Methanotrophic Bacteria Research Articles

Bioupcycling Methane and CO2 for Succinate Production by an Engineered Type I Methanotrophic Bacterium.

Methane, a byproduct of agricultural activities, has shown potential as a nonedible substrate for biomanufacturing. The production of succinate by a methanotrophic bacterium utilizing methane presents an innovative route for the sustainable synthesis of chemicals. In this study, Methylotuvimicrobium buryatense 5GB1S was genetically modified through the reconstruction of an artificial serine cycle to enable the bioconversion of both methane and CO2 into succinate. The 13C labeling analysis confirmed the CO2 fixing in M. buryatense 5GB1S, leading to a 46% improvement in carbon conversion efficiency and a 107% increase in succinate production compared to the wild-type strain. The transcriptome data on carbon metabolisms was assessed to guide future optimizations for strengthening the overall carbon flux from methane to succinate. Finally, the maximum succinate titer of 299.36 mg/L was achieved under oxygen-limited conditions in 3 L bioreactors, which resulted in the volumetric productivity of 199.60 mg/L/day, representing a 23-fold enhancement compared to the wild-type strain. This study offers a new strategy for upcycling greenhouse gases into succinate in a sustainable manner through methanotrophic-based biomanufacturing.

Effects of dietary nitrate, fumaric acid, and methanotrophic bacteria supplementation on rumino-intestinal nutrient metabolism and enteric gas exchange in dairy cows

The objective of the study was to investigate the effects of two approaches for enteric methane (CH4) mitigation and one approach for redirection of excess hydrogen (H2) resulting from CH4 inhibition, on dairy cows’ gas exchange and nutrient digestibility. Approaches for CH4 mitigation were methanotrophic bacteria supplementation and dietary nitrate, whereas the approach for H2 redirection was nitrate combined with fumaric acid. An incomplete 6 × 6 Latin square design experiment was conducted with 4 periods of 21 d using 6 rumen, duodenum, and ileum cannulated Danish Holstein cows with DIM of 123 ± 64.8 d (mean ± SD) and the milk yield was 33.6 ± 9.39 kg/d at beginning of experiment. The treatments were organized in a 2 × 3 factorial arrangement, where the first factor represented treatments without or with methanotrophic bacteria supplementation (MET), and the second factor represented 3 different dietary additive supplementations (DIET). These were a basal diet (BAS; no additives), a diet with nitrate (NIT; 10 g nitrate/kg dry matter (DM)), and a diet with nitrate combined with fumaric acid (NIT-F; NIT + 15 g fumaric acid/kg DM). Cows had ad libitum access to diets with a forage to concentrate ratio of 60 to 40 on dry matter basis. Following adaptation to experimental diets, samples of rumen fluid, digesta from duodenum and ileum, and feces were collected to estimate nutrient digestibility using Cr2O3 and TiO2 as external flow markers. Then, gas exchanges were measured in respiration chambers. There was no CH4 mitigating effect of MET. Nitrate reduced CH4 production (g/d), yield (g/kg DMI), and intensity (g/kg ECM) by 19.5, 11.9, and 17.2 %, respectively, whereas H2 yield (g/kg DMI) was increased by 261 %. Ruminal redox value was decreased by nitrate, and individual rumen volatile fatty acid proportions reflected a more reduced rumen environment although propionate proportions decreased. Nutrient digestibility was not affected by nitrate although microbial CP efficiency (g of microbial CP/kg true rumen digested organic matter) was decreased. Supplementing fumaric acid in combination with nitrate decreased H2 production by 26.8 % compared to nitrate fed cows, and this was associated with increased propionate concentrations. However, there was no effect on H2 emission when corrected for dry matter intake (DMI) or energy-corrected milk (ECM) yield. There were no effects of any of the treatments on DMI or ECM yield. In conclusion, the results demonstrated a CH4 mitigating effect of nitrate supplementation resulting in increased H2 emission. The effects on nutrient digestibility and rumen fermentation were minor. Fumaric acid supplementation redirected some H2 to propionate, although the efficiency was small. Supplementation of methanotrophic bacteria did not suppress CH4 emission.

Overexpression of native carbonic anhydrases increases carbon conversion efficiency in the methanotrophic biocatalyst Methylococcus capsulatus Bath.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4 and CO2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2 evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH4 into CO2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4 conversion bioprocesses.

