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A synthetic DNA fragment containing primer binding sites for the quantification of ten different microbial groups was constructed and evaluated as a reliable enumeration standard for quantitative real-time PCR (qPCR) analyses. This approach has been exemplary verified for the quantification of several methanogenic orders and families in a series of samples drawn from a mesophilic biogas plant. Furthermore, the total amounts of bacteria as well as the number of sulfate-reducing and propionic acid bacteria as potential methanogenic interaction partners were successfully determined. The obtained results indicated a highly dynamic microbial community structure which was distinctly affected by the organic loading rate, the substrate selection, and the amount of free volatile fatty acids in the fermenter. Methanosarcinales was the most predominant methanogenic order during the 3 months of observation despite fluctuating process conditions. During all trials, the modified quantification standard indicated a maximum of reproducibility and efficiency, enabling this method to open up a wide range of novel application options.

The importance of thorough experimental design, implementation, and interpretation of laboratory experiments is known but rarely considered in continuous anaerobic digestion experiments. Especially in the evaluation of small effects on gas yields, e.g., by substrate disintegration, the usual methods such as the use of single digesters or the mere comparison of mean values often reach their limits. A proper statistical interpretation will be demonstrated on four examples of semi‐continuous laboratory‐scale anaerobic digestion experiments that investigated the effects of substrate disintegration on biogas yields. Power analysis was performed for one exemplary experimental setup. In this particular case, the power analysis showed that, with digester duplicates per treatment, a minimum 6 % difference in biogas yield could be significantly detected.