Editorial: The microbiology of the biogas process

[Excerpt] The world is facing unprecedent challenges, related with energy crisis and climate change. Intensification of renewable energy production, with special focus on sustainable biogas and biomethane, is one of the front-line topics today. Biogas/biomethane will play a role in the transition toward climate-neutral and secure energy system. Ensuring that biomethane is produced from organic waste/wastewater is essential to support circularity and sustainability. Scaling up biomethane production and assuring its economic competitiveness are current key challenges. Biogas generation occurs in natural and engineered environments, such as anaerobic bioreactors, and involves a cascade of reactions catalyzed by complex microbial communities. To unlock and boost the full potential of waste-based biomethane production, bioprocess optimization is needed, which requires deep knowledge on microbial diversity and physiology, as well as on the complex microbial interactions and metabolic networks occurring in biogas processes. This was the motivation for launching the Research Topic “The Microbiology of the Biogas Process,” which comprises six original research articles by 48 authors, addressing different facets of the theme and resorting to diverse approaches, reflecting the complexity of the topic. [...]This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit.info:eu-repo/semantics/publishedVersio

Non-syntrophic reactions in anaerobic unsaturated LCFA conversion by methanogenic sludges

Lipids are energy-rich compounds. This energy can be conserved as biogas in anaerobic bioreactors but the process is frequently hindered by long-chain fatty acids (LCFA) accumulation. LCFA catabolism is thought to occur via beta-oxidation, performed by anaerobic bacteria that live in obligatory syntrophy with H2 consuming methanogens, but the initial steps of unsaturated LCFA biodegradation are still unclear. In this work we hypothesize that these initial steps do not depend on interspecies H2 transfer. To test this, six anaerobic bioreactors were continuously fed with saturated or unsaturated C16- and C18-LCFA, and operated in the presence or absence of bromoethanesulfonate, a selective inhibitor of methanogens. Intermediates of LCFA degradation including long- and medium-chain fatty acids, volatile fatty acids and methane were monitored. Bacterial community composition was analysed in the different bioreactors by denaturing gradient gel electrophoresis of 16S rRNA reverse transcriptase-PCR products. In the presence or absence of the inhibitor of methanogenesis, palmitate (C16:0) accumulated during the degradation of oleate (C18:1), accounting for more than 50% of total accumulated LCFA. Palmitoleate (C16:1) feeding resulted in the build-up of myristate (C14:0) and palmitate (C16:0). Accumulation of saturated intermediary-LCFA was two to four times higher in bioreactors in which methanogenesis was inhibited compared to methanogenic bioreactors. Beta-oxidation of saturated intermediates only occurred in methanogenic bioreactors. No catabolic activity was observed in the bioreactor fed with saturated LCFA when methanogenesis was inhibited. These results show that the first steps of unsaturated LCFA degradation are not obligatorily syntrophic, and suggest that beta-oxidation is the limiting step in the overall conversion of LCFA to methane

Anaerobic LCFA degradation: a role for non-syntrophic conversions?

For many years the focus of lipids/long-chain fatty-acids (LCFA) wastewater treatment was on technological and process developments. More recently, promising results on the anaerobic treatment of LCFA-containing wastewaters[1] widened the attention to the microbiology aspects as well. In anaerobic bioreactors, LCFA can be β-oxidized to acetate and H2 by acetogenic bacteria, in obligatory syntrophy with methanogens. Presently, 14 species have been described that grow on fatty-acids in syntrophy with methanogens, all belonging to the families Syntrophomonadaceae and Syntrophaceae[2]. Among these, only 4 species are able to degrade mono- and/or polyunsaturated LCFA. The reason why the degradation of unsaturated LCFA is not more widespread remains unknown. Early studies suggested that degradation of unsaturated LCFA requires complete chain saturation prior to β-oxidation[2]. Unsaturated LCFA, such as linoleate (C18:2) and oleate (C18:1), would be metabolized through a hydrogenation step yielding stearate (C18:0), then entering the β-oxidation cycle. However, this theory is inconsistent with the observed accumulation of palmitate (C16:0) in continuous bioreactors fed with oleate[1]. We hypothesize that LCFA chain saturation might be a non-syntrophic process, i.e. unsaturated LCFA can function as electron donors and acceptors, as protons released in a first β-oxidation step can be used to hydrogenate the unsaturated hydrocarbon. To test this, linoleate (C18:2), oleate (C18:1) and a mixture of stearate (C18:0) and palmitate (C16:0) were continuously fed to bioreactors with methanogenesis-active or -inhibited anaerobic sludge. In the reactors fed with linoleate and oleate, palmitate accumulated in methanogenesis-active and -inhibited bioreactors up to concentrations of approximately 2 mM and 8 mM, respectively. In methanogenesis-inhibited bioreactors fed with a mixture of saturated LCFA (stearate and palmitate) no biological activity occurred. These results suggest the occurrence of a non-syntrophic step during the degradation of unsaturated LCFA in anaerobic bioreactors. The identification of microbial communities involved in non-syntrophic linoleate/oleate to palmitate conversion will give more insights into this novel biochemical mechanism

