Biohydrogen Production: A Protein to Community Level Perspective Study

Excessive usage of traditional energy reserves leading to increased environmental pollution and global warming have strongly urged for alternative sustainable energy sources. Due to non-polluting nature and high energy yields, hydrogen (H₂) gas is considered as an ideal candidate for alternative fuel. Biohydrogen (bioH₂) production from organic wastes is a sustainable approach, addressing energy production through organic waste disposal. Organic wastes such as lignocellulosic biomass and industrial glycerol, a by-product of biodiesel manufacturing process, have been recently investigated for their bioconversion potential. However, bioconversion of such organic wastes is a challenge due to the presence of impurities, toxic degradation products and complex nature. In comparison to pure bacterial strains, natural microflora could be an ideal inoculum choice offering better adaptability, substrate utilization efficiency and bioconversion rates. Another challenge to ensure efficient fermentation is to optimize various physico-chemical factors such as pH, temperature, substrate selection and concentration, medium compounds, and H₂ removal and collection due to individual and interactive effects on microbial growth, metabolism and hydrogenase enzyme. Hydrogenases are metalloenzymes that reversibly catalyzes proton reduction to H₂, and are divided into three classes based on the metal cofactor at the active site, [Fe-Fe], [Ni-Fe] and [Fe] hydrogenase. Among the hydrogenase classes, [Fe-Fe] hydrogenases exhibit highest catalytic activity involving mostly in H₂ production. Apart from their pivotal role in fermentative H₂ production, [Fe-Fe] hydrogenases promise an alternative catalyst choice in fuel cells. However, in spite of their preference towards H₂ production, [Fe-Fe] hydrogenases are extremely prone to catalytic inactivation upon oxygen exposure. This is the major challenge, at the protein level, that hinders a cost-effective approach for biotechnological applications and suggests the requirement of targeted tools to investigate the inactivation process at the molecular level. The purpose of the present study was to investigate bioH₂ production in protein to community level perspective. More specifically the aims were to (1) establish an anaerobic biopanning procedure to enrich antibody binders specific against clostridial [Fe-Fe] hydrogenase protein, (2) develop and standardize a novel enrichment system, (3) implement the enrichment technique to enrich functional inoculum capable of degrading complex substrates, (4) enrich crude glycerol fermenting microbial community and finally, (5) optimize the physico-chemical factors influencing fermentative H₂ production for efficient bioprocess. In the present study, biopanning with synthetic ‘mixed’ single chain variable fragment (scFv) libraries against active and inactive clostridial [Fe-Fe] hydrogenases aided the enrichment of anti-hydrogenase antibodies. Out of ninety four (from inactive hydrogenase) and ninety two (from active hydrogenase) random clones screened, nine potential antibody clones with recognition specificity towards Clostridium acetobutylicum [Fe-Fe] hydrogenase were selected. The enriched binders also recognized [Fe-Fe] hydrogenase from C. butyricum. Based on the results from this study, it could be reasoned that the binders with generic specificity against closely related clostridial [Fe-Fe] hydrogenases can be used as novel molecular tools for quantitative monitoring [Fe-Fe] hydrogenases at the protein level. Another of-note observation was the specificity of the antibody binders towards active and inactive hydrogenases. Preliminary experiments indicated 7Ac binder (enriched against active hydrogenase) specificity towards the catalytically active [Fe-Fe] hydrogenase rather to the inactive state and 48In (enriched against inactive hydrogenase) recognized both catalytic states. These findings indicate the possibility to apply the isolated antibody clones for functional detection of clostridial [Fe-Fe] hydrogenases. The study progresses in investigating bioH₂ production in perspective of microbial community. The novel microbial enrichment system was developed and the proof-of-principle experiments conducted using artificial mixed microbial community and varied selection criteria allowed the enrichment of the best H₂ producer. The system was implemented in enriching cellobiose degrading H₂ producer from an environmental sample. The bacterial strain isolated by spread plate technique on agar plates containing CMC was affiliated with Citrobacter sp. and named as Citrobacter sp. CMC-1. Citrobacter sp. CMC-1 utilized glucose, cellobiose and CMC and followed mixed-acid fermentation profile producing H₂ and carbon dioxide (CO₂) as gaseous metabolites and acetate, formate, lactate and ethanol as liquid metabolites. At optimized values of cultivation conditions (pH 6.0 and 34 ˚C) the H₂ yield was 1.82 mol-H₂/mol-glucose. The isolate efficiently fermented monomeric hemi-cellulose sugars to H₂ (mol-H₂/mol-substrate): Galactose, 1.18; Mannose, 1.23; Xylose, 1.22; Arabinose, 0.94 and Rhamnose, 1.01). Except for arabinose, an increase in cultivation period improved the biomass and H₂ yield (mol-H₂/mol-substrate): Galactose, 1.68; Mannose, 1.93 and Xylose, 1.63) followed with observations of reduced formate accumulation in the medium, indicating that Citrobacter sp. CMC-1 produced H₂ from formate breakdown via the FHL complex. Microbial community pre-dominated with Clostridium spp. enriched from activated sludge fermented crude glycerol mainly to H₂, CO₂, acetate, butyrate and ethanol. Optimal bioprocess conditions for the enriched inoculum were experimentally observed to be pH 6.5, 40˚C and 1g/L crude glycerol. The H₂ yield from raw glycerol at optimal cultivation conditions was 1.1 mol-H₂/mol-glycerol consumed . At elevated crude glycerol concentrations, substrate utilization and H₂ production were limited due to the presence of impurities in the crude glycerol fraction. The bioconversion of crude glycerol to H₂ was further improved by statistical optimization of the growth medium composition. Initial screening with Plackett – Burman design identified NH₄Cl, K₂HPO and KH₂PO₄ with individual and interactive effects on H₂ yield. Among the three identified media components, NH₄Cl and KH₂PO₄ imparted the maximal significance and were optimized in scrutiny. A series of statistical models identified the optimal media composition for improved H₂ production from crude glycerol fermentations and were successful in improving the H₂ yield by 29% (1.42 mol-H₂/mol-glycerol consumed ) in comparison to previously reported value (1.1 mol-H₂/mol-glycerol consumed )

