(579h) A Novel Phenotype of Methanotroph Methylomicrobium Buryatense 5GB1 Induced By Extreme Oxidative Stress

Aerobic methanotrophs are bacteria that can utilize methane as the sole source of carbon and energy for growth. Methanotrophs play important roles in the global carbon and nitrogen cycles, including the control of emissions of anthropogenic and natural gas methane. Type I methanotrophs have drawn significant research interest in the last decade due to their potential for commercial methane bioconversion to value-added products such as fuels and chemicals.¹ Haloalkaliphilic methanotrophs, such as Methylomicrobium buryatense 5GB1 and Methylomicrobium alcaliphilum 20Z, are especially promising biocatalysts due to their high growth rate and high resistance to contamination under the preferred high pH and high salt growth conditions.^2.3

In our prior research, we found that for M. buryatense 5GB1, methane-limited phenotype yields higher organic compounds production than oxygen-limited phenotype.⁴ This is contrary to the common belief that oxygen-limited phenotype delivers increased production of organic compounds. In this work, we report a novel phenotype of haloalkaliphilic methanotrophs induced by extreme oxidative stress. By subjecting the cells (M. buryatense 5GB1) to extremely high oxidative stress (80% or 90% O₂), we discovered that the methanotrophs produced significant amount of formate. For M. buryatense 5GB1, the average organic carbon yield was 32% with formate making up the majority, compared to that of 1.1-1.5% under optimal oxygen conditions.⁵ It is worth noting that under the extreme oxygen condition, the formate excretion was increased by an order of magnitude compared to the reported values.⁶

In this talk, we discuss our efforts in characterizing this novel phenotype through both batch and continuous cultivations. Specifically, M. buryatense 5GB1 was cultivated under different oxygen conditions (20% and 80% O₂) with either batch or continuous operation. We analyzed the growth rates, carbon yield distribution, and transcriptomic profiles of cells grown under different conditions. These data depicted a downregulated methane oxidation pathway, except for formate dehydrogenase. Other up-regulated enzymes, such as methyltransferases and superoxide dismutase, confirm that the novel phenotype was a result of oxidative stress conditions.

References

1. Kalyuzhnaya, M. G., Puri, A. W., & Lidstrom, M. E. (2015). Metabolic engineering in methanotrophic bacteria. Metabolic engineering, 29, 142-152.
2. Fu, Y., He, L., Reeve, J., Beck, D. A., & Lidstrom, M. E. (2019). Core metabolism shifts during growth on methanol versus methane in the methanotroph Methylomicrobium buryatense 5GB1. MBio, 10(2), e00406-19.
3. Akberdin, I.R., Thompson, M., Hamilton, R., Desai, N., Alexander, D., Henard, C.A., Guarnieri, M.T. and Kalyuzhnaya, M.G. (2018). Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach. Scientific reports, 8(1), 1-13.
4. A. Stone, M.V. Hilliard, K. Badr, A.D. Bradford, Q.P. He, and J. Wang, Comparative study of oxygen-limited and methane-limited growth phenotypes of Methylomicrobium buryatense 5GB1, Biochemical Engineering Journal, 161 (2020), p.107707.
5. Gilman, A., Laurens, L.M., Puri, A.W., Chu, F., Pienkos, P.T. and Lidstrom, M.E., 2015. Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1. Microbial cell factories, 14(1), pp.1-8.
6. Gilman A, Fu Y, Hendershott M, Chu F, Puri AW, Smith AL, Pesesky M, Lieberman R, Beck DAC, Lidstrom ME., 2017. Oxygen-limited metabolism in the methanotroph Methylomicrobium buryatense 5GB1C. PeerJ 5:e3945