Search

Biogas from agricultural waste streams represents an important way to produce fossil-free energy, allow nutrient recycling and reduce greenhouse gas emissions. However, biogas production from agricultural substrates is currently far from reaching its full potential. In Sweden, the number of biogas plants and their output have increased in recent years, but they are still experiencing harsh economic conditions. A recent evaluation (2010–2015) of 31 farm-scale biogas production facilities in Sweden sought to identify parameters of importance for further positive development. In this paper, data on plant operation, gas yield and digestate quality for 27 of these plants are summarised and statistically analysed to investigate factors that could allow an increase in overall biogas production and in nutrient content in the digestate. The analysis showed that addition of co-substrates to manure results in higher gas production, expressed as both specific methane potential and volumetric gas production, than when manure is the sole substrate. Use of co-substrate was also found to be influential for the nutrient content of the digestate. These observed improvements caused by co-digestion should be considered when subsidy systems for manure-based biogas processes are being created, as they could also improve the economics of biogas production. However, to achieve higher efficiency in existing biogas plants and to improve the situation for future investments, a more detailed, long-term evaluation programme should also be considered. [ABSTRACT FROM AUTHOR]

The objective of the study was to evaluate the effect of cashew nut shell extract (CNSE) and glycerol (purity >99%) on enteric methane (CH4) production and microbial communities in an automated gas in vitro system. Microbial communities from the in vitro system were compared with samples from the donor cows, in vivo. Inoculated rumen fluid was mixed with a diet with a 60:40 forage:concentrate ratio and, in total, 5 different treatments were set up: 5 mg of CNSE (CNSE-L), 10 mg of CNSE (CNSE-H), 15 mmol of glycerol/L (glycerol-L), and 30 mmol of glycerol/L (glycerol-H), and a control without feed additive. Gas samples were taken at 2, 4, 8, 24, 32, and 48 h of incubation, and the CH4 concentration was measured. Samples of rumen fluid were taken for volatile fatty acid analysis and for microbial sequence analyses after 8, 24, and 48 h of incubation. In vivo rumen samples from the cows were taken 2 h after the morning feeding at 3 consecutive days to compare the in vitro system with in vivo conditions. The gas data and data from microbial sequence analysis (454 sequencing) were analyzed using a mixed model and principal components analysis. These analyses illustrated that CH4 production was reduced with the CNSE treatment, by 8 and 18%, respectively, for the L and H concentration. Glycerol instead increased CH4 production by 8 and 12%, respectively, for the L and H concentration. The inhibition with CNSE could be due to the observed shift in bacterial population, possibly resulting in decreased production of hydrogen or formate, the methanogenic substrates. Alternatively the response could be explained by a shift in the methanogenic community. In the glycerol treatments, no main differences in bacterial or archaeal population were detected compared with the in vivo control. Thus, the increase in CH4 production may be explained by the increase in substrate in the in vitro system. The reduced CH4 production in vitro with CNSE suggests that CNSE can be a promising inhibitor of CH4 formation in the rumen of dairy cows. [ABSTRACT FROM AUTHOR]

The effects of recirculating the liquid fraction of the digestate during mesophilic anaerobic co-digestion of steam-exploded Salix and cow manure were investigated in laboratory-scale continuously stirred tank reactors. An average organic loading rate of 2.6 g VS L−1 d−1 and a hydraulic retention time (HRT) of 30 days were employed. Co-digestion of Salix and manure gave better methane yields than digestion of manure alone. Also, a 16% increase in the methane yield was achieved when digestate was recirculated and used instead of water to dilute the feedstock (1:1 dilution ratio). The reactor in which the larger fraction of digestate was recirculated (1:3 dilution ratio) gave the highest methane yields. Ammonia and volatile fatty acids did not reach inhibitory levels, and some potentially inhibitory compounds released during steam explosion (i.e., furfural and 5-hydroxy methyl furfural) were only detected at trace levels throughout the entire study period. However, accumulation of solids, which was more pronounced in the recycling reactors, led to decreased methane yields in those systems after three HRTs. Refraining from the use of fresh water to dilute biomass with a high-solids content and obtaining a final digestate with increased dry matter content might offer important economic benefits in full-scale processes. To ensure long-term stability in such an approach, it would be necessary to optimize separation of the fraction of digestate to be recirculated and also perform proper monitoring to avoid accumulation of solids. [Copyright &y& Elsevier]