Draft genome sequence data of a psychrophilic tundra soil methanotroph, Methylobacter psychrophilus Z-0021 (DSM 9914).

Psychrophilic methanotrophic bacteria are abundant and play an important role in methane removal in cold methanogenic environments, such as boreal and arctic terrestrial and aquatic ecosystems. They could be also applied in the bioconversion of biogas and natural gas into value-added products (e.g., chemicals and single-cell protein) in cold regions. Hence, isolation and genome sequencing of psychrophilic methanotrophic bacteria are needed to provide important data on their functional capabilities. However, psychrophilic methanotroph isolates and consequently their genome sequences are rare. Fortunately, Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH was able to revive the long-extinct pure culture of a psychrophilic methanotrophic tundra soil isolate, Methylobacter psychrophilus Z-0021 (DSM 9914), from their stocks during 2022. Here, we describe the de novo assembled genome sequence of Methylobacter psychrophilus Z-0021 comprising a total of 4691082 bp in 156 contigs with a G+C content of 43.1% and 4074 coding sequences. The preliminary genome annotation analysis of Z-0021 identified genes encoding oxidation of methane, methanol and formaldehyde, assimilation of carbon and nitrate, and N2 fixation. In pairwise genome-to-genome comparisons with closely related methanotrophic strains, the strain Z-0021 had an average nucleotide identity (ANI) of 92.9% and 78.2% and a digital DNA-DNA hybridization (dDDH) value of 50.6% and 22% with a recently described psychrophilic, lake isolate, Methylobacter sp. S3L5C and a psychrotrophic, arctic wetland soil isolate, Methylobacter tundripaludum SV96, respectively. In addition, the respective similarities between genomes of the strains S3L5C and SV96 were 78.1% ANI and 21.8% dDDH. Comparison to widely used ANI and dDDH thresholds to delineate unique species (<95% ANI and <70% dDDH) suggests that Methylobacter psychrophilus Z-0021, Methylobacter tundripaludum SV96 and Methylobacter sp. S3L5C are different species. The draft genome of Z-0021 has been deposited at GenBank under the accession JAOEGU000000000

