Abstract
Methane, a potent greenhouse gas, and methanol, commonly called wood alcohol, are common by-products of modern industrial processes. They can, however, be consumed as a feedstock by bacteria known as methanotrophs, which can serve as useful vectors for biotransformation and bioproduction. Successful implementation in industrial settings relies upon efficient growth and bioconversion, and the optimization of culturing conditions for these bacteria remains an ongoing effort, complicated by the wide variety of characteristics present in the methanotroph culture collection. Here, we demonstrate the variable growth outcomes of five diverse methanotrophic strains – Methylocystis sp. Rockwell, Methylocystis sp. WRRC1, Methylosinus trichosporium OB3b, Methylomicrobium album BG8, and Methylomonas denitrificans FJG1 – grown on either methane or methanol, at three different concentrations, with either ammonium or nitrate provided as nitrogen source. Maximum optical density (OD), growth rate, and biomass yield were assessed for each condition. Further metabolite and fatty acid methyl ester (FAME) analyses were completed for Methylocystis sp. Rockwell and M. album BG8. The results indicate differential response to these growth conditions, with a general preference for ammonium-based growth over nitrate, except for M. denitrificans FJG1. Methane is also preferred by most strains, with methanol resulting in unreliable or inhibited growth in all but M. album BG8. Metabolite analysis points to monitoring of excreted formic acid as a potential indicator of adverse growth conditions, while the magnitude of FAME variation between conditions may point to strains with broader substrate tolerance. These findings suggest that methanotroph strains must be carefully evaluated before use in industry, both to identify optimal conditions and to ensure the strain selected is appropriate for the process of interest. Much work remains in addressing the optimization of growth strategies for these promising microorganisms since disregarding these important steps in process development could ultimately lead to inefficient or failed bioprocesses.
Highlights
Methane-oxidizing bacteria (MOB), or methanotrophs, oxidize single-carbon molecules, methane, to be used as their sole carbon and energy source
At 2.5 mmol methane, the cultures were found to be oxygen limited as the onset of stationary phase coincided with the depletion of oxygen, while methane remained in the gas headspace (Supplementary Figure S2)
Due to the mass transfer limitation of methane into the liquid medium, the apparent carbon availability to the culture is mediated by the surface area of the liquid–gas interface, whereas methanol is immediately available to the culture
Summary
Methane-oxidizing bacteria (MOB), or methanotrophs, oxidize single-carbon molecules, methane, to be used as their sole carbon and energy source. Within the Proteobacteria, which encompass the majority of currently cultured methanotrophs, MOB can be further classified as Alphaproteobacteria (Alpha-MOB and Type II) or Gammaproteobacteria (Gamma-MOB, Type I, or Type X), with each group having distinct physiological traits. Differentiating traits include their primary central carbon pathways (serine pathway in Alpha-MOB and ribulose monophosphate pathway in Gamma-MOB), orientation and distribution of intracytoplasmic membranes (ICMs), and composition of lipids in terms of fatty acid proportions (Hanson and Hanson, 1996). The related strain M. buryatense 5GB1 grew better on methane than on methanol in a bioreactor, but still demonstrated robust growth on methanol (Gilman et al, 2015), as did Methylomicrobium alcaliphilum 20Z (Akberdin et al, 2018)
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