ARC Training Centre for the Transformation of Australia's Biosolids Resource, RMIT University, Building 215, Level 3, Room 003-06, RMIT Bundoora West Campus, 225-245 Plenty Road, Bundoora, Victoria 3083, Australia.
ARC Training Centre for the Transformation of Australia's Biosolids Resource, RMIT University, Building 215, Level 3, Room 003-06, RMIT Bundoora West Campus, 225-245 Plenty Road, Bundoora, Victoria 3083, Australia.
Water Res. 2024 Apr 1;253:121354. doi: 10.1016/j.watres.2024.121354. Epub 2024 Feb 22.
DNA-based monitoring of microbial communities that are responsible for the performance of anaerobic digestion of sewage wastes has the potential to improve resource recoveries for wastewater treatment facilities. By treating sludge with propidium monoazide (PMA) prior to amplicon sequencing, this study explored how the presence of DNA from dead microbial biomass carried over with feed sludge may mislead process-relevant biomarkers, and whether primer choice impacts such assessments. Four common primers were selected for amplicon preparation, also to determine if universal primers have sufficient taxonomic or functional coverage for monitoring ecological performance; or whether two domain-specific primers for Bacteria and Archaea are necessary. Anaerobic sludges of three municipal continuously stirred-tank reactors in Victoria, Australia, were sampled at one time-point. A total of 240 amplicon libraries were sequenced on a Miseq using two universal and two domain-specific primer pairs. Untargeted metabolomics was chosen to complement biological interpretation of amplicon gene-based functional predictions. Diversity, taxonomy, phylogeny and functional potentials were systematically assessed using PICRUSt2, which can predict community wide pathway abundance. The two chosen universal primers provided similar diversity profiles of abundant Bacteria and Archaea, compared to the domain-specific primers. About 16 % of all detected prokaryotic genera covering 30 % of total abundances and 6 % of PICRUSt2-estimated pathway abundances were affected by PMA. This showed that dead biomass in the anaerobic digesters impacted DNA-based assessments, with implications for predicting active processes, such as methanogenesis, denitrification or the identification of organisms associated with biological foams. Hence, instead of running two sequencing runs with two different domain-specific primers, we propose conducting PMA-seq with universal primer pairs for routine performance monitoring. However, dead sludge biomass may have some predictive value. In principal component analysis the compositional variation of 239 sludge metabolites resembled that of 'dead-plus-alive' biomass, suggesting that dead organisms contributed to the potentially process-relevant sludge metabolome.
基于 DNA 的微生物群落监测可以提高污水处理厂资源回收效率，这些微生物群落负责污水的厌氧消化。通过在扩增子测序前用吖啶橙单叠氮化物（PMA）处理污泥，本研究探讨了进料污泥中携带的死微生物生物质的 DNA 存在如何误导与过程相关的生物标志物，以及引物选择是否会影响此类评估。选择了四种常见的引物用于扩增子制备，还确定通用引物是否具有足够的分类或功能覆盖范围来监测生态性能；或者是否需要针对细菌和古菌的两种特定于域的引物。在澳大利亚维多利亚州的三个市政连续搅拌罐式反应器中，在一个时间点采集了厌氧污泥。总共在 Miseq 上使用两对通用引物和两对特定于域的引物对 240 个扩增子文库进行了测序。选择非靶向代谢组学来补充扩增子基因功能预测的生物学解释。使用 PICRUSt2 系统地评估了多样性、分类学、系统发育和功能潜力，PICRUSt2 可以预测群落中途径丰度。与特定于域的引物相比，选择的两种通用引物提供了丰富细菌和古菌的相似多样性分布。约 16%的所有检测到的原核生物属，占总丰度的 30%，以及 PICRUSt2 估计的途径丰度的 6%，受到 PMA 的影响。这表明厌氧消化器中的死生物质会影响基于 DNA 的评估，这对预测甲烷生成、反硝化或识别与生物泡沫相关的生物等活性过程具有影响。因此，我们建议在进行常规性能监测时，使用通用引物对而不是运行两个带有两个不同特定于域的引物的测序运行。然而，死污泥生物质可能具有一定的预测价值。在主成分分析中，239 种污泥代谢物的组成变化与“死加活”生物质相似，这表明死生物对潜在的过程相关污泥代谢组有贡献。