Organic matter lability modifies the vertical structure of methane-related microbial communities in lake sediments

Eutrophication increases the input of labile, algae-derived, organic matter (OM) into lake sediments. This potentially increases methane (CH4) emissions from sediment to water through increased methane production rates and decreased methane oxidation efficiencyefficiency in sediments. However, the effecteffect of OM lability on the structure of methane oxidizing (methanotrophic) and methane producing (methanogenic) microbial communities in lake sediments is still understudied. We studied the vertical profilesprofiles of the sediment and porewater geochemistry and the microbial communities (16S rRNA gene amplicon sequencing) at fivefive profundal stations of an oligo-mesotrophic, boreal lake (Lake Paajarvi, Finland), varying in surface sediment OM sources (assessed via sediment C:N ratio). Porewater profilesprofiles of methane, dissolved inorganic carbon (DIC), acetate, iron, and sulfur suggested that sites with more autochthonous OM showed higher overall OM lability, which increased remineralization rates, leading to increased electron acceptor (EA) consumption and methane emissions from sediment to water. When OM lability increased, the abundance of anaerobic nitrite-reducing methanotrophs (Candidatus Methylomirabilis) relative to aerobic methanotrophs (Methylococcales) in the methane oxidation layer of sediment surface decreased, suggesting that Methylococcales were more competitive than Ca. Methylomirabilis under decreasing redox conditions and increasing methane availability due to their more diverse metabolism (fermentation and anaerobic respiration) and lower affinityaffinity for methane. Furthermore, when OM lability increased, the abundance of methanotrophic community in the sediment surface layer, especially Ca. Methylomirabilis, relative to the methanogenic community decreased. We conclude that increasing input of labile OM, subsequently affectingaffecting the redox zonation of sediments, significantlysignificantly modifies the methane producing and consuming microbial community of lake sediments

The role of organic matter and microbial community controlling nitrate reduction under elevated ferrous iron concentrations in boreal lake sediments

The nitrogen availability, that affects the greenhouse gas emission and the trophic level of lakes, is controlled mainly by microbial processes. We measured in a boreal nitrate and iron rich lake how the rates of potential denitrification and dissimilatory nitrate reduction to ammonia (DNRA) are affected by degradability of organic matter and availability of aqueous ferrous iron. We also investigated the microbial community by using 16S rRNA gene and shotgun metagenomic sequencing approach, which allows taxonomic analyses and detection of metagenome-assembled genomes (MAGs) containing genes for both nitrate reduction and iron oxidation. The results show that truncated denitrification, leading to release of nitrous oxide, is favored over dinitrogen production in conditions where the degradability of the organic matter is low. DNRA rates were always minor compared to denitrification and appeared to be independent of the degradability of organic carbon. Reduced iron stimulated nitrate reducing processes, although consistently only DNRA. However, the proportion of MAGs containing DNRA genes was low suggesting chemistry driven stimulation by reduced iron. Nevertheless, the metagenomic analyses revealed unique taxa genetically capable of oxidizing iron and reducing nitrate simultaneously. Overall, the results highlight the spatial variability in microbial community and nitrous oxide emissions in boreal lake sediments.Peer reviewe

Organic matter lability modifies the vertical structure of methane-related microbial communities in lake sediments

