Mathai Prince P, Zitomer Daniel H, Maki James S
1Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA.
2Department of Civil, Construction and Environmental Engineering, Water Quality Center, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA.
Microbiology (Reading). 2015 Jun;161(6):1189-97. doi: 10.1099/mic.0.000085. Epub 2015 Mar 26.
In methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H(2), acetate and CO(2) by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.
在产甲烷生境中，挥发性脂肪酸（VFA），如丙酸和丁酸，是有机物降解的主要中间产物。挥发性脂肪酸会被与产甲烷古菌共生的脂肪酸降解互营细菌（SFAB）进一步代谢为氢气、乙酸和二氧化碳。尽管它们在挥发性脂肪酸降解中发挥着不可或缺的作用，但对于脂肪酸降解互营细菌的丰度及其环境分布却知之甚少。为了促进生态学研究，我们开发了四种新型的属特异性定量PCR（qPCR）检测方法，其引物组靶向已知的脂肪酸降解互营细菌：互营杆菌属、史密斯氏菌属、泥杆菌属和互营单胞菌属。通过计算机模拟和实验（靶标对照、克隆文库和熔解曲线分析）方法确认了引物组的特异性。这些qPCR检测方法被应用于量化各种嗜温产甲烷生境中的脂肪酸降解互营细菌，包括实验室丙酸富集培养物、中试规模和全规模厌氧反应器、奶牛瘤胃、马粪便、实验性稻田土壤、沼泽溪流和沼泽沉积物。在丙酸富集培养物和厌氧反应器中发现了最高的脂肪酸降解互营细菌16S rRNA基因拷贝数，其次是沼泽溪流和沼泽沉积物样本。此外，还观察到脂肪酸降解互营细菌和产甲烷菌的丰度随反应器配置和底物特性而变化相。据我们所知，这项研究是首次使用基于qPCR的方法对产甲烷生境中的脂肪酸降解互营细菌进行全面量化的研究。这些分子工具将帮助研究人员更好地了解工程环境和自然环境中的互营微生物群落。
