Complete genome sequence of Citroniella saccharovorans DSM 29873, isolated from human fecal sample.

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.

A low-methane rice with high-yield potential realized via optimized carbon partitioning.

Global rice cultivation significantly contributes to anthropogenic methane emissions. The methane emissions are caused by methane-producing microorganisms (methanogenic archaea) that are favoured by the anoxic conditions of paddy soils and small carbon molecules released from rice roots. However, different rice cultivars are associated with differences in methane emission rates suggesting that there is a considerable natural variation in this trait. Starting from the hypothesis that sugar allocation within a plant is an important factor influencing both yields and methane emissions, the aim of this study was to produce high-yielding rice lines associated with low methane emissions. In this study, the offspring (here termed progeny lines) of crosses between a newly characterized low-methane rice variety, Heijing 5, and three high-yielding elite varieties, Xiushui, Huayu and Jiahua, were selected for combined low-methane and high-yield properties. Analyses of total organic carbon and carbohydrates showed that the progeny lines stored more carbon in above-ground tissues than the maternal elite varieties. Also, metabolomic analysis of rhizospheric soil surrounding the progeny lines showed reduced levels of glucose and other carbohydrates. The carbon allocation, from roots to shoots, was further supported by a transcriptome analysis using massively parallel sequencing of mRNAs that demonstrated elevated expression of the sugar transporters SUT-C and SWEET in the progeny lines as compared to the parental varieties. Furthermore, measurement of methane emissions from plants, grown in greenhouse as well as outdoor rice paddies, showed a reduction in methane emissions by approximately 70 % in the progeny lines compared to the maternal elite varieties. Taken together, we report here on three independent low-methane-emission rice lines with high yield potential. We also provide a first molecular characterisation of the progeny lines that can serve as a foundation for further studies of candidate genes involved in sugar allocation and reduced methane emissions from rice cultivation.

Characterisation of meropenem-resistant Bacillus sp. FW 1 isolated from biogas digestate.

Recently a Bacillus sp. strain FW 1 was isolated from biogas digestate and shown to have novel resistance to meropenem (MEM), of critical importance in human medicine. MEM-resistance has so far only been described for one species within the genus Bacillus, that is, Bacillus cereus. Bacillus is an abundant representative of the microbial community in biogas digesters and consequently, the finding indicates a risk of spreading such resistance when using the digestate as fertiliser. In this study, the Bacillus strain was characterised and classified as Heyndrickxia oleronia (previous Bacillus oleronius), previously not described to harbour MEM-resistance. The mechanism of resistance was explored by metallo-ß-lactamase (MBL) production, mapping of carbapenemase genes and genome analysis. The transferability of MEM-resistance in strain FW 1 was investigated by plasmid transformation/conjugation, combined with genome analysis. The results confirmed MBL production for both strain FW 1 and the type strain H. oleronia DSM 9356T . However, elevated MEM resistance was found for strain FW 1, which was suggested to be caused by the production of unclassified carbapenemase, or overexpression of MBL. Moreover, the results suggest that the MEM-resistance of strain FW 1 is not transferable, thus representing a limited risk of MEM-resistance spread to the environment when using digestate on arable land.

Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions.

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 H2 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.

Sulfide in engineered methanogenic systems - Friend or foe?

Sulfide ions are regarded to be toxic to microorganisms in engineered methanogenic systems (EMS), where organic substances are anaerobically converted to products such as methane, hydrogen, alcohols, and carboxylic acids. A vast body of research has addressed solutions to mitigate process disturbances associated with high sulfide levels, yet the established paradigm has drawn the attention away from the multifaceted sulfide interactions with minerals, organics, microbial interfaces and their implications for performance of EMS. This brief review brings forward sulfide-derived pathways other than toxicity and with potential significance for anaerobic organic matter degradation. Available evidence on sulfide reactions with organic matter, interventions with key microbial metabolisms, and interspecies electron transfer are critically synthesized as a guidance for comprehending the sulfide effects on EMS apart from the microbial toxicity. The outcomes identify existing knowledge gaps and specify future research needs as a step forward towards realizing the potential of sulfide-derived mechanisms in diversifying and optimizing EMS applications.

