Water Quality Measurements with a Simple Molecular Analysis (PCR-RFLP) of the Microbiome in a Metropolitan River System in Japan


  • Liswara NENENG Department of Biology Education, University of Palangka Raya, Indonesia http://orcid.org/0000-0001-9006-7329
  • Rudy Agung NUGROHO Animal Physiology, Development, and Molecular Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Mulawarman, Indonesia
  • Yukio KOMAI Department of Environmental Engineering, Osaka Institute of Technology, Japan
  • Naru TAKAYAMA Department of Environmental Engineering, Osaka Institute of Technology, Japan
  • Koji KAWAMURA Department of Environmental Engineering, Osaka Institute of Technology, Japan




Freshwater environment, ribosomal RNA, principal component analysis, water chemistry


Urbanization has affected natural freshwater environments by contamination with sewage, toxic chemicals, and excess nutrients, which cause algal bloom, pollution, and ecosystem degradation. To ensure sustainable use of natural waters, appropriate monitoring methods are required. This study aims to investigate the diversity of the microbial community in a metropolitan river system in Japan using a low-cost DNA-based approach, PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism), as a potential bioindicator of environmental change. Surface waters were sampled in seven sites in a river system. Water chemical parameters and concentrations of heavy metals were determined. Microbial DNA was extracted from the samples, ribosomal RNA was amplified with universal primers, and RFLP was scored by agarose gels. Water chemical analyses showed that surface water at the inflow point of a sewage treatment plant had signs of eutrophication. Heavy metal concentrations in surface water were low (< 0.01 ppm) in all sites. The PCR-RFLP analysis showed polymorphisms both in 16S and 18S rRNAs, indicating that the method can detect at least a part of the microbiome changes in a river system. Sequencing of some fragments found the sequence close to a ciliate isolated in wastewater treatment plants, implying contamination from sewage. Principal component analysis (PCA) identified the RFLPs associated with chemical water parameters, which could be bioindicators of environmental pollution. We also found the RFLPs independent of water quality parameters, suggesting that this simple DNA-based analysis can also detect biological changes in water ecosystems that are not quantified by chemical measurements of water quality.


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Author Biographies

Liswara NENENG, Department of Biology Education, University of Palangka Raya, Indonesia

Department of Biology Education, University of Palangka Raya, Indonesia

Yukio KOMAI, Department of Environmental Engineering, Osaka Institute of Technology, Japan

Osaka Institute of Technology, Osaka, Japan


S Suthar, J Sharma, M Chabukdhara and AK Nema. Water quality assessment of river Hindon at Ghaziabad, India: impact of industrial and urban wastewater. Environ. Monit. Assess 2010; 165, 103-12.

J Rousk and P Bengtson. Microbial regulation of global biogeochemical cycles. Front. Microbiol. 2014; 5, 103.

LH Zeglin. Stream microbial diversity in response to environmental changes: Review and synthesis of existing research. Front. Microbiol. 2015; 6, 454.

Y Bai, W Qi, J Liang and J Qu. Using high-throughput sequencing to assess the impacts of treated and untreated wastewater discharge on prokaryotic communities in an urban river. Appl. Environ. Microbiol. 2014; 98, 1841-51.

A Lapanje, D Drobne, S Nikcevic, A Perović, P Zidar, J Štrus, H Hollert and G Karaman. Bacterial community Structure analyses to assess pollution of water and sediments in the lake shkodra/skadar, Balkan Peninsula. Environ. Sci. Pollut. Res. Int. 2005; 12, 361-8.

S Tiquia. Metabolic diversity of the heterotrophic microorganisms and potential link to pollution of the Rouge River. J. Environ. Pollut. 2010; 158, 1435-43.

L Wang, J Zhang, H Li, H Yang, C Peng, Z Peng an L Lu. Shift in the microbial community composition of surface water and sediment along an urban river. Sci. Total Environ. 2018; 627, 600-12.

K Jordaan and C Bezuidenhout. Bacterial community composition of an urban river in the North West Province, South Africa, in relation to physico-chemical water quality. Environ. Sci. Pollut. Res. Int. 2016; 23, 5868-80.

Y Pei, Z Yu, J Ji, A Khan and X Li. Microbial community structure and function indicate the severity of chromium contamination of the Yellow River. Front. Microbiol. 2018; 9, 38.

JC Morse, YJ Bae and G Munkhjargal, N Sangpradub, K Tanida, TS Vshivkova, B Wang, LYang and CM Yule. Freshwater biomonitoring with macroinvertebrates in East Asia. Fornt. Ecol. Environ. 2007; 5, 33-42.

VH Resh and DM Rosenberg. Freshwater biomonitoring and benthic macroinvertebrates. Chapman & Hall, New York, 1993.

