Metatranscriptomics-guided genome-scale metabolic reconstruction reveals the carbon flux and trophic interaction in methanogenic communities.

BACKGROUND: Despite rapid advances in genomic-resolved metagenomics and remarkable explosion of metagenome-assembled genomes (MAGs), the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain largely uncertain, which has profound implications in biotechnology and biogeochemistry. RESULTS: In this study, we combined long-read sequencing and metatranscriptomics-guided metabolic reconstruction to provide a genome-wide perspective of carbon mineralization flow from polymers to methane in an anaerobic bioreactor. Our results showed that incorporating long reads resulted in a substantial improvement in the quality of metagenomic assemblies, enabling the effective recovery of 132 high-quality genomes meeting stringent criteria of minimum information about a metagenome-assembled genome (MIMAG). In addition, hybrid assembly obtained 51% more prokaryotic genes in comparison to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction unveiled the remarkable metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acid oxidation through syntrophic relationships with dominant methanogens Methanoregulaceae bin.74 and Methanothrix sp. bin.206. The activity of bin.206 preferring acetate as substrate exceeded that of bin.74 with increasing loading, reinforcing the substrate determinantal role. CONCLUSION: Overall, our study uncovered some key active anaerobic lineages and their metabolic functions in this complex anaerobic ecosystem, offering a framework for understanding carbon transformations in anaerobic digestion. These findings advance the understanding of metabolic activities and trophic interactions between anaerobic guilds, providing foundational insights into carbon flux within both engineered and natural ecosystems. Video Abstract.

New insights into syntrophic ethanol oxidation: Effects of operational modes and solids retention times.

Anaerobic ethanol oxidation relies on syntrophic interactions among functional microorganisms to become thermodynamically feasible. Different operational modes (sequencing batch reactors, SBRs, and continuous flow reactors, CFRs) and solids retention times (SRT, 25 days and 10 days) were employed in four ethanol-fed reactors, named as SBR25d, SBR10d, CFR25d, and CFR10d, respectively. System performance, syntrophic relationships, microbial communities, and metabolic pathways were examined. During the long-term operation, 2002.7 ± 56.0 mg COD/L acetate was accumulated in CFR10d due to the washout of acetotrophic methanogens. Microorganisms with high half-saturation constants were enriched in reactors of 25-day SRT. Moreover, ethanol oxidizing bacteria and acetotrophic methanogens with high half-saturation constants could be acclimated in SBRs. In SBRs, Syner-01 and Methanothrix dominated, and the low SRT of 10 days increased the relative abundance of Geobacter to 38.0%. In CFRs, the low SRT of 10 days resulted in an increase of Desulfovibrio among syntrophic bacteria, and CFR10d could be employed in enriching hydrogenotrophic methanogens like Methanobrevibacter.

Syntrophic Propionate Oxidation: One of the Rate-Limiting Steps of Organic Matter Decomposition in Anoxic Environments.

Syntrophic propionate oxidation is one of the rate-limiting steps during anaerobic decomposition of organic matter in anoxic environments. Syntrophic propionate-oxidizing bacteria (SPOB) are members of the "rare biosphere" living at the edge of the thermodynamic limit in most natural habitats. Hitherto, only 10 bacterial species capable of syntrophic propionate oxidization have been identified. SPOB employ different metabolisms for propionate oxidation (e.g., methylmalonyl-CoA pathway and C6 dismutation pathway) and show diverse life strategies (e.g., obligately and facultatively syntrophic lifestyle). The flavin-based electron bifurcation/confurcation (FBEB/C) systems have been proposed to help solve the thermodynamic dilemma during the formation of the low-potential products H2 and formate. Molecular ecological approaches, such as DNA stable isotope probing (DNA-SIP) and metagenomics, have been used to detect SPOB in natural environments. Furthermore, the biogeographical pattern of SPOB has been recently described in paddy soils. A comprehensive understanding of SPOB is essential for better predicting and managing organic matter decomposition and carbon cycling in anoxic environments. In this review, we described the critical role of syntrophic propionate oxidation in anaerobic decomposition of organic matter, phylogenetic and metabolic diversity, life strategies and ecophysiology, composition of syntrophic partners, and pattern of biogeographic distribution of SPOB in natural environments. We ended up with a few perspectives for future research.

