Methanotrophs use methane as a sole carbon source and thus play a critical role in its global consumption. Intensified interest in methanotrophs for their low-cost production of value-added products and large-scale industrialization has led to investigations of strain-to-strain variation in parameters for growth optimization and metabolic regulation. In this study, Methylocystis sp. Rockwell was grown with methane or methanol as a carbon source and ammonium or nitrate as a nitrogen source. The intracellular metabolomes and production of polyhydroxybutyrate, a bioplastic precursor, were compared among treatments to determine how the different combinations of carbon and nitrogen sources affected metabolite production. The methane-ammonium condition resulted in the highest growth, followed by the methane-nitrate, methanol-nitrate, and methanol-ammonium conditions. Overall, the methane-ammonium and methane-nitrate conditions directed metabolism toward energy-conserving pathways, while methanol-ammonium and methanol-nitrate directed the metabolic response toward starvation pathways. Polyhydroxybutyrate was produced at greater abundances in methanol-grown cells, independent of the nitrogen source. Together, the results revealed how Methylocystis sp. Rockwell altered its metabolism with different combinations of carbon and nitrogen source, with implications for production of industrially relevant metabolites.
Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp. Rockwell was grown on methane with three different nitrogen sources (ammonium, nitrate, and atmospheric nitrogen), and biomass samples were harvested at defined time points during lag, exponential, and stationary growth phases. PHB cell content was analyzed at these sampling points via a standard gas chromatography-flame ionization detector method, which requires hydrolysis of PHB and esterification of the resulting monomer under acidic conditions, and a novel, rapid, cost-effective approach based on fixation and staining of bacterial cells via Nile Blue A fluorescent dye enabling differential staining of cell membranes and intracellular PHB granules for single-cell analysis through fluorescence microscopy. Overall, the two PHB quantification approaches were in agreement at all stages of growth and in all three growing conditions tested. The PHB cell content was greatest with atmospheric nitrogen as a nitrogen source, followed by ammonium and nitrate. Under atmospheric nitrogen and ammonium conditions, PHB cell content decreased with growth progression, while under nitrate conditions PHB cell content remained unchanged in all growth phases. In addition to presenting a rapid, efficient method enabling in vivo quantification of PHB production, the present study highlights the impact of nitrogen source on PHB production by Methylocystis sp. Rockwell. Key points• A novel fluorescence microscopy method to quantify PHB in single cells was developed • The microscopy method was validated by the derivation/gas chromatography method • Methylocystis sp. Rockwell synthesizes PHB granules without nutrient stress Keywords Polyhydroxybutyrate • Methylocystis sp. Rockwell • Methanotroph • Fluorescence microscopy • Granule visualization • Gas chromatography Marina Lazic and Ravindra Gudneppanavar contributed equally to this work.
Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled the phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated hydroxylamine dehydrogenase ( haoAB ) and overall central metabolic activity, whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species. IMPORTANCE Methanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism varies across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.
Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled its phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated haoAB hydroxylamine dehydrogenase and the overall central metabolic activity; whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species.
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