Immediate Effects of Ammonia Shock on Transcription and Composition of a Biogas Reactor Microbiome.

The biotechnological process of biogas production from organic material is carried out by a diverse microbial community under anaerobic conditions. However, the complex and sensitive microbial network present in anaerobic degradation of organic material can be disturbed by increased ammonia concentration introduced into the system by protein-rich substrates and imbalanced feeding. Here, we report on a simulated increase of ammonia concentration in a fed batch lab-scale biogas reactor experiment. Two treatment conditions were used simulating total ammonia nitrogen concentrations of 4.9 and 8.0 g/L with four replicate reactors. Each reactor was monitored concerning methane generation and microbial composition using 16S rRNA gene amplicon sequencing, while the transcriptional activity of the overall process was investigated by metatranscriptomic analysis. This allowed investigating the response of the microbial community in terms of species composition and transcriptional activity to a rapid upshift to high ammonia conditions. Clostridia and Methanomicrobiales dominated the microbial community throughout the entire experiment under both experimental conditions, while Methanosarcinales were only present in minor abundance. Transcription analysis demonstrated clostridial dominance with respect to genes encoding for enzymes of the hydrolysis step (cellulase, EC 3.2.1.4) as well as dominance of key genes for enzymes of the methanogenic pathway (methyl-CoM reductase, EC 2.8.4.1; heterodisulfide reductase, EC 1.8.98.1). Upon ammonia shock, the selected marker genes showed significant changes in transcriptional activity. Cellulose hydrolysis as well as methanogenesis were significantly reduced at high ammonia concentrations as indicated by reduced transcription levels of the corresponding genes. Based on these experiments we concluded that, apart from the methanogenic archaea, hydrolytic cellulose-degrading microorganisms are negatively affected by high ammonia concentrations. Further, Acholeplasma and Erysipelotrichia showed lower abundance under increased ammonia concentrations and thus might serve as indicator species for an earlier detection in order to counteract against ammonia crises.

Long-term investigation of microbial community composition and transcription patterns in a biogas plant undergoing ammonia crisis.

Ammonia caused disturbance of biogas production is one of the most frequent incidents in regular operation of biogas reactors. This study provides a detailed insight into the microbial community of a mesophilic, full-scale biogas reactor (477 kWh h-1 ) fed with maize silage, dried poultry manure and cow manure undergoing initial process disturbance by increased ammonia concentration. Over a time period of 587 days, the microbial community of the reactor was regularly monitored on a monthly basis by high-throughput amplicon sequencing of the archaeal and bacterial 16S rRNA genes. During this sampling period, the total ammonia concentrations varied between 2.7 and 5.8 g l-1 [NH4 + -N]. To gain further inside into the active metabolic pathways, for selected time points metatranscriptomic shotgun analysis was performed allowing the quantification of marker genes for methanogenesis, hydrolysis and syntrophic interactions. The results obtained demonstrated a microbial community typical for a mesophilic biogas plant. However in response to the observed changing process conditions (e.g. increasing NH4 + levels, changing feedstock composition), the microbial community reacted highly flexible by changing and adapting the community composition. The Methanosarcina-dominated archaeal community was shifted to a Methanomicrobiales-dominated archaeal community in the presence of increased ammonia conditions. A similar trend as in the phylogenetic composition was observed in the transcription activity of genes coding for enzymes involved in acetoclastic methanogenesis and syntrophic acetate oxidations (Codh/Acs and Fthfs). In accordance, Clostridia simultaneously increased under elevated ammonia concentrations in abundance and were identified as the primary syntrophic interaction partner with the now Methanomicrobiales-dominated archaeal community. In conclusion, overall stable process performance was maintained during increased ammonia concentration in the studied reactor based on the microbial communities' ability to flexibly respond by reorganizing the community composition while remaining functionally stable.

BEAP profiles as rapid test system for status analysis and early detection of process incidents in biogas plants.

