Stable isotopes of C and H in methane fermentation of agriculture substrates at different temperature conditions

Agricultural substrates (maize silage and cattle manure) were used to carry out methane fermentation process in bioreactors under laboratory conditions. Identical mixtures of these substrates were incubated for 43 days at 20, 30 and 40ºC to determine how di erent temperature conditions a ect the 13C(CH4), H(CH4), and 13C(CO2) values. To ensure correct anaerobic digestion, the following parameters of the organic substrates and fermentation solutions were monitored: total organic carbon (TOC), volatile solids (VS), volatile fatty acids (VFA), chemical oxygen demand (COD) and carbon to nitrogen ratio (C/N). The variants with higher incubation temperature yielded higher amounts of biogas (20ºC=84.5, 30ºC=101.8 and 40ºC=133.3 dm3/kg VS). In the case of gas products of methane fermentation, it was observed that the higher temperature of incubation a ects the depletion in heavy isotopes. At 20ºC, 30ºC, and 40ºC mean values of 13C(CH4) reached −26.4, −29.7, and −35.4h, respectively. Mean values of 2H(CH4) were −311.6, −354.0, and −398.5permil, and of 13C(CO2) +8.9, +3.7, and −2.3h, respectively. Moreover, the apparent fractionation coe cient 13C(CO2-CH4) were calculated, which decreased when the temperature increased. This isotopic tool was used to identify acetoclastic reaction as a dominant methanogenesis pathway. Observed changes in the isotopic composition of gaseous products obtained at different incubation temperatures may indicate decomposition of di erent carbon sources (e.g. lactate, propionate) to acetate and its fermentation by acetoclastic methanogens. It is possible that this was also related to the observation of the various metabolic models due to the varied methanogenic community composition

A Special Issue (Part-II): mafic-ultramafic rocks andalkaline-carbonatitic magmatism and associated hydrothermalmineralization – dedication to Lia N. Kogarko

Thisisthesecondpartofatwo-volumespecialis- sue of Open Geoscience (formerly Central European Jour- nal of Geosciences) that aims to be instrumental in pro- viding an update of Mac-Ultramac Rocks and Alkaline

Trehalose, mannitol and arabitol as indicators of fungal metabolism in late Cretaceous and Miocene deposits

Trehalose, mannitol and arabitol are the main saccharides of extant fungal metabolism, but their occurrence and distribution in geological materials have rarely been considered. Here, we identify these sugars in Miocene lignites and for the first time in Late Cretaceous mudstones and coals. The co-occurrence of trehalose, mannitol and arabitol in the sedimentary rocks investigated suggests their fungal origin, because these three saccharides are major compounds present in most modern fungi, including the very common mycorrhizal and wood-rotting groups. Therefore, we conclude that these sugars should be treated as new fungal biomarkers (biomolecules) present in geological rocks. Trehalose and mannitol are major compounds in total extracts of the samples and a sum of their concentration reaches 4.6 μg/g of sample. The arabitol concentrations do not exceed 0.5 μg/g, but in contrast to trehalose, the concentration correlates well with mannitol (R2=0.94), suggesting that they have the same, translocatory role in fungi. Based on the trehalose vs. mannitol and arabitol distributions in Cretaceous samples and their comparison with data for modern fungi, we preliminarily conclude that the coal seams from the Rakowice Małe (SW Poland) section were formed during warmer climatic periods than the overlying sediments. Furthermore, no DNA could be isolated from the samples of lignites and overlying sediments, whereas it was abundant in the control samples of maple, birch and oak wood degraded by fungi. This indicates an absence of recent fungi responsible for decay in lignites and implies that the saccharide origin is connected with ancient fungi. Other sugar alcohols and acids like D-pinitol, quinic acid and shikimic acid, were found for the first time in sedimentary rocks, and their source is inferred to be from higher plants, most likely conifers. The preservation of mono- and disaccharides of fungal origins in pre-Palaeogene strata implies that compounds previously thought as unstable can survive for tens to hundreds of millions of years without structural changes in immature rocks unaffected by secondary processes

Methane-yielding microbial communities processing lactate-rich substrates : a piece of the anaerobic digestion puzzle