Methane biohydroxylation into methanol by Methylosinus trichosporium OB3b: possible limitations and formate use during reaction.

Methane (CH4) hydroxylation into methanol (MeOH) by methanotrophic bacteria is an attractive and sustainable approach to producing MeOH. The model strain Methylosinus trichosporium OB3b has been reported to be an efficient hydroxylating biocatalyst. Previous works have shown that regardless of the bioreactor design or operation mode, MeOH concentration reaches a threshold after a few hours, but there are no investigations into the reasons behind this phenomenon. The present work entails monitoring both MeOH and formate concentrations during CH4 hydroxylation, where neither a gaseous substrate nor nutrient shortage was evidenced. Under the assayed reaction conditions, bacterial stress was shown to occur, but methanol was not responsible for this. Formate addition was necessary to start MeOH production. Nuclear magnetic resonance analyses with 13C-formate proved that the formate was instrumental in regenerating NADH; formate was exhausted during the reaction, but increased quantities of formate were unable to prevent MeOH production stop. The formate mass balance showed that the formate-to-methanol yield was around 50%, suggesting a cell regulation phenomenon. Hence, this study presents the possible physiological causes that need to be investigated further. Finally, to the best of our knowledge, this study shows that the reaction can be achieved in the native bacterial culture (i.e., culture medium containing added methanol dehydrogenase inhibitors) by avoiding the centrifugation steps while limiting the hands-on time and water consumption.

Methanobactins: Structures, Biosynthesis, and Microbial Diversity.

Methanobactins (Mbns) are ribosomally synthesized and posttranslationally modified peptide natural products released by methanotrophic bacteria under conditions of copper scarcity. Mbns bind Cu(I) with high affinity via nitrogen-containing heterocycles and thioamide groups installed on a precursor peptide, MbnA, by a core biosynthetic enzyme complex, MbnBC. Additional stabilizing modifications are enacted by other, less universal biosynthetic enzymes. Copper-loaded Mbn is imported into the cell by TonB-dependent transporters called MbnTs, and copper is mobilized by an unknown mechanism. The machinery to biosynthesize and transport Mbn is encoded in operons that are also found in the genomes of nonmethanotrophic bacteria. In this review, we provide an update on the state of the Mbn field, highlighting recent discoveries regarding Mbn structure, biosynthesis, and handling as well as the emerging roles of Mbns in the environment and their potential use as therapeutics.

Influence of seasons and environmental variables on methane dynamics in the Muthukuda Mangrove sediments of Tamil Nadu

Aim: In order to understand the methane biochemistry in the Muthukuda mangrove ecosystem, this study aimed to report on methane production, oxidation, flux, and also to investigate the effect of seasons and environmental variables on methane dynamics. Methodology: Sediment analysis was carried out monthly for a year (2021-2022) from the Muthukuda mangrove, south-east coast of India, which is an unexplored region in terms of methane dynamics. Methane parameters methane concentration, methane oxidation, methane production and methanotrophic enumeration were analysed from the sediment samples collected by a PVC corer from the study site. The methane parameters were analysed using standard methods by gas chromatography. The physiological parameters were analysed on the site using portable probes. Results: The physio-chemical parameters varied with the changing seasons at different depths. The concentrations of organic parameters were higher for carbohydrates, followed by lipids and proteins. LOM was higher in the surface and mid sediments in all the seasons. The concentration of methane was higher during summer in surface sediments. Generally, higher methane oxidation was observed in the mid and bottom sediments during all the seasons. Methanotrophic enumeration ranged from 0.4 ±0.7 to 4.8±2.4 x108 g-1. Interpretation: The results suggest that the bacterial community was involved in both methane oxidation and production, though oxidation of methane was lesser than production contributing to the atmospheric methane, methanotrophic bacteria which oxidizes methane significantly playing a major role in the regulation of methane flux. Key words: Methane oxidation, Methanotrophs, Muthukuda mangrove

Effects of anaerobic oxidation of methane (AOM) driven by iron and manganese oxides on methane emissions in constructed wetlands and underlying mechanisms