RO Carey, GJ Hochmuth, CJ Martinez, TH Boyer, MD Dukes, GS Toor and JL Cisar. Evaluating nutrient impacts in urban watersheds: Challenges and research opportunities. Environ. Pollut. 2013; 173, 138-49.

N Zheng, Q Wang, Z Liang and D Zheng. Characterization of heavy metal concentrations in the sediments of three freshwater rivers in Huludao City, Northeast China. J. Environ. Pollut. 2008; 154, 135-42.

L Zinger, A Gobet and T Pommier. Two decades of describing the unseen majority of aquatic microbial diversity. Mol. Ecol. 2012; 21, 1878-96.

M Pfrender, C Hawkins, M Bagley, G Courtney, B Creutzburg, JH Epler, S Fend, D Schindel, LCJ Ferrington, PL Hartzell, S Jackson, DP Larsen, A Lévesque, JC Morse, MJ Petersen, D Ruiter and M Whiting. Assessing macroinvertebrate biodiversity in freshwater ecosystems: Advances and challenges in DNA-based approaches. Quart. Rev. Biol. 2010; 85, 319-40.

J Theron and T Cloete. Molecular techniques for determining microbial diversity and community structure in natural environments. Crit. Rev. Microbiol. 2000; 26, 37-57.

SYS der Moon‐van, GWV der Staay, L Guillou, D Vaulot, H Claustre and LK Medlin. Abundance and diversity of prymnesiophytes in the picoplankton coμmunity from the equatorial pacific ocean inferred from 18S rDNA sequences. Limnol. Oceanogr. 2000; 45, 98-109.

WG Weisburg, SM Barns, DA Pelletier and DJ Lane. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 1991; 173, 697-703.

T García-Armisen, Ö İnceoğlu, NK Ouattara, A Anzil, MA Verbanck, N Brion and P Servais. Seasonal variations and resilience of bacterial communities in a sewage polluted urban river. PLoS One 2014. 9, e92579.

W Sun, T Xiao, M Sun, Y Dong, Z Ning, E Xiao, S Tang and J Li. Diversity of the sediment microbial community in the Aha watershed (Southwest China) in response to acid mine drainage pollution gradients. Appl. Environ. Microbiol. 2015; 81, 4874-84.

H Yin, J Niu, Y Ren, J Cong, X Zhang, F Fan, Y Xiao, X Zhang, J Deng, M Xie, Z He, J Zhou, Y Liang and X Liu. An integrated insight into the response of sedimentary microbial communities to heavy metal contamination. Sci. Rep. 2015; 5, 14266.

K Nakamura and M Suto. Analysis of bacterivorous protozoa in the natural environment (in Japanese). J. Jap. Soc. Civil Eng. Ser. G 2012; 68, III_31-III_40.

J Fried, W Ludwig, R Psenner and KH Schleifer. Improvement of ciliate identification and quantification: A new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques. Syst. Appl. Microbiol. 2002; 25, 555-71.

N Fujimoto, E Matsuo and M Murata. Evaluation of the small-eukaryote community composition in a mesotrophic lake by sequencing the 18S rRNA genes. Jap. J. Water Treat Biol. 2014; 50, 85-94.

H Sekiguchi, M Watanabe, T Nakahara, B Xu and H Uchiyama. Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis. Appl. Environ. Microbiol. 2002; 68, 5142-50.

B Drury, E Rosi-Marshall and JJ Kelly. Wastewater treatment effluent reduces the abundance and diversity of benthic bacterial communities in urban and suburban rivers. Appl. Environ. Microbiol. 2013; 79, 1897-905.

J Zhu, J Zhang, Q Li, T Han, J Xie, Y Hu and L Chai. Phylogenetic analysis of bacterial community composition in sediment contaminated with multiple heavy metals from the Xiangjiang River in China. Mar. Pollut. Bull. 2013; 70, 134-9.

JC Cho and SJ Kim. Increase in bacterial community diversity in subsurface aquifers receiving livestock wastewater input. Appl. Environ. Microbiol. 2000; 66, 956-65.

H Yin, G Qiu, L Wu, M Xie, J Zhou, Z Dai, D Wang, L Kellogg, L Cao and X Liu. Microbial community diversity and changes associated with a mine drainage gradient at the Dexing copper mine, China. Aquat. Microb. Ecol. 2008; 51, 67-76.




How to Cite

NENENG, L. ., NUGROHO, R. A. ., KOMAI, Y. ., TAKAYAMA, N. ., & KAWAMURA, K. . (2019). Water Quality Measurements with a Simple Molecular Analysis (PCR-RFLP) of the Microbiome in a Metropolitan River System in Japan. Walailak Journal of Science and Technology (WJST), 17(3), 257–268. https://doi.org/10.48048/wjst.2020.5869