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This study aimed to examine the bacterial, methanogenic archaeal, and eukaryotic community structure in both the midgut and hindgut of Pachnoda marginata larvae using an amplicon sequencing approach. The goal was to investigate how various diets and the soil affect the composition of these three‐domain microbial communities within the gut of insect larvae. The results indicated a notable variation in the microbial community composition among the gut compartments. The majority of the bacterial community in the hindgut was composed of Ruminococcaceae and Christensenellaceae. Nocardiaceae, Microbacteriaceae, and Lachnospiraceae were detected in midgut samples from larvae feeding on the leaf diet, whereas Sphingomonadaceae, Rhodobacteraceae, and Promicromonasporaceae dominated the bacterial community of midgut of larvae feeding on the straw diet. The diet was a significant factor that influenced the methanogenic archaeal and eukaryotic community patterns. The methanogenic communities in the two gut compartments significantly differed from each other, with the midgut communities being more similar to those in the soil. A higher diversity of methanogens was observed in the midgut samples of both diets compared to the hindgut. Overall, the microbiota of the hindgut was more host‐specific, while the assembly of the midgut was more influenced by the environmental microorganisms. [ABSTRACT FROM AUTHOR]

With the increasing use of renewable energy resources for the power grid, the need for long-term storage technologies, such as power-to-gas systems, is growing. Biomethanation provides the opportunity to store energy in the form of the natural gas-equivalent biomethane. This study investigates a novel plug flow reactor that employs a helical static mixer for the biological methanation of hydrogen and carbon dioxide. In tests, the reactor achieved an average methane production rate of 2.5 L C H 4 L R ∗ d (methane production [LCH4] per liter of reactor volume [LR] per day [d]) with a maximum methane content of 94%. It demonstrated good flexibilization properties, as repeated 12 h downtimes did not negatively impact the process. The genera Methanothermobacter and Methanobacterium were predominant during the initial phase, along with volatile organic acid-producing, hydrogenotrophic, and proteolytic bacteria. The average ratio of volatile organic acid to total inorganic carbon increased to 0.52 ± 0.04, while the pH remained stable at an average of pH 8.1 ± 0.25 from day 32 to 98, spanning stable and flexible operation modes. This study contributes to the development of efficient flexible biological methanation systems for sustainable energy storage and management. [ABSTRACT FROM AUTHOR]

The increasing share of renewable electricity in the grid drives the need for sufficient storage capacity. Especially for seasonal storage, power-to-gas can be a promising approach. Biologically produced methane from hydrogen produced from surplus electricity can be used to substitute natural gas in the existing infrastructure. Current reactor types are not or are poorly optimized for flexible methanation. Therefore, this work proposes a new reactor type with a plug flow reactor (PFR) design. Simulations in COMSOL Multiphysics ® showed promising properties for operation in laminar flow. An experiment was conducted to support the simulation results and to determine the gas fraction of the novel reactor, which was measured to be 29%. Based on these simulations and experimental results, the reactor was constructed as a 14 m long, 50 mm diameter tube with a meandering orientation. Data processing was established, and a step experiment was performed. In addition, a kLa of 1 h−1 was determined. The results revealed that the experimental outcomes of the type of flow and gas fractions are in line with the theoretical simulation. The new design shows promising properties for flexible methanation and will be tested. [ABSTRACT FROM AUTHOR]

Power-to-X (P2X) technologies will play a more important role in the conversion of electric power to storable energy carriers, commodity chemicals and even food and feed. Among the different P2X technologies, microbial components form cornerstones of individual process steps. This review comprehensively presents the state-of-the-art of different P2X technologies from a microbiological standpoint. We are focusing on microbial conversions of hydrogen from water electrolysis to methane, other chemicals and proteins. We present the microbial toolbox needed to gain access to these products of interest, assess its current status and research needs, and discuss potential future developments that are needed to turn todays P2X concepts into tomorrow's technologies. [ABSTRACT FROM AUTHOR]

