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J Microbiol Methods. 2008 Mar;72(3):235-40. doi: 10.1016/j.mimet.2007.12.001. Epub 2007 Dec 15.

An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments.

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Department of Civil and Environmental Engineering, University of Connecticut, Storrs, CT 06269-2037, USA.


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.

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