ß-Dicarbonyls Facilitate Engineered Microbial Bromoform Biosynthesis.

Ruminant livestock produce around 24% of global anthropogenic methane emissions. Methanogenesis in the animal rumen is significantly inhibited by bromoform, which is abundant in seaweeds of the genus Asparagopsis. This has prompted the development of livestock feed additives based on Asparagopsis to mitigate methane emissions, although this approach alone is unlikely to satisfy global demand. Here we engineer a non-native biosynthesis pathway to produce bromoform in vivo with yeast as an alternative biological source that may enable sustainable, scalable production of bromoform by fermentation. ß-dicarbonyl compounds with low pKa values were identified as essential substrates for bromoform production and enabled bromoform synthesis in engineered Saccharomyces cerevisiae expressing a vanadate-dependent haloperoxidase gene. In addition to providing a potential route to the sustainable biological production of bromoform at scale, this work advances the development of novel microbial biosynthetic pathways for halogenation.

Sustained stoichiometric imbalance and its ecological consequences in a large oligotrophic lake.

Considerable attention is given to absolute nutrient levels in lakes, rivers, and oceans, but less is paid to their relative concentrations, their nitrogen:phosphorus (N:P) stoichiometry, and the consequences of imbalanced stoichiometry. Here, we report 38 y of nutrient dynamics in Flathead Lake, a large oligotrophic lake in Montana, and its inflows. While nutrient levels were low, the lake had sustained high total N: total P ratios (TN:TP: 60 to 90:1 molar) throughout the observation period. N and P loading to the lake as well as loading N:P ratios varied considerably among years but showed no systematic long-term trend. Surprisingly, TN:TP ratios in river inflows were consistently lower than in the lake, suggesting that forms of P in riverine loading are removed preferentially to N. In-lake processes, such as differential sedimentation of P relative to N or accumulation of fixed N in excess of denitrification, likely also operate to maintain the lake's high TN:TP ratios. Regardless of causes, the lake's stoichiometric imbalance is manifested in P limitation of phytoplankton growth during early and midsummer, resulting in high C:P and N:P ratios in suspended particulate matter that propagate P limitation to zooplankton. Finally, the lake's imbalanced N:P stoichiometry appears to raise the potential for aerobic methane production via metabolism of phosphonate compounds by P-limited microbes. These data highlight the importance of not only absolute N and P levels in aquatic ecosystems, but also their stoichiometric balance, and they call attention to potential management implications of high N:P ratios.

A Distinct, Flocculent, Acidogenic Microbial Community Accompanies Methanogenic Granules in Anaerobic Digesters.

The formation of dense, well-settling methanogenic granules is essential for the operation of high-rate, up-flow anaerobic bioreactors used for wastewater treatment. Granule formation (granulation) mechanisms have been previously proposed, but an ecological understanding of granule formation is still lacking. Additionally, much of the current research on granulation only examines the start-up phase of bioreactor operation, rather than monitoring the fate of established granules and how new granules emerge over time. This paper, therefore, attempts to provide an insight into the microbial ecology of granule formation outside the start-up phase of bioreactor operation and develop an ecological granulation model. The microbial communities of granules actively undergoing growth, breakage, and reformation were examined, and an ecological granulation model was proposed. A distinct pregranular microbial community, with a high proportion of acidogenic organisms, such as the Streptococcaceae, was identified and suggested to have a role in initiating granulation by providing simpler substrates for the methanogenic and syntrophic communities which developed during granule growth. After initial granule formation, deterministic influences on microbial community assembly increased with granule size and indicated that microbial community succession was influenced by granule growth, leading to the formation of a stepwise ecological model for granulation. IMPORTANCE Complex microbial communities in engineered environments can aggregate to form surface-attached biofilms. Others form suspended biofilms, such as methanogenic granules. The formation of dense, methanogenic granules underpins the performance of high-rate, anaerobic bioreactors in industrial wastewater treatment. Granule formation (granulation) has been well studied from a physico-chemical perspective, but the ecological basis is poorly understood. We identified a distinct, flocculent, microbial community, which was present alongside granules, comprising primary consumers likely key in providing simpler substrates to granules. This flocculent community is understudied in anaerobic digestion and may initiate, or perpetuate, granule formation. We propose that it may be possible to influence bioreactor performance (e.g., to regulate volatile fatty acid concentrations) by manipulating this community. The patterns of microbial community diversity and assembly revealed by the study indicate that cycles of granule growth and breakage lead to overall diversification of the bioreactor meta-community, with implications for bioreactor process stability.