In recent years, anaerobic oxidation of methane with iron and manganese oxides as electron acceptors has been confirmed to exist in electrochemical constructed wetlands (CW), and this process is an important way to reduce methane emissions. However, the internal mechanisms of emission reduction, microbial ecological networks, and electron transfer mechanisms remain unclear. In this study, we found that AOM driven by iron and manganese oxides (Fe/Mn-AOM) reduced methane emissions by more than 50.86% in constructed wetland – microbial fuel cell (CW-MFC) systems. In iron and manganese systems, the relative abundance of microorganisms associated with methane metabolism (methanotrophic bacteria) increased significantly. AOM-related microorganisms cooperated with other microorganisms to form a robust and stable microbial ecological network. In the process of methane metabolism, there was an increase in the proportion of reverse methanogenic genes in iron and manganese systems, with the proportion of mcr genes being 1.75 and 1.79 times that of control systems. The key genes (cyc1 and cox11) that influence cytochrome C synthesis in AOM process were significantly enriched, resulting in enhanced electron transfer process. The coexistence of iron and manganese oxides enabled the simultaneous biochemical processes of CH4 oxidation and CO2 reduction within a single system. Additionally, the increase in humic acid also facilitated electron transfer, thereby significantly enhancing the removal efficiency of COD, TN, and TP in the CW-MFC systems. This study reveals the potential mechanism of Fe/Mn-AOM for CW-MFC methane emission reduction, which can provide a theoretical basis for the collaborative treatment of sewage and methane control in CWs, so as to achieve the maximum environmental benefits of CW.

Replacement of fishmeal with a microbial single-cell protein induced enteropathy and poor growth outcomes in barramundi (Lates calcarifer) fry.

Fish meal (FM) replacement is essential for the sustainable expansion of aquaculture. This study focussed on the feasibility of replacing FM with a single-cell protein (SCP) derived from methanotrophic bacteria (Methylococcus capsulatus, Bath) in barramundi fry (Lates calcarifer). Three isonitrogenous and isoenergetic diets were formulated with 0%, 6.4% and 12.9% inclusion of the SCP, replacing FM by 0%, 25% and 50%. Barramundi fry (initial body weight 2.5 ± 0.1 g) were fed experimental diets for 21 days to assess growth performance, gut microbiome composition and gut histopathology. Our findings revealed that both levels of SCP inclusion induced detrimental effects in barramundi fry, including impaired growth and reduced survival compared with the control group (66.7% and 71.7% survival in diets replacing FM with SCP by 25% and 50%, respectively; p < .05). Both dietary treatments presented mild necrotizing enteritis with subepithelial oedema and accumulation of PAS positive, diastase resistant droplets within hepatocytes (ceroid hepatopathy) and pancreatic atrophy. Microbiome analysis revealed a marked shift in the gut microbial community with the expansion of potential opportunistic bacteria in the genus Aeromonas. Reduced overall performance in the highest inclusion level (50% SCP) was primarily associated with reduced feed intake, likely related to palatability issues, albeit pathological changes observed in gut and liver may also play a role. Our study highlights the importance of meticulous optimization of SCP inclusion levels in aquafeed formulations, and the need for species and life-stage specific assessments to ensure the health and welfare of fish in sustainable aquaculture practices.

Uncovering the microbial community structure and physiological profiles of terrestrial mud volcanoes: A comprehensive metagenomic insight towards their trichloroethylene biodegradation potentiality

Mud volcanoes are dynamic geological features releasing methane (CH4), carbon dioxide (CO2), and hydrocarbons, harboring diverse methane and hydrocarbon-degrading microbes. However, the potential application of these microbial communities in chlorinated hydrocarbons bioremediation purposes such as trichloroethylene (TCE) has not yet been explored. Hence, this study investigated the mud volcano's microbial diversity functional potentiality in TCE degradation as well as their eco-physiological profiling using metabolic activity. Geochemical analysis of the mud volcano samples revealed variations in pH, temperature, and oxidation-reduction potential, indicating diverse environmental conditions. The Biolog Ecoplate™ carbon substrates utilization pattern showed that the Tween 80 was highly consumed by mud volcanic microbial community. Similarly, MicroResp® analysis results demonstrated that presence of additive C-substrates condition might enhanced the cellular respiration process within mud-volcanic microbial community. Full-length 16 S rRNA sequencing identified Proteobacteria as the dominant phylum, with genera like Pseudomonas and Hydrogenophaga associated with chloroalkane degradation, and methanotrophic bacteria such as Methylomicrobium and Methylophaga linked to methane oxidation. Functional analysis uncovered diverse metabolic functions, including sulfur and methane metabolism and hydrocarbon degradation, with specific genes involved in methane oxidation and sulfur metabolism. These findings provide insights into the microbial diversity and metabolic capabilities of mud volcano ecosystems, which could facilitate their effective application in the bioremediation of chlorinated compounds.

