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Applied and Environmental Microbiology, March 2002, p. 1392-1402, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1392-1402.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

Tsukasa Ito,1 Satoshi Okabe,1* Hisashi Satoh,2 and Yoshimasa Watanabe1

Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628,1 Department of Civil Engineering Faculty of Engineering, Hachinohe Institute of Technology, Myo Hachinohe 031-8501, Japan2

Received 10 September 2001/ Accepted 28 November 2001

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 µM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 µm from the surface during all stages of biofilm development. The first sulfide production of 0.32 µmol of H2S m-2 s-1 was detected below ca. 500 µm in the 3rd week and then gradually increased to 0.70 µmol H2S m-2 s-1 in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 x 109 cells cm-3 and 3.6 x 109 cells cm-3, respectively, in the surface 400 µm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.

* Corresponding author. Mailing address: Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, North 13, West 8, Kita-ku, Sapporo 060-8626, Japan. Phone: 81-11-706-6266. Fax: 81-11-707-6585. E-mail: sokabe{at}eng.hokudai.ac.jp.

Applied and Environmental Microbiology, March 2002, p. 1392-1402, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1392-1402.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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