Selected issues of anaerobic digestion based on the studies on hydrogen- and methane yielding bioreactors

Anaerobic digestion of organic matter results from the metabolic activity of many groups of microorganisms. Interactions between microorganisms during acidogenesis, acetogenesis and methanogenesis, source of inoculum, type of feedstock and operational conditions determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. In innovative installations it is desirable to separate acidogenesis from acetogenesis and methanogenesis to favour respectively the production of biohydrogen or biomethane under controlled conditions

Searching for Metabolic Pathways of Anaerobic Digestion: A Useful List of the Key Enzymes

The general scheme of anaerobic digestion is well known. It is a complex process promoted by the interaction of many groups of microorganisms and has four major steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. The aim of the study was to prepare a systematized list of the selected enzymes responsible for the key pathways of anaerobic digestion based on the Kyoto Encyclopedia of Genes and Genomes database resource. The list contains (i) key groups of hydrolases involved in the process of degradation of organic matter; (ii) the enzymes catalyzing reactions leading to pyruvate formation; (iii) the enzymes of metabolic pathways of further pyruvate transformations; (iv) the enzymes of glycerol transformations; (v) the enzymes involved in transformation of gaseous or nongaseous products of acidic fermentations resulting from nonsyntrophic nutritional interactions between microbes; (vi) the enzymes of amino acid fermentations; (vii) the enzymes involved in acetogenesis; and (viii) the enzymes of the recognized pathways of methanogenesis. Searching for the presence and activity of the enzymes as well as linking structure and function of microbial communities allows to develop a fundamental understanding of the processes, leading to methane production. In this contribution, the present study is believed to be a piece to the enzymatic road map of anaerobic digestion research

Anaerobic Digestion: I. A Common Process Ensuring Energy Flow and the Circulation of Matter in Ecosystems. II. A Tool for the Production of Gaseous Biofuels

Anaerobic digestion, a process that ultimately generates methane and carbon dioxide, is common in natural anoxic ecosystems where concentrations of electron acceptors such as nitrate, the oxidized forms of metals and sulphate are low. It also occurs in landfill sites and wastewater treatment plants. The general scheme of anaerobic digestion is well known and comprises four major steps: (i) hydrolysis of complex organic polymers to monomers; (ii) acidogenesis that results in the formation of hydrogen and carbon dioxide as well as non-gaseous fermentation products that are further oxidized to hydrogen, carbon dioxide and acetate in (iii) acetogenesis based on syntrophic metabolism and (iv) methanogenesis. Approaches to the analysis of methane-yielding microbial communities and data acquisition are described. There is currently great interest in the development of new technologies for the production of biogas (primarily methane) from anaerobic digestion as a source of renewable energy. This includes the modernization of landfill sites and wastewater treatment plants and the construction of biogas plants. Moreover, research effort is being devoted to the idea of separating hydrolysis and acidogenesis from acetogenesis and methanogenesis under controlled conditions to favour biohydrogen and biomethane production, respectively. These two stages occur under different conditions and are carried out in separate bioreactors

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNArepair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes.Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. Theaim of this study was to assess the usefulness of these alkB mutants in different aspects of research onAlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes incells. Using of HK82 and BS87 strains, we found the following differences among these alkB−derivatives:(i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii)different specificity of Arg+revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv)the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, andnuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, mayresult in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis.In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearingseveral additional mutations in its genome, is not an exact derivative of the AB1157 strain, and showsadditional features that may disturb proper interpretation of obtained results

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

Inhibition of hydrogen-yielding dark fermentation by ascomycetous yeasts

Hydrogen-yielding fermentation conducted in bioreactors is an alternative method of hydrogen production. However, unfavourable processes can seriously inhibit bio-hydrogen generation during the acidogenic step of anaerobic digestion. Here, ascomycetous yeasts were identified as a major factor inhibiting the production of bio-hydrogen by fermentation.Changes in the performance of hydrogen-producing bioreactors including metabolic shift,quantitative changes in the fermentation products, decreased pH, instability of the microbialcommunity and consequently a dramatic drop in bio-hydrogen yield were observed following yeast infection. Ascomycetous yeasts from the genera Candida, Kazachstania and Geotrichum were isolated from hydrogen-producing bioreactors. Yeast metabolites secreted into the growth medium showed antibacterial activity. Our studies indicate that yeast infection of hydrogen-producing microbial communities is one of the serious obstacles to use dark fermentation as an alternative method of bio-hydrogen production. It also explains why studies on hydrogen fermentation are still limited to the laboratory or pilot-scale systems

Biohydrogen and Biomethane (Biogas) Production in the Consecutive Stages of Anaerobic Digestion of Molasses

Anaerobic digestion, whose final products are methane and carbon dioxide, has been used to produce biogas from waste biomass as an alternative energy source. For the purpose of innovative, modern technologies based on microbial processes, it is desirable to separate the hydrogen- (hydrolysis and acidogenesis) and methane-yielding (acetogenesis and methanogenesis) stages of anaerobic digestion to respectively favor the production of hydrogen and methane under controlled conditions. Previously, we developed a benchscale (3- and 3.5-litre bioreactors) two-stage anaerobic digestion system producing hydrogen (in stage 1)and methane (in stage 2) from sucrose-rich by-products of the sugar beet refining industry as the primary energy substrates under mesophilic conditions. Recently, the two-stage system for hydrogen and methane production has been successfully scaled up 10-fold (a pilot scale) and currently operates in one of the Polish sugar factories. The efficiency of hydrogen and methane production were directly proportional to the scale of installation. The obtained results led to the development objectives of further research that the end result will be an innovative solution for the sugar factory as a producer of gaseous biofuels

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

Dynamics of dark fermentation microbial communities in the light of lactate and butyrate production.

Abstract Background: This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. Results: MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0–6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. Conclusions: Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation