Klask Christian-Marco, Kliem-Kuster Nicolai, Molitor Bastian, Angenent Largus T
Environmental Biotechnology Group, Center for Applied Geoscience, University of Tübingen, Tübingen, Germany.
Max Planck Fellow Groups, Max Planck Institute for Developmental Biology, Tübingen, Germany.
Front Microbiol. 2020 May 6;11:724. doi: 10.3389/fmicb.2020.00724. eCollection 2020.
The pH-value in fermentation broth is a critical factor for 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 experiments. 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 achieved in bioreactors. Here, we present a self-built, comparatively cheap, and user-friendly multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. We tested the functionality of the MBS by cultivating the acetogen with CO and H at steady-state conditions (=chemostat). The experiments (total of 10 bioreactors) were addressing the two questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; and (2) does feeding nitrate influence the product spectrum under controlled pH conditions with CO and H? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. On the one hand, our data showed high reproducibility for gas-fermentation experiments with under standard cultivation conditions using the MBS. On the other hand, feeding nitrate as sole N-source improved growth by up to 62% and ethanol production by 2-3-fold. However, we observed differences in growth, and acetate and ethanol production rates between all nitrate bioreactors. We explained the different performances with a pH-buffering effect that resulted from the interplay between undissociated acetic acid production and ammonium production and because of stochastic inhibition events, which led to complete crashes at different operating times.
发酵液中的pH值是产乙酸菌代谢通量和生长行为的关键因素。在不受控制的pH条件下，如在瓶内实验中，由于未离解乙酸的积累，预计pH值会随时间下降。因此，代谢变化（如由于基因改造）的影响可能仍不清楚甚至未被揭示。相比之下，在生物反应器中可以实现pH控制条件。在此，我们展示了一种自行构建的、相对便宜且用户友好的多生物反应器系统（MBS），它由六个1升规模的pH控制生物反应器组成。我们通过在稳态条件下（即恒化器）用CO和H培养产乙酸菌来测试MBS的功能。这些实验（总共10个生物反应器）解决了两个问题：（1）MBS是否能为气体发酵实验提供可重复的数据？；（2）在CO和H的pH控制条件下，添加硝酸盐是否会影响产物谱？我们在每个实验中应用了四个不同的pH周期，范围从pH 6.0到pH 4.5。一方面，我们的数据表明，在使用MBS的标准培养条件下，气体发酵实验具有很高的可重复性。另一方面，以硝酸盐作为唯一氮源可使生长提高多达62%，乙醇产量提高2至3倍。然而，我们观察到所有硝酸盐生物反应器之间在生长、乙酸盐和乙醇生产率方面存在差异。我们用未离解乙酸产生与铵产生之间的相互作用以及随机抑制事件导致的pH缓冲效应来解释不同的性能，这些随机抑制事件在不同的运行时间导致了完全崩溃。
