Sanitization of Biomass in Agricultural Biogas Plants Depends on the Type of Substrates

**Background:** Large-scale pig farming produces vast amounts of slurry, which is often used as fertilizer but can harbor zoonotic pathogens such as Escherichia coli O157:H7, Salmonella spp., Enterococcus spp., and others. Anaerobic digestion (methane fermentation) in biogas plants is a promising method to sanitize this biomass while generating renewable energy. However, the impact of different types of pig slurry (from maternal vs. fattening farms) on sanitization efficiency had not been clarified. This study aimed to determine how the type of pig slurry substrate affects the reduction of microorganisms during methane fermentation.

**Methods:** The study was conducted at two agricultural biogas plants in Poland, both using the same technology and operating under thermophilic conditions. BP-F used slurry from a fattening farm (pigs 24–114 kg, annual production ~39,500 pigs), while BP-M used slurry from a maternal farm (1300 sows, 350 gilts, annual production >47,500 piglets). Samples of raw slurry, input biomass (before fermentation), and digestate (after fermentation) were collected monthly over 15 cycles, yielding 90 total samples. Physicochemical analyses included dry matter (DM), dry organic matter (DOM), crude ash (CA), and ammonium nitrogen (NH4-N). Microbiological analyses quantified total bacteria, Enterobacteriaceae, E. coli, Hafnia alvei, and Enterococcus spp. using selective media and biochemical tests. Reduction efficiency was calculated as the percentage decrease in bacterial counts. Statistical analyses used non-parametric Mann–Whitney U and Kruskal–Wallis tests.

**Key Results:** The slurry from the fattening farm had significantly higher DM (5.32% vs. 2.25%), DOM (74.4% vs. 66.0%), CA (1.31% vs. 0.600%), and NH4-N (3.44 vs. 1.91 g/kg) compared to the maternal farm slurry. Input biomass at BP-F also had higher DM (9.44% vs. 5.39%), DOM (85.2% vs. 80.8%), CA (1.38% vs. 0.991%), and NH4-N (3.12 vs. 2.01 g/kg). Fermentation parameters were more favorable at BP-F: temperature 50.2°C vs. 46.7°C, pH 7.96 vs. 7.51, and hydraulic retention time 73.6 vs. 121 days (all p ≤ 0.05). VFA concentrations were not significantly different (4542 vs. 3716 mg/L). Microbiological reduction was consistently higher at BP-F: total bacterial reduction 94.3% vs. 80.3% (p ≤ 0.05), Enterobacteriaceae reduction 99.3% vs. 92.9% (p ≤ 0.05), H. alvei reduction 99.9% vs. 90.1% (p ≤ 0.05). E. coli reduction was >99.9% at BP-F vs. 97.3% at BP-M (not statistically different). Enterococcus reduction was 97.0% vs. 94.8% (not statistically different). Notably, E. coli was completely eliminated in BP-F digestate (0.00 log CFU/mL) but not in BP-M.

**Clinical Implications:** The study demonstrates that the type of pig slurry substrate significantly influences the sanitization efficiency of agricultural biogas plants. Slurry from fattening farms, with higher dry matter and ammonium nitrogen content, supports more stable thermophilic fermentation conditions (higher temperature and pH), leading to greater pathogen reduction. This has direct implications for reducing zoonotic disease transmission when digestate is used as fertilizer. The authors recommend locating biogas plants near fattening farms to maximize sanitization. H. alvei is proposed as a potential indicator organism for monitoring sanitization efficiency in thermophilic biogas plants. The findings support integrated approaches to livestock waste management that combine renewable energy production with public health protection.