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Optimizing media and planktonic culture conditions for in vitro experimentation using free-living Bradyrhizobium japonicum USDA110

  • Author(s): Welch, Zoe Sabrina
  • Advisor(s): Holden, Patricia A
  • et al.
Abstract

Bradyrhizobium japonicum USDA110 is an agronomically important bacterium with the ability to live as both an N2-fixing, soybean symbiont and a free-living, heterotrophic cell. Free-living Bradyrhizobium japonicum USDA110 has been successfully cultured in lab settings since the 20th century, and has been used in a variety of planktonic growth-based studies using various culture media. Recent sequencing and annotation of the Bradyrhizobium japonicum USDA110 genome has enabled further “-omics” based advancements, thus increasing the attractiveness of envisioning Bradyrhizobium japonicum USDA110 as a model organism for in vitro work. Further, as numerous in planta studies have indicated that Bradyrhizobium japonicum USDA110 possesses an inherent sensitivity to a variety of environmental stressors, this tendency reifies a position held by some soil ecologists that rhizobia may be conceptualized of as indicator species for agricultural soil systems. As there is increasing interest in developing reliable, in vitro, high throughput (HTP) screening strategies for hazard assessment in environmental compartments, it is worthwhile to examine how testing with Bradyrhizobium japonicum USDA110 could be used towards this purpose for agricultural soil systems. However, in order to use it to test metal-based toxicants, it is necessary to optimize a reliable testing paradigm —inclusive of a Bradyrhizobium japonicum USDA110 growth medium that is simultaneously defined, environmentally-relevant, supportive of robust culture growth, and which has been designed towards maintaining consistent exposure conditions over the duration of culture growth. Previously used Bradyrhizobium japonicum USDA110 growth media are problematic, largely due to considerations relating to undefined or chemically incompatible composition. This work customizes a better Bradyrhizobium japonicum USDA110 growth medium by using a stepwise approach of literature review (to create a comprehensive database of rhizobium growth medium recipes and construct a medium foundation), in silico modeling (to predict chemical speciation thus allowing for a “modeling out” of precipitates) for further medium design, and empirical testing (Bradyrhizobium japonicum USDA110 growth studies in varying, single-alteration medium formulations) to confirm the ability of the finalized medium design (ZY medium) to support optimal growth of Bradyrhizobium japonicum USDA110 within the tested constraints. I found that ZY supports robust Bradyrhizobium japonicum USDA110 population growth, and that microtiter culture conditions enabled high reproducibility of growth results as determined by specific growth rate and maximum population yield metrics calculated from hourly absorbance measurements. Optimized growth occurred with glycerol as the sole C source, nitrate as the sole N source, and with thiamine and biotin being absent from the medium. Further, I found evidence in support of P being a limiting nutrient for Bradyrhizobium japonicum USDA110. Preliminary Cu salt exposure studies show high reproducibility and possible dose-dependent effects, but also indicate that further system characterization is likely needed. This thesis work establishes a defined and environmentally-relevant USDA110 culture medium (ZY) and reliable testing methodology that facilitates planktonic growth and improved in vitro toxicity testing capacity.

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