A method was developed to quantify the performance of microorganisms involved in different digestion levels in biogas plants. The test system was based on the addition of butyrate (BCON), ethanol (ECON), acetate (ACON) or propionate (PCON) to biogas sludge samples and the subsequent analysis of CH4 formation in comparison to control samples. The combination of the four values was referred to as BEAP profile. Determination of BEAP profiles enabled rapid testing of a biogas plant's metabolic state within 24 h and an accurate mapping of all degradation levels in a lab-scale experimental setup. Furthermore, it was possible to distinguish between specific BEAP profiles for standard biogas plants and for biogas reactors with process incidents (beginning of NH4+-N inhibition, start of acidification, insufficient hydrolysis and potential mycotoxin effects). Finally, BEAP profiles also functioned as a warning system for the early prediction of critical NH4+-N concentrations leading to a drop of CH4 formation.

Increase of methane formation by ethanol addition during continuous fermentation of biogas sludge.

Very recently, it was shown that the addition of acetate or ethanol led to enhanced biogas formation rates during an observation period of 24 h. To determine if increased methane production rates due to ethanol addition can be maintained over longer time periods, continuous reactors filled with biogas sludge were developed which were fed with the same substrates as the full-scale reactor from which the sludge was derived. These reactors are well reflected conditions of a full-scale biogas plant during a period of 14 days. When the fermenters were pulsed with 50-100 mM ethanol, biomethanation increased by 50-150 %, depending on the composition of the biogas sludge. It was also possible to increase methane formation significantly when 10-20 mM pure ethanol or ethanolic solutions (e.g. beer) were added daily. In summary, the experiments revealed that "normal" methane production continued to take place, but ethanol led to production of additional methane.

Short-term effect of acetate and ethanol on methane formation in biogas sludge.

Biochemical processes in biogas plants are still not fully understood. Especially, the identification of possible bottlenecks in the complex fermentation processes during biogas production might provide potential to increase the performance of biogas plants. To shed light on the question which group of organism constitutes the limiting factor in the anaerobic breakdown of organic material, biogas sludge from different mesophilic biogas plants was examined under various conditions. Therefore, biogas sludge was incubated and analyzed in anaerobic serum flasks under an atmosphere of N2/CO2. The batch reactors mirrored the conditions and the performance of the full-scale biogas plants and were suitable test systems for a period of 24 h. Methane production rates were compared after supplementation with substrates for syntrophic bacteria, such as butyrate, propionate, or ethanol, as well as with acetate and H2+CO2 as substrates for methanogenic archaea. Methane formation rates increased significantly by 35 to 126 % when sludge from different biogas plants was supplemented with acetate or ethanol. The stability of important process parameters such as concentration of volatile fatty acids and pH indicate that ethanol and acetate increase biogas formation without affecting normally occurring fermentation processes. In contrast to ethanol or acetate, other fermentation products such as propionate, butyrate, or H2 did not result in increased methane formation rates. These results provide evidence that aceticlastic methanogenesis and ethanol-oxidizing syntrophic bacteria are not the limiting factor during biogas formation, respectively, and that biogas plant optimization is possible with special focus on methanogenesis from acetate.

Quantification of methanogenic heterodisulfide reductase activity in biogas sludge.

Methanogenic archaea are essential for the production of methane in biogas plants. Here we present enzymatic test systems for the analysis of the metabolic activity of methanogens based on the heterodisulfide reductase reaction. The first rapid test shows that heterodisulfide reductase can be detected in 1 g of biogas sludge after sonication and centrifugation. The resulting cell lysate used reduced methylviologen for heterodisulfide reduction, a reaction that is specifically catalyzed by methanogenic heterodisulfide reductase. In the second test cell lysate from 60 g of biogas sludge was separated by ultracentrifugation. Both, cytoplasmic membrane and cytoplasmic fractions revealed heterodisulfide reductase activity, indicating the presence of hydrogenotrophic and aceticlastic methanogens, respectively.