Characterisation of a low methane emission rice cultivar suitable for cultivation in high latitude light and temperature conditions.

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.

Improved bioenergy value of residual rice straw by increased lipid levels from upregulation of fatty acid biosynthesis.

BACKGROUND: Rice (Oryza sativa) straw is a common waste product that represents a considerable amount of bound energy. This energy can be used for biogas production, but the rate and level of methane produced from rice straw is still low. To investigate the potential for an increased biogas production from rice straw, we have here utilized WRINKLED1 (WRI1), a plant AP2/ERF transcription factor, to increase triacylglycerol (TAG) biosynthesis in rice plants. Two forms of Arabidopsis thaliana WRI1 were evaluated by transient expression and stable transformation of rice plants, and transgenic plants were analyzed both for TAG levels and biogas production from straw. RESULTS: Both full-length AtWRI1, and a truncated form lacking the initial 141 amino acids (including the N-terminal AP2 domain), increased fatty acid and TAG levels in vegetative and reproductive tissues of Indica rice. The stimulatory effect of the truncated AtWRI1 was significantly lower than that of the full-length protein, suggesting a role for the deleted AP2 domain in WRI1 activity. Full-length AtWRI1 increased TAG levels also in Japonica rice, indicating a conserved effect of WRI1 in rice lipid biosynthesis. The bio-methane production from rice straw was 20% higher in transformants than in the wild type. Moreover, a higher producing rate and final yield of methane was obtained for rice straw compared with rice husks, suggesting positive links between methane production and a high amount of fatty acids. CONCLUSIONS: Our results suggest that heterologous WRI1 expression in transgenic plants can be used to improve the metabolic potential for bioenergy purposes, in particular methane production.

Co-substrate composition is critical for enrichment of functional key species and for process efficiency during biogas production from cattle manure.

Cattle manure has a low energy content and high fibre and water content, limiting its value for biogas production. Co-digestion with a more energy-dense material can improve the output, but the co-substrate composition that gives the best results in terms of degree of degradation, gas production and digestate quality has not yet been identified. This study examined the effects of carbohydrate, protein and fat as co-substrates for biogas production from cattle manure. Laboratory-scale semi-continuous mesophilic reactors were operated with manure in mono-digestion or in co-digestion with egg albumin, rapeseed oil, potato starch or a mixture of these, and chemical and microbiological parameters were analysed. The results showed increased gas yield for all co-digestion reactors, but only the reactor supplemented with rapeseed oil showed synergistic effects on methane yield. The reactor receiving potato starch indicated improved fibre degradation, suggesting a priming effect by the easily accessible carbon. Both these reactors showed increased species richness and enrichment of key microbial species, such as fat-degrading Syntrophomonadaceae and families known to include cellulolytic bacteria. The addition of albumin promoted enrichment of known ammonia-tolerant syntrophic acetate- and potential propionate-degrading bacteria, but still caused slight process inhibition and less efficient overall degradation of organic matter in general, and of cellulose in particular.

Current trends in waste valorization.

This paper presents the scientific breakthroughs made in bioprocess engineering and microbial biotechnology for the conversion of wastes into products with added value and/or biofuels. The significant results obtained in the emerging fields of hybrid electrosynthesis, the role of enzymes in the degradation of plastics, polyhydroxyalkanoate and 5-aminolevulinic acid production, fermentation technology and the application of molecular engineering tools to bioprocess technology are highlighted.

Absence of oxygen effect on microbial structure and methane production during drying and rewetting events.

