Succinic acid production with Actinobacillus succinogenes: rate and yield analysis of chemostat and biofilm cultures

Abstract

Background: Succinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof.

Results: The volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g(-1), while the ratios for biofilm runs started at 2.4 g.g(-1) and increased up to 3.3 g.g(-1) as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g(-1) for chemostat runs, while the yields varied between 0.63 g.g(-1) and 0.74 g.g(-1) for biofilm runs. Specific growth rates (μ) were shown to be severely inhibited by the formation of organic acids, with μ only 12% of μ(max) at a succinic acid titre of 7 g.L(-1). Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases.

Conclusions: The novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.

Figure 1

Growth rates vs. C _SA of prominent batch studies on A. succinogenes employing Glc as substrate [ 12 , 18 - 27 ]. The μ values were estimated from the reported biomass/SA vs. time profiles. Severe product inhibition is evident with growth ceasing between 8 g.L⁻¹ and 14 g.L⁻¹ of SA. The blue data cloud covers the majority of the experimental measurements.

Figure 2

Chemostat steady-state measurements of ΔGlc, SA and biomass plotted against D. High shear conditions prevented biomass attachment. Extrapolation of data indicates that μ_max lies between 0.8 h⁻¹ and 0.85 h⁻¹.

Figure 3

Chemostat mass-based yield ratios (SA/AA, FA/AA and Y _Glc,SA ) plotted against D. The average Y_Glc,SA is relatively stable at 0.48 ± 0.047 g.g⁻¹. FA/AA is close to the equimolar value (0.77 g.g⁻¹) at a D of 0.8 h⁻¹, decreasing to zero as D is decreased. SA/AA is in the vicinity of 1.4 g.g⁻¹ for D values below 0.5 h⁻¹, while a slight decrease is observed at higher D values.

Figure 4

Chemostat SA productivities (q _SA and r _SA ) plotted against D. The r_SA vs. D curve is expected to follow a straight line fit with the slope giving Y_x,SA ^true and the intercept giving m_SA. The straight line trend is observed for all values except the highest values of D. This is most likely connected to inaccurate measurements at low biomass concentrations, where small amounts of insoluble material inflate the measurements. Accordingly, only the r_SA measurements at the D values between 0.1 h⁻¹ and 0.5 h⁻¹ were considered for the linear regression. Regression model given by equation (1).

Figure 5

Chemostat growth rates (μ) plotted against SA concentration. Good agreement with the data cloud in Figure 1 is obtained. Inhibition model given by equation (2) with μ_max at 0.82 h⁻¹.

Figure 6

Chemostat and biofilm volumetric productivities (q _SA ) plotted against D. The comparative q_SA values for the chemostat and biofilm reactors indicate the order of magnitude difference in volumetric productivity. Differences can be attributed to total biomass quantities (see Table 1 and Table 2).

Figure 7

Biofilm reactor mass-based yield ratios (SA/AA, FA/AA and Y _Glc,SA ) plotted against ΔGlc. When compared to the comparative graph for the chemostat (Figure 3), the Y_Glc,SA is significantly higher (up to 50% more). The SA/AA ratio of the biofilm runs is more than double that of the chemostat runs (Figure 3). The FA/AA ratio is close to equimolar (0.77 g.g⁻¹) at low ΔGlc but decreases with increasing ΔGlc.

Figure 8

Breakdown of SA production rate (r _SA ). Measured total rates are given by the red triangles with the empirical fit given by equation (3). The growth contribution to rate is given by the red curve - obtained from the product of the growth function (equation (2)) and Y_x,SA ^true (Figure 4). The maintenance contribution to rate is given by the green curve - obtained by subtracting the growth contribution from the total rate.

Figure 9

The bioreactor setup used for both the chemostat and biofilm experiments. The reactor section is shown in bold with an in-line gas trap. The reactor section consists of a 3 mm silicone tube (approximately 5 m length) with an active volume of 50–60 mL, depending on the liquid level in the gas trap.