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The potential of a mesophilic-thermophilic-mesophilic anaerobic digestion system was investigated with respect to improvement of both digestion and sanitation efficiencies during treatment of liquid cattle manure. The pilot plant produced a high methane yield from liquid dairy cattle manure of 0.24 m³ (kg VSfed)-1. Considering the low system loading rate of 1.4–1.5 kg VS (m³ d)-1, digestion efficiency compared to conventional processes did not appear improved. The minimum guaranteed retention time in the tubular thermophilic reactor was increased compared to a continuously stirred tank reactor. Levels of intestinal enterococci in raw liquid manure as determined with cultivation methods were reduced by 2.5–3 log units to a level of around 10² cfu/mL. This sanitizing effect was achieved both during mesophilic-thermophilic-mesophilic and thermophilic-mesophilic treatment, provided the thermophilic digester was operated at 53–55°C. A change in feeding interval from 1 h to 4 h did not significantly alter methane yield and sanitation efficiency. It was proposed that a two-stage, thermophilic-mesophilic anaerobic digestion system would be able to achieve the same sanitizing effect and equal or better digestion efficiency at lower costs. [ABSTRACT FROM AUTHOR]

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples. [ABSTRACT FROM AUTHOR]

We developed a suitable system of DNA extraction and real-time quantitative polymerase chain reaction (qPCR) for the specific and sensitive quantification of pathogens and other relevant (indicator) organisms in recalcitrant material such as cattle manure. PCR inhibition by coextraction of humic compounds was minimized in this system, resulting in detection sensitivity of one target DNA copy per reaction well. Data from qPCR analysis for Escherichia coli agreed with cultivation based results, but orders of magnitude more fecal enterococci, Enterobacteriaceae and Campylobacter jejuni, were determined by qPCR than by cultivation. These bacteria may have been in a potentially hazardous active but non-cultivable state. The qPCR system is much less time consuming than conventional cultivation, highly specific, can detect non-cultivable organisms, provides high measurement throughput, and is cost attractive. It should be considered as an alternative in various application areas for (prescribed routine) cultivation based assays, e.g. for biosafety and hygiene monitoring. [ABSTRACT FROM AUTHOR]