Natural environments with frequent drainage experience drying and rewetting events that impose fluctuations in water availability and oxygen exposure. These relatively dramatic cycles profoundly impact microbial activity in the environment and subsequent emissions of methane and carbon dioxide. In this study, we mimicked drying and rewetting events by submitting methanogenic communities from strictly anaerobic environments (anaerobic digestors) with different phylogenetic structures to consecutive desiccation events under aerobic (air) and anaerobic (nitrogen) conditions followed by rewetting. We showed that methane production quickly recovered after each rewetting, and surprisingly, no significant difference was observed between the effects of the aerobic or anaerobic desiccation events. There was a slight change in the microbial community structure and a decrease in methane production rates after consecutive drying and rewetting, which can be attributed to a depletion of the pool of available organic matter or the inhibition of the methanogenic communities. These observations indicate that in comparison to the drying and rewetting events or oxygen exposure, the initial phylogenetic structure and the organic matter quantity and quality exhibited a stronger influence on the methanogenic communities and overall microbial community responses. These results change the current paradigm of the sensitivity of strict anaerobic microorganisms to oxygen exposure.

Microbial community development during syngas methanation in a trickle bed reactor with various nutrient sources.

Microbial community development within an anaerobic trickle bed reactor (TBR) during methanation of syngas (56% H2, 30% CO, 14% CO2) was investigated using three different nutrient media: defined nutrient medium (241 days), diluted digestate from a thermophilic co-digestion plant operating with food waste (200 days) and reject water from dewatered digested sewage sludge at a wastewater treatment plant (220 days). Different TBR operating periods showed slightly different performance that was not clearly linked to the nutrient medium, as all proved suitable for the methanation process. During operation, maximum syngas load was 5.33 L per L packed bed volume (pbv) & day and methane (CH4) production was 1.26 L CH4/Lpbv/d. Microbial community analysis with Illumina Miseq targeting 16S rDNA revealed high relative abundance (20-40%) of several potential syngas and acetate consumers within the genera Sporomusa, Spirochaetaceae, Rikenellaceae and Acetobacterium during the process. These were the dominant taxa except in a period with high flow rate of digestate from the food waste plant. The dominant methanogen in all periods was a member of the genus Methanobacterium, while Methanosarcina was also observed in the carrier community. As in reactor effluent, the dominant bacterial genus in the carrier was Sporomusa. These results show that syngas methanation in TBR can proceed well with different nutrient sources, including undefined medium of different origins. Moreover, the dominant syngas community remained the same over time even when non-sterilised digestates were used as nutrient medium. KEY POINTS: • Independent of nutrient source, syngas methanation above 1 L/Lpbv/D was achieved. • Methanobacterium and Sporomusa were dominant genera throughout the process. • Acetate conversion proceeded via both methanogenesis and syntrophic acetate oxidation.

AcetoBase Version 2: a database update and re-analysis of formyltetrahydrofolate synthetase amplicon sequencing data from anaerobic digesters.

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.

Identifying targets for increased biogas production through chemical and organic matter characterization of digestate from full-scale biogas plants: what remains and why?

BACKGROUND: This study examines the destiny of macromolecules in different full-scale biogas processes. From previous studies it is clear that the residual organic matter in outgoing digestates can have significant biogas potential, but the factors dictating the size and composition of this residual fraction and how they correlate with the residual methane potential (RMP) are not fully understood. The aim of this study was to generate additional knowledge of the composition of residual digestate fractions and to understand how they correlate with various operational and chemical parameters. The organic composition of both the substrates and digestates from nine biogas plants operating on food waste, sewage sludge, or agricultural waste was characterized and the residual organic fractions were linked to substrate type, trace metal content, ammonia concentration, operational parameters, RMP, and enzyme activity. RESULTS: Carbohydrates represented the largest fraction of the total VS (32-68%) in most substrates. However, in the digestates protein was instead the most abundant residual macromolecule in almost all plants (3-21 g/kg). The degradation efficiency of proteins generally lower (28-79%) compared to carbohydrates (67-94%) and fats (86-91%). High residual protein content was coupled to recalcitrant protein fractions and microbial biomass, either from the substrate or formed in the degradation process. Co-digesting sewage sludge with fat increased the protein degradation efficiency with 18%, possibly through a priming mechanism where addition of easily degradable substrates also triggers the degradation of more complex fractions. In this study, high residual methane production (> 140 L CH4/kg VS) was firstly coupled to operation at unstable process conditions caused mainly by ammonia inhibition (0.74 mg NH3-N/kg) and/or trace element deficiency and, secondly, to short hydraulic retention time (HRT) (55 days) relative to the slow digestion of agricultural waste and manure. CONCLUSIONS: Operation at unstable conditions was one reason for the high residual macromolecule content and high RMP. The outgoing protein content was relatively high in all digesters and improving the degradation of proteins represents one important way to increase the VS reduction and methane production in biogas plants. Post-treatment or post-digestion of digestates, targeting microbial biomass or recalcitrant protein fractions, is a potential way to achieve increased protein degradation.