Detection of Thermoacidophiles from Yellowstone Hot Spring

Aerobic methanotrophic bacteria maintain an unrivalled capability of utilizing methane as their sole carbon and energy source and have been retrieved from various environments. Phylogenetically, true aerobic thermoacidophilic methane oxidizers capable of growing below pH 3 have hitherto been associated only with the phylum Verrucomicrobia. In this report, the initial detection of a moderately thermoacidophilic Methylococcus-like Type Ib methanotroph of the class Gammaproteobacteria from an acidic thermal spring (50oC and pH 2.8) in the Yellowstone National Park, USA is presented. The isolate, termed YT-MC, was identified in a methane enrichment (55°C), which may represent a novel strain in the family Methylococcaceae Type Ib. The existence of this bacterium in the enrichments was demonstrated by the detection of pmoA gene, Southern blotting technique, phase-contrast, and electron microscopy. The coccus-typed cells showed tubular membranes instead of intracytoplasmic membrane systems (ICM). The soluble methane monooxygenase (sMMO) was not detected by PCR, indicating that the biotransformation of methane to methanol is oxidized by the particulate methane monooxygenase (pMMO). Moreover, YT-MC performs in a formerly undiscovered active biological methane sink in geothermal acidic environments, magnifying our knowledge of its ecological role in methane cycling, diversity, and coexistence of aerobic methanotrophy. Furthermore, the present study also reports the isolation and identification of an alphaproteobacterial heterotroph (strain YT-AC) and a verrucomicrobial methanotroph (strain YT-VM) from the same environment. Bangladesh J Microbiol, Volume 40, Number 2, December 2023, pp 86-94

Biosynthesis of polyhydroxybutyrate from methane and carbon dioxide using type II methanotrophs

Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.

Evolutionary ecology of denitrifying methanotrophic NC10 bacteria in the deep-sea biosphere.

The NC10 phylum links anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophic pathway. Although numerous amplicon-based studies revealed the distribution of this phylum, comprehensive genomic insights and niche characterization in deep-sea environments were still largely unknown. In this study, we extensively surveyed the NC10 bacteria across diverse deep-sea environments, including waters, sediments, cold seeps, biofilms, rocky substrates, and subseafloor aquifers. We then reconstructed and analysed 38 metagenome-assembled genomes (MAGs), and revealed the extensive distribution of NC10 bacteria and their intense selective pressure in these harsh environments. Isotopic analyses combined with gene expression profiling confirmed that active nitrite-dependent anaerobic methane oxidation (n-DAMO) occurs within deep-sea sediments. In addition, the identification of the Wood-Ljungdahl (WL) and 3-hydroxypropionate/4-hydroxybutyrat (3HB/4HP) pathways in these MAGs suggests their capability for carbon fixation as chemoautotrophs in these deep-sea environments. Indeed, we found that for their survival in the oligotrophic deep-sea biosphere, NC10 bacteria encode two branches of the WL pathway, utilizing acetyl-CoA from the carbonyl branch for citric acid cycle-based energy production and methane from the methyl branch for n-DAMO. The observed low ratios of non-synonymous substitutions to synonymous substitutions (pN/pS) in n-DAMO-related genes across these habitats suggest a pronounced purifying selection that is critical for the survival of NC10 bacteria in oligotrophic deep-sea environments. These findings not only advance our understanding of the evolutionary adaptations of NC10 bacteria but also underscore the intricate coupling between the carbon and nitrogen cycles within deep-sea ecosystems, driven by this bacterial phylum.

Impact of Rhizospheric Microbiome on Rice Cultivation.

