International Journal of Environmental Bioremediation & Biodegradation
ISSN (Print): 2333-8628 ISSN (Online): 2333-8636 Website: Editor-in-chief: Apply for this position
Open Access
Journal Browser
International Journal of Environmental Bioremediation & Biodegradation. 2014, 2(4), 197-201
DOI: 10.12691/ijebb-2-4-7
Open AccessArticle

Amplified Ribosomal DNA Restriction Analysis as a Tool to Characterize Microbial Community Structure of Activated Sludge of Common Effluent Treatment Plant

M. Shah1,

1Industrial Waste Water Research Laboratory Division of Applied & Environmental Microbiology Enviro Technology Limited Gujarat, India

Pub. Date: August 06, 2014

Cite this paper:
M. Shah. Amplified Ribosomal DNA Restriction Analysis as a Tool to Characterize Microbial Community Structure of Activated Sludge of Common Effluent Treatment Plant. International Journal of Environmental Bioremediation & Biodegradation. 2014; 2(4):197-201. doi: 10.12691/ijebb-2-4-7


ARDRA (Amplified ribosomal DNA restriction analysis) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed.

Amplified ribosomal DNA restriction analysis wastewater treatment plants 16S ribosomal DNA · Activated sludge

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit


[1]  Seviour R, Nielsen PH (2010) Microbial ecology of activated sludge. London: IWA Publishing Company. 688 p. 2. Bitton G (2011) Wastewater microbiology. Hoboken, NJ: John Wiley and Sons. 746 p.
[2]  Graham DW, Smith VH (2004) Designed ecosystem services: Application of ecological principles in wastewater treatment engineering. Front Ecol Environ 4: 199-206.
[3]  Wang X, Wen X, Yan H, Ding K, Zhao F, et al. (2011) Bacterial community dynamics in a functionally stable pilot-scale wastewater treatment plant. Bioresour Technol 102: 2352-2357.
[4]  Curtis TP, Head IM, Graham DW (2003) Theoretical ecology in engineering biology. Environ Sci Technol 37: 64A-70A.
[5]  Wells GF, Park HD, Eggleston B, Francis CA, Criddle CS (2011) Fine-scale bacterial community dynamics and the taxa-time relationship within a full-scale activated sludge bioreactor. Water Res 45: 5476-5488.
[6]  Wagner M, Loy A, Nogueira R, Purkhold U, Lee N, et al. (2002) Microbial community composition and function in wastewater treatment plants. Antonie van Leeuwenhoek 81: 665 680.
[7]  Kaewpipat K, Grady CPL (2002) Microbial population dynamics in laboratory scale activated sludge reactors. Water Sci Technol 46: 19-27.
[8]  Padayachee P, Ismail A, Bux F (2006) Elucidation of the microbial community structure within a laboratory-scale activated sludge process using molecular techniques. Water SA 32: 679-686.
[9]  Jenkins D (2008) From total suspended solids to molecular biology tools a personal view of biological wastewater treatment process population dynamics. Water Environ Res 80: 677-687.
[10]  Saikaly PE, Stroot PG, Oerther DB (2005) Use of 16S rRNA gene terminal restriction fragment analysis to assess the impact of solids retention time on the bacterial diversity of activated sludge. Appl Environ Microbiol 71: 5814-5822.
[11]  Ofit¸ eru ID, Lunn M, Curtis TP, Wells GF, Criddle CS, et al. (2010) Combined niche and neutral effects in a microbial wastewater treatment community. Proc Natl Acad Sci U S A 107: 15345-15350.
[12]  Sanapareddy N, Hamp TJ, Gonzalez LC, Hilger HA, Fodor AA, et al. (2009) Molecular diversity of a North Carolina wastewater treatment plant as revealed by pyrosequencing. Appl Environ Microbiol 75: 1688-1696.
[13]  Fierer N, Lennon JT (2011) The generation and maintenance of diversity in microbial communities. Am J Bot 98: 439-448.
[14]  Nemergut DR, Costello EK, Hamady M, Lozupone C, Jiang L, et al. (2011) Global patterns in the biogeography of bacterial taxa. Environ Microbiol 13: 135-144.
[15]  Benedict RG, Carlson DA (1971) Aerobic heterotrophic bacteria in activated sludge. Water Res 5: 1023-1030.
[16]  Dias FF, Bhat JV (1964) Microbial ecology of activated sludge. Appl Microbiol 12: 412-417.
[17]  Lighthart B, Oglesby RT (1969) Bacteriology of an activated sludge wastewater treatment plant: A guide to methodology. J Water Pollut Control Fed 41: R267-R281.
[18]  van Veen W (1973) Bacteriology of activated sludge, in particular the filamentous bacteria. Antonie van Leeuwenhoek 39: 189-205.
[19]  Eschenhagen M, Schuppler M, Ro¨ske I (2003) Molecular characterization of the microbial community structure in two activated sludge systems for the advanced treatment of domestic effluents. Water Res 37: 3224-3232.
[20]  Snaidr J, Amann R, Huber I, Ludwing W, Schleifer K (1997) Phylogenetic analysis and in-situ identification of bacteria in activated sludge. Appl Environ Microbiol 63: 2884-2896.
