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

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

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

Serial anaerobic digestion improves protein degradation and biogas production from mixed food waste

Optimization of the biogas generation process is important to achieve efficient degradation and high methane yield, and to reduce methane emissions from the digestate. In this study, serial digester systems with two or three biogas reactors were compared with a single reactor, with the aim of improving degree of degradation and methane yield from food waste and assessing adaptation of microbial communities to different reactor steps. All systems had the same total organic load (2.4 g VS/(L d)) and hydraulic retention time (55 days). Serial systems increased methane yield by >5% compared with the single reactor, with the majority of the methane being obtained from the first-step reactors. Improved protein degradation was also obtained in serial systems, with >20% lower outgoing protein concentration compared with the single reactor and increasing NH4+-N concentration with every reactor step. This resulted in separation of high ammonia (>384 mg NH3-N/L) levels from the main methane production, reducing the risk of methanogen inhibition. Methanosarcina dominated the methanogenic community in all reactors, but increases in the hydrogenotrophic genera Methanoculleus and Methanobacterium were observed at higher ammonia levels. Potential syntrophic acetate-oxidizing bacteria, such as MBA03 and Dethiobacteraceae, followed the same trend as the hydrogenotrophic methanogens. Phylum Bacteroidota family Paludibacteraceae was highly abundant in the first steps and then decreased abruptly, potentially linked to an observed decrease in degradation in the last-step reactors. Nevertheless, the results indicated a trend of increasing relative abundance of the potentially proteolytic genera Proteiniphilum and Fastidiosipila with successive reactor steps

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

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

Acetate and Lactate Production During Two-Stage Anaerobic Digestion of Food Waste Driven by Lactobacillus and Aeriscardovia

Background: In a previous study, single-stage processes were compared with two-stage processes, using either food waste alone or mixed with thin stillage as substrate. Overall methane yield increased (by 12%) in two-stage compared with single-stage digestion when using food waste, but decreased when food waste was co-digested with thin stillage (50:50 on VS basis). The obtained difference in methane yield was likely caused by a higher acetate level in the first stage reactor operating with food waste alone (around 20 g/L) compared to the reactor also treating thin stillage (around 8 g /L). The present study sought to shed additional light on possible causes of the large difference in methane yield by scrutinizing the microbial community in the first- and second-stage reactors, using a combined Illumina sequencing and qPCR approach. Results: In the first-stage process, acid-tolerant Aeriscardovia and Lactobacillus formed a highly efficient consortium. For food waste with high levels of acetate (20 g/L, equal to 0.14 g acetate/g VS) was produced but when thin stillage was added the pH was lower (<4), resulting in lactate production exceeding acetate production. This difference in hydrolysate composition between the reactors resulted in development of slightly different communities in the second-stage, for both hydrolysis, fermentation, and acetogenesis. High acetate concentration appeared to promote proliferation of different syntrophic consortia, such as various syntrophic acetate oxidizers, members of the genus Syntrophomonas and candidate phylum Cloacimonetes, likely explaining the higher methane yields with two-step compared with single-stage digestion of food waste.Conclusion: Using food waste as sole substrate resulted in enrichment of Lactobacillus and Aeriscardovia and high acetate yields in the first-stage reactor. This was beneficial for biogas yield in two-stage digestion, where efficient acid-degrading syntrophic consortia developed. Addition of thin stillage contributed to low pH and higher lactate production, which resulted in decreased methane yield in the two-stage process compared with using food waste as sole substrate

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

Anaerobic Digestion of Animal Manure and Influence of Organic Loading Rate and Temperature on Process Performance, Microbiology, and Methane Emission From Digestates

Biogas production from manure is of particular value in regard of lowering greenhouse gas emissions and enhancing nutrient re-circulation. However, the relatively low energy content and the characteristics of manure often result in low degradation efficiency, and the development of operating strategies is required to improve the biogas yield and the economic benefits. In this study, the potential to enhance the performance of two full-scale biogas plants operating with cattle manure, in mono-digestion or combined with poultry manure, was investigated. Four continuously fed laboratory-scale reactors were operated in sets of two, in which the temperature in one reactor in each set was increased from 37-42 degrees C to 52 degrees C. The potential to increase the capacity was thereafter assessed by increasing the organic loading rate (OLR), from ca 3 to 5 kg volatile solids (VS)/ m(3) and day. The processes were evaluated with both chemical and microbiological parameters, and in addition, the residual methane potential (RMP) was measured to evaluate the risk of increased methane emissions from the digestate. The results showed that both processes could be changed from mesophilic to themophilic temperature without major problems and with a similar shift in the microbial community profile to a typical thermophilic community, e.g., an increase in the relative abundance of the phylum Firmicutes. However, the temperature increase in the reactor co-digesting cattle and poultry manure caused a slight accumulation of fatty acids (2 g/l) and reduced the specific methane production, most likely due to ammonia inhibition (0.4-0.7 g NH3/l). Still, during operation at higher OLR, thermophilic as compared to mesophilic temperature slightly increased the methane yield and specific methane production, in both investigated processes. However, the higher OLR decreased the overall degree of degradation in all processes, and this showed a positive correlation with increased RMP values. Chemical analyses suggested that high RMP values (40-98 Nml gVS(-1)) were related to the degradation of cellulose, hemicellulose, and volatile fatty acid enriched in the digestate. Conclusively, increased temperature and load can increase the methane yield from manure but can result in less efficient degradation and increased risks for methane emissions during storage and handling of the digestate

Draft Genome Sequence of the Cellulolytic Strain Clostridium sp. Bc-iso-3 Isolated from an Industrial-Scale Anaerobic Digester

Clostridium sp. Bc-iso-3 is a cellulolytic strain isolated from a Swedish industrial-scale biogas digester. Here, we present the draft genome sequence of this strain, which consists of four contigs with a total length of 4,327,139 bp and an average coverage of 312.97