Flux Balance Analysis Indicates that Methane Is the Lowest Cost Feedstock for Microbial Cell Factories

Abstract

The low cost of natural gas has driven significant interest in using C₁ carbon sources (e.g. methane, methanol, CO, syngas) as feedstocks for producing liquid transportation fuels and commodity chemicals. Given the large contribution of sugar and lignocellulosic feedstocks to biorefinery operating costs, natural gas and other C1 sources may provide an economic advantage. To assess the relative costs of these feedstocks, we performed flux balance analysis on genome-scale metabolic models to calculate the maximum theoretical yields of chemical products from methane, methanol, acetate, and glucose. Yield calculations were performed for every metabolite (as a proxy for desired products) in the genome-scale metabolic models of three organisms: Escherichia coli (bacterium), Saccharomyces cerevisiae (yeast), and Synechococcus sp. PCC 7002 (cyanobacterium). The calculated theoretical yields and current feedstock prices provided inputs to create comparative feedstock cost surfaces. Our analysis shows that, at current market prices, methane feedstock costs are consistently lower than glucose when used as a carbon and energy source for microbial chemical production. Conversely, methanol is costlier than glucose under almost all price scenarios. Acetate feedstock costs could be less than glucose given efficient acetate production from low-cost syngas using nascent biological gas to liquids (BIO-GTL) technologies. Our analysis suggests that research should focus on overcoming the technical challenges of methane assimilation and/or yield of acetate via BIO-GTL to take advantage of low-cost natural gas rather than using methanol as a feedstock.

Fig. 1

Formaldehyde assimilation pathways. The ribulose mono-phosphate (RuMP) pathway is a bacterial formaldehyde assimilation pathway that uses ribulose-5-phosphate as a substrate for formaldehyde assimilation. The dihydroxyacetone (DHA) pathway is a fungal formaldehyde assimilation pathway that uses xylulose-5-phosphate as a substrate for formaldehyde assimilation. The serine pathway is a bacterial pathway that uses glycine to assimilate formaldehyde. The serine pathway also assimilates one carbon dioxide for every two formaldehydes assimilated. All three pathways produce glycolytic intermediates. The abbreviations are defined as follows: CH4 – methane, MeOH – methanol, CHO – formaldehyde, CO2 – carbon dioxide, H6P – hexulose 6-phosphate, F6P – fructose 6-phosphate, FBP – fructose 1,6 bisphosphate, DHAP – dihydroxyacetone phosphate, G3P – glyceraldehyde 3-phosphate, E4P – eyrthrose 4-phosphate, S7P – septulose 7-phosphate, R5P – ribose 5-phosphate, Xu5P – xylulose 5-phosphate, Ru5P – ribulose 5-phosphate, DHA – dihydroxyacetone, Ser – serine, HPyr – hydroxypyruvate, Glyc – glycerate, 2PG – 2-phosphoglycerate, 3PG –3-phosphoglycerate, PEP – phosphoenolpyruvate, HCO3 – bicarbonate, OAc – oxaloacetate, MAL – malate, MALC – malyl-CoA, AcC – acetyl-CoA, GLX – glyoxylate, GLY – glycine.

Fig. 2

The comparative product yields for alternative carbon sources compared to glucose are shown with individual products (points) as well as the least squares linear fit through the origin (lines). Equations for each alternative carbon source can be found in Supplemental Table 7. A, B, and C: Comparative yields for E. coli, S. cerevisiae, and S. PCC7002 respectively are shown for acetate, methanol, and methane carbon sources (E. coli also shows results for glycerol and xylose). D:E. coli comparative yields normalized on a per carbon basis on multiple substrates. E:E. coli comparative yields with a minimum of 10% of the maximum biomass growth rate (dashed lines) compared to no biomass requirement (solid lines). F: Comparative yields for a subset of data points from A as well as three heterologous pathway target products, as listed in Table 1, in comparison to the linear regressions determined in A.

Fig. 3

A–C: C₁ assimilation pathway usage across different substrates and organisms. These box plots summarize the yield of each metabolite from the model and carbon source listed as a ratio to the yield from the full model using all three formaldehyde assimilation pathways. The base model has no formaldehyde assimilation pathways. The box plot whiskers are determined using Tukey method and outliers are not shown (outliers shown in Supplemental Figure 1). D: A comparison of different pathways for carbon efficient assimilation of glycolytic substrates in E. coli. All pathways provided similar yield ratios (using linear regression) and were tested only feeding the substrate listed below each set of bars. (Base = Oxidative Glycolysis only, NOG = Non-oxidative Glycolysis, WLI = Wood-Ljungdhal Pathway, 3HP4HB = 3-hydroxypropionate 4-hydroxybutyrate Pathway).

Fig. 4

Surfaces of price adjusted yield ratios calculated over a range of methane, methanol, and glucose substrate prices. The black box represents the range of current market prices (we did not estimate market prices for acetate price). Values above one indicate higher alternate carbon source profitability, while values below one indicate higher glucose profitability.