Quantification of Sulfate-reducing Bacteria in Industrial Wastewater, by Real-time Polymerase Chain Reaction (PCR) Using dsrA and apsA Genes

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5′-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 × 102 and 9.5 × 106 copies/μL and between 1.2 × 103 and 1.2 × 107 copies/μL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 107–108 targets mL−1) and lower during the winter (about 104–105 targets mL−1). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.

[1]  W. Dilling,et al.  Aerobic respiration in sulfate‐reducing bacteria* , 1990 .

[2]  J. Leloup,et al.  Molecular quantification of sulfate-reducing microorganisms (carrying dsrAB genes) by competitive PCR in estuarine sediments. , 2004, FEMS microbiology ecology.

[3]  E. Knodel,et al.  Immunological Cross-Reactivities of Adenosine-5′-Phosphosulfate Reductases from Sulfate-Reducing and Sulfide-Oxidizing Bacteria , 1991, Applied and environmental microbiology.

[4]  Gerard Muyzer,et al.  Molecular methods to study the organization of microbial communities , 1995 .

[5]  D. B. Nedwell,et al.  Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR , 2004 .

[6]  J. Trevors,et al.  Microbial gene expression in soil: methods, applications and challenges. , 2005, Journal of microbiological methods.

[7]  M. Friedrich.  Phylogenetic Analysis Reveals Multiple Lateral Transfers of Adenosine-5′-Phosphosulfate Reductase Genes among Sulfate-Reducing Microorganisms , 2002, Journal of bacteriology.

[8]  F. Widdel,et al.  Dissimilatory Sulfate- and Sulfur-Reducing Prokaryotes , 2006 .

[9]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..

[10]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[11]  C. Holliger,et al.  Development of a real-time PCR method for quantification of the three genera Dehalobacter, Dehalococcoides, and Desulfitobacterium in microbial communities. , 2004, Journal of microbiological methods.

[12]  A. Ogram,et al.  Phylogeny of sulfate‐reducing bacteria , 2000 .

[13]  T. Tourova,et al.  Copy Number of Ribosomal Operons in Prokaryotes and Its Effect on Phylogenetic Analyses , 2004, Microbiology.

[14]  C. Bottema,et al.  Characterization of polymerase chain reaction amplification of specific alleles. , 1990, Analytical biochemistry.

[15]  C. Levenson,et al.  Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. , 1990, Nucleic acids research.

[16]  Rekha Seshadri,et al.  The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough , 2004, Nature Biotechnology.

[17]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[18]  R. Bartha,et al.  The Sulphate-Reducing Bacteria , 1979 .

[19]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[20]  Michael Wagner,et al.  Phylogeny of Dissimilatory Sulfite Reductases Supports an Early Origin of Sulfate Respiration , 1998, Journal of bacteriology.

[21]  K. Daly,et al.  Development of oligonucleotide probes and PCR primers for detecting phylogenetic subgroups of sulfate-reducing bacteria. , 2000, Microbiology.

[22]  A. E. Greenberg,et al.  Standard methods for the examination of water and wastewater : supplement to the sixteenth edition , 1988 .

[23]  R. Lillebaek.  Application of antisera raised against sulfate-reducing bacteria for indirect immunofluorescent detection of immunoreactive bacteria in sediment from the German Baltic Sea , 1995, Applied and environmental microbiology.

[24]  S. Stubner.  Enumeration of 16S rDNA of Desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen detection. , 2002, Journal of microbiological methods.

[25]  Glenn R. Gibson,et al.  Sulphate-reducing Bacteria: List of Contributors , 2007 .

[26]  Turova Tp,et al.  Copy number of ribosomal operons in prokaryotes and its effect on phylogenic analyses , 2003 .

[27]  R. Amann,et al.  Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR. , 2003, Environmental microbiology.

[28]  R. Amann,et al.  Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms , 1992, Applied and environmental microbiology.

[29]  K. Stetter,et al.  Adenylylsulfate reductases from archaea and bacteria are 1:1 αβ‐heterodimeric iron–sulfur flavoenzymes – high similarity of molecular properties emphasizes their central role in sulfur metabolism , 2000, FEBS letters.

[30]  D. Jain.  Evaluation of the semisolid Postgate's B medium for enumerating sulfate-reducing bacteria , 1995 .

[31]  N. Siboni,et al.  Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity , 2006, Applied and Environmental Microbiology.

[32]  S. Stubner.  Quantification of Gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR. , 2004, Journal of microbiological methods.

[33]  Daniel B. Oerther,et al.  The oligonucleotide probe database , 1996, Applied and environmental microbiology.

[34]  G. Muyzer,et al.  Aerobic Organic Carbon Mineralization by Sulfate-Reducing Bacteria in the Oxygen-Saturated Photic Zone of a Hypersaline Microbial Mat , 2005, Microbial Ecology.

[35]  M. Wagner,et al.  Lateral Gene Transfer of Dissimilatory (Bi)Sulfite Reductase Revisited , 2005, Journal of bacteriology.

[36]  P. Levett.  Anaerobic microbiology :a practical approach , 1991 .

[37]  D. J. Stewart.  The sulphate-reducing bacteria, 2nd ed.: By J. R. Postgate. Pp. 208. Cambridge University Press, 1984. £20.00 ($29.50) , 1984 .

[38]  Linda L. Blackall,et al.  Multiple Lateral Transfers of Dissimilatory Sulfite Reductase Genes between Major Lineages of Sulfate-Reducing Prokaryotes , 2001, Journal of bacteriology.

[39]  Awwa,et al.  Standard Methods for the examination of water and wastewater , 1999 .

[40]  L. Forney,et al.  Molecular microbial ecology: land of the one-eyed king. , 2004, Current opinion in microbiology.

[41]  W. Hamilton,et al.  Sulphate-reducing bacteria and anaerobic corrosion. , 1985, Annual review of microbiology.

[42]  B. Jørgensen,et al.  Microsensor Measurements of Sulfate Reduction and Sulfide Oxidation in Compact Microbial Communities of Aerobic Biofilms , 1992, Applied and environmental microbiology.

[43]  V. Zachar,et al.  Absolute quantification of target DNA: a simple competitive PCR for efficient analysis of multiple samples. , 1993, Nucleic acids research.

[44]  J. Odom.  Industrial and Environmental concerns with sulfate-reducing bacteria. , 1990 .

[45]  O. Clarisse,et al.  Dynamics of Sulfate-Reducing Microorganisms (dsrAB genes) in Two Contrasting Mudflats of the Seine Estuary (France) , 2005, Microbial Ecology.

[46]  L Raeymaekers,et al.  Basic principles of quantitative PCR , 2000, Molecular biotechnology.

[47]  T. Nakagawa,et al.  Molecular Characterization of Community Structures and Sulfur Metabolism within Microbial Streamers in Japanese Hot Springs , 2003, Applied and Environmental Microbiology.

[48]  R. Tanner.  Monitoring sulfate-reducing bacteria: comparison of enumeration media , 1989 .

[49]  M. Höfle,et al.  Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea , 2004, Applied and Environmental Microbiology.

[50]  G. Muyzer,et al.  Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments , 1996, Applied and environmental microbiology.