged between 58-31%. At the phylum level, in all samples, Euryarchaeota was the most dominant phylum. Proteobacteria (14.8-21.97%) and Actinobacteria (5.53-15.94%) phyla were high in rate. Furthermore, members of Spirochaeotes (0.63-4.82%) and Bacteroidetes (1.72-2.38%) were analyzed in the samples. This study revealed both bacterial and archaeal populations in the reactor of high-concentration organic sugar wastewater. These results will help in the development of more efficient anaerobic treatment systems.

Keywords

Anaerobic reactor, 16S rRNA gene cloning, FISH, microbial diversity, metagenomic analysis

Thanks

I would like to thank Prof. Dr. Mehmet Burcin MUTLU (Eskişehir-Turkey) for his scientific comments on this work.

References

• 1. Aird, D., Ross, M. G., Chen, W. S., Danielsson, M., Fennell, T., Russ, C., Jaffe B.D., Nusbaum C. & Gnirke, A. 2011. Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biology, 12(2): 1-14. https://doi.org/10.1186/gb-2011-12-2-r18
• 2. Albertsen, M., Hugenholtz, P., Skarshewski, A., Nielsen, K.L., Tyson, G.W. & Nielsen, P.H. 2013. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nature Biotechnology, 31(6): 533-538. https://doi.org/10.1038/nbt.2579
• 3. Amann, R.I., Ludwig, W. & Schleifer, K.H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59(1): 143-169. https://doi.org/10.1128/MMBR.59.1.143-169.1995
• 4. Ambuchi, J.J., Liu, J., Wang, H., Shan, L., Zhou, X., Mohammed, M.O. & Feng, Y. 2016. Microbial community structural analysis of an expanded granular sludge bed (EGSB) reactor for beet sugar industrial wastewater (BSIW) treatment. Applied Microbiology and Biotechnology, 100(10): 4651-4661. https://doi.org/10.1007/s00253-015-7245-2
• 5. 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(10): 1136-1142. https://doi.org/10.5897/AJB2013.12228
• 6. Andersson, S. 2009. Characterization of bacterial biofilms for wastewater treatment (Doctoral dissertation, Kungliga Tekniska Högskolan). KTH, School of Biotechnology, Environmental Microbiology, Stockholm, Swedish.
• 7. Bragg, L. & Tyson, G.W. 2014. Metagenomics using next-generation sequencing. In: Environmental microbiology: methods and protocols, vol. 1096. 2nd ed, Paulsen IT., Holmes AJ, (eds.). New York City: Humana Press; 183-201. https://doi.org/10.1007/978-1-62703-712-9_15
• 8. Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., & Knight, R. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5): 335-336. https://doi.org/10.1038/nmeth.f.303
• 9. Chen, Y., Cheng, J.J. & Creamer, K.S. 2008. Inhibition of anaerobic digestion process: a review. Bioresource Technology, 99(10): 4044-4064. https://doi.org/10.1016/j.biortech.2007.01.057
• 10. Daims, H., Stoecker, K. & Wagner, M. 2005. Fluorescence in situ hybridization for the detection of prokaryotes. In Molecular Microbial Ecology: 208-228. Osborn, A.M., Smith, C.J., (eds.) Taylor & Francis, Abingdon, UK.
• 11. Gao, R., Cao, Y., Yuan, X., Zhu, W., Wang, X. & Cui, Z. 2012. Microbial diversity in a full-scale anaerobic reactor treating high concentration organic cassava wastewater. African Journal of Biotechnology, 11(24): 6494-6500. https://doi.org/10.5897/AJB11.3142
• 12. Gilbride, K.A., Lee, D.Y. & Beaudette, L.A. 2006. Molecular techniques in wastewater: understanding microbial communities, detecting pathogens, and real-time process control. Journal of Microbiological Methods, 66(1): 1-20. https://doi.org/10.1016/j.mimet.2006.02.016
• 13. Guo, J., Peng, Y., Ni, B.J., Han, X., Fan, L. & Yuan, Z. 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(1): 33-44. https://doi.org/10.1186/s12934-015-0218-4
• 14. Jukes, T.H. & Cantor, C.R. 1969. Evolution of protein molecules. Mammalian protein metabolism, Munro, H.N. (ed.). New York: Academic Press. 3: 21-132. https://doi.org/10.1016/B978-1-4832-3211-9.50009-7
