Adaptation of Methylomicrobium album BG8 to growth at low pH

Abstract

Methanotrophic bacteria are capable of converting single carbon sources such as methane or methanol, two common industrial waste products, into value-added compounds like bioplastics or biofuels. These microorganisms have significant potential for the mitigation of these low-value industrial by-products considered as waste in many industries, including energy and pulp and paper. The implementation of these bacteria has proven difficult outside of the laboratory as they grow slowly and demonstrate decreased production under industrial conditions. One potential solution to alleviate these issues is to apply adaptive evolution strategies to improve growth characteristics of the organism. In this study, the methanotrophic bacterium Methylomicrobium album BG8 was adapted to growth at low pH. The growth of M. album BG8 was screened over a range of pH in both nitrate- and ammonium-based media. Since growth was not conducive at lower pH in ammonium- based medium, the study focused on adaptation in nitrate-based medium. Cells were adapted by sequential growth in media with decreasing pH, ranging from pH 6.86 (standard conditions) down to pH 3.80 when grown on methane and to pH 3.85 when grown on methanol. The growth of adapted cells was compared to that of unadapted cells at low and standard pH conditions. Adapted cells showed greatly improved performance at low pH and no alteration to performance at standard pH. Cell adaptation remained stable after passaging from low pH to standard pH and returned to low pH. Again, no loss or change in performance was observed upon passaging, suggesting the adaptation was stable and at least not solely phenotypic. Transmission Electron Microscopy of cells showed minor differences in the cell shape and structure of M. album BG8 growing at low ii and standard pH in both adapted and unadapted cells. However, one significant difference was the loss of production of outer membrane vesicles (OMVs) in adapted cells. DNA analysis showed numerous mutations and Single nucleotide polymorphisms (SNPs) between adapted cells and the reference M. album BG8 genome of the parental strain. Adaptation in methane or methanol resulted in many of the same differences from the parental strain with a handful of different mutations for each carbon source. There were a number of mutations and SNPs located in genes related to membrane composition and functions, DNA/RNA synthesis and repair, and transposases – all of which can have a function in adapting cells to new environments. This work provides a template for adaptation of microorganisms to harsh environments and a starting point for adaption of M. album BG8 to growth in specific waste stream conditions, which could provide the potential to speed up industrial implementation of methanotrophs, decrease risks associated with moving from the lab to industry, and improve the performance of the industrial process.