Sequential Mixed Cultures: From Syngas to Malic Acid

Abstract

Synthesis gas (syngas) fermentation using acetogenic bacteria is an approach for production of bulk chemicals like acetate, ethanol, butanol, or 2,3-butandiol avoiding the fuel vs. food debate by using carbon monoxide, carbon dioxide, and hydrogen from gasification of biomass or industrial waste gases. Suffering from energetic limitations, yields of C4-molecules produced by syngas fermentation are quite low compared with ABE fermentation using sugars as a substrate. On the other hand, fungal production of malic acid has high yields of product per gram metabolized substrate but is currently limited to sugar containing substrates. In this study, it was possible to show that Aspergilus oryzae is able to produce malic acid using acetate as sole carbon source which is a main product of acetogenic syngas fermentation. Bioreactor cultivations were conducted in 2.5 L stirred tank reactors. During the syngas fermentation part of the sequential mixed culture, Clostridium ljungdahlii was grown in modified Tanner medium and sparged with 20 mL/min of artificial syngas mimicking a composition of clean syngas from entrained bed gasification of straw (32.5 vol-% CO, 32.5 vol-% H2, 16 vol-% CO2, and 19 vol-% N2) using a microsparger. Syngas consumption was monitored via automated gas chromatographic measurement of the off-gas. For the fungal fermentation part gas sparging was switched to 0.6 L/min of air and a standard sparger. Ammonia content of medium for syngas fermentation was reduced to 0.33 g/L NH4Cl to meet the requirements for fungal production of dicarboxylic acids. Malic acid production performance of A. oryzae in organic acid production medium and syngas medium with acetate as sole carbon source was verified and gave YP∕S values of 0.28 g/g and 0.37 g/g respectively. Growth and acetate formation of C. ljungdahlii during syngas fermentation were not affected by the reduced ammonia content and 66 % of the consumed syngas was converted to acetate. The overall conversion of CO and H2 into malic acid was calculated to be 3.5 g malic acid per mol of consumed syngas or 0.22 g malic acid per gram of syngas.

Keywords: Aspergillus oryzae; Clostridium ljungdahlii; acetate; fermentation; malic acid; process coupling; syngas.

Figure 1

Basic scheme of process setup for anaerobic syngas fermentation (left) and aerobic fungal fermentation (right). pHICR, pH indicate, control and record; TICR, temperature indicate, control and record; ORPICR, ORP indicate, control and record; AF, anti-foam; GC, gas chromatograph. For aerobic fungal fermentation the microsparger had to be turned sideways to make room for the standard sparger. No pH adjustment was conducted during fungal fermentation.

Figure 2

Malic acid production (c_{Malic acid}) using acetic acid (c_{Acetic acid}) as carbon source in fungal organic acid production medium. Fumaric acid (c_{Fumaric acid}) is the most important side product during A. oryzae fermentation. All concentrations are given as average of three independent experiments ± standard deviation.

Figure 3

Malic acid production (c_{Malic acid}) using acetic acid (c_{Acetic acid}) as carbon source in fermented syngas fermentation medium. Fumaric acid (c_{Fumaric acid}) is the most important side product during A. oryzae fermentation. All concentrations are given as average of three independent experiments ± standard deviation.

Figure 4

Mean online and offline values for syngas fermentation part of sequential mixed culture (three replicas). Upper part: Average amount of substance flow rates for hydrogen (red), carbon monoxide (yellow), and carbon dioxide (green) in the off-gas. The lightly colored areas around the average lines show minimum and maximum variance between bioreactors. Bottom part: Average values for ORP (blue line), BDM (black dots), fructose concentration (light green squares), acetate concentration (blue-green diamonds), and ethanol concentration (orange half-filled diamonds).

Figure 5

Overall concentration of consumed substrates and consumption rate for hydrogen (H₂, red), carbon monoxide (CO, yellow), and carbon dioxide (CO₂, green). Values are shown in absolute values c_{i, R(t)} and ${\Delta \stackrel{\cdot }{n}}_{\text{i}}$ (solid lines) and in percent of the amount gone into the bioreactor at each specific time (E_i, dotted lines) and in percent of the rate of the ingoing amount of substance (e_i, dotted lines). The lightly colored areas around the average lines show minimum and maximum variance between three bioreactors.

Figure 6

Malic acid production (c_{Malic acid}), and acetic acid (c_{Acetic acid}) consumption in the three bioreactors A, B, and C from triple approach for syngas fermentation after 96 h of fermentation. Fumaric acid (c_{Fumaric acid}) is the main side product during A. oryzae fermentation.