Journal of Applied & Environmental Microbiology
ISSN (Print): 2373-6747 ISSN (Online): 2373-6712 Website: http://www.sciepub.com/journal/jaem Editor-in-chief: Sankar Narayan Sinha
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Journal of Applied & Environmental Microbiology. 2014, 2(5), 220-230
DOI: 10.12691/jaem-2-5-4
Open AccessArticle

Effect of 3-Chloroaniline in Microbial Community Structure of Activated Sludge

M. Shah1,

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

Pub. Date: July 16, 2014

Cite this paper:
M. Shah. Effect of 3-Chloroaniline in Microbial Community Structure of Activated Sludge. Journal of Applied & Environmental Microbiology. 2014; 2(5):220-230. doi: 10.12691/jaem-2-5-4

Abstract

Present work evaluated the effects on activated-sludge reactor functions of a 3-chloroaniline (3-CA) pulse and bioaugmentation by inoculation with the 3-CA-degrading strain Pseudomonas stuzeri. Changes in functions such as nitrification, carbon removal, and sludge compaction were studied in relation to the sludge community structure, in particular the nitrifying populations. Denaturing gradient gel electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization (FISH) were used to characterize and enumerate the ammonia-oxidizing microbial community immediately after a 3-CA shock load. Two days after the 3-CA shock, ammonium accumulated, and the nitrification activity did not recover over a 12-day period in the non bioaugmented reactors. In contrast, nitrification in the bioaugmented reactor started to recover on day 4. The DGGE patterns and the FISH and real-time PCR data showed that the ammonia-oxidizing microbial community of the bioaugmented reactor recovered in structure, activity, and abundance, while the number of ribosomes of the ammonia oxidizers in the nonbioaugmented reactor decreased drastically and the community composition changed and did not recover. The settle ability of the activated sludge was negatively influenced by the 3-CA addition, with the sludge volume index increasing by a factor of 2.3. Two days after the 3-CA shock in the non bioaugmented reactor, chemical oxygen demand (COD) removal efficiency decreased by 36% but recovered fully by day 4. In contrast, in the bioaugmented reactor, no decrease of the COD removal efficiency was observed. This study demonstrates that bioaugmentation of wastewater reactors to accelerate the degradation of toxic chlorinated organic such as 3-CA protected the nitrifying bacterial community, thereby allowing faster recovery from toxic shocks.

Keywords:
chloroaniline Pseudomonas stutzeri COD ribosome

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References:

[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 settle ability 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]  Boon, N., J. Goris, P. De Vos, W. Verstraete, and E. M. Top. 2000. Bioaugmentation of activated sludge by an indigenous 3-chloroaniline degrading Comamonas testosteroni strain, I2 gfp. Appl. Environ. Microbiol. 66: 2906-2913.
 
[42]  Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1992. Standard methods for the examination of water and wastewater, 18th ed. American Public Health Association, American Water Works Association, and Water Environment Federation, Washington D.C.
 
[43]  Boon, N., J. Goris, P. De Vos, W. Verstraete, and E. M. Top. 2001. Genetic diversity among 3-chloroaniline and aniline degrading strains of the Comamonadaceae. Appl. Environ. Microbiol. 67: 1107-1115.
 
[44]  Amann, R. I. 1995. In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes, p. 1-15. In A. D. L. Akkermans, J. D. van Elsas, and F. J. de Bruijn (ed.), Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
 
[45]  Griffiths, R. I., A. S. Whiteley, A. G. O’Donnell, and M. J. Bailey. 2000. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA-and rRNA-based microbial community composition. Appl. Environ. Microbiol. 66: 5488-5491.
 
[46]  Kowalchuk, G. A., P. L. E. Bodelier, G. H. J. Heilig, J. R. Stephen, and H. J. Laanbroek. 1998. Community analysis of ammonia-oxidising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotide probe hybridisation. FEMS Microbiol. Ecol. 27: 339-350.
 