Metatranscriptional characterization of metabolic dynamics in anaerobic membrane bioreactor producing methane from low-strength wastewater.

An anaerobic membrane bioreactor (AnMBR) with media is an emerging carbon-neutral biotechnology for low-strength wastewater (LSWW) treatment and methane recovery. Understanding metabolic dynamics among methanogens and syntrophic bacteria is important in optimizing the design and operation of AnMBR. However, little is known about it, especially in media-attached microbial communities. This study explored metabolic dynamics to compare media-attached and suspended conditions. Accordingly, metagenomes and metatranscriptomes from AnMBRs with polymeric media and fed with different influent concentrations (350 and 700 mg-COD/L) were analyzed. Metabolic dynamics were profoundly influenced by the different growth habitats and influent conditions, although the applied influent concentrations are within the range of typical LSWW. Metabolic dynamics prediction results suggest that media-attached-growth habitats may have provided a more favorable microenvironment for methanogens to grow and produce methane, especially under low influent conditions. These findings provide significant implications for optimizing floating media design and operation of AnMBR-producing methane from LSWW.

Distribution of extracellular amino acids and their potential functions in microbial cross-feeding in anaerobic digestion systems.

Anaerobic digestion is a prevalent bioenergy production process relying on a complex network of symbiotic interactions, where the nutrient based cross-feeding is an essential microbial mechanism. Here, the cross-feeding function was assessed by analyzing extracellular polymeric substances-associated amino acids in microbial aggregates collected from 14 lab-scale anaerobic digesters, as well as deciphering their genetically biosynthetic potential by syntrophic bacteria and methanogens. The total concentration of essential amino acids ranged from 1.2 mg/g VSS to 174.0 mg/g VSS. The percentages of glutamic acid (8.5 âˆ¼ 37.6%), lysine (2.7 âˆ¼ 22.6%), alanine (5.6 âˆ¼ 13.2%), and valine (3.0 âˆ¼ 10.4%) to the total amount of detected amino acids were the highest in most samples. Through metagenomics analysis, several investigated syntrophs (i.e., Smithella, Syntrophobacter, Syntrophomonas, and Mesotoga) and methanogens (i.e., Methanothrix and Methanosarcina) were auxotrophies, but the genetic ability of syntrophs and methanogens to synthesize some essential amino acids could be complementary, implying potential cross-feeding partnership.

Characterization of trace metal impact on organic acid metabolism and functional microbial community in treating dairy processing wastewater with thermophilic anaerobic membrane bioreactor.

The anaerobic digestion (AD) of dairy processing wastewater (DPW) to produce bioenergy is considered promising but also associated with the possibility of an unbalanced organic matter and trace metal (TM) content. In this study, the TM content and its impact on AD were determined in an anaerobic membrane bioreactor operated to treat DPW. The results indicated that a deficiency in TMs resulted in the slow deterioration of the process, reducing biogas production, disrupting the buffer system, and the massive accumulation of organic acid. The deficiency of Co/Ni was significant, while iron fluctuated due to microbial and chemical effects. Syntrophic propionate oxidizing bacteria and methanogen were the main groups suppressed under the TM deficient environment, resulting in AD failure. No inhibitory effect on the lactic acid metabolism was observed. Hence, supplying theoretical TM dosage to DPW was necessary to realize the efficient and stable AD process and robust microbial community.

Methanogenic treatment of dairy product wastewater by thermophilic anaerobic membrane bioreactor: Ammonia inhibition and microbial community.