Highlights: [•] 10 out of 13 randomly selected biogas plants showed syntrophic acetate oxidation (SAO). [•] High SAO bacteria (SAOB) abundance despite differences in operation parameters. [•] Stable levels of SAOB under variable operating conditions. [•] SAOB correlated positively with low abundance of aceticlastic methanogens. [•] The thermophilic SAOB T. phaeum correlated with high ammonia levels. [Copyright &y& Elsevier]

Highlights: [•] Co-digestion of steam exploded or non-pretreated wheat straw with cattle manure. [•] Impact of operational temperatures on methane production. [•] No improved biogas yield were obtained when co-digesting wheat straw with manure. [•] Steam exploded straw did not result in higher methane yield. [•] Higher process temperature did not result in higher methane yields. [ABSTRACT FROM AUTHOR]

During anaerobic digestion of organic waste, both energy-rich biogas and a nutrient-rich digestate are produced. The digestate can be used as a fertiliser in agricultural soils if the levels of hazardous compounds and pathogens are low. This article reviews the main findings about phenols in anaerobic digestion processes degrading organic solid wastes, and examines the effect of process temperature on the anaerobic degradation of phenols, the microbial community and the quality of the digestate. The degradation efficiency of a number of different phenols has been shown to be correlated to the process temperature. Higher degradation efficiency is observed at mesophilic process temperature than at thermophilic temperature. Possible explanations for this variation in the degradation of phenols include differences in diversity, particularly of the phenol-degrading bacteria, and/or the presence of temperature-sensitive enzymes. Chemical analysis of digestate from bioreactors operating at thermophilic temperature detected a higher content of phenols compared to mesophilic bioreactors, verifying the degradation results. Digestate with the highest phenol content has the greatest negative impact on soil microbial activity. [Copyright &y& Elsevier]

Stricter nutrient discharge limits at wastewater treatment plants (WWTPs) are increasing the demand for external carbon sources for denitrification, especially at cold temperatures. Production of carbon sources at WWTP by fermentation of sewage sludge often results in low yields of soluble carbon and volatile fatty acids (VFA) and high biogas losses, limiting its feasibility for full-scale application. This study investigated the overall impact of thermal hydrolysis pre-treatment (THP) on the production of VFA for denitrification through the fermentation of municipal sludge and digestate. Fermentation products and yields, denitrification efficiency and potential impacts on methane yield in the downstream process after carbon source separation were evaluated. Fermentation of THP substrates resulted in 37-70 % higher soluble chemical oxygen demand (sCOD) concentrations than fermentation of untreated substrates but did not significantly affect VFA yield after fermentation. Nevertheless, THP had a positive impact on the denitrification rates and on the methane yields of the residual solid fraction in all experiments. Among the different carbon sources tested, the one produced from the fermentation of THP-digestate showed an overall better potential as a carbon source than other substrates (e.g. sludge). It obtained a relatively high carbon solubilisation degree (39 %) and higher concentrations of sCOD (19 g sCOD/L) and VFA (9.8 g VFA COD /L), which resulted in a higher denitrification rate (8.77 mg NOx-N/g VSS∙h). After the separation of the carbon source, the solid phase from this sample produced a methane yield of 101 mL CH 4 /g VS. Furthermore, fermentation of a 50:50 mixture of THP-substrate and raw sludge produced also resulted in a high VFA yield (283 g VFA COD /kg VS in ) and denitrification rate of 8.74 mg NOx-N/g VSS∙h, indicating a potential for reduced treatment volumes. Calculations based on a full-scale WWTP (Käppala, Stockholm) demonstrated that the carbon sources produced could replace fossil-based methanol and meet the nitrogen effluent limit (6 mg/L) despite their ammonium content. Fermentation of 50-63 % of the available sludge at Käppala WWTP in 2028 could produce enough carbon source to replace methanol, with only an 8-20 % reduction in methane production, depending on the production process. Additionally, digestate production would be sufficient to generate 81 % of the required carbon source while also increasing methane production by 5 % if a portion of the solid residues were recirculated to the digester., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