[21]  Wagner M, Amann R, Lemmer H, Schleifer K (1993) Probing activated sludge with oligonucleotides specific for proteobacteria: Inadequacy of culture dependent methods for describing microbial community structure. Appl Environ Microbiol 59: 1520-1525.
[22]  Watanabe K, Yamamoto S, Hino S, Harayama S (1998) Population dynamics of phenol-degrading bacteria in activated sludge determined by gyrB-targeted quantitative PCR. Appl Environ Microbiol 64: 1203-1209.
[23]  Green JL, Bohannan BJM (2006) Spatial scaling of microbial biodiversity. Trends Ecol Evol 21: 501-507.
[24]  Martiny JBH, Bohannan BJM, Brown JH, Colwell RK, Fuhrman JA, et al. (2006) Microbial biogeography: putting microorganisms on the map. Nat Rev Microbiol 4: 102-112.
[25]  van der Gast CJ, Jefferson B, Reid E, Robinson T, Bailey MJ, et al. (2006) Bacterial diversity is determined by volume in membrane bioreactors. Environ Microbiol 8: 1048-1055.
[26]  Wang X, Wen X, Criddle C, Wells G, Zhang J, et al. (2010) Community analysis of ammonia-oxidizing bacteria in activated sludge of eight wastewater treatment systems. J Environ Sci (China) 22: 627-634.
[27]  Loy A, Daims H, Wagner M (2002) Activated sludge: molecular techniques for determining community composition. In: Bitton G, editor. The Encyclopedia of environmental microbiology. Hoboken, NJ: John Wiley and Sons. 26-43.
[28]  Onuki M, Satoh H, Mino T, Matsuo T (2000) Application of molecular methods to microbial community analysis of activated sludge. Water Sci Technol 42: 17-22.
[29]  Wilderer PA, Bungartz HJ, Lemmer H, Wagner M, Keller J, et al. (2002) Modern scientific methods and their potential in wastewater science and technology. Water Res 36: 370-393.
[30]  de los Reyes FL III (2010) Challenges in determining causation in structure function studies using molecular biological techniques. Water Res 44: 4948-4957.
[31]  Jones PA, Schuler AJ (2010) Seasonal variability of biomass density and activated sludge settleability in full-scale wastewater treatment systems. Chem Eng J 164: 16-22.
[32]  Carvalho G, Lemos PC, Oehmen A, Reis MAM (2007) Denitrifying phosphorus removal: Linking the process performance with the microbial community structure. Water Res 41: 4383-4396.
[33]  Briones A, Raskin L (2003) Diversity and dynamics of microbial communities in engineered environments and their implications for process stability. Curr Opin Biotechnol 14: 270-276.
[34]  Gentile ME, Jessup CM, Nyman JL, Criddle CS (2007) Correlation of functional instability and community dynamics in denitrifying dispersed-growth reactors. Appl Environ Microbiol 73: 680-690.
[35]  Curtis TP, Sloan WT (2006) Towards the design of diversity: stochastic models for community assembly in wastewater treatment plants. Water Sci Technol 54: 227-236.
[36]  Daims H, Taylor MW, Wagner M (2006) Wastewater treatment: a model system for microbial ecology. Trends Biotechol 24: 483-489.
[37]  Prosser JI, Bohannan BJ, Curtis TP, Ellis RJ, Firestone MK, et al. (2007) The role of ecological theory in microbial ecology. Nat Rev Microbiol 5: 384-392.
[38]  Acinas SG, Rodríguez-Valera F, Pedrós-Alió C (1997) Spatial and temporal variation in marine bacterioplancton diversity as shown by RFLP fingerprinting of PCR amplified 16S rDNA. FEMS Microbiol Ecol 24: 27-40
[39]  Martínez-Murcia AJ, Acinas SG, Rodríguez-Valera F (1995) Evaluation of prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified from hipersaline environments. FEMS Microbiol Ecol 17: 247-256
[40]  Moyer CL, Dobbs FC, Karl DM (1994) Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Appl Environ Microbiol 60: 871-879
[41]  APHA, AWWA and WEF (1992) Standard methods for the examination of water and wastewater. American Public Health Assoc., Washington, DC
[42]  Moore DD (1996) Purification and concentration of DNA from aqueous solutions. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Steidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology.
[43]  Zhou JZ, Fries MR, Chee-Sanford J, Tiedje JM (1995) Phylogenetic analysis of a new group of denitrifiers capable of anaerobic growth on toluene: description of Azoarcus tolulyticus sp. nov. Int J Syst Bacteriol 45: 500-506 John Wiley and Sons, New York
[44]  Sneath PHA, Sokal RR (1973) The estimation of taxonomic resemblance. In: Kennedy D, Park RB (eds) Numerical taxonomy. The principles and practice of numerical classification. Freeman, San Francisco, pp 129-132
[45]  Ehlers MM, Cloete TE (1999) Comparing the protein profiles of 21 different activated sludge systems after SDS-PAGE. Water Res 33: 1181-1186.