Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis

The structures of the bacterial and archaeal communities in an anaerobic digester were monitored over a 2 year period. The study was performed on a fluidized bed reactor fed with vinasse. The objective was to characterize the population dynamics over a long time period under constant environmental parameters. Total bacterial and archaeal populations were measured independently by fluorescence-based polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) analysis using an automated DNA sequencer. With the current level of accuracy, the technique was able to monitor 45 bacterial and seven archaeal 16S rDNA molecules. The community dynamics were compared with molecular inventories of the microbial community based on 16S rDNA sequences done at the beginning of the study. The six archaeal and the 22 most frequent bacterial operational taxonomic units (OTUs) identified were associated with their SSCP peak counterparts. Overall, the data indicated that, throughout the period of the study, rapid significant shifts in the species composition of the bacterial community occurred, whereas the archaeal community remained relatively stable.     

Single strand conformation polymorphism monitoring of 16S rDNA Archaea during start-up of an anaerobic digester

A laboratory-scale continuously mixed anaerobic digester was inoculated with a mix of anaerobic sludge and fed with glucose. The start-up strategy was progressive and chemical analyses were done to evaluate digester performance from day 1 to day 107. In parallel, Archaeal community dynamics were monitored by SSCP analysis of the V3 region of 16S rDNA genes and further characterized by partial sequencing of 16S rDNA genes. At day 1 the inoculum contained at least five distinct Archaeal peaks close to known methanogenic species. The dominant peak was very close to Methanosaeta concilli, the remaining species being members of the Methanobacteriales and Methanomicrobiales. A rapid shift of the Archaeal population was observed during the experiment. At day 21 Methanobacterium formicicum, which was not detected at day 1, became the dominant methanogenic species in the bioreactor and remained so until the end of the experiment.     

Bacterial community dynamics during production of registered designation of origin Salers cheese as evaluated by 16S rRNA gene single-strand conformation polymorphism analysis

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.     

Quantifying bacterial population dynamics in compost using 16S rRNA gene probes

Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h. The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total. The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8. During this period the relative abundance of taxon-specific gene targets accounted for only 17–33% of the total bacterial rRNA gene targets. Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h. Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased. Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes. However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).     

Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystem

The dynamics of bacterial and archaeal populations of a laboratory-scale anaerobic digestor were investigated during a crisis period of the process reflected by an accumulation of acetate. A culture-independent approach based on single strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products was used. A spirochete and a Synergistes sp. showed high and changing activity levels during the study. A Clostridium sp. showed a transient increase in presence and activity concomitant with the highest acetate concentrations. A major shift in the most active archaeal populations from hydrogenotrophic to acetoclastic methanogens preceded the recovery of the reactor.     

Characterization of the predominant bacterial population of different mangrove rhizosphere soils using 16S rRNA gene-based single-strand conformation polymorphism (SSCP)

Variations in chemical parameters and bacterial populations in mangrove rhizosphere samples were noted for different sites. The C, N, P and K contents as well as pH, EC and salinity showed variation between sites. Significant differences in soil properties were also found in sampling sites. Two types of soil were noted among sites. Guesthouse had significantly higher organic matter and nutrient content (N) than other three sites suggesting that human discharges, litter deposition and surface runoff were major nutrient inputs. This contaminated site was located at the landward edges. Positive correlations between organic matter, N, P and K contents were found suggesting that these nutrients were from similar input sources. Effects of sampling sites on microbial diversity were also analyzed via SSCP. Porteresia coarctata and Rhizophora mucronata did not show any variation in the banding patterns among replicates sampled in short distance within site. But Sonneratia apetala showed variation among replicates sampled in distance within site. A significant variation was noted in the SSCP profile among replicates between sites. The majority of dominant SSCP band sequences were related to bacterial genera of root and root-free soil environments, namely Bacillus, Planococcus, Planomicrobium, low G+C Gram-positive bacterium, glacial ice bacterium and unidentified bacteria. In the analysis of 16S rRNA sequences, members belonging to the phylum Firmicutes dominated the sequence collection. The phylogenetic analysis of 16S rRNA gene sequences showed close relationships to a wide range of clones or bacterial species of phylum Firmicutes and unidentified bacteria.     

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.     

16S rDNA sequence diversity of a culture-accessible part of an anaerobic digestor bacterial community

A bacterial culture-based inventory with 16S rDNA identification of the isolates was carried out on an anaerobic digestor microbial ecosystem to compare to the 16S rDNA sequences directly retrieved from the ecosystem by a molecular inventory previously made in our laboratory. Twenty OTUs (Operational Taxonomic Units) belonging to five of the major bacterial groups were identified from 338 isolated colonies. The sequences of 13 of the 20 OTUs were not closely related to any hitherto published sequences (less than 96% sequence identity). Six OTUs out of 20 were found to have sequences similar to sequences of the molecular inventory. Despite the biases expected to be associated with the molecular and culture-based methods, the distribution of the isolated OTUs into the different bacterial phyla was similar to that of the molecular OTUs.     

Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.     

Advanced computational algorithms for microbial community analysis using massive 16S rRNA sequence data

With the aid of next-generation sequencing technology, researchers can now obtain millions of microbial signature sequences for diverse applications ranging from human epidemiological studies to global ocean surveys. The development of advanced computational strategies to maximally extract pertinent information from massive nucleotide data has become a major focus of the bioinformatics community. Here, we describe a novel analytical strategy including discriminant and topology analyses that enables researchers to deeply investigate the hidden world of microbial communities, far beyond basic microbial diversity estimation. We demonstrate the utility of our approach through a computational study performed on a previously published massive human gut 16S rRNA data set. The application of discriminant and topology analyses enabled us to derive quantitative disease-associated microbial signatures and describe microbial community structure in far more detail than previously achievable. Our approach provides rigorous statistical tools for sequence-based studies aimed at elucidating associations between known or unknown organisms and a variety of physiological or environmental conditions.     

Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism

The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.     

Dynamics of bacterial community in solid-state fermented feed revealed by 16S rRNA

Aims:  The aim of the present study was to monitor the changes in the composition of microbiota in solid-state fermented feed and to evaluate their biosafety.
Methods and Results:  In the solid-state fermentation, six probiotic bacteria strains were used as inoculum and soybean meal were used as carbohydrate source. At 0, 1, 2, 3, 5, 7, 10 and 15 days, samples were collected for further analysis. Denaturing gradient gel electrophoresis (DGGE) analysis showed that Bifidobacterium bifidum and Bacillus licheniformis were always present throughout the fermentation. Bifidobacterium bifidum , Enterococcus faecalis and Lactobacillus acidophilum were dominant throughout the entire fermentation as monitored by Lactobacillus -specific PCR–DGGE. Probiotics supplementation could reduce the levels of the pathogenic bacteria such as Staphylococcus aureus and enterotoxigenic Escherichia coli (ETEC) by species-specific real-time PCR. And Salmonella spp. was not detected throughout the entire fermentation.
Conclusions:  Probiotics supplemented are always dominant throughout the whole period of solid-state fermentation and effective in preventing the growth of pathogens.
Significance and Impact of the Study:  Based on the results, the high quality, stable solid-state fermented feed could be produced and applied in the pigs to improve the animal performances.     

Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood

Bacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individuals by PCR under conditions involving 30 cycles that did not produce any visible products from negative control saline. Even from control samples, PCR involving 35-40 cycles yielded visible bands. Major clones detected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonas subgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. No clone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gastrointestinal flora.     

Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA.

The 16S rRNA gene of the magnetotactic magnetogen Aquaspirillum magnetotacticum MS1 was amplified by a polymerase chain reaction, using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that A. magnetotacticum MS1 belongs to the alpha-group of proteobacteria. This assignment offers perspective on the biochemical properties of A. magnetotacticum, since this organism is expected to have the general properties that are common to this phylogenetic group.     

Quantification of bacterial morphotypes within anaerobic digester granules from transmission electron micrographs using image analysis

Summary Image analysis was applied to sequential transmission electron micrographs of an ultrathin section from the central region of an anaerobic digester granule to quantify the constituent bacterial morphotypes present. Our experience indicates that this procedure is suitable for the determination of populations of small spherical granules only and that it would be a useful technique for monitoring granule development. The cell area data determined in this study should permit rapid future quantification of Methanothrix- and Methanobacterium-like cells from cell counts derived from transmission electron micrographs.     

Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR

Benzene is a common groundwater pollutant that is often recalcitrant under the anaerobic conditions that prevail at hydrocarbon-contaminated aquifers. Thus, determining the potential for anaerobic benzene degradation is important to assess the feasibility of intrinsic bioremediation. In this work we developed a 16S rRNA biomarker to estimate the concentration of putative benzene degraders in a methanogenic consortium that has been enriched on benzene for several years. Primers were designed based on phylogenetic information from this consortium. The primers and probe were obtained by sequencing the dominant denaturing gradient gel electrophoresis band of this consortium, which corresponded to Desulfobacterium sp. clone OR-M2. No hybridization was observed with DNA samples from negative controls (i.e. toluene-degrading and dehalorespiring methanogenic consortia that do not degrade benzene). Samples from an anaerobic aquifer column that was bioaugmented with this benzene-degrading consortium showed a strong correlation between benzene degradation activity and the concentration of the target organism. Although our data do not prove that Desulfobacterium sp. is a benzene degrader, its enrichment as a result of benzene consumption and its correlation to anaerobic benzene degradation activity suggest that it either initiates benzene degradation or is a critical (commensal) partner. Therefore, the utility of this primers and probe set to assess anaerobic benzene degradation potential was demonstrated. This is the first report of the use of real-time quantitative PCR for forensic analysis of anaerobic benzene degradation. Whether this biomarker will be adequately selective and broadly applicable to assess benzene degradation potential under strongly anaerobic (sulfate reducing and methanogenic) conditions will require further research.