• 15. Khan, M.A., Ashar, N.N., Ganesh, A.G., Rais, N., Faheem, S.M. & Khan, S.T. 2019. Bacterial Community Structure in Anaerobic Digesters of a Full Scale Municipal Wastewater Treatment Plant‒Case Study of Dubai, United Arab Emirates. Journal of Sustainable Development of Energy, Water and Environment Systems, 7(3): 385-398. https://doi.org/10.13044/j.sdewes.d6.0222
• 16. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6): 1547-1549. https://doi.org/10.1093/molbev/msy096
• 17. Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L. & Pace, N.R. 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proceedings of the National Academy of Sciences, 82(20): 6955-6959. https://doi.org/10.1073/pnas.82.20.6955
• 18. Lee, C., Kim, J., Hwang, K., O'Flaherty, V. & Hwang, S. 2009. Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters. Water Research, 43(1): 157-165. https://doi.org/10.1016/j.watres.2008.09.032
• 19. Mutlu, M.B. & Güven, K. 2015. Bacterial diversity in Çamaltı saltern, Turkey. Polish Journal of Microbiology, 64(1): 37-45. https://doi.org/10.33073/pjm-2015-005
• 20. Nakasaki, K., Kwon, S.H. & Takemoto, Y. 2015. An interesting correlation between methane production rates and archaea cell density during anaerobic digestion with increasing organic loading. Biomass and Bioenergy, 78: 17-24. https://doi.org/10.1016/j.biombioe.2015.04.004
• 21. Narihiro, T. & Sekiguchi, Y. 2007. Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update. Current Opinion in Biotechnology, 18(3): 273-278. https://doi.org/10.1016/j.copbio.2007.04.003
• 22. Nayak, B.S., Levine, A.D., Cardoso, A. & Harwood, V.J. 2009. Microbial population dynamics in laboratory‐scale solid waste bioreactors in the presence or absence of biosolids. Journal of Applied Microbiology, 107(4): 1330-1339. https://doi.org/10.1111/j.1365-2672.2009.04319.x
• 23. Oksanen, J. 2013. Vegan: ecological diversity. R Project, 368 pp.
• 24. Raskin, L., Stromley, J.M., Rittmann, B.E. & Stahl, D.A. 1994. Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Applied and Environmental Microbiology, 60(4): 1232-1240. https://doi.org/10.1128/AEM.60.4.1232-1240.1994
• 25. Saitou, N. & Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4): 406-425.
• 26. Singka, D., Kumdhitiahutsawakul, L., Rekkriangkrai, P. & Pathom-Aree, W. 2012. A simple method for DNA extraction from activated sludge. Chiang Mai Journal of Science, 39(1): 111-118.
• 27. 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. https://doi.org/10.1016/j.procbio.2010.05.017
• 28. Vaneechoutte, M., Rossau, R., De Vos, P., Gillis, M., Janssens, D., Paepe, N., De Ruck, A., Fiers, T., Claeys, G. & Kersters, K. 1992. Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA). FEMS Microbiology Letters, 93(3): 227-233. https://doi.org/10.1111/j.1574-6968.1992.tb05102.x
• 29. Vanwonterghem, I., Jensen, P.D., Ho, D.P., Batstone, D.J. & Tyson, G.W. 2014. Linking microbial community structure, interactions and function in anaerobic digesters using new molecular techniques. Current Opinion in Biotechnology, 27: 55-64. https://doi.org/10.1016/j.copbio.2013.11.004
• 30. Venkiteshwaran, K., Bocher, B., Maki, J. & Zitomer, D. 2015. Relating anaerobic digestion microbial community and process function. Microbiology Insights, 8(2): 37-44. https://doi.org/10.4137/MBI.S33593
• 31. Ye, L., Zhang, T., Wang, T. & Fang, Z. 2012. Microbial structures, functions, and metabolic pathways in wastewater treatment bioreactors revealed using high-throughput sequencing. Environmental Science and Technology, 46(24): 13244-13252. https://doi.org/10.1021/es303454k
• 32. Yıldız, S., Namal, O.Ö. & Çekim, M. 2013. Atık su arıtma teknolojilerindeki tarihsel gelişimler. Selçuk Üniversitesi Mühendislik, Bilim ve Teknoloji Dergisi, 1(1): 55-67 (In Turkish).