Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

Abstract

Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S. wolfei LYB was closely related to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, of which the majority was accounted for by the genus Syntrophomonas.

FIG. 1

Membrane template used for the specificity study (Fig. 4). OCM, Oregon Collection of Methanogens; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; ATCC, American Type Culture Collection.

FIG. 2

Unrooted phylogenetic tree for the family Syntrophomonadaceae, inferred from comparisons of SSU rRNA sequences with a maximum-likelihood algorithm. The tree was constructed with the evolutionary distances shown in Table 2.

FIG. 3

T_d studies for probes S-F-Synm-0700-a-A-23, S-G-Synm-0126-a-A-19, S-S-S.bry-0181-a-A-21, S-S-S.sap-0181-a-A-20, and S-S-S.wol-0180-a-A-21. Adjacent to the probe dissociation results are SSU rRNA sequences of target and nontarget species and probe sequences. The top SSU rRNA sequences for each list of organisms are those of the target organisms. Dashes in the succeeding sequences signify identical nucleotides. A superscript a indicates that an unlabeled version of the competitive probe was used with the labeled probe; X represents the universal base analogue N₅. A superscript b indicates organisms that were not included in the experimental evaluation of probe specificity. A superscript c indicates that the organism was not included in the experimental evaluation of probe specificity due to three significant mismatches.

FIG. 4

Results of the probe specificity study. Membrane hybridization results were analyzed with a PhosphorImager and were scanned and printed with Adobe Photoshop 3.0 (Adobe, Seattle, Wash.). (a) Probe S-∗-Univ-1390-a-A-18; (b) probe S-F-Synm-0700-a-A-23; (c) probe S-G-Synm-0126-a-A-19; (d) probe S-S-S.bry-0181-a-A-21; (e) probe S-S-S.sap-0181-a-A-20; (f) probe S-S-S.wol-0180-a-A-21.