USE OF METHANE IN CLOSED-LOOP LIFE SUPPORT SYSTEMS FOR SPACE MISSIONS

The paper discusses the use of methane (generated in the process of oxygen recovery from carbon dioxide released by the crew during its hydrogenation in the Sabatier reaction, with subsequent extraction of 61% of oxygen through electrolysis of the resultant water) in a regenerative life support system for crews on space missions. It demonstrates that the methane resulting from Sabatier reaction can be used both for pyrolysis in order to return the resulting hydrogen into this reaction so as to extract 100% of oxygen from carbon dioxide, and for producing food protein for life support in space. The use of methane pyrolysis was enabled by new technologies which allowed lowering the process temperature down to 500–700°C and obtaining the easy-to-remove carbon. It provides recommendations for designing space systems for methane pyrolysis. The paper makes the case for use of the existing processes for industrial production of protein from methane using methanotrophic bacteria in the production of food protein for space food rations, determines the balance of a closed-loop methanotrophic reaction, provides calculation basis and recommendations for designing space systems for methanotrophic production of food protein. Development of a system for food protein production from methane will enable its use as one of the systems for providing food on the Moon and Mars, as well as a backup system in space transportation missions. Key words: space missions, crew life support, СО2 hydration, methane pyrolysis, methanotrophic bacteria, food protein.

Nitrogen (N2) fixation activity under different oxygen concentration of methanotrophic bacteria isolated from rice fields and their molecular identification

Rice fields are a significantly sources of atmospheric methane. Methanotrophic bacteria are unique in their ability to utilize methane as a sole carbon source and their ability to fix N2. This research successfully characterized N2 fixation activity under different oxygen concentrations of methanotrophic bacteria isolated from rice fields. From 19 tested isolates, four isolates performed activity to fix N2. They could fix N2 on different concentration of air saturation (10 % up to 100%). The growth of methanotrophs is not directly corelated with the N2 fixation activity, and their N2 fixation activities are affected by O2 concentrations. The BGM 3 and BGM 9 isolates had very good N2 fixation activity. Their activities were increased by increasing air saturation up to 50% (approximately 10% O2), but then decrease by increasing air saturation from 50% (approximately 10% O2) to 100% (approximately 20% O2). However, the highest N2 fixation activity was performed by the BGM 9 isolate at 30% air saturation (approximately 6% O2), and the isolate was identified as Methylococcus capsulatus. This information can support application of the isolates to achieve sustainable and environmentally friendly agricultural system.

Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production

Background As methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry. Results Through three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24–410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with PrpmB-cadA and PDnaA-cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply. Conclusions The tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory. Graphical Abstract

Multiple groups of methanotrophic bacteria mediate methane oxidation in anoxic lake sediments

Freshwater lakes represent an important source of the potent greenhouse gas methane (CH4) to the atmosphere. Methane emissions are regulated to large parts by aerobic (MOx) and anaerobic (AOM) oxidation of methane that are important sinks in lakes. In contrast to marine benthic environments, our knowledge about the modes of AOM and the related methanotrophic microorganisms in anoxic lake sediments is still rudimentary. Here we demonstrate the occurrence of AOM in the anoxic sediments of Lake Sempach (Switzerland), with maximum in situ AOM rates observed within the surface sediment layers in presence of multiple groups of methanotrophic bacteria and various oxidants known to support AOM. However, substrate-amended incubations (with NO2-, NO3-, SO42-, Fe3+ and Mn4+) revealed that none of the electron acceptors previously reported to support AOM enhanced methane turnover in Lake Sempach sediments under anoxic conditions. In contrast, the addition of oxygen to the anoxic sediments resulted in an approximately tenfold increase in methane oxidation relative to the anoxic incubations. Phylogenetic and isotopic evidence indicate that both Type I and Type II aerobic methanotrophs were growing on methane under both oxic and anoxic conditions, although methane assimilation rates were an order of magnitude higher under oxic conditions. While the anaerobic electron acceptor responsible for AOM could not be identified, these findings expand our understanding of the metabolic versatility of canonically aerobic methanotrophs under anoxic conditions, with important implications for future investigations to identify methane oxidation processes. Bacterial AOM by facultative aerobic methane oxidizers might be of much larger environmental significance in reducing methane emissions than previously thought.

Seasonal dynamics of methanotrophic bacteria in a boreal oil sands end-pit lake

Base Mine Lake (BML) is the first full-scale demonstration end pit lake for the oil sands mining industry in Canada. We examined aerobic methanotrophic bacteria over all seasons for five years in this dimictic lake. Methanotrophs comprised up to 58% of all bacterial reads in 16S rRNA gene amplicon sequencing analyses (median 2.8%), and up to 2.7 × 10 4 cells mL −1 of water (median 0.5 × 10 3 ) based on qPCR of pmoA genes. Methanotrophic activity and populations in the lake water were highest during fall turnover, and remained high through the winter ice-covered period into spring turnover. They declined during summer stratification, especially in the epilimnion. Three methanotroph genera ( Methylobacter , Methylovulum , and Methyloparacoccus ) cycled seasonally, based on both relative and absolute abundance measurements. Methylobacter and Methylovulum populations peaked in winter/spring, when methane oxidation activity was psychrophilic. Methyloparacoccus populations increased in the water column through summer and fall, when methane oxidation was mesophilic, and also predominated in the underlying tailings sediment. Other, less abundant genera grew primarily during summer, possibly due to distinct CH 4 /O 2 microniches created during thermal stratification. These data are consistent with temporal and spatial niche differentiation based on temperature, CH 4 and O 2 . This pit lake displays methane cycling and methanotroph population dynamics similar to natural boreal lakes. Importance statement: The study examined methanotrophic bacteria in an industrial end pit lake, combining molecular DNA methods (both quantitative and descriptive) with biogeochemical measurements. The lake was sampled over 5 years, in all four seasons, as often as weekly, and included sub-ice samples. The resulting multi-season and multi-year dataset is unique in its size and intensity, and allowed us to document clear and consistent seasonal patterns of growth and decline of three methanotroph genera ( Methylobacter , Methylovulum , and Methyloparacoccus ). Laboratory experiments suggested that one major control of this succession was niche partitioning based on temperature. The study helps to understand microbial dynamics in engineered end-pit lakes, but we propose that the dynamics are typical of boreal stratified lakes, and widely applicable in microbial ecology and limnology. Methane oxidising bacteria are important model organisms in microbial ecology, and have implications for global climate change.