(160ap) Investigation of Co-Substrate Utilization By E. Limosum for Biofuel Applications

Methanol is a desirable carbon source for bioprocessing due to its existing industrial infrastructure and availability from conventional or renewable sources that do not compete with food crops. Certain acetogenic bacteria are able to convert methanol to acetic acid at exceptionally high yield, fixing carbon dioxide in the process. We are researching the metabolism of Eubacterium limosum, a methylotrophic acetogen, with the aim of engineering it to use methanol to produce higher-value chemicals. Though the yield is high, the growth and substrate uptake rates are slow (specific growth rate is 0.08 h^-1 and methanol uptake rate is 9 mmol/gDCW/h). It has been observed that in the presence of glucose, the specific growth rate and methanol uptake rate are higher than with either substrate individually (7.5 times higher and 33% higher, respectively).¹ These increases suggest that providing glucose as a co-substrate with methanol could enhance the organism’s utilization of methanol, making methanol and its associated benefits more viable for industrial applications. To understand the mechanism behind the synergistic effect of this co-utilization, we are employing a combination of experimental and computational approaches.

First, the eVOLVER reactor system² was adapted into “AneVO,” to support anaerobic growth on methanol with continuous feeding of glucose. This adapted reactor system allows for growth and monitoring of continuous anaerobic cultures, each with a separate feed rate, and the ability to individually control dilution rate, agitation, and temperature. AneVO was used to perform co-substrate utilization experiments in which E. limosum was grown with methanol alone, glucose alone, and methanol with a range of glucose feed rates. The cultures were initiated with methanol, then glucose was slowly delivered during active growth at six different carbon ratios ranging from 0.05 up to 32 (cumulative carbon-moles of glucose to initial carbon-moles of methanol). Based on substrate uptake and confirmed by isotopic labeling, co-metabolism was observed for ratios of 2 and below. Interestingly, the specific growth rate was approximately 6 times higher for the culture grown at a ratio of 0.6 C-moles of glucose to methanol, relative to methanol-only grown cultures. Liquid Chromatography Mass Spectrometry (LC-MS) and the growth data will be used to perform Metabolic Flux Analysis (MFA), which will determine the amount (or flux) and source of carbon that passes through each metabolic step, including pathway intermediates. Differences in the flux maps may show preferred paths that are enabled by each substrate condition. Conversely, differences in the flux maps may also show metabolic bottlenecks that are overcome by co-metabolism. The MFA will provide insight into the mechanism of methanol-glucose co-metabolism and its benefits, which may be used to engineer methylotrophic biocatalysts. Preliminary results from these initial investigations will be shared.

Citations

1. Loubiere, P., Gros, E., Paquet, V. & Lindley, N. D. Kinetics and physiological implications of the growth behaviour of Eubacterium limosum on glucose/methanol mixtures. J. Gen. Microbiol. 138, 979–985 (1992).
2. Wong, B. G., Mancuso, C. P., Kiriakov, S., Bashor, C. J. & Khalil, A. S. Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER. Nat. Biotechnol. 36, 614–623 (2018).