Reprint of: Contribution of enrichments and resampling for sulfate reducing bacteria diversity assessment by high-throughput cultivation.

The development of new high-throughput cultivation methods aims to increase the isolation efficiency as compared to standard techniques that often require enrichment procedures to compensate the low microbial recovery. In the current study, estuarine sulfate-reducing bacteria were isolated using an anaerobic isolation procedure in 384-well microplates. Ninety-nine strains were recovered from initial sediments. Isolates were identified according to their partial 16S rRNA sequences and clustered into 13 phylotypes. Besides, the increase in species richness obtained through enrichments or resampling was investigated. Forty-four enrichment procedures were conducted and shifts in sulfate-reducing bacterial communities were investigated through dsrAB gene fingerprinting. Despite efforts in conducting numerous enrichment conditions only few of them were statistically different from initial sample. The cultural diversity obtained from 3 of the most divergent enrichments, as well as from resampled sediments equally contributed to raise the sulfate-reducing diversity up to 22 phylotypes. Enrichments (selection of metabolism) or resampling (transient populations and micro-heterogeneity) may still be helpful to assess new microbial phylotypes. Nevertheless, all the newly cultivated strains were all representatives of minor Operational Taxonomic Units and could eventually be recovered by maintaining high-throughput isolation effort from the initial sediments.

Contribution of enrichments and resampling for sulfate reducing bacteria diversity assessment by high-throughput cultivation.

The development of new high-throughput cultivation methods aims to increase the isolation efficiency as compared to standard techniques that often require enrichment procedures to compensate the low microbial recovery. In the current study, estuarine sulfate-reducing bacteria were isolated using an anaerobic isolation procedure in 384-well microplates. Ninety-nine strains were recovered from initial sediments. Isolates were identified according to their partial 16S rRNA sequences and clustered into 13 phylotypes. Besides, the increase in species richness obtained through enrichments or resampling was investigated. Forty-four enrichment procedures were conducted and shifts in sulfate-reducing bacterial communities were investigated through dsrAB gene fingerprinting. Despite efforts in conducting numerous enrichment conditions only few of them were statistically different from initial sample. The cultural diversity obtained from 3 of the most divergent enrichments, as well as from resampled sediments equally contributed to raise the sulfate-reducing diversity up to 22 phylotypes. Enrichments (selection of metabolism) or resampling (transient populations and micro-heterogeneity) may still be helpful to assess new microbial phylotypes. Nevertheless, all the newly cultivated strains were all representatives of minor Operational Taxonomic Units and could eventually be recovered by maintaining high-throughput isolation effort from the initial sediments.

Combination of high throughput cultivation and dsrA sequencing for assessment of sulfate-reducing bacteria diversity in sediments.

Improving the knowledge on sulfate-reducing bacteria (SRB) diversity and ecophysiology will permit a better understanding on their key roles in aquatic ecosystems. Therefore, their diversity was evaluated in estuarine sediments by a polyphasic approach including dsrA gene cloning and sequencing (156 clones) and high-throughput isolations in 384-well microplates (177 strains). Using the related thresholds of 95% (DsrA amino acid sequences) and 97% (16S rRNA gene sequences) for sequence similarity, SRB were grouped into 60 and 22 operational taxonomic units, respectively. Both approaches poorly overlapped and rather complemented each other. The clone library was dominated by sequences related to the Desulfobacteraceae, while only one isolate belonged to this family. Most of the strains were affiliated to the genera Desulfopila and Desulfotalea within the Desulfobulbaceae. Desulfopila-related strains exhibited a high phylogenetic microdiversity and represented numerically significant populations. In contrast, Desulfovibrio isolates were less abundant but displayed a high phylogenetic diversity. Three hundred and eighty-four-well microplate isolations enhanced significantly the number of isolates handled. As a consequence, 15 new taxa sharing less than 98% sequence similarity (16S rRNA gene) with their closest relatives were obtained. This polyphasic approach allowed to obtain a high phylogenetic diversity and thus a better view of sulfate-reducing communities in intertidal sediments.