Dairy product wastewater contains high-strength organic matter suitable for anaerobic treatment, but excessive protein degradation may lead to an ammonia inhibition problem. This work studied protein-rich dairy product wastewater treatment in the anaerobic membrane bioreactor. The results showed that a temporary self-detoxification phase of ammonia inhibition from the change of pH buffer system was vital for rapid reactor recovery by substrate dilution. The ammonia washout from the reactor was simulated by a kinetic model. After ammonia inhibition, the relative abundance of syntrophic lactic and propionic acids oxidising bacteria significantly reduced along with fermentative bacteria involved in mixed organic acids production. Nevertheless, the relative abundance of the protein degradation bacteria producing acetic acid and H2/CO2 increased. A potential metabolic process change was proposed by profiling the functional community. To conclude, substrate dilution is essential for overcoming ammonia inhibition in the anaerobic treatment of protein-rich dairy product wastewater.

Techno-economic and Life Cycle Analysis of MixAlco® Processes for Mixed Alcohol Production from Brown Algae.

The need for producing renewable fuels from biomass has increased due to depleting fossil resources and environmental concerns. However, the low fraction of biomass carbon converted to product is an undeniable drawback for most current biofuel productions from fermentation due to undecomposed lignin in biomass composition and carbon loss as CO2. In this work, two main production routes of the MixAlco® process, the ketonization route (KR) and esterification route (ER) are evaluated for the mixed alcohol production by brown algae, a third-generation biomass without lignin. A novel fermentation process using syntrophic bacteria consortia (SBC) is developed to produce acetic acid from waste gas produced by KR and ER process. The paper investigates the integrated flowsheet for these alternative routes, using techno-economic and life cycle analysis to compare the minimum selling price and environmental impacts. From TEA, we find that the overall costs for KR and ER are lower than the SBC processes. The cost of ketonization routes is lower than esterification routes. The capital cost and operating cost for the ER+SBC process are the highest. Raw materials and utilities are the two major costs for all the processing routes examined. The MSP for the ER+SBC process is the lowest out of all four routes. ER process performs the best in terms of environmental impacts except in water depletion compared with other processes, while the KR process performs the worst regarding the environmental metrics.

Electron transfer and mechanism of energy production among syntrophic bacteria during acidogenic fermentation: A review.

Volatile fatty acids (VFAs) production plays an important role in the process of anaerobic digestion (AD), which is often the critical factor determining the metabolic pathways and energy recovery efficiency. Fermenting bacteria and acetogenic bacteria are in syntrophic relations during AD. Thus, clear elucidation of the interspecies electron transfer and energetic mechanisms among syntrophic bacteria is essential for optimization of acidogenic. This review aims to discuss the electron transfer and energetic mechanism in syntrophic processes between fermenting bacteria and acetogenic bacteria during VFAs production. Homoacetogenesis also plays a role in the syntrophic system by converting H2 and CO2 to acetate. Potential applications of these syntrophic activities in bioelectrochemical system and value-added product recovery from AD of organic wastes are also discussed. The study of acidogenic syntrophic relations is in its early stages, and additional investigation is required to better understand the mechanism of syntrophic relations.

Microbial Symbiosis: A Network towards Biomethanation.

Biomethanation through anaerobic digestion (AD) is the most reliable energy harvesting process to achieve waste-to-energy. Microbial communities, including hydrolytic and fermentative bacteria, syntrophic bacteria, and methanogenic archaea, and their interspecies symbioses allow complex metabolisms for the volumetric reduction of organic waste in AD. However, heterogeneity in organic waste induces community shifts in conventional anaerobic digesters treating sewage sludge at wastewater treatment plants globally. Assessing the metabolic roles of individual microbial species in syntrophic communities remains a challenge, but such information has important implications for microbially enhanced energy recovery. This review focuses on the alterations in digester microbiome and intricate interspecies networks during substrate variation, symbiosis among the populations, and their implications for biomethanation to aid stable operation in real-scale digesters.

