title: An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments.
abstract: Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using beta-agarase I). The results indicated that the enzymatic method was much gentler on IOB cells and yielded an approximately 5000-fold higher DNA mass than the published method. The 16S rRNA gene clone library developed from the beta-agarase I treated IOB enrichments indicated a high IOB community diversity with sequences in alpha-, beta-, gamma-, delta-, epsilon-Proteobacteria, unclassified Proteobacteria, unclassified Bacteroidetes and unclassified Bacteria. In contrast, the published method resulted in mainly gamma-Proteobacterial clone sequences. In addition, only the cells recovered by agarase treatment were amenable to direct fluorescence in situ hybridization (FISH). Therefore, we propose that the agarase method is a better IOB cell recovery method, because it is simpler, faster, and retains more genetic diversity.
authors.affiliation: Department of Civil and Environmental Engineering, University of Connecticut, Storrs, CT 06269-2037, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Yu
authors.forename: Joerg
authors.initials: J
authors.lastname: Graf
authors.forename: Barth F
authors.initials: BF
authors.lastname: Smets
mesh_terms: D044822:Biodiversity; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D007501:Iron / Q000378:metabolism; D010084:Oxidation-Reduction; D020560:Proteobacteria / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D007501:Iron