Production of biogas from different organic materials is a most interesting source of renewable energy.The biomethane potential (BMP) of these materials has to be determined to get insight in design parameters for anaerobic digesters. Although several norms and guidelines for BMP tests exist, inter-laboratory tests regularly show high variability of BMPs for the same substrate. A workshop was held in June 2015, in Leysin, Switzerland, with over 40 attendees from 30 laboratories around the world, to agree on common solutions to the conundrum of inconsistent BMP test results. This paper presents the consensus of the intense roundtable discussions and cross-comparison of methodologies used in respective laboratories. Compulsory elements for the validation of BMP results were defined. They include the minimal number of replicates, the request to carry out blank and positive control assays, a criterion for the test duration, details on BMP calculation, and last but not least criteria for rejection of the BMP tests. Finally, recommendations on items that strongly influence the outcome of BMP tests such as inoculum characteristics, substrate preparation, test setup, and data analysis are presented to increase the probability of obtaining validated and reproducible results.

Ammonia-rich substrates inhibit the anaerobic digestion (AD) process and constitute the main reason for low energy recovery in full-scale reactors. It is estimated that many full-scale AD reactors are operating in ammonia induced "inhibited steady-state" with significant losses of the potential biogas production yield. To date there are not any reliable methods to alleviate the ammonia toxicity effect or to efficiently digest ammonia-rich waste. In the current study, bioaugmentation as a possible method to alleviate ammonia toxicity effect in a mesophilic continuously stirred-tank reactor (CSTR) operating under "inhibited steady state" was tested. A fast growing hydrogenotrophic methanogen (i.e., Methanoculleus bourgensis MS2(T)) was bioaugmented in the CSTR reactor at high ammonia levels (5 g NH4(+)-N L(-1)). A second CSTR reactor was used as control with no bioaugmentation. The results derived from this study clearly demonstrated a 31.3% increase in methane production yield in the CSTR reactor, at steady-state, after bioaugmentation. Additionally, high-throughput 16S rRNA gene sequencing analysis showed a 5-fold increase in relative abundance of Methanoculleus spp. after bioaugmentation. On the contrary to all methods used today to alleviate ammonia toxicity effect, the tested bioaugmentation process performed without interrupting the continuous operation of the reactor and without replacing the ammonia-rich feedstock.

Methanogenesis from acetate (aceticlastic methanogenesis or syntrophic acetate oxidation (SAO) coupled with hydrogenotrophic methanogenesis) is the most important step for the biogas process. The major environmental factors influencing methanogenesis are volatile fatty acids, ammonia, pH, and temperature. In our study, the effect of acetate and ammonia concentration on the methanogenic pathway from acetate and on the methanogenic communities was elucidated in two experiments: one where inocula were gradually exposed to increasing concentrations of acetate or ammonia, and another with direct exposure to different ammonia concentrations. The methanogenic pathway was determined by following the production of 14CH4 and 14CO2 from acetate labeled in the methyl group (C‐2). Microbial communities' composition was determined by fluorescence in situ hybridization. Upon acclimatization to acetate and ammonia, thermophilic cultures clearly shifted their acetate bioconversion pathway from SAO with subsequent hydrogenotrophic methanogenesis (mediated by Methanobacteriales spp. and/or Methanomicrobiales spp.) to aceticlastic methanogenesis (mediated by Methanosarcinaceae spp.). On the contrary, acclimatization process resulted in no pathway shift with the mesophilic acclimatized culture. When nonacclimatized thermophilic culture was exposed to high ammonia levels (7 g NH4 +‐N L−1), aceticlastic Methanosarcinaceae spp. was found to be the dominant methanogen.

Ammonia is a major environmental factor influencing biomethanation in full-scale anaerobic digesters. In this study, the effect of different ammonia levels on methanogenic pathways and methanogenic community composition of full-scale biogas plants was investigated. Eight full-scale digesters operating under different ammonia levels were sampled, and the residual biogas production was followed in fed-batch reactors. Acetate, labelled in the methyl group, was used to determine the methanogenic pathway by following the 14 CH 4 and 14 CO 2 production. Fluorescence in situ hybridisation was used to determine the methanogenic communities' composition. Results obtained clearly demonstrated that syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis was the dominant pathway in all digesters with high ammonia levels (2. ? -N L -1 ), while acetoclastic methanogenic pathway dominated at low ammonia (\1.21 g NH 4? -N L -1 ). Thermophilic Methanomicrobiales spp. and mesophilic Methanobacteriales spp. were the most abundant methanogens at free ammonia concentrations above 0.44 g NH 3 -N L -1 and total ammonia concentrations above 2.8 g NH 4? -N L -1 , respectively. Meanwhile, in anaerobic digesters with low ammonia (\1.21 g NH 4? -N L -1 ) and free ammonia (\0.07 g NH 3 -N L -1 ) levels, mesophilic and thermophilic Methanosaetaceae spp. were the most abundant methanogens.