The rhizosphere niche is extremely important for the overall growth and development of plants. Evidently, it is necessary to understand the complete mechanism of plant microbe interactions of the rhizosphere for sustainable and low input productivity. To meet the increasing global food demand, rice (Oryza sativa L.) agriculture seeks optimal conditions. The unique oxic-anoxic interface of rice-growing soil has invited divergent microbes with dynamic biogeochemical cycles. This review provides the systematic analysis of microbes associated with the major biogeochemical cycles with the aim to generate better management strategies of rhizospheric microbiome in the field of rice agriculture. For instance, several methanogenic and methanotrophic bacteria in the rice rhizosphere make an equilibrium for methane concentration in the environment. The carbon sequestration in paddy soil is again done through many rhizospheric microorganisms that can directly assimilate CO2 with their photoautotrophic mode of nutrition. Also the phosphate solubilizing microbes remain to be the most important keys for the PGPR activity of the paddy ecosystem. In addition, rhizospheric microbiome remain crucial in degradation and solubilization of organo-sulfur and insoluble inorganic sulfides which can be taken by the plants. Further, this review elucidates on the advantages of using metagenomic and metaproteomic approaches as an alternative of traditional approaches to understand the overall metabolic pathways operational in paddy-field. These knowledges are expected to open new possibilities for designing the balanced microbiome used as inoculum for intensive farming and will eventually lead to exert positive impacts on rice cultivation.

Leafcutter ants enhance microbial drought resilience in tropical forest soil.

We conducted a research campaign in a neotropical rainforest in Costa Rica throughout the drought phase of an El-Nino Southern Oscillation event to determine microbial community dynamics and soil C fluxes. Our study included nests of the leafcutter ant Atta cephalotes, as soil disturbances made by these ecosystem engineers may influence microbial drought response. Drought decreased the diversity of microbes and the abundance of core microbiome taxa, including Verrucomicrobial bacteria and Sordariomycete fungi. Despite initial responses of decreasing diversity and altered composition, 6 months post-drought the microbiomes were similar to pre-drought conditions, demonstrating the resilience of soil microbial communities to drought events. A. cephalotes nests altered fungal composition in the surrounding soil, and reduced both fungal mortality and growth of Acidobacteria post-drought. Drought increased CH4 consumption in soils due to lower soil moisture, and A. cephalotes nests decrease the variability of CH4 emissions in some soil types. CH4 emissions were tracked by the abundance of methanotrophic bacteria and fungal composition. These results characterize the microbiome of tropical soils across both time and space during drought and provide evidence for the importance of leafcutter ant nests in shaping soil microbiomes and enhancing microbial resilience during climatic perturbations.

New Solutions in Single-Cell Protein Production from Methane: Construction of Glycogen-Deficient Mutants of Methylococcus capsulatus MIR

The biotechnology of converting methane to single-cell protein (SCP) implies using fast-growing thermotolerant aerobic methanotrophic bacteria. Among the latter, members of the genus Methylococcus received significant research attention and are used in operating commercial plants. Methylococcus capsulatus MIR is a recently discovered member of this genus with the potential to be used for the purpose of SCP production. Like other Methylococcus species, this bacterium stores carbon and energy in the form of glycogen, particularly when grown under nitrogen-limiting conditions. The genome of strain MIR encodes two glycogen synthases, GlgA1 and GlgA2, which are only moderately related to each other. To obtain glycogen-free cell biomass of this methanotroph, glycogen synthase mutants, ΔglgA1, ΔglgA2, and ΔglgA1ΔglgA2, were constructed. The mutant lacking both glycogen synthases exhibited a glycogen-deficient phenotype, whereas the intracellular glycogen content was not reduced in strains defective in either GlgA1 or GlgA2, thus suggesting functional redundancy of these enzymes. Inactivation of the glk gene encoding glucokinase also resulted in a sharp decrease in glycogen content and accumulation of free glucose in cells. Wild-type strain MIR and the mutant strain ΔglgA1ΔglgA2 were also grown in a bioreactor operated in batch and continuous modes. Cell biomass of ΔglgA1ΔglgA2 mutant obtained during batch cultivation displayed high protein content (71% of dry cell weight (DCW) compared to 54% DCW in wild-type strain) as well as a strong reduction in glycogen content (10.8 mg/g DCW compared to 187.5 mg/g DCW in wild-type strain). The difference in protein and glycogen contents in biomass of these strains produced during continuous cultivation was less pronounced, yet biomass characteristics relevant to SCP production were slightly better for ΔglgA1ΔglgA2 mutant. Genome analysis revealed the presence of glgA1-like genes in all methanotrophs of the Gammaproteobacteria and Verrucomicrobia, while only a very few methanotrophic representatives of the Alphaproteobacteria possessed these determinants of glycogen biosynthesis. The glgA2-like genes were present only in genomes of gammaproteobacterial methanotrophs with predominantly halo- and thermotolerant phenotypes. The role of glycogen in terms of energy reserve is discussed.