In order to partially mimic the efficient lignocellulose pretreatment process performed naturally in the gut system of Pachnoda marginata larvae, two wheat straw pretreatments were evaluated: a mechanical pretreatment via cutting the straw into two different sizes and an alkaline pretreatment with calcium hydroxide. After pretreatment, gut enrichment cultures on wheat straw at alkaline pH were inoculated and kept at mesophilic conditions over 45 days. The methanogenic community was composed mainly of the Methanomicrobiaceae and Methanosarcinaceae families. The combined pretreatment, size reduction and alkaline pretreatment, was the best condition for methane production. The positive effect of the straw pretreatment was higher in the midgut cultures, increasing the methane production by 192%, while for hindgut cultures the methane production increased only by 149% when compared to non-pretreated straw. Scanning electron microscopy (SEM) showed that the alkaline pretreatment modified the surface of the wheat straw fibers, which promoted biofilm formation and microbial growth. The enrichment cultures derived from larva gut microbiome were able to degrade larger 1 mm alkaline treated and smaller 250 µm but non-pretreated straw at the same efficiency. The combination of mechanical and alkaline pretreatments resulted in increased, yet not superimposed, methane yield. [ABSTRACT FROM AUTHOR]

The Pachnoda marginata larva have complex gut microbiota capable of the effective conversion of lignocellulosic biomass. Biotechnological utilization of these microorganisms in an engineered system can be achieved by establishing enrichment cultures using a lignocellulosic substrate. We established enrichment cultures from contents of the midgut and hindgut of the beetle larva using wheat straw in an alkaline medium at mesophilic conditions. Two different inoculation preparations were used: procedure 1 (P1) was performed in a sterile bench under oxic conditions using 0.4% inoculum and small gauge needles. Procedure 2 (P2) was carried out under anoxic conditions using more inoculum (4%) and bigger gauge needles. Higher methane production was achieved with P2, while the highest acetic acid concentrations were observed with P1. In the enrichment cultures, the most abundant bacterial families were Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae, and Marinilabiliaceae. Further, the most abundant methanogenic genera were Methanobrevibacter, Methanoculleus, and Methanosarcina. Our observations suggest that in samples processed with P1, the volatile fatty acids were not completely converted to methane. This is supported by the finding that enrichment cultures obtained with P2 included acetoclastic methanogens, which might have prevented the accumulation of acetic acid. We conclude that differences in the inoculum preparation may have a major influence on the outcome of enrichment cultures from the P. marginata larvae gut. [ABSTRACT FROM AUTHOR]

Livestock manure emits reduced sulfur compounds and methane, which affect nature and the climate. These gases are efficiently mitigated by addition of a tannic acid-sodium fluoride combination inhibitor (TA-NaF), and to some extent by acidification. In this paper, TA-NaF treatment was performed on swine manure to study the treatment influence on methanogenic pathways and sulfur transformation pathways in various laboratory experiments. Stable carbon isotope labeling revealed that both untreated and TA-NaF treated swine manures were dominated by hydrogenotrophic methanogenesis. However, in supplementary experiments in wastewater sludge, TA-NaF clearly inhibited acetoclastic methanogenesis, whereas acidification inhibited hydrogenotrophic methanogenesis. In swine manure, TA-NaF inhibited s-amino acid catabolism to a larger extent than sulfate reduction. Conversely, acidification reduced sulfate reduction activity more than s-amino acid degradation. TA-NaF treatment had no significant effect on methanogenic community structure, which was surprising considering clear effects on isotope ratios of methane and carbon dioxide. Halophile sulfate reducers adapted well to TA-NaF treatment, but the community change also depended on temperature. The combined experimental work resulted in a proposed inhibition scheme for sulfur transformations and methanogenic pathways as affected by TA-NaF and acidification in swine manure and in other inocula. [ABSTRACT FROM AUTHOR]