Characterization of an anaerobic thermophilic glycerol-degrading enrichment culture

Background: The glycerol market was totally changed by the biodiesel industry, which resulted in the production of an excess of this compound as an industrial by-product. As a consequence, the price of glycerol dropped and a huge interest in alternatives for its valorisation emerged since then. In the field of Biotechnology research, glycerol is an attractive compound for the microbial production of chemical building blocks. Objectives: The aim of this work was to investigate thermophilic anaerobic communities capable of conversion of glycerol. Methods: Thermophilic sludge from a lab-scale anaerobic reactor fed with skim milk and sodium oleate (50:50% chemical oxygen demand) was incubated at 55°C in closed bottles containing bicarbonate-buffered medium supplemented with 10mM glycerol. Periodic successive transfers of the glycerol-converting enrichment culture, combined with serial dilutions were performed. After eight generations a highly enriched, low diversity (microscopic observations and 16s rRNA DGGE profiling) microbial culture was obtained. Conclusions: The enriched culture converted glycerol mainly to methane (6mM) and acetate (7mM) within 6 days of incubation. A yet unknown organic compound was also produced. Sequencing results obtained on the Illumina platform showed the bacterial predominance of an uncultured Thermotoga species (75 % of the retrieved sequences), an uncultured Anaerobaculum species (13 %) and a close relative to Thermoanaerobacter pseudethanolicus (5 %). Isolation of the new uncultured Thermotoga and Anaerobaculum species is ongoing and their role in glycerol degradation will be assessed

Exploring the interrelatedness of risk factors for child maltreatment: A network approach

Background: Theories on the etiology of child maltreatment generally focus on the interaction between multiple risk and protective factors. Moreover, the quadratic model of cumulative risk describes a threshold at which the risk of child maltreatment increases exponentially, suggesting a synergistic effect between risk factors. Objective: This study explored the interrelatedness of risk factors for child maltreatment. Participants and Setting: The sample consisted of risk assessments performed for both high-risk families (n = 2,399; child protection services) and lower risk families (n = 1,904; community outreach services). Methods : Network analyses were performed on parental risk factors. Three networks were constructed: a cross-sample network, a high-risk network, and a lower risk network. The relations between risk factors were examined, as well as the centrality of each risk factor in these networks. Additionally, the networks of the two samples were compared. Results : The networks revealed that risk factors for child maltreatment were highly interrelated, which is consistent with Belsky’s multi-dimensional perspective on child maltreatment. As expected, risk factors were generally stronger related to each other in the high-risk sample than in the lower risk sample. Centrality analyses showed that the following risk factors play an important role in the development of child maltreatment: “Caregiver was maltreated as a child”, “History of domestic violence”, and “Caregiver is emotionally absent”. Conclusions : We conclude that studying the interrelatedness of risk factors contributes to knowledge on the etiology of child maltreatment and the improvement of both risk assessment procedures and interventions for child maltreatment

Competition and coexistence of sulfate-reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol−1, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol−1, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community

The stable isotopic signature of biologically produced molecular hydrogen (H₂)

