International Journal of Environmental Bioremediation & Biodegradation
ISSN (Print): 2333-8628 ISSN (Online): 2333-8636 Website: http://www.sciepub.com/journal/ijebb Editor-in-chief: Apply for this position
Open Access
Journal Browser
Go
International Journal of Environmental Bioremediation & Biodegradation. 2014, 2(3), 100-111
DOI: 10.12691/ijebb-2-3-2
Open AccessArticle

Evaluation and Analysis of Bacterial Communities from Different Waste Water Treatment Plants by Denaturing Gradient Gel Electrophoresis with Group Specific 16s rRNA

Maulin P Shah1,

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

Pub. Date: May 07, 2014

Cite this paper:
Maulin P Shah. Evaluation and Analysis of Bacterial Communities from Different Waste Water Treatment Plants by Denaturing Gradient Gel Electrophoresis with Group Specific 16s rRNA. International Journal of Environmental Bioremediation & Biodegradation. 2014; 2(3):100-111. doi: 10.12691/ijebb-2-3-2

Abstract

The diversity of different bacterial groups of activated sludge samples that received wastewater from four different types of industry was investigated by a nested PCR-DGGE (denaturing gradient gel electrophoresis) approach. Specific 16S rRNA primers were chosen for large bacterial groups (Bacteria and α-Proteobacteria in particular), which dominate activated sludge communities, as well as for actinomycetes, ammonium oxidizers and methanotrophs (Types I and II). In addition primers for the new Acidobacterium group were used to observe their community structure in activated sludge. After this first PCR amplification, a second PCR with Bacterial primers yielded 16S rRNA gene fragments that were subsequently separated by DGGE, thus generating “group specific DGGE patterns”. The community structure and diversity of the bacterial groups from the different samples was further analyzed using different techniques, such as statistical analysis and Shannon diversity index evaluation of the band patterns. By combining the seven DGGE gels, cluster analysis, Multidimensional scaling (MDS) and Principal Component Analysis (PCA) clearly clustered two of the four activated sludge types separately. It was shown that the combination of molecular and statistical methods can be very useful to differentiate activated sludge microbial communities.

Keywords:
DGGE 16s rRNA Waste Water Treatment PCR

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]  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.
 
[2]  Aleshchenkova, Z. M., A. S. Samsonova, and N. F. Semochkina 1999. Effects of degrading microorganisms on purification of wastewater from Lavsan production enterprises with the use of activated sludge. Appl. Biochem. Microbiol. 35: 404-406.
 
[3]  Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
 
[4]  Curtis, T. P., and N. G. Craine 1998. The comparison of the diversity of activated sludge plants. Water Sci. Tech. 37: 71-78.
 
[5]  da Silva, E. M., A. M. V. M. Soares, and A. J. M. Moreno 1998. The use of the mitochondrial transmembrane electric potential as an effective biosensor in ecotoxicological research. Chemosphere. 36: 2375-2390.
 
[6]  Amann, R., F. O. Glockner, and A. Neef 1997. Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes. FEMS Microbiol. Rev. 20: 191-200.
 
[7]  Blackall, L. L., P. C. Burrell, H. Gwilliam, D. Bradford, P. L. Bond, and P. Hugenholtz 1998. The use of 16S rDNA clone libraries to describe the microbial diversity of activated sludge communities. Water Sci. Tech. 37: 451-454.
 
[8]  Amann, R. I., W. Ludwig, and K. H. Schleifer 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143-169.
 
[9]  Wagner, M., R. Amann, H. Lemmer, and K. H. Schleifer 1993. Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culturedependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59: 1520-1525.
 
[10]  Wagner, M., R. Erhart, W. Manz, R. Amann, H. Lemmer, D. Wedi, and K. H. Schleifer 1994. Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl. Environ. Microbiol. 60: 792-800.
 
[11]  Dahlberg, C., M. Bergström, and M. Hermansson 1998. In situ detection of high levels of horizontal plasmid transfer in marine bacterial communities. Appl. Environ. Microbiol. 64: 2670-2675.
 
[12]  Eichner, C. A., R. W. Erb, K. N. Timmis, and I. Wagner-Döbler 1999. Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community. Appl. Environ. Microbiol. 65: 102-109.
 
[13]  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.
 
[14]  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. Meth. 32: 133-154.
 
[15]  Heuer, H., M. Krsek, P. Baker, K. Smalla, and E. M. H. Wellington 1997. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63: 3233-3241.
 
[16]  Henckel, T., M. Friedrich, and R. Conrad 1999. Molecular analyses of the methane oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl. Environ. Microbiol. 65: 1980-1990.
 
[17]  Gomes, N. C. M., H. Heuer, J. Schönfeld, R. Costa, L. Mendonça-Hagler, and K. Smalla 2001. Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil studied by temperature gradient gel electrophoresis. Plant Soil. 232: 167-180.
 
[18]  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.
 
[19]  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.
 
[20]  Smit, E., P. Leeflang, B. Glandorf, J. D. van Elsas, and K. Wernars 1999. Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis. Appl. Environ. Microbiol. 65: 2614-2621.
 
[21]  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, I2gfp. Appl. Environ. Microbiol. 66: 2906-2913.
 
[22]  Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
 
[23]  Pearson, K. 1926. On the coefficient of radical likeliness. Biometrika. 18: 105-117.
 
[24]  Ward, J. H. 1963. Hierarchical grouping to optimize an objective function. J. Am. Stat. Assoc. 58: 236-244.
 
[25]  Shannon, C. E., and W. Weaver 1963. The mathematical theory of communication. University of Illinois Press, Urbana.
 