Enzymatic Pretreatment of Byproducts from Soapstock Splitting and Glycerol Processing for Improvement of Biogas Production.

This study investigated acid splitting wastewater (ASW) and interphase (IF) from soapstock splitting, as well as matter organic non glycerol (MONG) from glycerol processing, as potential substrates for biogas production. Batch and semicontinuous thermophilic anaerobic digestion experiments were conducted, and the substrates were preliminary treated using commercial enzymes kindly delivered by Novozymes A/C. The greatest enhancement in the batch digestion efficiency was achieved when three preparations; EversaTransform, NovoShape, and Lecitase were applied in the hydrolysis stage, which resulted in the maximum methane yields of 937 NL/kg VS and 915 NL/kg VS obtained from IF and MONG, respectively. The co-digestion of 68% ASW, 16% IF, and 16% MONG (wet weight basis) performed at an organic loading rate (OLR) of 1.5 kg VS/m3/day provided an average methane yield of 515 NLCH4/kg VSadded and a volatile solid reduction of nearly 95%. A relatively high concentration of sulfates in the feed did not significantly affect the digestion performance but resulted in an increased hydrogen sulfide concentration in the biogas with the peak of 4000 ppm.

Dynamic Effect of Operational Regulation on the Mesophilic BioMethanation of Grape Marc.

Wine production annually generates an estimated 11 million metric tonnes of grape marc (GM) worldwide. The diversion of this organic waste away from landfill and towards its use in the generation of renewable energy has been investigated. This study aimed to evaluate the effectiveness of operational parameters relating to the treatment regime and inoculum source in the extraction of methane from GM under unmixed anaerobic conditions at 35 °C. The study entailed the recirculation of a previously acclimated sludge (120 days) as downstream inoculum, an increased loading volume (1.3 kg) and a low substrate-to-inoculum ratio (10:3 SIR). The results showed that an incorporation of accessible operational controls can effectively enhance cumulative methane yield (0.145 m3 CH4 kg-1 VS), corresponding to higher amounts of digestible organics converted. The calculated average volumetric methane productivity equalled 0.8802 L CH4 LWork-1 d-1 over 33.6 days whilst moderate pollutant removal (43.50% COD removal efficiency) was achieved. Molecular analyses identified Firmicutes and Bacteroidetes phyla as core organisms for hydrolytic and fermentative stages in trophic relationships with terminal electron acceptors from the methane-producing Methanosarcina genus. Economic projections established that the cost-effective operational enhancements were sustainable for valorisation from grape marc by existing wineries and distilleries.

The rumen microbiome inhibits methane formation through dietary choline supplementation.

Enteric fermentation from ruminants is a primary source of anthropogenic methane emission. This study aims to add another approach for methane mitigation by manipulation of the rumen microbiome. Effects of choline supplementation on methane formation were quantified in vitro using the Rumen Simulation Technique. Supplementing 200 mM of choline chloride or choline bicarbonate reduced methane emissions by 97-100% after 15 days. Associated with the reduction of methane formation, metabolomics analysis revealed high post-treatment concentrations of ethanol, which likely served as a major hydrogen sink. Metagenome sequencing showed that the methanogen community was almost entirely lost, and choline-utilizing bacteria that can produce either lactate, ethanol or formate as hydrogen sinks were enriched. The taxa most strongly associated with methane mitigation were Megasphaera elsdenii and Denitrobacterium detoxificans, both capable of consuming lactate, which is an intermediate product and hydrogen sink. Accordingly, choline metabolism promoted the capability of bacteria to utilize alternative hydrogen sinks leading to a decline of hydrogen as a substrate for methane formation. However, fermentation of fibre and total organic matter could not be fully maintained with choline supplementation, while amino acid deamination and ethanolamine catabolism produced excessive ammonia, which would reduce feed efficiency and adversely affect live animal performance.

Applying PICRUSt and 16S rRNA functional characterisation to predicting co-digestion strategies of various animal manures for biogas production.