Biologically produced molecular hydrogen (H₂) is characterised by a very strong depletion in deuterium. Although the biological source to the atmosphere is small compared to photochemical or combustion sources, it makes an important contribution to the global isotope budget of H₂. Large uncertainties exist in the quantification of the individual production and degradation processes that contribute to the atmospheric budget, and isotope measurements are a tool to distinguish the contributions from the different sources. Measurements of &delta; D from the various H₂ sources are scarce and for biologically produced H₂ only very few measurements exist. <br><br> Here the first systematic study of the isotopic composition of biologically produced H₂ is presented. In a first set of experiments, we investigated &delta; D of H₂ produced in a biogas plant, covering different treatments of biogas production. In a second set of experiments, we investigated pure cultures of several H₂ producing microorganisms such as bacteria or green algae. A Keeling plot analysis provides a robust overall source signature of &delta; D = &minus;712&permil; (±13&permil;) for the samples from the biogas reactor (at 38 °C, &delta; D_H2O= +73.4&permil;), with a fractionation constant &varepsilon;_H2-H2O of −689&permil; (±20&permil;) between H₂ and the water. The five experiments using pure culture samples from different microorganisms give a mean source signature of &delta; D = &minus;728&permil; (±28&permil;), and a fractionation constant &varepsilon;_H2-H2O of −711&permil; (±34&permil;) between H₂ and the water. The results confirm the massive deuterium depletion of biologically produced H₂ as was predicted by the calculation of the thermodynamic fractionation factors for hydrogen exchange between H₂ and water vapour. Systematic errors in the isotope scale are difficult to assess in the absence of international standards for &delta; D of H₂. <br><br> As expected for a thermodynamic equilibrium, the fractionation factor is temperature dependent, but largely independent of the substrates used and the H₂ production conditions. The equilibrium fractionation coefficient is positively correlated with temperature and we measured a rate of change of 2.3&permil; / °C between 45 °C and 60 °C, which is in general agreement with the theoretical prediction of 1.4&permil; / °C. Our best experimental estimate for &varepsilon;_H2-H2O at a temperature of 20 °C is −731&permil; (±20&permil;) for biologically produced H₂. This value is close to the predicted value of −722&permil;, and we suggest using these values in future global H₂ isotope budget calculations and models with adjusting to regional temperatures for calculating &delta; D values

Hydrogen producing microbial communities of the biocathode in a microbial electrolysis cell

In the search for alternatives for fossil fuels and the reuse of the energy from waste streams, the microbial electrolysis cell is a promising technique. The microbial electrolysis cell is a two electrode system in which at the anode organic substances, including waste water, are used by microorganisms that release the terminal electrons to the electrode. These electrons are subsequently used at the cathode resulting in the production of a current. By addition of a small voltage, hydrogen gas can be produced by combining electrons and protons at the cathode. To catalyse the hydrogen evolution reaction at the cathode, expensive catalysts such as platinum are required. Recently, the use of biocathodes has shown great potential as an alternative for platinum. The microbial community responsible for the hydrogen evolution in such systems is, however, not well understood. In this study we focused on the characterization of the microbial communities of the microbial electrolysis cell biocathode using molecular techniques. The results show that the microbial community consists of 44% Proteobacteria, 27% Firmicutes, 18% Bacteriodetes and 12% related to other phyla. Within the major phylogenetic groups we found several clusters of uncultured species belonging to novel taxonomic groups at genus level. These novel taxonomic groups developed under environmentally unusual conditions and might have properties that have not been described before. Therefore it is of great interest to study those novel groups further. Within the Proteobacteria a major cluster belonged to the Deltaproteobacteria and based on the known characteristics of the closest related cultured species, we suggest a mechanism for microbial electron transfer for the production of hydrogen at the cathode

Novel anaerobe obtained from a hexadecane-degrading consortium

Background: Aliphatic hydrocarbons (AHC) are abundant in crude oil and fuels, and are frequent contaminants of water, soil and sediments. There is potential for AHC bioremediation using sulfate as electron acceptor, due to its abundance in marine environments and natural presence in soils and groundwater. Objectives: In this work sulfate-reducing anaerobic microorganisms involved in AHC biodegradation were studied. Methods: Anaerobic sludge was incubated at 37ºC with hexadecane (1mM) and sulfate (20mM) in serum vials. Cultures were successively transferred to fresh medium until a stable enrichment was obtained (monitored by microscopy and PCR-DGGE of 16S rRNA gene). For isolation of AHC-degrading bacteria, serial dilutions and successive transfers are now running using palmitate (1mM) as an easier substrate. Conclusions: Cultures growing on palmitate show two main bacterial cell types: a rod-shaped bacterium closely related to Desulfomonile limimaris (94% identity) was predominant in the first 30 days of incubation, when 83% of the added palmitate was degraded coupled to 4 mM sulfate reduction (suggesting stoichiometric palmitate conversion to acetate); and an oval-shaped bacterium related to Desulforhabdus amnigena (99% identity) that mainly developed when incubations where extended and a total of 11.5 mM sulfate was reduced. Growth of Desulforhabdus was stimulated when incubated with acetate. The role of the Desulfomonile in AHC degradation will be further discussed in the presentation, as well as its halorespiring ability, a characteristic of the Desulfomonile genera. Further characterization of this novel bacterium is important due to its high potential for bioremediation of hydrocarbons, fats and halogenated pollutants