Characterization of the genes involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

Junqin PANG, Masami MATSUDA, Masashi KURODA, Daisuke INOUE, Kazunari SEI, Kei NISHIDA, Michihiko IKE

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Front. Environ. Sci. Eng. ›› 2016, Vol. 10 ›› Issue (4) : 7. DOI: 10.1007/s11783-016-0846-x
RESEARCH ARTICLE
RESEARCH ARTICLE

Characterization of the genes involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

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Abstract

Nitrogen-cycling microbial communities in municipal WWTPs were characterized.

Numbers of amoA, nirK and nirS genes were quantified by MPN-PCR.

Diversity of whole nitrogen-cycling communities was analyzed with DNA microarray.

CAS process retained diverse nitrogen cycling populations.

Specific, limited populations may be dominated in nitrogen removal processes.

To improve nitrogen removal performance of wastewater treatment plants (WWTPs), it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods:most probable number-polymerase chain reaction (MPN-PCR) and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional activated sludge processes (CAS) are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern:the nirS and the bacterial amoA genes were more abundant than the nirK and archaeal amoA genes, respectively. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes (AO), whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific.

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Keywords

DNA microarray analysis / Nitrogen cycling functional genes / Most probable number-polymerase chain reaction (MPN-PCR) / Wastewater treatment plants (WWTPs)

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Junqin PANG, Masami MATSUDA, Masashi KURODA, Daisuke INOUE, Kazunari SEI, Kei NISHIDA, Michihiko IKE. Characterization of the genes involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR. Front. Environ. Sci. Eng., 2016, 10(4): 7 https://doi.org/10.1007/s11783-016-0846-x