An estimated 25 million tons of animal manure is produced globally every year, causing considerable impact to the environment. These impacts can be managed through the use of anaerobic digestion (AD) This process achieves waste degradation through enzymatic activity, the efficiency of the AD process is directly related to microorganisms that produce these enzymes. Biomethane potential (BMP) assays remain the standard theoretical framework to pre-determine biogas yield and have been used to determine the feasibility of substrates or their combination for biogas production. However, an integrated approach that combines substrate choice and co-digestion would provide an improvement to the current predictive models. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) addresses the limitations of assays in this regard. In this paper, the biochemical functions of horse, cow, and pig manures are predicted. A total of 135 predicted KEGG Orthologies (KOs) showed amino acids, carbohydrate, energy, lipid, and xenobiotic metabolisms in all the samples. Linear discriminant analysis (LDA) combined with the effect size measurements (LEfSe), showed that fructose, mannose, amino acid and nucleotide sugar, phosphotransferase (PST) as well as starch and sucrose metabolisms were significantly higher in horse manure samples. 36 of the KOs were related to the acidogenesis and/or acetogenesis AD stages. Extended bar plots showed that 11 significant predictions were observed for horse-cow, while 5 were predicted for horse-pig and for cow-pig manures. Based on these predictions, the AD process can be enhanced through co-digestion strategies that takes into account the predicted metabolic contributions of the manure samples. The results supported the BMP calculations for the samples in this study. Biogas yields can be improved if this combined approach is employed in routine analysis before co-digesting different substrates.

Active microbial ecosystem in glacier basal ice fuelled by iron and silicate comminution-derived hydrogen.

The basal zone of glaciers is characterized by physicochemical properties that are distinct from firnified ice due to strong interactions with underlying substrate and bedrock. Basal ice (BI) ecology and the roles that the microbiota play in biogeochemical cycling, weathering, and proglacial soil formation remain poorly described. We report on basal ice geochemistry, bacterial diversity (16S rRNA gene phylogeny), and inferred ecological roles at three temperate Icelandic glaciers. We sampled three physically distinct basal ice facies (stratified, dispersed, and debris bands) and found facies dependent on biological similarities and differences; basal ice character is therefore an important sampling consideration in future studies. Based on a high abundance of silicates and Fe-containing minerals and, compared to earlier BI literature, total C was detected that could sustain the basal ice ecosystem. It was hypothesized that C-fixing chemolithotrophic bacteria, especially Fe-oxidisers and hydrogenotrophs, mutualistically support associated heterotrophic communities. Basal ice-derived rRNA gene sequences corresponding to genera known to harbor hydrogenotrophic methanogens suggest that silicate comminution-derived hydrogen can also be utilized for methanogenesis. PICRUSt-predicted metabolism suggests that methane metabolism and C-fixation pathways could be highly relevant in BI, indicating the importance of these metabolic routes. The nutrients and microbial communities release from melting basal ice may play an important role in promoting pioneering communities establishment and soil development in deglaciating forelands.

Mineral status and enteric methane production in dairy cows during different stages of lactation.

BACKGROUND: Lactating dairy cows are the greatest livestock contributor of methane, a major global greenhouse gas (GHG). However, good feeding management with adequate mineral intake can offers an effective approach to maintaining high levels of milk production and the health of dairy cows over the entire course of lactation, while also helping to reduce methane emission. The study described here investigated the plasma concentrations of both macroelements (Ca, Na, K, Mg, P) and microelements (Zn, Cu, Fe, Mn), as well as enteric methane emission and milk composition in high-yielding dairy cows in different lactation periods. The experiment was performed on Holstein-Friesian dairy cows with the average milk yield of 41 (± 9) L/day in a Polish commercial farm with modern dairy systems. A total of thirty high-yielding dairy cows were randomly assigned into three groups differing by lactation stage: early stage (Early, days 25-100), middle stage (Middle, days 101-250), and late stage (Late, day 250 and later). Dietary treatment for all cows was a total mixture ration (TMR) with maize and alfalfa silage the main forage components. RESULTS: The greatest milk yield and methane production were recorded in early-stage lactating cows, but the greatest methane intensity per kg of corrected milk was recorded in the late stage of lactation. Plasma concentrations of macroelements and microelements did not differ by lactation stages, but increased plasma concentrations of Zn and Fe and decreased plasma levels of Mg were noted during lactation. A positive correlation was found between plasma levels of Mg and other macroelements (Ca, Na, K), and between the concentrations of Fe and Zn, P in plasma, but no correlation between methane emission and mineral status was detected in the different lactation stages. CONCLUSIONS: Our results showed different mineral requirements and enteric methane emissions in each lactation stage. The feeding strategy and mineral utilization were adequate to maintain the health, mineral status, and milk production of the Holstein cows during the entire lactation period, and suggest an effective way of reducing methane emission.