High ammonia concentrations in anaerobic degradation systems cause volatile fatty acid accumulation and reduced methane yield, which often derive from restricted activity of syntrophic acid-oxidising bacteria and hydrogenotrophic methanogens. Inclusion of additives that facilitate the electron transfer or increase cell proximity of syntrophic species by flocculation can be a suitable strategy to counteract these problems, but its actual impact on syntrophic interactions has yet to be determined. In this study, microbial cultivation and molecular and microscopic analysis were performed to evaluate the impact of conductive (graphene, iron oxide) and non-conductive (zeolite) additives on the degradation rate of acetate and propionate to methane by highly enriched ammonia-tolerant syntrophic cultures derived from a biogas process. All additives had a low impact on the lag phase but resulted in a higher rate of acetate (except graphene) and propionate degradation. The syntrophic bacteria 'Candidatus Syntrophopropionicum ammoniitolerans', Syntrophaceticus schinkii and a novel hydrogenotrophic methanogen were found in higher relative abundance and higher gene copy numbers in flocculating communities than in planktonic communities in the cultures, indicating benefits to syntrophs of living in close proximity to their cooperating partner. Microscopy and element analysis showed precipitation of phosphates and biofilm formation in all batches except on the graphene batches, possibly enhancing the rate of acetate and propionate degradation. Overall, the concordance of responses observed in both acetate- and propionate-fed cultures highlight the suitability of the addition of iron oxide or zeolites to enhance acid conversion to methane in high-ammonia biogas processes. KEY POINTS: • All additives promoted acetate (except graphene) and propionate degradation. • A preference for floc formation by ammonia-tolerant syntrophs was revealed. • Microbes colonised the surfaces of iron oxide and zeolite, but not graphene., (© 2024. The Author(s).)

A complete genome was recovered from Citroniella saccharovorans, strain DSM 29873, using Oxford Nanopore Technologies. The genome assembly contains 1,413,868 bp with 30.23% G+C content. The species belongs to the family Peptoniphilaceae and, as of yet, is the only cultivated representative of the genus Citroniella ., Competing Interests: The authors declare no conflict of interest.

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name 'Candidatus Thermosyntrophopropionicum ammoniitolerans' was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H 2 and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L -1 day -1 at hydrogen partial pressure 4-30 Pa and available energy was around -20 mol -1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems., (© 2023. The Author(s).)

Rice cultivation on paddy soil is commonly associated with emissions of methane, a greenhouse gas, but rice varieties may differ in their actual level of emissions. This study analysed methane emissions associated with 22 distinct rice genotypes, using gas chromatography, and identified the cultivar Heijing 5 from northern China as a potential low-methane rice variety. To confirm this and to examine whether Heijing 5 can perform similarly at higher latitudes, Heijing 5 was cultivated in field trials in China (lat. 32° N) and Sweden (lat. 59° N) where (i) methane emissions were measured, (ii) methanogen abundance in the rhizosphere was determined using quantitative PCR, and (iii) the concentrations of nutrients in water and of heavy metals in rice grain and paddy soil were analysed. The results demonstrated that the low-methane rice cultivar Heijing 5 can successfully complete an entire growth period at high-latitude locations such as central Sweden. Massively parallel sequencing of mRNAs identified candidate genes involved in day length and cold acclimatisation. Cultivation of Heijing 5 in central Sweden was also associated with relatively low heavy metal accumulation in rice grains and lowered nutrient losses to neighbouring water bodies., (© 2023. The Author(s).)