Eutrophication increases the input of labile, algae-derived, organic matter (OM) into lake sediments. This potentially increases methane (CH4) emissions from sediment to water through increased methane production rates and decreased methane oxidation efficiency in sediments. However, the effect of OM lability on the structure of methane oxidizing (methanotrophic) and methane producing (methanogenic) microbial communities in lake sediments is still understudied. We studied the vertical profiles of the sediment and porewater geochemistry and the microbial communities (16S rRNA gene amplicon sequencing) at five profundal stations of an oligo-mesotrophic, boreal lake (Lake Pääjärvi, Finland), varying in surface sediment OM sources (assessed via sediment C:N ratio). Porewater profiles of methane, dissolved inorganic carbon (DIC), acetate, iron, and sulfur suggested that sites with more autochthonous OM showed higher overall OM lability, which increased remineralization rates, leading to increased electron acceptor (EA) consumption and methane emissions from sediment to water. When OM lability increased, the abundance of anaerobic nitrite-reducing methanotrophs (Candidatus Methylomirabilis) relative to aerobic methanotrophs (Methylococcales) in the methane oxidation layer of sediment surface decreased, suggesting that Methylococcales were more competitive than Ca. Methylomirabilis under decreasing redox conditions and increasing methane availability due to their more diverse metabolism (fermentation and anaerobic respiration) and lower affinity for methane. Furthermore, when OM lability increased, the abundance of methanotrophic community in the sediment surface layer, especially Ca. Methylomirabilis, relative to the methanogenic community decreased. We conclude that increasing input of labile OM, subsequently affecting the redox zonation of sediments, significantly modifies the methane producing and consuming microbial community of lake sediments

