Department of Soil Science, University of Saskatchewan , Saskatoon, Saskatchewan S7N 5A8, Canada.
Department of Civil and Environmental Engineering, University of Alberta , Edmonton, Alberta T6G 1H9, Canada.
Environ Sci Technol. 2018 Feb 20;52(4):1773-1786. doi: 10.1021/acs.est.7b05773. Epub 2018 Feb 5.
Phosphorus availability and cycling in microbial communities is a key determinant of bacterial activity. However, identifying organisms critical to P cycling in complex biodegrading consortia has proven elusive. Here we assess a new DNA stable isotope probing (SIP) technique using heavy oxygen-labeled phosphate (PO) and its effectiveness in pure cultures and a nitrate-reducing benzene-degrading consortium. First, we successfully labeled pure cultures of Gram-positive Micrococcus luteus and Gram-negative Bradyrhizobium elkanii and separated isotopically light and heavy DNA in pure cultures using centrifugal analyses. Second, using high-throughput amplicon sequencing of 16S rRNA genes to characterize active bacterial taxa (C-labeled), we found taxa like Betaproteobacteria were key in denitrifying benzene degradation and that other degrading (nonhydrocarbon) inactive taxa (PO-labeled) like Staphylococcus and Corynebacterium may promote degradation through production of secondary metabolites (i.e., "helper" or "rock miner" bacteria). Overall, we successfully separated active and inactive taxa in contaminated soils, demonstrating the utility of PO-DNA SIP for identifying actively growing bacterial taxa. We also identified potential "miner" bacteria that choreograph hydrocarbon degradation by other microbes (i.e., the "hunters") without directly degrading contaminants themselves. Thus, while several taxa degrade benzene under denitrifying conditions, microbial benzene degradation may be enhanced by both direct degraders and miner bacteria.
微生物群落中的磷供应和循环是细菌活性的关键决定因素。然而，确定复杂生物降解共生体中对 P 循环至关重要的生物却一直难以捉摸。在这里，我们评估了一种新的 DNA 稳定同位素探测 (SIP) 技术，该技术使用重氧标记的磷酸盐 (PO)，并在纯培养物和硝酸盐还原苯降解共生体中评估其有效性。首先，我们成功地标记了革兰氏阳性微球菌和革兰氏阴性慢生根瘤菌的纯培养物，并使用离心分析在纯培养物中分离出同位素轻和重的 DNA。其次，我们使用 16S rRNA 基因高通量扩增子测序来表征活性细菌分类群（C 标记），发现像β变形菌这样的分类群是反硝化苯降解的关键，而其他降解（非碳氢化合物）的非活性分类群（PO 标记），如葡萄球菌和棒状杆菌，可能通过产生次生代谢物（即“助手”或“岩石矿工”细菌）来促进降解。总的来说，我们成功地在污染土壤中分离出活性和非活性分类群，证明了 PO-DNA SIP 用于鉴定活跃生长的细菌分类群的有效性。我们还确定了潜在的“矿工”细菌，它们通过其他微生物（即“猎人”）协调烃类的降解，而自身并不直接降解污染物。因此，虽然有几个分类群在反硝化条件下降解苯，但微生物苯降解可能会因直接降解菌和矿工菌而增强。