Uncovering antimicrobial resistance in three agricultural biogas plants using plant-based substrates.

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.

Miniphocaeibacter halophilus sp. nov., an ammonium-tolerant acetate-producing bacterium isolated from a biogas system.

An anaerobic bacterial strain, designated AMB_01T, recovered from mesophilic propionate enrichment of a high-ammonia biogas digester, was characterised using phenotypic and molecular taxonomic methods. Cells of AMB_01T 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 NH4Cl, with optimum growth at 37-42 °C and pH 8.0. AMB_01T 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-C17 : 1I and/or anteiso-C17 : 1B), C18 : 1ω7, C14 : 0, C16 : 0 and summed feature 3 (C16 : 1ω7 and/or iso-C15 : 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_01T 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_01T represents a novel species. Analysis of genomic, chemotaxonomic, biochemical and physiological data confirmed that strain AMB_01T represents a novel species, for which the name Miniphocaeibacter halophilus sp. nov. is proposed. The type strain is AMB_01T (=DSM 110247T=JCM 39107 T).

Influence of cysteine, serine, sulfate, and sulfide on anaerobic conversion of unsaturated long-chain fatty acid, oleate, to methane.

This study aims to elucidate the role of sulfide and its precursors in anaerobic digestion (i.e., cysteine, representing sulfur-containing amino acids, and sulfate) on microbial oleate conversion to methane. Serine, with a similar structure to cysteine but with a hydroxyl group instead of a thiol, was included as a control to assess potential effects on methane formation that were not related to sulfur functionalities. The results showed that copresence of sulfide and oleate in anaerobic batch assays accelerated the methane formation compared to assays with only oleate and mitigated negative effect on methane formation caused by increased sulfide level. Nuclear magnetic resonance spectroscopy of sulfide-exposed oleate suggested that sulfide reaction with oleate double bonds likely contributed to negation of the negative effect on the methanogenic activity. Methane formation from oleate was also accelerated in the presence of cysteine or serine, while sulfate decreased the cumulative methane formation from oleate. Neither cysteine nor serine was converted to methane, and their accelerating effects was associated to different mechanisms due to establishment of microbial communities with different structures, as evidenced by high-throughput sequencing of 16S rRNA gene. These outcomes contribute with new knowledge to develop strategies for optimum use of sulfur- and lipid-rich wastes in anaerobic digestion processes.

Microbiological Surveillance of Biogas Plants: Targeting Acetogenic Community.

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.

Profiling temporal dynamics of acetogenic communities in anaerobic digesters using next-generation sequencing and T-RFLP.

Acetogens play a key role in anaerobic degradation of organic material and in maintaining biogas process efficiency. Profiling this community and its temporal changes can help evaluate process stability and function, especially under disturbance/stress conditions, and avoid complete process failure. The formyltetrahydrofolate synthetase (FTHFS) gene can be used as a marker for acetogenic community profiling in diverse environments. In this study, we developed a new high-throughput FTHFS gene sequencing method for acetogenic community profiling and compared it with conventional terminal restriction fragment length polymorphism of the FTHFS gene, 16S rRNA gene-based profiling of the whole bacterial community, and indirect analysis via 16S rRNA profiling of the FTHFS gene-harbouring community. Analyses and method comparisons were made using samples from two laboratory-scale biogas processes, one operated under stable control and one exposed to controlled overloading disturbance. Comparative analysis revealed satisfactory detection of the bacterial community and its changes for all methods, but with some differences in resolution and taxonomic identification. FTHFS gene sequencing was found to be the most suitable and reliable method to study acetogenic communities. These results pave the way for community profiling in various biogas processes and in other environments where the dynamics of acetogenic bacteria have not been well studied.