Biohydrogen Production: A Protein to Community Level Perspective Study

Excessive usage of traditional energy reserves leading to increased environmental pollution and global warming have strongly urged for alternative sustainable energy sources. Due to non-polluting nature and high energy yields, hydrogen (H₂) gas is considered as an ideal candidate for alternative fuel. Biohydrogen (bioH₂) production from organic wastes is a sustainable approach, addressing energy production through organic waste disposal. Organic wastes such as lignocellulosic biomass and industrial glycerol, a by-product of biodiesel manufacturing process, have been recently investigated for their bioconversion potential. However, bioconversion of such organic wastes is a challenge due to the presence of impurities, toxic degradation products and complex nature. In comparison to pure bacterial strains, natural microflora could be an ideal inoculum choice offering better adaptability, substrate utilization efficiency and bioconversion rates. Another challenge to ensure efficient fermentation is to optimize various physico-chemical factors such as pH, temperature, substrate selection and concentration, medium compounds, and H₂ removal and collection due to individual and interactive effects on microbial growth, metabolism and hydrogenase enzyme. Hydrogenases are metalloenzymes that reversibly catalyzes proton reduction to H₂, and are divided into three classes based on the metal cofactor at the active site, [Fe-Fe], [Ni-Fe] and [Fe] hydrogenase. Among the hydrogenase classes, [Fe-Fe] hydrogenases exhibit highest catalytic activity involving mostly in H₂ production. Apart from their pivotal role in fermentative H₂ production, [Fe-Fe] hydrogenases promise an alternative catalyst choice in fuel cells. However, in spite of their preference towards H₂ production, [Fe-Fe] hydrogenases are extremely prone to catalytic inactivation upon oxygen exposure. This is the major challenge, at the protein level, that hinders a cost-effective approach for biotechnological applications and suggests the requirement of targeted tools to investigate the inactivation process at the molecular level. The purpose of the present study was to investigate bioH₂ production in protein to community level perspective. More specifically the aims were to (1) establish an anaerobic biopanning procedure to enrich antibody binders specific against clostridial [Fe-Fe] hydrogenase protein, (2) develop and standardize a novel enrichment system, (3) implement the enrichment technique to enrich functional inoculum capable of degrading complex substrates, (4) enrich crude glycerol fermenting microbial community and finally, (5) optimize the physico-chemical factors influencing fermentative H₂ production for efficient bioprocess. In the present study, biopanning with synthetic ‘mixed’ single chain variable fragment (scFv) libraries against active and inactive clostridial [Fe-Fe] hydrogenases aided the enrichment of anti-hydrogenase antibodies. Out of ninety four (from inactive hydrogenase) and ninety two (from active hydrogenase) random clones screened, nine potential antibody clones with recognition specificity towards Clostridium acetobutylicum [Fe-Fe] hydrogenase were selected. The enriched binders also recognized [Fe-Fe] hydrogenase from C. butyricum. Based on the results from this study, it could be reasoned that the binders with generic specificity against closely related clostridial [Fe-Fe] hydrogenases can be used as novel molecular tools for quantitative monitoring [Fe-Fe] hydrogenases at the protein level. Another of-note observation was the specificity of the antibody binders towards active and inactive hydrogenases. Preliminary experiments indicated 7Ac binder (enriched against active hydrogenase) specificity towards the catalytically active [Fe-Fe] hydrogenase rather to the inactive state and 48In (enriched against inactive hydrogenase) recognized both catalytic states. These findings indicate the possibility to apply the isolated antibody clones for functional detection of clostridial [Fe-Fe] hydrogenases. The study progresses in investigating bioH₂ production in perspective of microbial community. The novel microbial enrichment system was developed and the proof-of-principle experiments conducted using artificial mixed microbial community and varied selection criteria allowed the enrichment of the best H₂ producer. The system was implemented in enriching cellobiose degrading H₂ producer from an environmental sample. The bacterial strain isolated by spread plate technique on agar plates containing CMC was affiliated with Citrobacter sp. and named as Citrobacter sp. CMC-1. Citrobacter sp. CMC-1 utilized glucose, cellobiose and CMC and followed mixed-acid fermentation profile producing H₂ and carbon dioxide (CO₂) as gaseous metabolites and acetate, formate, lactate and ethanol as liquid metabolites. At optimized values of cultivation conditions (pH 6.0 and 34 ˚C) the H₂ yield was 1.82 mol-H₂/mol-glucose. The isolate efficiently fermented monomeric hemi-cellulose sugars to H₂ (mol-H₂/mol-substrate): Galactose, 1.18; Mannose, 1.23; Xylose, 1.22; Arabinose, 0.94 and Rhamnose, 1.01). Except for arabinose, an increase in cultivation period improved the biomass and H₂ yield (mol-H₂/mol-substrate): Galactose, 1.68; Mannose, 1.93 and Xylose, 1.63) followed with observations of reduced formate accumulation in the medium, indicating that Citrobacter sp. CMC-1 produced H₂ from formate breakdown via the FHL complex. Microbial community pre-dominated with Clostridium spp. enriched from activated sludge fermented crude glycerol mainly to H₂, CO₂, acetate, butyrate and ethanol. Optimal bioprocess conditions for the enriched inoculum were experimentally observed to be pH 6.5, 40˚C and 1g/L crude glycerol. The H₂ yield from raw glycerol at optimal cultivation conditions was 1.1 mol-H₂/mol-glycerol consumed . At elevated crude glycerol concentrations, substrate utilization and H₂ production were limited due to the presence of impurities in the crude glycerol fraction. The bioconversion of crude glycerol to H₂ was further improved by statistical optimization of the growth medium composition. Initial screening with Plackett – Burman design identified NH₄Cl, K₂HPO and KH₂PO₄ with individual and interactive effects on H₂ yield. Among the three identified media components, NH₄Cl and KH₂PO₄ imparted the maximal significance and were optimized in scrutiny. A series of statistical models identified the optimal media composition for improved H₂ production from crude glycerol fermentations and were successful in improving the H₂ yield by 29% (1.42 mol-H₂/mol-glycerol consumed ) in comparison to previously reported value (1.1 mol-H₂/mol-glycerol consumed )