Antimicrobial resistance (AMR) is becoming an increasing global concern and the anaerobic digestion (AD) process represents a potential transmission route when digestates are used as fertilizing agents. AMR contaminants, e.g. antibiotic-resistant bacteria (ARB) and plasmid-mediated antibiotic resistance genes (ARGs) have been found in different substrates and AD systems, but not yet been investigated in plant-based substrates. AMR transfer from soils to vegetable microbiomes has been observed, and thus crop material potentially represents a so far neglected AMR load in agricultural AD processes, contributing to AMR spread. In order to test this hypothesis, this study examined the AMR situation throughout the process of three biogas plants using plant-based substrates only, or a mixture of plant-based and manure substrates. The evaluation included a combination of culture-independent and -dependent methods, i.e., identification of ARGs, plasmids, and pathogenic bacteria by DNA arrays, and phylogenetic classification of bacterial isolates and their phenotypic resistance pattern. To our knowledge, this is the first study on AMR in plant-based substrates and the corresponding biogas plant. The results showed that the bacterial community isolated from the investigated substrates and the AD processing facilities were mainly Gram-positive Bacillus spp. Apart from Pantoea agglomerans, no other Gram-negative species were found, either by bacteria culturing or by DNA typing array. In contrast, the presence of ARGs and plasmids clearly indicated the existence of Gram-negative pathogenic bacteria, in both substrate and AD process. Compared with substrates, digestates had lower levels of ARGs, plasmids, and culturable ARB. Thus, digestate could pose a lower risk of spreading AMR than substrates per se. In conclusion, plant-based substrates are associated with AMR, including culturable Gram-positive ARB and Gram-negative pathogenic bacteria-associated ARGs and plasmids. Thus, the AMR load from plant-based substrates should be taken into consideration in agricultural biogas processing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

AcetoBase is a public repository and database of formyltetrahydrofolate synthetase (FTHFS) sequences. It is the first systematic collection of bacterial FTHFS nucleotide and protein sequences from genomes and metagenome-assembled genomes and of sequences generated by clone library sequencing. At its publication in 2019, AcetoBase (Version 1) was also the first database to establish connections between the FTHFS gene, the Wood-Ljungdahl pathway and 16S ribosomal RNA genes. Since the publication of AcetoBase, there have been significant improvements in the taxonomy of many bacterial lineages and accessibility/availability of public genomics and metagenomics data. The update to the AcetoBase reference database described here (Version 2) provides new sequence data and taxonomy, along with improvements in web functionality and user interface. The evaluation of this latest update by re-analysis of publicly accessible FTHFS amplicon sequencing data previously analysed with AcetoBase Version 1 revealed significant improvements in the taxonomic assignment of FTHFS sequences. Database URL: https://acetobase.molbio.slu.se., (© The Author(s) 2022. Published by Oxford University Press.)

An anaerobic bacterial strain, designated AMB_01 T , recovered from mesophilic propionate enrichment of a high-ammonia biogas digester, was characterised using phenotypic and molecular taxonomic methods. Cells of AMB_01 T are coccus-shaped and often occur arranged as diplococci or sarcina. Growth occurred at 20-45 °C, initial pH 5.5-8.5 and with up to 0.7 M NH 4 Cl, with optimum growth at 37-42 °C and pH 8.0. AMB_01 T achieved high cell density and highest acetate production when grown on carbohydrates, including monomers, disaccharides and polysaccharides, such as glucose, maltose, cellobiose and starch. The strain was also able to use amino acids and some organic acids and alcoholic compounds for growth. Acetate was formed as the main product and yeast was not required for growth. The major cellular fatty acids were summed feature 4 (iso-C 17 : 1 I and/or anteiso-C 17 : 1 B), C 18 : 1 ω7, C 14 : 0 , C 16 : 0 and summed feature 3 (C 16 : 1 ω7 and/or iso-C 15 : 0 2OH). The highest 16S rRNA gene sequence similarity found was with Miniphocaeibacter massiliensis (96.6 %), within the family Peptoniphilaceae , phylum Bacillota (Firmicutes). The genomic DNA G+C content was 29.0 mol%. An almost complete set of genes for the acetyl-CoA pathway was found. Genome comparisons between AMB_01 T and close relatives showed highest digital DNA-DNA hybridisation to Finegoldia magna (23 %), highest average nucleotide identity with genome nucleotide and amino acid sequences to M. massiliensis (72 and 73 %, respectively) and highest average nucleotide identity (87 %) with Schnuerera ultunensis , indicating that AMB_01 T represents a novel species. Analysis of genomic, chemotaxonomic, biochemical and physiological data confirmed that strain AMB_01 T represents a novel species, for which the name Miniphocaeibacter halophilus sp. nov. is proposed. The type strain is AMB_01 T (=DSM 110247 T =JCM 39107 T ).