References

[1]
Smith V H, Tilman G D, Nekola J C. Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems. Environmental Pollution, 1999, 100(1-3): 179–196
CrossRef Google scholar
[2]
Ahn Y H. Sustainable nitrogen elimination biotechnologies: a review. Process Biochemistry, 2006, 41(8): 1709–1721
CrossRef Google scholar
[3]
Richardson D J, Watmough N J. Inorganic nitrogen metabolism in bacteria. Current Opinion in Chemical Biology, 1999, 3(2): 207–219
CrossRef Google scholar
[4]
Zhu G B, Peng Y Z, Li B K, Guo J H, Yang Q, Wang S Y. Biological removal of nitrogen from wastewater. Reviews of Environmental Contamination and Toxicology, 2008, 192: 159–195
CrossRef Google scholar
[5]
Song B, Tobias C R. Molecular and stable isotope methods to detect and measure anaerobic ammonium oxidation (anammox) in aquatic ecosystems. Methods in Enzymology, 2011, 496: 63–89
CrossRef Google scholar
[6]
Kampschreur M J, Temmink H, Kleerebezem R, Jetten M S M, van Loosdrecht C M. Nitrous oxide emission during wastewater treatment. Water Research, 2009, 43(17): 4093–4103
CrossRef Google scholar
[7]
Soda S, Arai T, Inoue D, Ishigaki T, Ike M, Yamada M. Statistical analysis of global warming potential, eutrophication potential, and sludge production of wastewater treatment in Japan. Journal of Sustainable Energy and Environment, 2013, 4(1): 33–40
[8]
Amann R I, Ludwing W, Schleifer K H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 1995, 59(1): 143–169
[9]
Prosser J I, Embley T M. Cultivation-based and molecular approaches to characterization of terrestrial and aquatic nitrifiers. Antonie van Leeuwenhoek, 2002, 81(1): 165–179
CrossRef Google scholar
[10]
Smith C J, Osborn A M. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiology Ecology, 2009, 67(1): 6–20
CrossRef Google scholar
[11]
He Z, Deng Y, Zhou J. Development of functional gene microarrays for microbial community analysis. Current Opinion in Biotechnology, 2012, 23(1): 49–55
CrossRef Google scholar
[12]
Inoue D, Pang J, Matsuda M, Sei K, Nishida K, Ike M. Development of a whole community genome amplification-assisted DNA microarray method to detect functional genes involved in the nitrogen cycle. World Journal of Microbiology & Biotechnology, 2014, 30(11): 2907–2915
CrossRef Google scholar
[13]
Hashimoto K, Matsuda M, Inoue D, Ike M. Bacterial community dynamics in a full-scale municipal wastewater treatment plant employing conventional activated sludge process. Journal of Bioscience and Bioengineering, 2014, 118(1): 64–71
CrossRef Google scholar
[14]
Iwamoto T, Tani K, Nakamura K, Suzuki Y, Kitagawa M, Eguchi M, Nasu M. Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE. FEMS Microbiology Ecology, 2000, 32(2): 129–141
CrossRef Google scholar
[15]
Rotthauwe J H, de Boe W, Liesack W. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology, 1997, 63(12): 4704–4712
[16]
Sang N, Soda S, Sei K, Ike M. Effect of aeration on stabilization of organic solid waste and microbial population dynamics in lab-scale landfill bioreactors. Journal of Bioscience and Bioengineering, 2008, 106(5): 425–432
CrossRef Google scholar
[17]
Francis C A, Roberts K J, Michael J, Santoro A E, Oakley B B. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(41): 16683–14688
CrossRef Google scholar
[18]
Wu L, Thompson D K, Li G, Hurt R A, Tiedje J M, Zhou J. Development and evaluation of functional gene arrays for detection of selected genes in the environment. Applied and Environmental Microbiology, 2001, 67(12): 5780–5790
CrossRef Google scholar
[19]
Wu L, Liu X, Christopher W S, Zhou J. Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole community genome amplification. Applied and Environmental Microbiology, 2006, 72(7): 4931–4941
CrossRef Google scholar
[20]
Geets J, de Cooman M, Wittebolle L, Heylen K, Vanparys B, de Vos P, Verstraete W, Boon N. Real-time PCR assay for the simultaneous quantification of nitrifying and denitrifying bacteria in activated sludge. Applied Microbiology and Biotechnology, 2007, 75(1): 211–221
CrossRef Google scholar
[21]
Wang Z, Zhang X X, Lu X, Liu B, Li Y, Long C, Li A. Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing. PLoS ONE, 2014, 9(11): e113603
CrossRef Google scholar
[22]
Shu D, He Y, Yue H, Wang Q. Microbial structures and community functions of anaerobic sludge in six full-scale wastewater treatment plants as revealed by 454 high-throughput pyrosequencing. Bioresource Technology, 2015, 186: 163–172
CrossRef Google scholar
[23]
Daims H, Wagner M. The microbiology of nitrogen removal. In: Seviour RJ, Nielsen PH, eds. Microbial Ecology of Activated Sludge. London: IWA Publishing, 2010,259–280
[24]
Kayee P, Sonthiphand P, Rongsayamanont C, Limpiyakorn T. Archaeal amoA genes outnumber bacterial amoA genes in municipal wastewater treatment plants in Bangkok. Microbial Ecology, 2011, 62(4): 776–788
CrossRef Google scholar
[25]
Limpiyakorn T, Sonthiphand P, Rongsayamanont C, Polprasert C. Abundance of amoA genes of ammonia-oxidizing archaea and bacteria in activated sludge of full-scale wastewater treatment plants. Bioresource Technology, 2011, 102(4): 3694–3701
CrossRef Google scholar
[26]
Wu Y J, Whang L M, Fukushima T, Chang S H. Responses of ammonia-oxidizing archaeal and betaproteobacterial populations to wastewater salinity in a full-scale municipal wastewater treatment plant. Journal of Bioscience and Bioengineering, 2013, 115(4): 424–432
CrossRef Google scholar
[27]
Burgin A J, Hamilton S K. Have we overemphasized the role of denitrification in aquatic ecosystems? A review of nitrate removal pathways. Frontiers in Ecology and the Environment, 2007, 5(2): 89–96
CrossRef Google scholar
[28]
Kraft B, Strous M, Tegetmeyer H E. Microbial nitrate respiration—genes, enzymes and environmental distribution. Journal of Biotechnology, 2011, 155(1): 104–117
CrossRef Google scholar
[29]
Rütting T, Boeckx P, Müller C, Klemedtsson L. Assessment of the importance of dissimilatory nitrate reduction to ammonium for the terrestrial nitrogen cycle. Biogeosciences, 2011, 8(7): 1779–1791
CrossRef Google scholar
[30]
van den Berg E M, van Dongen U, Abbas B, van Loosdrecht M C M. Enrichment of DNRA bacteria in a continuous culture. ISME Journal, 2015, 9(10): 2153–2161
CrossRef Google scholar
[31]
Strous M, van Gerven E, Kuenen J G, Jetten M. Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (anammox) sludge. Applied and Environmental Microbiology, 1997, 63(6): 2446–2448
[32]
Matsuda M, Inoue D, Anami Y, Tsutsui H, Sei K, Soda S, Ike M. Comparative analysis of DNA-based microbial community composition and substrate utilisation patterns of activated sludge microorganisms from wastewater treatment plants operated under different conditions. Water Science and Technology, 2010, 61(11): 2843–2851
CrossRef Google scholar
[33]
Takada K, Hashimoto K, Soda S, Ike M, Yamashita K, Hashimoto T. Characterization of microbial community in membrane bioreactors treating domestic wastewater. Journal of Water and Environment Technology, 2014, 12(2): 99–107
CrossRef Google scholar
[34]
Ma Q, Qu Y, Shen W, Zhang Z, Wang J, Liu Z, Li D, Li H, Zhou J. Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina high-throughput sequencing. Bioresource Technology, 2015, 179: 436–443
CrossRef Google scholar

Acknowledgements

This study was partly supported by the Global COE Program, “Evaluation of Research and Education on Integrated River Basin Management in Asian Region” of the University of Yamanashi, Japan, which is sponsored by the Japan Society for the Promotion of Science (JSPS), and a Kurita Water Environment Foundation grant.

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11783-016-0846-x and is accessible for authorized users.
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