High precision of measurement of methane potential is important for the economic operation of biogas plants in the future. The biochemical methane potential (BMP) test based on the VDI 4630 protocol is the state-of-the-art method to determine the methane potential in Germany. The coefficient of variation (CV) of methane yield was >10% in several previous inter-laboratory tests. The aim of this work was to investigate the effects of inoculum and the digestion system on the measurement variability. Methane yield and methane percentage of five substrates were investigated in a Hohenheim biogas yield test (D-HBT) by using five inocula, which were used several times in inter- laboratory tests. The same substrates and inocula were also tested in other digestion systems. To control the quality of the inocula, the effect of adding trace elements (TE) and the microbial community was investigated. Adding TE had no influence for the selected, well- supplied inocula and the community composition depended on the source of the inocula. The CV of the specific methane yield was <4.8% by using different inocula in one D-HBT (D-HBT1) and <12.8% by using different digestion systems compared to D-HBT1. Incubation time between 7 and 14 days resulted in a deviation in CV of <4.8%.

The increasing demand for renewable energy sources and demand-oriented electricity provision makes anaerobic digestion (AD) one of the most promising technologies. In addition to energy crops, the use of lignocellulosic residual and waste materials from agriculture is becoming increasingly important. However, AD of such feedstocks is often associated with difficulties due to the high content of lignocellulose and its microbial persistence. In the present work, the effect of hydrothermal pretreatment (HTP) on the digestibility of wheat straw is investigated and evaluated. Under different HTP temperatures (160–180 °C) and retention times (15–45 min), a significant increase in biomethane potential (BMP) can be observed in all cases. The highest BMP (309.64 mL CH4 g⁻¹ volatile solid (VS) is achieved after pretreatment at 160 °C for 45 min, which corresponds to an increase of 19% of untreated wheat straw. The results of a multiple linear regression model show that the solubilization of organic materials is influenced by temperature and time. Furthermore, using two different first-order kinetic models, an enhancement of AD rate during hydrolysis due to pretreatment is observed. However, the increasing intensity of pretreatment conditions is accompanied by a decreasing trend in the conversion of intermediates to methane.

Lignocellulosic biomass is an abundant organic material, which can be utilised in biogas plants for sustainable production of biogas. Since these substrates usually have high lignin contents and consist of rather elongated particles, a special pretreatment is required for an economical and process-stable utilisation in the biogas plant. The mechanical pretreatment of horse manure was carried out with the prototype of a ball mill at different speeds. The aim of ball milling is to comminute the substrate and disintegrate the lignocellulosic bond. Mechanical pretreatment in the ball mill resulted in a significant increase in specific methane yield of more than 37% in anaerobic batch digestion (up to 243 L_CH4 kg_VS⁻¹) of horse manure. The kinetics of the methane gas formation process was analysed by a modified Gompertz model fitting and showed a higher methane production potential and maximum daily methane production rate as well as a lower duration of the lag phase after pretreatment at 6 rpm. This was further confirmed by sieve analyses, which showed a significant reduction of particle size compared to the untreated variant. Thus, the use of the ball mill increases the specific methane yield and improves the fermentation of lignocellulosic substrates such as horse manure. Graphical Abstract

Agricultural residues, especially horse manure, represent an underutilised resource for biogas production. This study investigated mechanical ball mill pretreatment effects on substrate mixtures containing high proportion of horse manure and other agricultural residues in full-scale continuous stirred tank reactors. Experiments compared pretreated and untreated substrate mixtures under continuous and shock feeding scenarios at different organic loading rates. Results showed excellent process stability with consistent pH (7.8-8.0) and ratios of volatile fatty acids to total alkalinity of carbonates below 0.25, indicating robust buffering capacity despite challenging input substrates. Pretreatment significantly improved specific methane yields (from 216 ± 75 L_CH4 kg_VS^-1 to 312 ± 52 L_CH4 kg_VS^-1) and enhanced degradation kinetics. Economic evaluation demonstrated positive energy balances (165.7 to 332.0 kWh t_VS^-1) across all operating conditions despite energy input for pretreatment. This validates agricultural residues as viable alternatives to conventional energy crops for demand-oriented biogas production, contributing to sustainable renewable energy generation.

Efforts to integrate biogas plants into bioeconomy concepts will lead to an expansion of manure-based (small) biogas plants, while their operation is challenging due to critical characteristics of some types of livestock manure. For a better process understanding, in this study, three manure-based small biogas plants were investigated with emphasis on microbiome diversity. Due to varying digester types, feedstocks, and process conditions, 16S rRNA gene amplicon sequencing showed differences in the taxonomic composition. Dynamic variations of each investigated biogas plant microbiome over time were analyzed by terminal restriction fragment length polymorphism (TRFLP), whereby nonmetric multidimensional scaling (NMDS) revealed two well-running systems, one of them with a high share of chicken manure, and one unstable system. By using Threshold Indicator Taxa Analysis (TITAN), community-level change points at ammonium and ammonia concentrations of 2.25 g L⁻¹ and 193 mg L⁻¹ or volatile fatty acid concentrations of 0.75 g L⁻¹were reliably identified which are lower than the commonly reported thresholds for critical process stages based on chemical parameters. Although a change in the microbiome structure does not necessarily indicate an upcoming critical process stage, the recorded community-level change points might be a first indication to carefully observe the process.

There are almost 9500 biogas plants in Germany, which are predominantly operated with energy crops and residues from livestock husbandry over the last two decades. In the future, biogas plants must be enabled to use a much broader range of input materials in a flexible and demand-oriented manner. Hence, the microbial communities will be exposed to frequently varying process conditions, while an overall stable process must be ensured. To accompany this transition, there is the need to better understand how biogas microbiomes respond to management measures and how these responses affect the process efficiency. Therefore, 67 microbiomes originating from 49 agricultural, full-scale biogas plants were taxonomically investigated by 16S rRNA gene amplicon sequencing. These microbiomes were separated into three distinct clusters and one group of outliers, which are characterized by a specific distribution of 253 indicative taxa and their relative abundances. These indicative taxa seem to be adapted to specific process conditions which result from a different biogas plant operation. Based on these results, it seems to be possible to deduce/assess the general process condition of a biogas digester based solely on the microbiome structure, in particular on the distribution of specific indicative taxa, and without knowing the corresponding operational and chemical process parameters. Perspectively, this could allow the development of detection systems and advanced process models considering the microbial diversity.