Background Biogas production is a very complex process due to the high complexity in diversity and interactions of the microorganisms mediating it, and only limited and diffuse knowledge exists about the variation of taxonomic and functional patterns of microbiomes across different biogas reactors, and their relationships with the metabolic patterns. The present study used metagenomic sequencing and radioisotopic analysis to assess the taxonomic, functional, and metabolic patterns of microbiomes from 14 full-scale biogas reactors operated under various conditions treating either sludge or manure.ResultsThe results from metagenomic analysis showed that the dominant methanogenic pathway revealed by radioisotopic analysis was not always correlated with the taxonomic and functional compositions. It was found by radioisotopic experiments that the aceticlastic methanogenic pathway was dominant, while metagenomics analysis showed higher relative abundance of hydrogenotrophic methanogens. Principal coordinates analysis showed the sludge-based samples were clearly distinct from the manure-based samples for both taxonomic and functional patterns, and canonical correspondence analysis showed that the both temperature and free ammonia were crucial environmental variables shaping the taxonomic and functional patterns. The study further the overall patterns of functional genes were strongly correlated with overall patterns of taxonomic composition across different biogas reactors.ConclusionsThe discrepancy between the metabolic patterns determined by metagenomic analysis and metabolic pathways determined by radioisotopic analysis was found. Besides, a clear correlation between taxonomic and functional patterns was demonstrated for biogas reactors, and also the environmental factors that shaping both taxonomic and functional genes patterns were identified.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0465-6) contains supplementary material, which is available to authorized users.

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Ammonium chloride (NHCl) was usually used as a model ammonia source to simulate ammonia inhibition during anaerobic digestion (AD) of nitrogen-rich feedstocks. However, ammonia in AD originates mainly from degradation of proteins, urea and nucleic acids, which is distinct from NHCl. Thus, in this study, the inhibitory effect of a "natural" ammonia source (urea) and NHCl, on four pure methanogenic strains (aceticlastic: Methanosarcina thermophila, Methanosarcina barkeri; hydrogenotrophic: Methanoculleus bourgensis, Methanoculleus thermophilus), was assessed under mesophilic (37 °C) and thermophilic (55 °C) conditions. The results showed that urea hydrolysis increased pH significantly to unsuitable levels for methanogenic growth, while NHCl had a negligible effect on pH. After adjusting initial pH to 7 and 8, urea was significantly stronger inhibitor with longer lag phases to methanogenesis compared to NHCl. Overall, urea seems to be more toxic on both aceticlastic and hydrogenotrophic methanogens compared to NHCl under the same total and free ammonia levels.

Ammonia-rich substrates can cause inhibition on anaerobic digestion process. Syntrophic acetate-oxidizing bacteria (SAOB) and hydrogenotrophic methanogens are important for the ammonia inhibitory mechanism on anaerobic digestion. The roles and interactions of SAOB and hydrogenotrophic methanogens to ammonia inhibition effect are still unclear. The aim of the current study was to determine the ammonia toxicity levels of various pure strains of SAOB and hydrogenotrophic methanogens. Moreover, ammonia toxicity on the syntrophic-cultivated strains of SAOB and hydrogenotrophic methanogens was tested. Thus, four hydrogenotrophic methanogens (i.e. Methanoculleus bourgensis, Methanobacterium congolense, Methanoculleu thermophilus and Methanothermobacter thermautotrophicus), two SAOB (i.e. Tepidanaerobacter acetatoxydans and Thermacetogenium phaeum) and their syntrophic cultivation were assessed under 0.26, 3, 5 and 7 g NH4 (+)-N L(-1). The results showed that some hydrogenotrophic methanogens were equally, or in some cases, more tolerant to high ammonia levels compared to SAOB. Furthermore, a mesophilic hydrogenotrophic methanogen was more sensitive to ammonia toxicity compared to thermophilic methanogens tested in the study, which is contradicting to the general belief that thermophilic methanogens are more vulnerable to high ammonia loads compared to mesophilic. This unexpected finding underlines the fact that the complete knowledge of ammonia inhibition effect on hydrogenotrophic methanogens is still absent.