Ex situ biomethanation allows the conversion of hydrogen produced from surplus electricity to methane. The flexibility of the process was recently demonstrated, yet it is unknown how intermittent hydrogen feeding impacts the functionality of the microbial communities. We investigated the effect of starvation events on the hydrogen consumption and methane production rates (MPRs) of two different methanogenic communities that were fed with hydrogen and carbon dioxide. Both communities showed functional resilience in terms of hydrogen consumption and MPRs upon starvation periods of up to 14 days. The origin of the inoculum, community structure and dominant methanogens were decisive for high gas conversion rates. Thus, pre-screening a well performing inoculum is essential to ensure the efficiency of biomethanation systems operating under flexible gas feeding regimes. Our results suggest that the type of the predominant hydrogenotrophic methanogen (here: Methanobacterium) is important for an efficient process. We also show that flexible biomethanation of hydrogen and carbon dioxide with complex microbiota is possible while avoiding the accumulation of acetate, which is relevant for practical implementation. In our study, the inoculum from an upflow anaerobic sludge blanket reactor treating wastewater from paper industry performed better compared to the inoculum from a plug flow reactor treating cow manure and corn silage. Therefore, the implementation of the power-to-gas concept in wastewater treatment plants of the paper industry, where biocatalytic biomass is readily available, may be a viable option to reduce the carbon footprint of the paper industry. [ABSTRACT FROM AUTHOR]

The aim of this review is to give a summary of natural lignocellulose-degrading systems focusing mainly on animal digestive tracts of wood-feeding insects and ruminants in order to find effective strategies that can be applied to improve anaerobic digestion processes in engineered systems. Wood-feeding animals co-evolved with symbiotic microorganisms to digest lignocellulose-rich biomass in a very successful way. Considering the similarities between these animal gut systems and the lignocellulose-based biotechnological processes, the gut with its microbial consortium can be a perfect model for an advanced lignocellulose-degrading biorefinery. The physicochemical properties and structure of the gut may provide a scheme for the process design, and the microbial consortium may be applied as genetic resource for the up-scaled bioreactor communities. Manipulation of the gut microbiota is also discussed in relation to the management of the reactor communities. [ABSTRACT FROM AUTHOR]

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis is developed for detecting and typing of 'Dehalococcoides' species. The studies have shown SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.

Abstract: Three different bioaugmentation cultures enriched from natural and engineered cellulolytic environments (cow and goat rumen, a biogas reactor digesting sorghum biomass) were compared for their enhancement potential on the anaerobic digestion of wheat straw. Methane yields were determined in batch tests using the Automatic Methane Potential Test System operated for 30 days under mesophilic conditions. All cultures had positive effects on substrate degradation, and higher methane yields were observed in the bioaugmented reactors compared to control reactors set up with standard inoculum. However, the level of enhancement differed according to the type of the enrichment culture. Methane yield in batch reactors augmented with 2% cow rumen derived enrichment culture was increased by only 6%. In contrast, reactors amended with 2% goat rumen derived enrichment culture or with the bioaugmentation culture obtained from the biogas reactor digesting sorghum biomass produced 27 and 20% more methane, respectively. The highest methane yield was recorded in reactors amended with 6% goat rumen derived enrichment culture, which represented an increase by 36%. The microbial communities were quite similar at the end of the batch tests independently of the bioaugmentation sources, indicating that the introduced microbial communities of the enrichment cultures did not dominate the reactors. [ABSTRACT FROM AUTHOR]

Abstract: Anaerobic digestion of poultry manure poses many challenges due to ammonia inhibition by nitrogen‐containing compounds. Here, the effect of NH4Cl or urea‐induced inhibition was investigated in bioreactors fed with maize silage or cellulose and compared to previous experiments with chicken manure as ammonia source. The hydrogen and carbon stable isotope compositions of biogas samples were evaluated to assess the shifts in predominant methanogenic pathways during increasing ammonia inhibition. Due to the higher free ammonia concentrations, stronger inhibition was observed in the urea added reactors compared to the NH4Cl treated ones. Collectively, the results suggested that ammonium salts are not really appropriate to investigate ammonia inhibition during anaerobic digestion of manure. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies. [ABSTRACT FROM AUTHOR]

