An open-source multiple-bioreactor system for replicable gas-fermentation experiments: Nitrate feed results in stochastic inhibition events, but improves ethanol production of Clostridium ljungdahlii with CO2 and H2

Abstract

The pH-value in fermentation broth has a large impact on the metabolic flux and growth behavior of acetogens. A decreasing pH level throughout time due to undissociated acetic acid accumulation is anticipated under uncontrolled pH conditions such as in bottle experiment. As a result, the impact of changes in the metabolism (e.g., due to a genetic modification) might remain unclear or even unrevealed. In contrast, pH-controlled conditions can be easily achieved in commercially available bioreactors. However, their acquisition is costly and their operation is time consuming, and therefore the experiment is often limited to a single bioreactor run. Here, we present a self-built, relatively cheap, and easy to handle open-source multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. The functionality of the MBS was tested in three experiments by cultivating the acetogen Clostridium ljungdahlii with CO2 and H2 at steady-state conditions (=chemostat). The experiments were addressing the questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; (2) does feeding acetate alter the production rate of ethanol; and (3) does feeding nitrate influence the product spectrum under controlled pH conditions with CO2 and H2? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. Our data show high reproducibility for gas-fermentation experiments with C. ljungdahlii, using the MBS. We found that feeding acetate did not improve ethanol production, but rather impaired growth and reduced acetate production. Using nitrate as sole N-source, on the other hand, enhanced biomass production even at a low pH. However, we observed differences in growth, acetate, and ethanol production rates between triplicate bioreactors (n=3). We explained the different performances because of stochastic inhibition events, which we observed through the accumulation of nitrite, and which led to complete crashes at different operating times. One of these bioreactors recovered after the crash and showed enhanced ethanol production rates while simultaneously producing less acetate. The MBS offers a great opportunity to perform bench-scale bioreactor experiments at steady-state conditions with replicates, which is especially attractive for academia.