The anaerobic digestion microbiome has been puzzling us since the dawn of molecular methods for mixed microbial community analysis. Monitoring of the anaerobic digestion microbiome can either take place via a non-targeted holistic evaluation of the microbial community through fingerprinting or by targeted monitoring of selected taxa. Here, we compared four different microbial community fingerprinting methods, i.e., amplicon sequencing, metaproteomics, metabolomics and cytomics, in their ability to characterise the full-scale anaerobic digestion microbiome. Cytometric fingerprinting through cytomics reflects a, for anaerobic digestion, novel, single cell-based approach of direct microbial community fingerprinting by flow cytometry. Three different digester types, i.e., sludge digesters, digesters treating agro-industrial waste and dry anaerobic digesters, each reflected different operational parameters. The α-diversity analysis yielded inconsistent results, especially for richness, across the different methods. In contrast, β-diversity analysis resulted in comparable profiles, even when translated into phyla or functions, with clear separation of the three digester types. In-depth analysis of each method's features i.e., operational taxonomic units, metaproteins, metabolites, and cytometric traits, yielded certain similar features, yet, also some clear differences between the different methods, which was related to the complexity of the anaerobic digestion process. In conclusion, cytometric fingerprinting through flow cytometry is a reliable, fast method for holistic monitoring of the anaerobic digestion microbiome, and the complementary identification of key features through other methods could give rise to a direct interpretation of anaerobic digestion process performance.

自从混合微生物群落分析的分子方法出现以来，厌氧消化微生物组就一直困扰着我们。厌氧消化微生物组的监测既可以通过指纹识别对微生物群落进行非针对性的整体评估，也可以通过对选定分类群的针对性监测来进行。在这里，我们比较了四种不同的微生物群落指纹图谱方法，即扩增子测序、宏蛋白质组学、代谢组学和细胞组学，它们表征全面厌氧消化微生物组的能力。通过细胞组学的细胞指纹图谱反映了一种用于厌氧消化的新型、基于单细胞的流式细胞术直接微生物群落指纹图谱方法。三种不同的消化器类型，即、污泥消化器、处理农业工业废物的消化器和干式厌氧消化器，每个都反映了不同的操作参数。α-多样性分析在不同的方法中产生了不一致的结果，特别是对于丰富度。相比之下，β-多样性分析产生了可比较的特征，即使在转化为门或功能时，三种消化器类型也有明显的分离。深入分析每种方法的特点，即、可操作的分类单位、元蛋白、代谢物和细胞特征，产生了某些相似的特征，但不同方法之间也有一些明显的差异，这与厌氧消化过程的复杂性有关。总之，流式细胞术的细胞指纹图谱是一种可靠、快速的厌氧消化微生物组整体监测方法，通过其他方法对关键特征的补充鉴定可以直接解释厌氧消化过程的性能。