Background: During strategic planning of a biogas plant, the local availability of resources for start-up and operation should be taken into consideration for a cost-efficient process. Because most bioethanol/sugar industries in Brazil are located in remote areas, the use of fresh cattle manure from local farms could be a solution for the inoculation of the biogas process. This study investigated the diversity and dynamics of bacterial and archaeal communities and the performance of biogas reactors inoculated with manure and a mixed inoculum from different biogas reactors as for a controlled start-up until steady state. Results: Laboratory-scale biogas reactors were fed semi-continuously with sugarcane filter cake alone (mono-digestion) or together with bagasse (co-digestion). At the initial start-up, the reactors inoculated with the mixed inoculum displayed a less diverse taxonomic composition, but with higher presence of significant abundances compared to reactors inoculated with manure. However, in the final steady state, the communities of the differently inoculated reactors were very similarly characterized by predominance of the methanogenic genera Methanosarcina and Methanobacterium, the bacterial families Bacteroidaceae, Prevotellaceae and Porphyromonadaceae (phylum Bacteroidetes) and Synergistaceae (phylum Synergistetes). In the mono-digestion reactors, the methanogenic communities varied greater than in the co-digestion reactors independently of the inoculation strategy. Conclusion: The microbial communities involved in the biogas production from waste products of the Brazilian bioethanol/sugar industry were relatively similar and stable at the reactor's steady phase independently of the inoculum source (manure or mixed inoculum). Therefore, the locally available manure can be used as inoculum for start-up of the biogas process, since it also contains the microbial resources needed. The strong fluctuation of methanogenic communities in mono-digestion reactors indicates higher risk of process instability than in co-digestion reactors. [ABSTRACT FROM AUTHOR]

The anaerobic digestion of filter cake and its co-digestion with bagasse, and the effect of gradual increase of the organic loading rate (OLR) from start-up to overload were investigated. Understanding the influence of environmental and technical parameters on the development of particular methanogenic pathway in the biogas process was an important aim for the prediction and prevention of process failure. The rapid accumulation of volatile organic acids at high OLR of 3.0 to 4.0 gvs.L-1.day-1 indicated strong process inhibition. Methanogenic community dynamics of the reactors was monitored by stable isotope composition of biogas and molecular biological analysis. A potential shift toward the aceticlastic methanogenesis was observed along with the OLR increase under stable reactor operating conditions. Reactor overloading and process failure were indicated by the tendency to return to a predominance of hydrogenotrophic methanogenesis with rising abundances of the orders Methanobacteriales and Methanomicrobiales and drop of the genus Methanosarcina abundance. [ABSTRACT FROM AUTHOR]

Biogas production from sugarcane waste has large potential for energy generation, however, to enable the optimization of the anaerobic digestion (AD) process each substrate characteristic should be carefully evaluated. In this study, the kinetic challenges for biogas production from different types of sugarcane waste were assessed. Samples of vinasse, filter cake, bagasse, and straw were analyzed in terms of total and volatile solids, chemical oxygen demand, macronutrients, trace elements, and nutritional value. Biochemical methane potential assays were performed to evaluate the energy potential of the substrates according to different types of sugarcane plants. Methane yields varied considerably (5-181 Nm3.tonFM-1), mainly due to the different substrate characteristics and sugar and/or ethanol production processes. Therefore, for the optimization of AD on a large-scale, continuous stirred-tank reactor with long hydraulic retention times (>35 days) should be used for biogas production from bagasse and straw, coupled with pre-treatment process to enhance the degradation of the fibrous carbohydrates. Biomass immobilization systems are recommended in case vinasse is used as substrate, due to its low solid content, while filter cake could complement the biogas production from vinasse during the sugarcane offseason, providing a higher utilization of the biogas system during the entire year. [ABSTRACT FROM AUTHOR]