[47]  Boon, N., W. De Windt, W. Verstraete, and E. M. Top. 2002. Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol. Ecol. 39: 101-112.
 
[48]  Muyzer, G., E. C. de Waal, and A. Uitterlinden. 1993. Profiling of complex microbial populations using denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59: 695-700.
 
[49]  Colwell, R. K. 1997. EstimateS: statistical estimation of species richness and shared species from samples, version 5. User’s Guide and application published at http://viceroy.eeb.uconn.edu/estimates.
 
[50]  Colwell, R. K., and J. A. Coddington. 1994. Estimating terrestrial biodiversity through extrapolation. Philos. Trans. R. Soc. London Ser. B 345: 101-118.
 
[51]  Hughes, B. J., J. J. Hellmann, T. H. Ricketts, and B. J. M. Bohannan. 2001. Counting the uncountable: statistical approaches to estimating microbial diversity. Appl. Environ. Microbiol. 67: 4399-4406.
 
[52]  Suzuki, M., and S. Giovannoni. 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62: 625-630.
 
[53]  Heid, C., J. Stevens, K. Livak, and P. Williams. 1996. Real time quantitative PCR. Genome Res. 6: 986-994.
 
[54]  Biesterfeld, S., L. Figueroa, M. Hernandez, and P. Russell. 2001. Quantification of nitrifying bacterial populations in a full-scale trickling filter using fluorescent in situ hybridisation. Water Environ. Res. 73: 329-338.
 
[55]  Daims, H., N. B. Ramsing, K. H. Schleifer, and M. Wagner. 2001. Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization. Appl. Environ. Microbiol. 67: 5810-5818.
 
[56]  Logemann, S., J. Schantl, S. Bijvank, M. van Loosdrecht, J. G. Kuenen, and M. S. M. Jetten. 1998. Molecular microbial diversity in a nitrifying reactor system without sludge retention. FEMS Microbiol. Ecol. 27: 239-249.
 
[57]  Bollmann, A., and H. J. Laanbroek. 2001. Continuous culture of ammoniaoxidising bacteria at low ammonium concentrations. FEMS Microbiol. Ecol. 37: 211-221.
 
[58]  Purkhold, U., A. Pommerening-Roser, S. Juretschko, M. C. Schmid, H. P. Koops, and M. Wagner. 2000. Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: Implications for molecular diversity surveys. Appl. Environ. Microbiol. 66: 5368-5382.
 
[59]  Aakra, A., J. B. Utaker, A. Pommerening-Roser, H. P. Koops, and I. F. Nes. 2001. Detailed phylogeny of ammonia-oxidizing bacteria determined by rDNA sequences and DNA homology values. Int. J. Syst. Evol. Microbiol. 51: 2021-2030.
 
[60]  Head, I. M., W. D. Hiorns, T. M. Embley, A. J. McCarthy, and J. R. Saunders. 1993. The phylogeny of autotrophic ammonia-oxidising bacteria as determined by analysis of 16S ribosomal RNA gene sequences. J. Gen. Microbiol. 139: 1147-1153.
 
[61]  Ficara, E., and A. Rozzi. 2001. pH-stat titration to assess nitrification inhibition. J. Environ. Eng. 127: 698-704.
 
[62]  McCarthy, G. W. 1999. Modes of action of nitrification inhibitors. Biol. Fertil. Soils 29: 1-9.
 
[63]  Gheewala, S. H., and A. P. Annachhatre. 1997. Biodegradation of aniline. Water Sci. Technol. 36: 53-63.
 
[64]  Lekang, O.-I., and H. Kleppe. 2000. Efficiency of nitrification in trickling filters using different filter media. Aquacult. Eng. 21: 181-199.
 
[65]  Villaverde, S., F. Fdz-Olanco, and P. A. Garcia. 2000. Nitrifying biofilm acclimation to free ammonia in submerged biofilters. Water Res. 34: 602-610.
 