Effluent solids recirculation to municipal sludge digesters enhances long-chain fatty acids degradation capacity.

BACKGROUND: Slow degradation kinetics of long-chain fatty acids (LCFA) and their accumulation in anaerobic digesters disrupt methanogenic activity and biogas production at high loads of waste lipids. In this study, we evaluated the effect of effluent solids recirculation on microbial LCFA (oleate) degradation capacity in continuous stirred-tank sludge digesters, with the overall aim of providing operating conditions for efficient co-digestion of waste lipids. Furthermore, the impacts of LCFA feeding frequency and sulfide on process performance and microbial community dynamics were investigated, as parameters that were previously shown to be influential on LCFA conversion to biogas. RESULTS: Effluent solids recirculation to municipal sludge digesters enabled biogas production of up to 78% of the theoretical potential from 1.0 g oleate l-1 day-1. In digesters without effluent recirculation, comparable conversion efficiency could only be reached at oleate loading rates up to 0.5 g l-1 day-1. Pulse feeding of oleate (supplementation of 2.0 g oleate l-1 every second day instead of 1.0 g oleate l-1 every day) did not have a substantial impact on the degree of oleate conversion to biogas in the digesters that operated with effluent recirculation, while it marginally enhanced oleate conversion to biogas in the digesters without effluent recirculation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea revealed that pulse feeding resulted in prevalence of fatty acid-degrading Smithella when effluent recirculation was applied, whereas Candidatus Cloacimonas prevailed after pulse feeding of oleate in the digesters without effluent recirculation. Combined oleate pulse feeding and elevated sulfide level contributed to increased relative abundance of LCFA-degrading Syntrophomonas and enhanced conversion efficiency of oleate, but only in the digesters without effluent recirculation. CONCLUSIONS: Effluent solids recirculation improves microbial LCFA degradation capacity, providing possibilities for co-digestion of larger amounts of waste lipids with municipal sludge.

Enrichment and description of novel bacteria performing syntrophic propionate oxidation at high ammonia level.

Inefficient syntrophic propionate degradation causes severe operating disturbances and reduces biogas productivity in many high-ammonia anaerobic digesters, but propionate-degrading microorganisms in these systems remain unknown. Here, we identified candidate ammonia-tolerant syntrophic propionate-oxidising bacteria using propionate enrichment at high ammonia levels (0.7-0.8 g NH3 L-1 ) in continuously-fed reactors. We reconstructed 30 high-quality metagenome-assembled genomes (MAGs) from the propionate-fed reactors, which revealed two novel species from the families Peptococcaceae and Desulfobulbaceae as syntrophic propionate-oxidising candidates. Both MAGs possess genomic potential for the propionate oxidation and electron transfer required for syntrophic energy conservation and, similar to ammonia-tolerant acetate degrading syntrophs, both MAGs contain genes predicted to link to ammonia and pH tolerance. Based on relative abundance, a Peptococcaceae sp. appeared to be the main propionate degrader and has been given the provisional name "Candidatus Syntrophopropionicum ammoniitolerans". This bacterium was also found in high-ammonia biogas digesters, using quantitative PCR. Acetate was degraded by syntrophic acetate-oxidising bacteria and the hydrogenotrophic methanogenic community consisted of Methanoculleus bourgensis and a yet to be characterised Methanoculleus sp. This work provides knowledge of cooperating syntrophic species in high-ammonia systems and reveals that ammonia-tolerant syntrophic propionate-degrading populations share common features, but diverge genomically and taxonomically from known species.