Nitrous oxide respiration in acidophilic methanotrophs

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.

Optimization of electroporation method and promoter evaluation for type-1 methanotroph, Methylotuvimicrobium alcaliphilum.

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted vs. wild-type M. alcaliphilum strains (DASS vs. DSM19304). Overall, a ∼ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.

Holed up, but thriving: Impact of multitrophic cryoconite communities on glacier elemental cycles

Cryoconite holes (water and sediment-filled depressions), found on glacier surfaces worldwide, serve as reservoirs of microbes, carbon, trace elements, and nutrients, transferring these components downstream via glacier hydrological networks. Through targeted amplicon sequencing of carbon and nitrogen cycling genes, coupled with functional inference-based methods, we explore the functional diversity of these mini-ecosystems within Antarctica and the Himalayas. These regions showcase distinct environmental gradients and experience varying rates of environmental change influenced by global climatic shifts. Analysis revealed a diverse array of photosynthetic microorganisms, including Stramenopiles, Cyanobacteria, Rhizobiales, Burkholderiales, and photosynthetic purple sulfur Proteobacteria. Functional inference highlighted the high potential for carbohydrate, amino acid, and lipid metabolism in the Himalayan region, where organic carbon concentrations surpassed those in Antarctica by up to 2 orders of magnitude. Nitrogen cycling processes, including fixation, nitrification, and denitrification, are evident, with Antarctic cryoconite exhibiting a pronounced capacity for nitrogen fixation, potentially compensating for the limited nitrate concentrations in this region. Processes associated with the respiration of elemental sulfur and inorganic sulfur compounds such as sulfate, sulfite, thiosulfate, and sulfide suggest the presence of a complete sulfur cycle. The Himalayan region exhibits a higher potential for sulfur cycling, likely due to the abundant sulfate ions and sulfur-bearing minerals in this region. The capability for complete iron cycling through iron oxidation and reduction reactions was also predicted. Methanogenic archaea that produce methane during organic matter decomposition and methanotrophic bacteria that utilize methane as carbon and energy sources co-exist in the cryoconite, suggesting that these niches support the complete cycling of methane. Additionally, the presence of various microfauna suggests the existence of a complex food web. Collectively, these results indicate that cryoconite holes are self-sustaining ecosystems that drive elemental cycles on glaciers and potentially control carbon, nitrogen, sulfur, and iron exports downstream.

High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation

BackgroundMethane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products.ResultsIn this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high–cell–density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane.ConclusionsThis study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Living emission abolish filters (LEAFs) for methane mitigation: design and operation

As one of the most potent greenhouse gases, methane is a critical target for the near-term mitigation of global warming. Efficient, scalable, easy-to-implement, and robust mitigation technologies are urgently needed to assist in reaching methane abolishment. The goal of this research was to test the applicability of active, extremophilic methanotrophic cells as a baseline concept for engineered systems aiming at methane capturing. The system, named living emission abolish filters (LEAFs), represents an array of immobilized biomaterials capable of capturing methane directly from vent streams. The biomaterials were made using cells of Methylotuvimicrobium alcaliphilum 20ZR, a robust halophilic methanotrophic bacterium with the ability to consume methane gas at low concentrations. Several critical parameters were tested, including (i) the composition of the matrix and optimal immobilization to increase catalyst load, (ii) the stability of methanotrophic cells, and (iii) the toxicity of trace gases (i.e. CO). We found that hydrogels coated with 2.3 mg cell dry weight/cm3 methanotrophic cells represent the best-performing biomaterials. The methane reduction potential of LEAFs fluctuated from 20% to 95% and depended on the methane concentration in the gas stream and the stream flow rates. The potential for commercial-scale deployment and emissions reductions was also evaluated. Total greenhouse gas emissions (combined using the global warming potential GWP100) from an example using a ventilation air methane source over a one-year period was shown to be reduced in two LEAF scenarios by 51% and 75%. Over longer time horizons, more significant reductions are possible as consistent methane consumption can be sustained. The study highlights the overall potential of the liquid-free bio-based composite methane mitigation system. Further improvements essential for system assembly and implementations should include (a) optimization of the cell immobilization protocols to improve cell load and the shelf-life of the system and (b) implementation of matrix moldings for cell immobilization to achieve optimal gas flow and increase the cell-gas interface.