Conversion of methane to organic acids is a widely found trait among gammaproteobacterial methanotrophs of freshwater lake and pond ecosystems

Aerobic gammaproteobacterial methanotrophs (gMOB) are key organisms controlling methane fluxes at the oxic-anoxic interfaces of freshwater ecosystems. Under hypoxic environments, gMOB may shift their aerobic metabolism to fermentation, resulting in the production of extracellular organic acids. We recently isolated a gMOB strain representing the Methylobacter spp. of boreal lake water columns (i.e., Methylobacter sp. S3L5C) and demonstrated that it converts methane to organic acids (acetate, formate, malate, and propionate) under hypoxic conditions. Annotation for putative genes encoding organic acid production within the isolate's genome and in environmental metagenome-assembled genomes (MAGs) representing Methylobacter spp. suggests that the potential for methane conversion into organic acids is widely found among Methylobacter spp. of freshwater ecosystems. However, it is not known yet whether the capability to convert methane to organic acids is restricted to Methylobacter spp. or ubiquitously present among other freshwater gMOB genera. Therefore, we isolated representatives of two additional gMOB genera from the boreal lake water columns, i.e., Methylomonas paludis S2AM and Methylovulum psychrotolerans S1L, and demonstrated similar bioconversion capacities. These genera could convert methane to organic acids, including acetate, formate, succinate, and malate. Additionally, S2AM produced lactate. Furthermore, we detected genes encoding organic acid production within their genomes and in MAGs representing Methylomonas spp. and Methylovulum spp. of lake and pond ecosystems. Altogether, our results demonstrate that methane conversion to various organic acids is a widely found trait among lake and pond gMOB, highlighting their role as pivotal mediators of methane carbon into microbial food webs of freshwater lake and pond ecosystems. IMPORTANCE Aerobic gammaproteobacterial methanotrophic bacteria (gMOB) play an important role in reducing methane emissions from freshwater ecosystems. In hypoxic conditions prevalent near oxic-anoxic interfaces, gMOB potentially shift their metabolism to fermentation, resulting in the conversion of methane to extracellular organic acids, which would serve as substrates for non-methanotrophic microbes. We intended to assess the prevalence of fermentation traits among freshwater gMOB. Therefore, we isolated two strains representing relevant freshwater gMOB genera, i.e., Methylovulum and Methylomonas, from boreal lakes, experimentally showed that they convert methane to organic acids and demonstrated via metagenomics that the fermentation potential is widely dispersed among lake and pond representatives of these genera. Combined with our recent study showing coherent results from another relevant freshwater gMOB genus, i.e., Methylobacter, we conclude that the conversion of methane to organic acids is a widely found trait among freshwater gMOB, highlighting their role as pivotal mediators of methane carbon into microbial food webs.Peer reviewe

Co-production of 1,3 propanediol and long-chain alkyl esters from crude glycerol

Crude glycerol is an excellent carbon source for bacterial production systems. Bacterial fermentation often generates by-products that can offer an additional carbon pool to improve the product profile for optimal valorization. In this study, the properties of two phylogenetically distinct bacteria, Acinetobacter baylyi ADP1 and Clostridium butyricum, were coupled in a one-pot batch process to co-produce 1,3 propanediol (PDO) and long-chain alkyl esters (wax esters, WEs) from crude glycerol. In the process, A. baylyi deoxidized the growth medium allowing glycerol fermentation and PDO production by C. butyricum. Reaeration of the co-cultivations enabled A. baylyi to metabolize the fermentation by-products, acetate and butyrate, and synthesize intracellular WEs. To improve PDO production and A. baylyi growth, carbon and macronutrients in the growth medium were screened and optimized using Plackett-Burman and Box-Behnken models. The validation experiment revealed a good correlation between the observed and predicted values. The salting-out method recovered 89.5% PDO from the fermentation broth and in vacuo extraction resulted in a PDO content of 5.3 g L-1. Nuclear magnetic resonance revealed a WE content and yield of 34.4 ± 1.4 mg L-1 and 34.2 ± 3.2 mg WE g-1 dry cell weight, respectively. A molar yield of 0.65 mol PDO mol-1 and 0.62 µmol WE mol-1 crude glycerol was achieved with the synthetic consortium. This work emphasizes the strength of response surface methodology in improving production processes from the mutualistic association of divergent bacterial species in consortium. The co-production of PDO and WEs from crude glycerol is demonstrated for the first time in this study.acceptedVersionPeer reviewe

