Toxicity reduction in continuous, high productivity ethanol fermentation by Parageobacillus thermoglucosidasius using in situ microbubble gas stripping

Abstract

Ethanol concentrations above 4% (v/v) are required for economic bioethanol production due to the cost of recovery from dilute solutions. Although thermophilic bacteria have many potential advantages over Saccharomyces cerevisiae as process organisms for second generation bioethanol production, they are known to be less tolerant to ethanol, typically to concentrations less than 4% (v/v). To address this issue we have investigated the application of in situ gas-stripping of ethanol using microbubbles to increase the surface area per unit volume of gas, using fed-batch and continuous cultures of the engineered ethanologenic thermophile Parageobacillus thermoglucosidasius TM242. By using microbubbles generated at room temperature using a Desai-Zimmerman Fluid Oscillator, we initially operated a mixed batch and fed-batch fermentation, followed by a continuous fermentation and finally a chemostat fermentation, under conditions which would have generated in excess of 4% (v/v) ethanol. In all cases, gas stripping maintained the actual dissolved ethanol concentration below, or close to toxic levels. As the focus of this study was on demonstrating the efficiency of in situ microbubble gas stripping, to simplify the operation the latter two processes involved a combination of produced and supplemented ethanol, with the chemostat culture producing a nominal maximum 7.1% v/v based on glucose used (5.1-5.3% (v/v) based on ethanol recovered). This offers a practical way to produce second generation bio-ethanol from thermophiles.

Fig. 1

Bioreactor modified for microbubble stripping (a) Dense microbubble plume exiting the diffusers in fermentation broth (note the mushroom shape bubble plume, indicating oscillation of input gas) and (b) a close-up view of the diffuser assembly and gas inlet tubes in water

Fig. 2

Schematic flow diagram of the continuous fermentation with fluidic oscillator (DZFO) generated microbubble aeration and ethanol in situ product extraction. Thick arrows show the direction of media or waste flow, while dashed arrows show the directional movement of gases.  = pump;  = ice bath;  = sterile filter;  = media flow;  = gas flow.

Fig. 3

Fed-batch fermentation with in situ microbubble ethanol extraction without mechanical mixing. A low gas input of 0.52 vvm (15.3% oxygen, 84.7% nitrogen) was used. 2 batches (A, 150 mL and B 100 mL) of 250 g/L glucose media were added at 19.5 and 22 h. Arrow marked F indicates start of media feed

Fig. 4

Cell (OD₆₀₀), ethanol and residual glucose concentrations in the bioreactor during continuous culture (D = 0.1 /h) of P. thermoglucosidasisus TM242 at 60 °C with a medium feed of 35 g/L glucose and 7.7% (v/v) ethanol with in situ microbubble ethanol extraction at a total gas input of 1.13 vvm (O:N ratio = 0.126). Dashed arrow indicates start of media feed

Fig. 5

Continuous culture fermentation of TM242 at D = 0.1 /h, redox − 280 mV (Additional file 5) with a feed of 35 g/L glucose + 5% v/v ethanol and in situ microbubble stripping at 1.13 vvm (at an O:N ratio of 0.126.) Dashed arrow indicates start of continuous media feed

Fig. 6

Composition of bioreactor exhaust vapour condensate samples taken at different time points (“as collected”) from the condenser trap, collated samples of this condensate, and Drechsel bottles A to C