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline ( pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA / mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. M ethanothermobacter and M ethanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera . The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion. [ABSTRACT FROM AUTHOR]

An enrichment culture dominated by one type of Dehalococcoides sp. (83% of clones) was characterised. This culture, originally derived from contaminated groundwater from the area of Bitterfeld- Wolfen ( Saxony- Anhalt, Germany), dehalogenates chlorinated ethenes to ethene. Further, the culture also dehalogenated vinyl bromide ( VB) and 1,2-dichloroethane ( DCA) to ethene, 1,2,3,4- and 1,2,3,5-tetrachlorobenzene ( Te CB), penta- and hexachlorobenzene ( Pe CB and HCB) to trichlorobenzenes ( TCB), lindane to monochlorobenzene ( MCB) and pentachlorophenol ( PCP) to 2,3,4,6-tetrachlorophenol ( Te CP). Growth was proven by quantitative PCR for all active cultures, except for those with Te CB, lindane and PCP. The growth yields obtained ranged from (2.9 ± 0.7) × 107 cells μmol−1 Br− released on VB to (34.8 ± 5.4) × 107 cells μmol−1 Cl− released on VC. Genes coding for nine putative reductive dehalogenases, the enzymes that mediate the respiratory process of dehalogenation, were identified. Phylogenetic analysis revealed eight reductive dehalogenases with similar sequences in other Dehalococcoides strains and one unique sequence. [ABSTRACT FROM AUTHOR]

Abstract: The description of microbial community structure is always biased by the selectivity of the methods applied. Although TA cloning of PCR amplified community DNA is one of the most widely used techniques in bacterial community analysis, no thorough comparative testing has been carried out on different TA cloning systems. In this study, we measured and compared the selectivity of two widely used TA-cloning kits in experimental setups where the length heterogeneity of the inserts modeled the natural length variation of the 16S rRNA gene and the 16S–23S intergenic spacer region. Both TOPO TA (Invitrogen, CA USA) and pGem-T vector system (Promega, WI USA) cloning kits showed significant and reproducible insert size related selectivity. The effect of ligation time and temperature was also studied in case of the pGem-T vector system. We compared the performance of the two cloning kits on an environmental sample, along with a semiquantitative community fingerprinting method to gain reference data free of cloning bias. The two clone libraries showed significantly different compositions, and were also differing from the community structure revealed by length heterogeneity PCR. [Copyright &y& Elsevier]

A Dehalococcoides-dominated culture coupling reductive dechlorination of tetrachloroethene (PCE) to ethene to growth was enriched from a European field site for the first time. Microcosms were set up using groundwater from a chlorinated ethene-contaminated anaerobic aquifer in Bitterfeld (Germany). Active, lactate-amended microcosms capable of PCE dechlorination to ethene without the accumulation of intermediates were used for further enrichment. After three transfers on lactate as an electron donor and PCE as an electron acceptor, the enrichment was transferred to parallel cultures with one of the chlorinated ethenes as an electron acceptor and acetate and hydrogen as the carbon and energy source, respectively. After three more transfers, a highly purified culture was derived that was capable of dechlorinating PCE with hydrogen and acetate as the electron donor and carbon source, respectively. PCR, followed by denaturing gradient gel electrophoresis, cloning and sequencing revealed that this culture was dominated by a Dehalococcoides sp. belonging to the Pinellas group. Investigation of substrate specificity in the parallel cultures suggested the presence of a novel Dehalococcoides that can couple all dechlorination steps, from PCE to ethene, to energy conservation. Quantitative real-time PCR confirmed growth with PCE, cis-dichloroethene, 1,1-dichloroethene or vinyl chloride as electron acceptors. The culture was designated BTF08 due to its origin in Bitterfeld. [ABSTRACT FROM AUTHOR]

Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was inves- tigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3'-terminal mismatch has a consid- erable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples. [ABSTRACT FROM AUTHOR]