Background: Anaerobic digestion, whose final products are methane and carbon dioxide, ensures energy flow and circulation of matter in ecosystems. This naturally occurring process is used for the production of renewable energy from biomass. Lactate, a common product of acidic fermentation, is a key intermediate in anaerobic digestion of biomass in the environment and biogas plants. Effective utilization of lactate has been observed in many experimen‑tal approaches used to study anaerobic digestion. Interestingly, anaerobic lactate oxidation and lactate oxidizers as a physiological group in methane‑yielding microbial communities have not received enough attention in the context of the acetogenic step of anaerobic digestion. This study focuses on metabolic transformation of lactate during the acetogenic and methanogenic steps of anaerobic digestion in methane‑yielding bioreactors.Results: Methane‑yielding microbial communities instead of pure cultures of acetate producers were used to process artificial lactate‑rich media to methane and carbon dioxide in up‑flow anaerobic sludge blanket reactors. The media imitated the mixture of acidic products found in anaerobic environments/digesters where lactate fermentation dominates in acidogenesis. Effective utilization of lactate and biogas production was observed. 16S rRNA profiling was used to examine the selected methane‑yielding communities. Among Archaea present in the bioreactors, the order Methanosarcinales predominated. The acetoclastic pathway of methane formation was further confirmed by analysis of the stable carbon isotope composition of methane and carbon dioxide. The domain Bacteria was represented by Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, Actinobacteria, Spirochaetes, Tenericutes, Caldithrix, Verrucomicro-bia, Thermotogae, Chloroflexi, Nitrospirae, and Cyanobacteria. Available genome sequences of species and/or genera identified in the microbial communities were searched for genes encoding the lactate‑oxidizing metabolic machinery homologous to those of Acetobacterium woodii and Desulfovibrio vulgaris. Furthermore, genes for enzymes of the reductive acetyl‑CoA pathway were present in the microbial communities.Conclusions: The results indicate that lactate is oxidized mainly to acetate during the acetogenic step of AD and this comprises the acetotrophic pathway of methanogenesis. The genes for lactate utilization under anaerobic conditions are widespread in the domain Bacteria. Lactate oxidation to the substrates for methanogens is the most energetically attractive process in comparison to butyrate, propionate, or ethanol oxidation

Lignite biodegradation under conditions of acidic molasses fermentation

Lignite is difficult to degrade, thus stimulation of the autochthonous lignite microflora and introduction of additional microorganisms are required for lignite decomposition. Here, a packed bed reactor, filled with lignite samples from the Konin region (central Poland) was supplied continuously with M9 medium, supplemented with molasses (a by-product from the sugar industry), for 124 days to stimulate the autochthonous lignite microflora. Acidic fermentation of molasses was observed in the bioreactor. The simultaneous decomposition of lignite occurred under this acidic molasses fermentation condition. Our results show decay of free (non-bound) organic compounds during anaerobic lignite biodegradation. The concentrations of n-alkanes, n-alkanols, n-alkanoic acids, diterpenoids, triterpenoids and steroids present in non-biodegraded samples decreased significantly (some compounds to zero) during biodegradation. Interestingly, other compound classes like phenols, ketones and certain organic compounds increased. We interpret this phenomenon as a gradual decomposition of polymers, lignin and cellulose, present in the lignite. These changes resulted from microbial activity since they were not observed in pure solutions of short-chain fatty acids. The 16SrRNA profiling of the microbial community selected in the bioreactor revealed that the dominant bacteria belonged to the Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes, furthermore representatives of 16 other phyla were also found. All the known taxa of lignocellulolytic bacteria were represented in the microbial community. Synergistic relations between bacteria fermenting molasses and bacteria degrading lignite are assumed. The results confirm lignin degradation in acidic medium by bacteria under anaerobic conditions

Ocena migracji składników gazu ziemnego w złożu Borzęcin na podstawie wyników analiz izotopów trwałych wodoru i węgla

Jedna z pierwszych instalacji EGR (ang. Enhanced Gas Recovery – wzmożonego wydobycia gazu) na świecie funkcjonuje na złożu Borzęcin (SW Polska). Instalacja umożliwia zebranie doświadczenia w rozwoju technologii CCS (ang. Carbon Capture and Storage – wychwyt, transport oraz trwała izolacja CO2) na skalę przemysłową. Ze względu na niewielkie rozmiary złoża technologia ta daje możliwość rozwoju badawczego EGR, przy czym wszystkie procesy związane z zatłaczaniem ditlenku węgla i siarkowodoru są możliwe do obserwacji w stosunkowo niedługiej skali czasu. Głównym celem badań była próba oceny migracji zatłaczanych gazów poprzez pobór i analizę izotopów trwałych wodoru i węgla w gazach z poszczególnych otworów wiertniczych. Dzięki uzyskanym wynikom możliwa jest ocena migracji składników gazu ziemnego w złożu Borzęcin. W rezultacie przeprowadzonych badań otrzymano nowe narzędzie do oceny efektywności wzmożonej eksploatacji gazu, które następnie może być zastosowane w technologiach związanych z CCS. Skład izotopowy wodoru (n = 63) w metanie mieścił się w zakresie od –159,3‰ do –85,2‰ (średnia –114,6‰), a skład izotopowy węgla (n = 92) w tym metanie wyniósł od –46,93‰ do –17,87‰ (średnia –35,22‰). Skład izotopowy węgla (n = 88) w ditlenku węgla osiągnął wartość od –23,33‰ do –3,11‰ (średnia –13,35‰). One of the first installations in the World, based on the EGR (Enhanced Gas Recovery) operates in Borzecin (SW Poland). Thus creating the opportunity to gather experience in the development of CCS (Carbon Capture and Storage) on an industrial scale. Due to the small size of the deposit, the technology allows great research opportunity to develop Enhanced Gas Recovery and all the processes associated with the injection of CO2 and H2S are possible to observe in a relatively short time scale. The main goal of the research is tracing migration fronts of injected gases threw sampling and isotopic analysis of gases from numeral boreholes. Gathered results gave the opportunity to get an appraisal of gas migration in the Borzecin półdeposit. Consequently a new tool for Enhanced Gas Recovery efficiency might be constructed with the potential of being applied in CCS technologies. Isotopic composition of hydrogen (n = 63) in methane varied from –159.3 to –85.2‰ (average –114.6‰) and isotopic composition of carbon including methane (n = 92) varied from –46.93 to –17.87‰ (average –35.22‰). Isotopic composition of carbon in carbon dioxide (n = 88) varied from –23.33 to –3.11‰ (average –13.35‰)