Acetogens play a very important role in anaerobic digestion and are essential in ensuring process stability. Despite this, targeted studies of the acetogenic community in biogas processes remain limited. Some efforts have been made to identify and understand this community, but the lack of a reliable molecular analysis strategy makes the detection of acetogenic bacteria tedious. Recent studies suggest that screening of bacterial genetic material for formyltetrahydrofolate synthetase (FTHFS), a key marker enzyme in the Wood-Ljungdahl pathway, can give a strong indication of the presence of putative acetogens in biogas environments. In this study, we applied an acetogen-targeted analyses strategy developed previously by our research group for microbiological surveillance of commercial biogas plants. The surveillance comprised high-throughput sequencing of FTHFS gene amplicons and unsupervised data analysis with the AcetoScan pipeline. The results showed differences in the acetogenic community structure related to feed substrate and operating parameters. They also indicated that our surveillance method can be helpful in the detection of community changes before observed changes in physico-chemical profiles, and that frequent high-throughput surveillance can assist in management towards stable process operation, thus improving the economic viability of biogas plants. To our knowledge, this is the first study to apply a high-throughput microbiological surveillance approach to visualise the potential acetogenic population in commercial biogas digesters., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Singh, Moestedt, Berg and Schnürer.)

Biogas, containing energy-rich methane, is produced by microbial decomposition of organic material under anaerobic conditions. Under controlled conditions, this process can be used for the production of energy and a nutrient-rich residue suitable for use as a fertilising agent. The biogas can be used for production of heat, electricity or vehicle fuel. Different substrates can be used in the process and, depending on substrate character, various reactor technologies are available. The microbiological process leading to methane production is complex and involves many different types of microorganisms, often operating in close relationships because of the limited amount of energy available for growth. The microbial community structure is shaped by the incoming material, but also by operating parameters such as process temperature. Factors leading to an imbalance in the microbial community can result in process instability or even complete process failure. To ensure stable operation, different key parameters, such as levels of degradation intermediates and gas quality, are often monitored. Despite the fact that the anaerobic digestion process has long been used for industrial production of biogas, many questions need still to be resolved to achieve optimal management and gas yields and to exploit the great energy and nutrient potential available in waste material. This chapter discusses the different aspects that need to be taken into consideration to achieve optimal degradation and gas production, with particular focus on operation management and microbiology.

Syntrophic acetate-oxidizing bacteria have been identified as key organisms for efficient biogas production from protein-rich materials. They normally grow as lithotrophs or heterotrophs, producing acetate through the Wood-Ljungdahl pathway, but when growing in syntrophy with methanogens, they reportedly reverse this pathway and oxidize acetate to hydrogen and carbon dioxide. However, the biochemical and regulatory mechanisms behind the shift and the way in which the bacteria regain energy remain unknown. In a genome-walking approach, starting with degenerated primers, we identified those gene clusters in Syntrophaceticus schinkii, Clostridium ultunense, and Tepidanaerobacter acetatoxydans that comprise the formyltetrahydrofolate synthetase gene (fhs), encoding a key enzyme of the Wood-Ljungdahl pathway. We also discovered that the latter two harbor two fhs alleles. The fhs genes are phylogenetically separated and in the case of S. schinkii functionally linked to sulfate reducers. The T. acetatoxydans fhs1 cluster combines features of acetogens, sulfate reducers, and carbon monoxide oxidizers and is organized as a putative operon. The T. acetatoxydans fhs2 cluster encodes Wood-Ljungdahl pathway enzymes, which are also known to be involved in C1 carbon metabolism. Isolation of the enzymes illustrated that both formyltetrahydrofolate synthetases of T. acetatoxydans were functionally active. However, only fhs1 was expressed, confirming bidirectional usage of the pathway., (© 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)