Microbial assemblages and methanogenesis pathways impact methane production and foaming in manure deep-pit storages.

Foam accumulation in swine manure deep-pits has been linked to explosions and flash fires that pose devastating threats to humans and livestock. It is clear that methane accumulation within these pits is the fuel for the fire; it is not understood what microbial drivers cause the accumulation and stabilization of methane. Here, we conducted a 13-month field study to survey the physical, chemical, and biological changes of pit-manure across 46 farms in Iowa. Our results showed that an increased methane production rate was associated with less digestible feed ingredients, suggesting that diet influences the storage pit's microbiome. Targeted sequencing of the bacterial 16S rRNA and archaeal mcrA genes was used to identify microbial communities' role and influence. We found that microbial communities in foaming and non-foaming manure were significantly different, and that the bacterial communities of foaming manure were more stable than those of non-foaming manure. Foaming manure methanogen communities were enriched with uncharacterized methanogens whose presence strongly correlated with high methane production rates. We also observed strong correlations between feed ration, manure characteristics, and the relative abundance of specific taxa, suggesting that manure foaming is linked to microbial community assemblage driven by efficient free long-chain fatty acid degradation by hydrogenotrophic methanogenesis.

Suppressing peatland methane production by electron snorkeling through pyrogenic carbon in controlled laboratory incubations.

Northern peatlands are experiencing more frequent and severe fire events as a result of changing climate conditions. Recent studies show that such a fire-regime change imposes a direct climate-warming impact by emitting large amounts of carbon into the atmosphere. However, the fires also convert parts of the burnt biomass into pyrogenic carbon. Here, we show a potential climate-cooling impact induced by fire-derived pyrogenic carbon in laboratory incubations. We found that the accumulation of pyrogenic carbon reduced post-fire methane production from warm (32 °C) incubated peatland soils by 13-24%. The redox-cycling, capacitive, and conductive electron transfer mechanisms in pyrogenic carbon functioned as an electron snorkel, which facilitated extracellular electron transfer and stimulated soil alternative microbial respiration to suppress methane production. Our results highlight an important, but overlooked, function of pyrogenic carbon in neutralizing forest fire emissions and call for its consideration in the global carbon budget estimation.

Early response of methanogenic archaea to H2 as evaluated by metagenomics and metatranscriptomics.

BACKGROUND: The molecular machinery of the complex microbiological cell factory of biomethane production is not fully understood. One of the process control elements is the regulatory role of hydrogen (H2). Reduction of carbon dioxide (CO2) by H2 is rate limiting factor in methanogenesis, but the community intends to keep H2 concentration low in order to maintain the redox balance of the overall system. H2 metabolism in methanogens becomes increasingly important in the Power-to-Gas renewable energy conversion and storage technologies. RESULTS: The early response of the mixed mesophilic microbial community to H2 gas injection was investigated with the goal of uncovering the first responses of the microbial community in the CH4 formation and CO2 mitigation Power-to-Gas process. The overall microbial composition changes, following a 10 min excessive bubbling of H2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composition and taxonomic abundance of the biogas producing anaerobic community did not change appreciably 2 hours after the H2 treatment, indicating that this time period was too short to display differences in the proliferation of the members of the microbial community. There was, however, a substantial increase in the expression of genes related to hydrogenotrophic methanogenesis of certain groups of Archaea. As an early response to H2 exposure the activity of the hydrogenotrophic methanogenesis in the genus Methanoculleus was upregulated but the hydrogenotrophic pathway in genus Methanosarcina was downregulated. The RT-qPCR data corroborated the metatranscriptomic RESULTS: H2 injection also altered the metabolism of a number of microbes belonging in the kingdom Bacteria. Many Bacteria possess the enzyme sets for the Wood-Ljungdahl pathway. These and the homoacetogens are partners for syntrophic community interactions between the distinct kingdoms of Archaea and Bacteria. CONCLUSIONS: External H2 regulates the functional activity of certain Bacteria and Archaea. The syntrophic cross-kingdom interactions in H2 metabolism are important for the efficient operation of the Power-to-Gas process. Therefore, mixed communities are recommended for the large scale Power-to-Gas process rather than single hydrogenotrophic methanogen strains. Fast and reproducible response from the microbial community can be exploited in turn-off and turn-on of the Power-to-Gas microbial cell factories.