Comparison of the microbial communities in anaerobic digesters treating high alkalinity synthetic wastewater at atmospheric and high-pressure (11 bar).

High-pressure anaerobic digestion is an appealing concept since it can upgrade biogas directly within the reactor. However, the decline of pH caused by the dissolution of CO2 is the main barrier that prevents a good operating high-pressure anaerobic digestion process. Therefore, in this study, a high-pressure anaerobic digestion was studied to treat high alkalinity synthetic wastewater, which could not be treated in a normal-pressure anaerobic digester. In the high-pressure reactor, the pH value was 7.5 ~ 7.8, and the CH4 content reached 88% at 11 bar. Unlike its normal-pressure counterpart (2285 mg/L acetic acid), the high-pressure reactor ran steadily (without volatile fatty acids inhibition). Furthermore, the microbial community changed in the high-pressure reactor. Specifically, key microbial guilds (Syntrophus (11.2%), Methanosaeta concilii (50.9%), and Methanobrevibacter (26.8%)) were dominant in the high-pressure reactor at 11 bar, indicating their fundamental roles under high-pressure treating high alkalinity synthetic wastewater.

Isolation and characterization of a new fructophilic Lactobacillus plantarum FPL strain from honeydew.

In the present study, a Lactobacillus plantarum FPL strain exhibiting fructophilic behavior has been isolated for the first time from honeydew. It is a probably syntrophic bacterium inhabiting the gastrointestinal tract of Coccus hesperidum L. and taking part in sugar metabolism. The promising growth characteristics and biochemical properties of Lb. plantarum FPL indicate that this may be a facultatively fructophilic species, whose properties are not associated with the loss of the alcohol/acetaldehyde dehydrogenase gene. The article attempts to classify the peculiar behavior of this strain by means of tests that are characteristic for FLAB as well as through a classic identification approach. In this study, we used a reference strain Lb. plantarum NRRL B-4496, which showed no fructophilic properties. With the FLAB group, the new strain shares the habit, such as a fructose-rich environment, the preference of this sugar for growth, and similar growth curves. However, it exceeds FLAB in terms of osmotolerance to high sugar content. The fructophilic Lb. plantarum FPL strain can proliferate and grow on a medium wherein the sugar concentration is 45 and 50% (w/v). Our findings indicate that honeydew can be a promising source of new fructophilic lactic acid bacteria.

Archaeal and Bacterial Community Structure in an Anaerobic Digestion Reactor (Lagoon Type) Used for Biogas Production at a Pig Farm.

Biogas production from animal waste is an economically viable way to reduce environmental pollution and produce valuable products, i.e., methane and a nutrient-rich organic waste product. An anaerobic digestion reactor for biogas production from pig waste was sampled at the entrance, middle (digestion chamber), and exit of a digester, while the bacterial and archaeal community structure was studied by 16S rRNA gene metagenomics. The number of bacterial operational taxonomic units (OTU)-97% was 3-7 times larger than that of archaeal ones. Bacteria and Archaea found in feces of animals (e.g., Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Methanosarcina, Methanolobus, Methanosaeta, and Methanospirillum) dominated the entrance of the digester. The digestion chamber was dominated by anaerobic sugar-fermenting OP9 bacteria and the syntrophic bacteria Candidatus Cloacamonas (Waste Water of Evry 1; WWE1). The methanogens dominant in the digestion chamber were the acetoclastic Methanosaeta and the hydrogenothrophic Methanoculleus and Methanospirillum. Similar bacterial and archaeal groups that dominated in the middle of the digestion chamber were found in the waste that left the digester. Predicted functions associated with degradation of xenobiotic compounds were significantly different between the sampling locations. The microbial community found in an anaerobic digestion reactor loaded with pig manure contained microorganisms with biochemical capacities related to the 4 phases of methane production.