Evaluation of acidogenesis products’ effect on biogas production performed with metagenomics and isotopic approaches

Anna Detman, Michał Bucha, Laura Treu, Aleksandra Chojnacka, Łukasz Pleśniak, Agnieszka Salamon, Ewa Łupikasza, Robert Gromadka, Jan Gawor, Agnieszka Gromadka, Wojciech Drzewicki, Marta Jakubiak, Marek Janiga, Irena Matyasik, Mieczysław K. Błaszczyk, Mariusz Orion Jędrysek, Stefano Campanaro, Anna Sikora.Background: During the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system. Results: Four methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%–67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities. Conclusions: In this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock

Stable isotopes of C and H in methane fermentation of agriculture substrates at different temperature conditions

Agricultural substrates (maize silage and cattle manure) were used to carry out methane fermentation process in bioreactors under laboratory conditions. Identical mixtures of these substrates were incubated for 43 days at 20, 30 and 40ºC to determine how different temperature conditions affect the δ13C(CH4), δH(CH4), and δ13C(CO2) values. To ensure correct anaerobic digestion, the following parameters of the organic substrates and fermentation solutions were monitored: total organic carbon (TOC), volatile solids (VS), volatile fatty acids (VFA), chemical oxygen demand (COD) and carbon to nitrogen ratio (C/N). The variants with higher incubation temperature yielded higher amounts of biogas (20ºC=84.5, 30ºC=101.8 and 40ºC=133.3 dm3/kg VS). In the case of gas products of methane fermentation, it was observed that the higher temperature of incubation affects the depletion in heavy isotopes. At 20ºC, 30ºC, and 40ºC mean values of δ13C(CH4) reached −26.4, −29.7, and −35.4‰, respectively. Mean values of δ2H(CH4) were −311.6, −354.0, and −398.5permil, and of δ13C(CO2) +8.9, +3.7, and −2.3‰, respectively. Moreover, the apparent fractionation coefficient α13C(CO2-CH4) were calculated, which decreased when the temperature increased. This isotopic tool was used to identify acetoclastic reaction as a dominant methanogenesis pathway. Observed changes in the isotopic composition of gaseous products obtained at different incubation temperatures may indicate decomposition of different carbon sources (e.g. lactate, propionate) to acetate and its fermentation by acetoclastic methanogens. It is possible that this was also related to the observation of the various metabolic models due to the varied methanogenic community composition

Occurrence and significance of mono-, di- and anhydrosaccharide biomolecules in Mesozoic and Cenozoic lignites and fossil wood

Mono-, di- and polysaccharides are common constituents of living organisms, but their occurrence and state of preservation in geological materials have only rarely been considered. Here, we present the monosaccharide, and for the first time the di- and anhydrosaccharide, identifications and distributions in Middle Miocene lignite and Middle Jurassic fossil wood samples. Detritic lignites contain fructose and glucose as dominant monosaccharides, and sucrose and trehalose as important disaccharides. Xylites contain monosaccharides (arabinose, arabinofuranose, glucose, and minor xylose and fructose), saccharols (erythritol, arabitol and mannitol), and also some disaccharides. The Middle Jurassic fossil wood samples contain glucose, glucofuranose and levoglucosan. The high content of holocellulose (up to 55 wt%) and co-occurrence of characteristic monosaccharides as arabinose, xylose and mannose in xylites suggests that not only cellulose, but also hemicellulose was preserved in samples as old as 13 Ma. Compounds like trehalose and mannitol appear to be products of wood-degrading fungi. Surprisingly, glucose, the most stable monosaccharide, and levoglucosan can occur in much older organic matter (ca. 168 Ma) as products from cellulose degradation, and possibly a remnant from wildfire burning of wood, respectively. Our findings confirm that saccharides can be preserved under favorable conditions in sedimentary organic matter of the Mesozoic to the Cenozoic eras, and can be used as specific biomarkers of cellulose and hemicellulose degradation, fungal metabolism, and wildfire events. However, we cannot exclude the possibility that at least part of the saccharides may be preserved in sedimentary rocks as the free compounds, common in plants and microorganisms