Pretreatment, Anaerobic Codigestion, or Both? Which Is More Suitable for the Enhancement of Methane Production from Agricultural Waste?

Pretreatment and codigestion are proven to be effective strategies for the enhancement of the anaerobic digestion of lignocellulosic residues. The purpose of this study is to evaluate the effects of pretreatment and codigestion on methane production and the hydrolysis rate in the anaerobic digestion of agricultural wastes (AWs). Thermal and different thermochemical pretreatments were applied on AWs. Sewage sludge (SS) was selected as a cosubstrate. Biochemical methane potential tests were performed by mixing SS with raw and pretreated AWs at different mixing ratios. Hydrolysis rates were estimated by the best fit obtained with the first-order kinetic model. As a result of the experimental and kinetic studies, the best strategy was determined to be thermochemical pretreatment with sodium hydroxide (NaOH). This strategy resulted in a maximum enhancement in the anaerobic digestion of AWs, a 56% increase in methane production, an 81.90% increase in the hydrolysis rate and a 79.63% decrease in the technical digestion time compared to raw AWs. On the other hand, anaerobic codigestion (AcoD) with SS was determined to be ineffective when it came to the enhancement of methane production and the hydrolysis rate. The most suitable mixing ratio was determined to be 80:20 (Aws/SS) for the AcoD of the studied AWs with SS in order to obtain the highest possible methane production without any antagonistic effect.

Aerobic bacterial methane synthesis.

Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.

Feeding cashew nut shell liquid decreases methane production from feces by altering fecal bacterial and archaeal communities in Thai local ruminants.

The effect of feeding cashew nut shell liquid (CNSL) on fecal fermentation products and microbiota was investigated in Thai native cattle and swamp buffaloes. Four of each animal were fed rice straw and concentrate diet with control pellets without CNSL for 4 weeks, followed by the same diet with pellets containing CNSL for another 4 weeks, so that CNSL was administered at a level of 4 g/100 kg body weight. Feces were collected the last 2 days in each feeding period. CNSL alkyl phenols were recovered from feces (16%-28%) in a similar proportion to those in the diet, indicating that most functional anacardic acid was not selectively removed throughout the digestive tract. In vitro production of gas from feces, particularly methane, decreased with CNSL feeding. The proportion of acetate in feces decreased with CNSL feeding, whereas that of propionate increased, without affecting total short-chain fatty acid concentration. CNSL feeding changed fecal microbial community, particularly in swamp buffaloes, which exhibited decreases in the frequencies of Treponema, unclassified Ruminococcaceae, and Methanomassiliicoccaceae. These results suggest that CNSL feeding alters not only rumen fermentation but also hindgut fermentation via modulation of the microbial community, thereby potentially attenuating methane emission from the feces of ruminant animals.

The influence of combined low-strength ultrasonics and micro-aerobic pretreatment process on methane generation and sludge digestion: Lipase enzyme, microbial activation, and energy yield.

