Okabe S, Ito T, Satoh H
Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, Kita-13 Nishi-8, 060-8628, Kita-ku, Sapporo, Japan.
Appl Microbiol Biotechnol. 2003 Dec;63(3):322-34. doi: 10.1007/s00253-003-1395-3. Epub 2003 Jul 17.
The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga- Flexibacter- Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H(2) as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2-4x10(9) cells cm(-3) and 5x10(7) filaments cm(-3), respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB.
通过结合分子技术、钼酸盐抑制批次实验和微电极测量，研究了在微需氧条件下生长的废水生物膜中硫酸盐还原菌（SRB）的群落结构以及SRB对碳矿化的贡献。从生物膜样品构建了细菌种群的16S rDNA克隆文库。分析的102个克隆被分为53个操作分类单元（OTU），克隆分布如下：噬纤维菌-屈挠杆菌-拟杆菌门（41%）、变形菌门（41%）、低G+C革兰氏阳性菌（18%）和其他门（3%）。还从以丙酸盐、乙酸盐和H₂作为电子供体的SRB富集培养物中构建了另外三个细菌克隆文库，以进一步研究由于不同碳源的添加导致的SRB群落结构差异。这些文库表明，SRB克隆在系统发育上具有多样性，并且与变形菌门δ亚类中的六个主要SRB属相关。荧光原位杂交（FISH）分析表明，脱硫球茎菌属和脱硫线菌属是该生物膜中最丰富的SRB物种，并且在生物膜表面检测到这种较高的丰度（分别约为2 - 4×10⁹个细胞 cm⁻³和5×10⁷个丝状体 cm⁻³）。微电极测量表明，当生物膜在以乙酸盐作为唯一碳源的合成培养基中培养时，高硫酸盐还原活性集中在位于有氧/缺氧界面正下方的一个狭窄区域。相反，当生物膜在初沉池出水的上清液中培养时，在整个缺氧层中发现了一个较宽的硫酸盐还原区。这可能是因为有机碳源从主体水扩散到生物膜中，并且在生物膜中产生了未知量的挥发性脂肪酸。分子技术和使用特定抑制剂（钼酸盐）的批次实验相结合的方法清楚地表明，脱硫球茎菌属是SRB种群中数量上重要的成员，并且是该生物膜中丙酸盐氧化为乙酸盐的主要贡献者。然而，乙酸盐优先被硝酸盐还原菌利用，而不是被利用乙酸盐的SRB利用。
