Controlling Ethanol Use in Chain Elongation by CO2 Loading Rate

Abstract

Chain elongation is an open-culture biotechnological process which converts volatile fatty acids (VFAs) into medium chain fatty acids (MCFAs) using ethanol and other reduced substrates. The objective of this study was to investigate the quantitative effect of CO₂ loading rate on ethanol usages in a chain elongation process. We supplied different rates of CO₂ to a continuously stirred anaerobic reactor, fed with ethanol and propionate. Ethanol was used to upgrade ethanol itself into caproate and to upgrade the supplied VFA (propionate) into heptanoate. A high CO₂ loading rate (2.5 L_CO2·L^-1·d^-1) stimulated excessive ethanol oxidation (EEO; up to 29%) which resulted in a high caproate production (10.8 g·L^-1·d^-1). A low CO₂ loading rate (0.5 L_CO2·L^-1·d^-1) reduced EEO (16%) and caproate production (2.9 g·L^-1·d^-1). Heptanoate production by VFA upgrading remained constant (∼1.8 g·L^-1·d^-1) at CO₂ loading rates higher than or equal to 1 L_CO2·L^-1·d^-1. CO₂ was likely essential for growth of chain elongating microorganisms while it also stimulated syntrophic ethanol oxidation. A high CO₂ loading rate must be selected to upgrade ethanol (e.g., from lignocellulosic bioethanol) into MCFAs whereas lower CO₂ loading rates must be selected to upgrade VFAs (e.g., from acidified organic residues) into MCFAs while minimizing use of costly ethanol.

Figure 1

Graphical summary of the effect of CO₂ loading rate on reactor performance with net production and consumption rates over time. At the red stars, samples for bacterial community analysis were taken. At the green star, a sample for archaeal community analysis was taken. T = 30 °C, pH 6.8, HRT = 17 h, V = 1 L.

Figure 2

(a) Net production and consumption rates of fatty acids and ethanol at different CO₂ loading rates (bars indicate standard deviations but are often too small to be visual), (b) carbon flux of EEO, caproate production and methane production at different CO₂ loading rates, (c) rate of methane vs EEO. Presented values at 0 L_CO2·L^–1·d^–1 are not steady state values but averages. Regression line = —.

Figure 3

Heatmap of bacterial community at different CO₂ loading rates in granular and suspended sludge. Numbers indicate percentage relative abundance. “Other” are specified in Figure S3. The ethanol loading rate at 2.5 L_CO2·L^–1·d^–1 was 32.2 g·L^–1·d^–1 whereas the ethanol loading rate at 1.0 and 0.0 L_CO2·L^–1·d^–1 was 16.3 g·L^–1·d^–1. IBC = initial bacterial community.

Figure 4

Sankey diagram with observed and calculated carbon fluxes at high (2.5 L_CO2·L^–1·d^–1) and low (0.5 L_CO2·L^–1·d^–1) CO₂ loading rate. Widths of arrows are proportional to carbon fluxes. Three functional groups of microorganisms (hydrogenotrophic methanogens, ethanol oxidizers, and chain elongating microorganisms) are indicated as green shapes. Calculations on carbon fluxes and numerical values are shown in SI Table S2. (I) Total CO₂ use, (I a) CO₂ use by methanogens, (I b) unidentified CO₂ use (i.e., biomass), (II) total ethanol use, (II a) excessive ethanol oxidation (EEO), (II b) ethanol oxidation through the reverse β-oxidation pathway, (II c) ethanol use for elongation of fatty acids through the reverse β-oxidation pathway (even), (II d) ethanol use for elongation of fatty acids through the reverse β-oxidation pathway (odd), (III) propionate use for VFA upgrading, (IV) (interspecies) hydrogen transfer, (V) acetate use for ethanol upgrading, (VI) methane production, (VII) hydrogen production, (VIII) unidentified acetate use (i.e., biomass), (IX) acetate production, (X) butyrate, caproate and caprylate production by ethanol upgrading, (XI) valerate and heptanoate production by VFA upgrading.