Low-frequency ultrasonics is a potential technology to reduce the hydrolysis phase period in anaerobic digestion process. In this study, theinfluence of combined low frequency ultrasonics and micro-aerobic (MA) pretreatment on sewage sludge solubilization, enzyme activity and anaerobic digestion were assessed. Initially, the effect of ultrasonic density (0.012, 0.014, 0.016, 0.018, 0.1, 0.12 and 0.14 W/mL) and irradiation time (1, 3, 5, 8, 9, 10 and 12 min) of 20 kHz frequency waves were investigated. Accordingly, the effect of micro-aerobic pretreatment (Air flow rate (AFR) = 0.1, 0.2, 0.3 and 0.5 VVM) within 20, 30, 40.48 and 60 h were examined. In addition, the effect of combined pretreatment on COD solubilization, lipase enzyme activation, ATP, percentage of live bacteria and methane gas production during the anaerobic process were examined. The results showed that the highest lipase activity (14.9 Umol/mL) was obtained under the effect of ultrasonic density of 0.1 W/ml within 9 min. The highest solubilization (65%) was observed under optimal micro-aerobic conditions: AFR = 0.2 (VVM) and micro-aerobic time: 40 h. Combined ultrasonic and micro-aerobic (US + MA) pretreatment increases the solubilization (70%), microbial activity (2080%) and lipase enzymatic activity (129%) compared to individual pretreatment. The Biogas production during anaerobic digestion pretreated with combined methods increased by 193% compared to the control, while the elevated values of biogas production in reactors pretreated by ultrasonic and micro-aerobic pretreatment alone were observed to be 101% and 165%, respectively. The net energy in reactor with the combined pre-treatment methods was calculated to be 1.26 kWh, while this value for control, pretreated ultrasonic and micro-aerobic reactors were obtained to be 0.56, 0.67 and 1.2 kWh, respectively.

[Functional Exams in the gastroenterology - new developments and tips for the common practice]. / Gastroenterologische Funktionsdiagnostik ­ neue Entwicklungen und Tipps für die Praxis.

The functional gastrointestinal disorders (FGIDs) have a high prevalence and are associated with high healthcare costs. The diagnosis of these diseases could be difficult and require func-tional tests such as high-resolution manometry (HRM) of the esophagus, anorectal manometry and H2-Breathtests. Due to the COVID-19 Pandemic and the fear of infections there was a marked reduction in the number of performed exams in the last months - nevertheless some exams are necessary, in order to exclude or to diagnose important and dangerous diseases like Achalasia. Goal of this article is to present some new and relevant developments in the field. The HRM of the esophagus is the diagnostic standard for Achalasia, a rare clinical condi-tion associated to dysphagia - new European guidelines suggests a safe strategy in perform-ing the pneumatic dilatation.The intestinal methanogen overgrowth (IMO) is a clinical condition caused by a high production of methane in the small intestine due to overgrowth of Methanobrevibacter smithii, this condition could be in some patients associated with irritable bowel syndrome.

A comparative analysis of biogas production from tomato bio-waste in mesophilic batch and continuous anaerobic digestion systems.

Annually, agricultural activity produces an enormous amount of plant biomass by-product. Many studies have reported the biomethane potential of agro-industrial wastes, but only a few studies have investigated applying the substrates in both batch and continuous mode. Tomato is one of the most popular vegetables globally; its processing releases a substantial amount of by-product, such as stems and leaves. This study examined the BMP of tomato plant (Solanum lycopersicum Mill. L. cv. Alfred) waste. A comparative test revealed that the BMPs of corn stover, tomato waste,and their combination were approximately the same, around 280 mL methane/g Volatile Solid. In contrast, the relative biogas production decreased in the presence of tomato waste in a continuous mesophilic anaerobic digestion system; the daily biogas productions were 860 ± 80, 290 ± 50, and 570 ± 70 mL biogas/gVolatile Solid/day in the case of corn stover, tomato waste, and their mixture, respectively. The methane content of biogas was around 46-48%. The fermentation parameters of the continuous AD experiments were optimal in all cases; thus, TW might have an inhibitory effect on the microbial community. Tomato plant materials contain e.g. flavonoids, glycoalkaloids (such as tomatine and tomatidine), etc. known as antimicrobial and antifungal agents. The negative effect of tomatine on the biogas yield was confirmed in batch fermentation experiments. Metagenomic analysis revealed that the tomato plant waste caused significant rearrangements in the microbial communities in the continuously operated reactors. The results demonstrated that tomato waste could be a good mono-substrate in batch fermentations or a co-substrate with corn stover in a proper ratio in continuous anaerobic fermentations for biogas production. These results also point to the importance of running long-term continuous fermentations to test the suitability of a novel biomass substrate for industrial biogas production.