[66]  Suwa, Y., Y. Imamura, T. Suzuki, T. Tashiro, and Y. Urushigawa. 1994. Ammonia-oxidizing bacteria with different sensitivities to (NH4)2SO4 in activated sludges. Water Res. 28: 1523-1532.
 
[67]  Lemmer, H., G. Lind, E. Muller, M. Schade, and B. Ziegelmayer. 2000. Scum in activated sludge plants: Impact of non-filamentous and filamentous bacteria. Acta Hydrochim. Hydrobiol. 28: 34-40.
 
[68]  Wanner, J., I. Ruzickova, P. Jetmarova, O. Krhutkova, and J. Paraniakova. 1998. A national survey of activated sludge separation problems in the Czech Republic: Filaments, floc characteristics and activated sludge metabolic properties. Water Sci. Technol. 37: 271-279.
 
[69]  Madoni, P., D. Davoli, and G. Gibin. 2000. Survey of filamentous microorganisms from bulking and foaming activated-sludge plants in Italy. Water Res. 34: 1767-1772.
 
[70]  Nielsen, J. L., L. H. Mikkelsen, and P. H. Nielsen. 2001. In situ detection of cell surface hydrophobicity of probe-defined bacteria in activated sludge. Water Sci. Technol. 43: 97-103.
 
[71]  McCarthy, A. J., and S. T. Williams. 1992. Actinomycetes as agents of biodegradation in the environment—a review. Gene 115: 189-192.
 
[72]  Curtis, T. P., and N. G. Craine. 1998. The comparison of the diversity of activated sludge plants. Water Sci. Technol. 37: 71-78.
 
[73]  Eichner, C. A., R. W. Erb, K. N. Timmis, and I. Wagner-Do¨bler. 1999. Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community. Appl. Environ. Microbiol. 65: 102-109.
 
[74]  Muyzer, G., and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Leeuwenhoek 73: 127-141.
 
[75]  van Elsas, J. D., G. F. Duarte, A. S. Rosado, and K. Smalla. 1998. Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment. J. Microbiol. Methods 32: 133-154.
 
[76]  Cilia, V., B. Lafay, and R. Christen. 1996. Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level. Mol. Biol. Evol. 13: 451-461.
 
[77]  Nubel, U., B. Engelen, A. Felske, J. Snaidr, A. Wieshuber, R. I. Amann, W. Ludwig, and H. Backhaus. 1996. Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J. Bacteriol. 178: 5636-5643.
 
[78]  Rainey, F. A., N. L. WardRainey, P. H. Janssen, H. Hippe, and E. Stackebrandt. 1996. Clostridium paradoxum DSM 7308(T) contains multiple 16S rRNA genes with heterogeneous intervening sequences. Microbiology 142: 2087-2095.
 
[79]  Vallaeys, T., E. Topp, G. Muyzer, V. Macheret, G. Laguerre, A. Rigaud, and G. Soulas. 1997. Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs. FEMS Microbiol. Ecol. 24: 279-285.
 
[80]  Josephson, K. L., C. P. Gerba, and T. L. Pepper. 1993. Polymerase chain reaction of nonviable bacterial pathogens. Appl. Environ. Microbiol. 59: 3513-3515.
 
[81]  Øvreas, L., L. Forney, F. L. Daae, and V. Torsvik. 1997. Distribution of bacterioplankton in meromictic lake Saelevannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl. Environ. Microbiol. 63: 3367-3373.
 
[82]  Mobarry, B., M. Wagner, V. Urbain, B. Rittmann, and D. Stahl. 1996. Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl. Environ. Microbiol. 62: 2156-2162.
 
[83]  Amann, R. I., B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux, and D. A. Stahl. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56: 1919-1925.
 
[84]  Daims, H., A. Bruhl, R. Amann, K. H. Schleifer, and M. Wagner. 1999. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22: 434-444.