[26]  Kennedy, A. C., and K. L. Smith 1995. Soil microbial diversity and the sustainability of agricultural soils. Plant Soil. 170: 75-86.
 
[27]  Phillips, C. J., D. Harris, S. L. Dollhopf, K. L. Gross, J. I. Prosser, and E. A. Paul 2000. Effects of agronomic treatments on structure and function of ammonia-oxidizing communities. Appl. Environ. Microbiol. 66: 5410-5418.
 
[28]  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.
 
[29]  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.
 
[30]  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.
 
[31]  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.
 
[32]  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.
 
[33]  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.
 
[34]  LaPara, T. M., C. H. Nakatsu, L. Pantea, and J. E. Alleman 2000. Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Appl. Environ. Microbiol. 66: 3951-3959.
 
[35]  Mudaly, D. D., B. W. Atkinson, and F. Bux 2001. 16S rRNA in situ probing for the determination of the family level community structure implicated in enhanced biological nutrient removal. Water Sci. Technol. 43: 91-98.
 
[36]  Neef, A., R. Witzenberger, and P. Kampfer 1999. Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes. J. Ind. Microbiol. Biotechnol. 23: 261-267.
 
[37]  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.
 
[38]  Barns, S. M., S. L. Takala, and C. R. Kuske 1999. Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment. Appl. Environ. Microbiol. 65: 1731-1737.
 
[39]  Sievert, S. M., J. Kuever, and G. Muyzer 2000. Identification of 16S ribosomal DNA-defined bacterial populations at a shallow submarine hydrothermal vent near Milos Island (Greece). Appl. Environ. Microbiol. 66: 3102-3109.
 
[40]  Ludwig, W., S. H. Bauer, M. Bauer, I. Held, G. Kirchhof, R. Schulze, I. Huber, S. Spring, A. Hartmann, and K. H. Schleifer 1997. Detection and in situ identification of representatives of a widely distributed new bacterial phylum. FEMS Microbiol. Lett. 153: 181-190.
 
[41]  Radajewski, S., P. Ineson, N. R. Parekh, and J. C. Murrell 2000. Stable-isotope probing as a tool in microbial ecology. Nature. 403: 646-649.
 
[42]  Davenport, R. J., T. P. Curtis, M. Goodfellow, F. M. Stainsby, and M. Bingley 2000. Quantitative use of fluorescent in situ hybridization to examine relationships between mycolic acid-containing actinomycetes and foaming in activated sludge plants. Appl. Environ. Microbiol. 66: 1158-1166.
 
[43]  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.
 
[44]  Seong, C. N., Y. S. Kim, K. S. Baik, S. D. Lee, Y. C. Hah, S. B. Kim, and M. Goodfellow 1999. Mycolic acid-containing actinomycetes associated with activated sludge foam. J. Microbiol. 37: 66-72.
 
[45]  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.
 
[46]  Aleshchenkova, Z. M., A. S. Samsonova, and N. F. Semochkina 1999. Effects of degrading microorganisms on purification of wastewater from Lavsan production enterprises with the use of activated sludge. Appl. Biochem. Microbiol. 35: 404-406.
 
[47]  Colquhoun, K. O. 1994. A proposed pathway for the biodegradation of hexamethylenetetramine. Water Sci. Technol. 30: 95-101.
 
[48]  Gisi, D., L. Willi, H. Traber, T. Leisinger, and S. Vuilleumier 1998. Effects of bacterial host and dichloromethane dehalogenase on the competitiveness of methylotrophic bacteria growing with dichloromethane. Appl. Environ. Microbiol. 64: 1194-1202.
 
[49]  Bowman, J., S. McCammon, and J. Skerratt 1997. Methylosphaera hansonii gen. nov., sp. nov., a psychrophilic, group I methanotroph from Antarctic marine-salinity, meromictic lakes. Microbiology. 143: 1451-1459.
 
[50]  Iwamoto, T., K. Tani, K. Nakamura, Y. Suzuki, M. Kitagawa, M. Eguchi, and M. Nasu 2000. Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE. FEMS Microbiol. Ecol. 32: 129-141.
 
[51]  Seghers, D., K. Verthé, D. Reheul, R. Bulcke, S. D. Siciliano, W. Verstraete, and E. M. Top Effects of long-term herbicide applications on the structure and diversity of bacterial communities in an agricultural soil revealed by 16S rRNA gene fingerprints, FAME analysis and activity assays.: Submitted.
 
[52]  Ballinger, S. J., I. M. Head, T. P. Curtis, and A. R. Godley 1998. Molecular microbial ecology of nitrification in an activated sludge process treating refinery wastewater. Water Sci. Technol. 37: 105-108.
 
[53]  Xiong, X. J., M. Hirata, H. Takanashi, M. G. Lee, and T. Hano 1998. Analysis of acclimation behavior against nitrification inhibitors in activated sludge processes. J. Ferment. Bioeng. 86: 207-214.
 
[54]  Schafer, H., L. Bernard, C. Courties, P. Lebaron, P. Servais, R. Pukall, E. Stackebrandt, M. Troussellier, T. Guindulain, J. Vives-Rego, and G. Muyzer 2001. Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations. FEMS Microbiol. Ecol. 34: 243-253.
 
[55]  Müller, A., K. Westergaard, S. Christensen, and S. J. Sørensen 2001. The effect of long-term mercury pollution on the soil microbial community. FEMS Microbiol. Ecol. 36: 11-19.
 
[56]  Rasmussen, L. D., and S. J. Sorensen 2001. Effects of mercury contamination on the culturable heterotrophic, functional and genetic diversity of the bacterial community in soil. FEMS Microbiol. Ecol. 36: 1-9.
 
[57]  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.