Metabolic pairing of aerobic and anaerobic production in a one-pot batch cultivation

Abstract Background The versatility of microbial metabolic pathways enables their utilization in vast number of applications. However, the electron and carbon recovery rates, essentially constrained by limitations of cell energetics, are often too low in terms of process feasibility. Cocultivation of divergent microbial species in a single process broadens the metabolic landscape, and thus, the possibilities for more complete carbon and energy utilization. Results In this study, we integrated the metabolisms of two bacteria, an obligate anaerobe Clostridium butyricum and an obligate aerobe Acinetobacter baylyi ADP1. In the process, a glucose-negative mutant of A. baylyi ADP1 first deoxidized the culture allowing C. butyricum to grow and produce hydrogen from glucose. In the next phase, ADP1 produced long chain alkyl esters (wax esters) utilizing the by-products of C. butyricum, namely acetate and butyrate. The coculture produced 24.5 ± 0.8 mmol/l hydrogen (1.7 ± 0.1 mol/mol glucose) and 28 mg/l wax esters (10.8 mg/g glucose). Conclusions The cocultivation of strictly anaerobic and aerobic bacteria allowed the production of both hydrogen gas and long-chain alkyl esters in a simple one-pot batch process. The study demonstrates the potential of ‘metabolic pairing’ using designed microbial consortia for more optimal electron and carbon recovery

Characterization and genome analysis of a psychrophilic methanotroph representing a ubiquitous Methylobacter spp. cluster in boreal lake ecosystems

Lakes and ponds are considered as a major natural source of CH4 emissions, particularly during the ice-free period in boreal ecosystems. Aerobic methane-oxidizing bacteria (MOB), which utilize CH4 using oxygen as an electron acceptor, are one of the dominant microorganisms in the CH4-rich water columns. Metagenome-assembled genomes (MAGs) have revealed the genetic potential of MOB from boreal aquatic ecosystems for various microaerobic/anaerobic metabolic functions. However, experimental proof of these functions, i.e., organic acid production via fermentation, by lake MOB is lacking. In addition, psychrophilic (i.e., cold-loving) MOB and their CH4-oxidizing process have rarely been investigated. In this study, we isolated, provided a taxonomic description, and analyzed the genome of Methylobacter sp. S3L5C, a psychrophilic MOB, from a boreal lake in Finland. Based on phylogenomic comparisons to MAGs, Methylobacter sp. S3L5C represented a ubiquitous cluster of Methylobacter spp. in boreal aquatic ecosystems. At optimal temperatures (3–12 °C) and pH (6.8–8.3), the specific growth rates (µ) and CH4 utilization rate were in the range of 0.018–0.022 h−1 and 0.66–1.52 mmol l−1 d−1, respectively. In batch cultivation, the isolate could produce organic acids, and the concentrations were elevated after replenishing CH4 and air into the headspace. Up to 4.1 mM acetate, 0.02 mM malate, and 0.07 mM propionate were observed at the end of the test under optimal operational conditions. The results herein highlight the key role of Methylobacter spp. in regulating CH4 emissions and their potential to provide CH4-derived organic carbon compounds to surrounding heterotrophic microorganisms in cold ecosystems.publishedVersionPeer reviewe