Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrB genes as molecular markers

Abstract

Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.

FIG. 1.

(A) DGGE analysis of 16S rRNA gene fragments using DNA and RNA samples from different sulfidogenic anaerobic bioreactors as templates. Lane 1, DNA sample from reactor A; lane 2, RNA sample from reactor A; lane 3, DNA sample from reactor B; lane 4, RNA sample from reactor B; lane 5, DNA sample from reactor C; lane 6, RNA sample from reactor C; lane 7, DNA sample from reactor D; lane 8, RNA sample from reactor D; lane 9, DNA sample from reactor E; lane 10, RNA sample from reactor E; lane 11, DNA sample from reactor F; lane 12, RNA sample from reactor F. Bands indicated with numbers were excised from the gels and sequenced. (B) Phylogenetic tree based on 16S rRNA gene sequences obtained from the DGGE bands. Sequences determined in this study are in boldface; the band number is preceded by 16S. The sequence accession numbers are shown in parentheses. The sequence of Archaeoglobus fulgidus was used as an outgroup but was pruned from the tree. A black dot indicates a bootstrap value of between 90 and 100%. The scale bar indicates 10% sequence difference.

FIG. 2.

(A) Negative image of a perpendicular denaturing gradient gel of PCR-amplified dsrB fragments from Desulfobulbus propionicus obtained with primer pair DSRp2060F-GC and DSR4R. The black dot indicates a urea-formamide concentration of 46%. The white dots indicate the urea-formamide concentrations (30% and 65%) used for the “time travel” experiment. (B) “Time travel” experiment with PCR-amplified dsrB fragments from Desulfobulbus propionicus (1) and Desulfomicrobium escambience (2). A mixture of the two fragments was loaded onto the gel every 15 min for a total of 240 min. The electrophoresis conditions were 150 V for 4 h.

FIG. 3.

(A) DGGE patterns of dsrB gene fragments using DNA and RNA samples from different sulfidogenic anaerobic bioreactors as templates (see the legend to Fig. 1A for specifications of the samples). Bands indicated with numbers were excised from the gels and sequenced. (B) Phylogenetic consensus tree for dsrAB amino acid sequences deduced from nearly full-length dsrAB sequences. Branching orders that were not supported by all treeing methods are shown as multifurcations. Partial sequences were individually added to the reconstructed consensus tree by applying parsimony criteria without allowing changes in the overall tree topology. Sequences determined in this study are in boldface; the band number is preceded by DSR. The sequence accession numbers are shown in parentheses. The sequences of Thermodesulfovibrio islandicus and Thermodesulfovibrio yellowstonii were used as an outgroup but were pruned from the tree. A black dot indicates a bootstrap value of between 90 and 100%. Bootstrap values were calculated only for nearly full-length dsrAB sequences. The scale bar indicates 10% sequence difference.

FIG. 4.

Whole-cell hybridization of reactor samples A to F with probe DSBA1017 (specific for Desulfobacca acetoxidans and labeled with Fluos) (green), probe DSR660 (specific for Desulfobulbus and labeled with Cy3) (red), and probe DSV827 (specific for Desulfovibrio and labeled with Cy5) (blue). Bar, 20 μm.