Revisiting Self-cycling Fermentation – New Characterization, Scheme, and Application
Creators: Tan, Yusheng
Publisher: name: University of Alberta Library
Year: 2022

<p>Self-cycling fermentation (SCF) is an advanced, automated, semi-continuous fermentation strategy that is used to improve the volumetric productivity of bioproduction. Typically, a microbial culture is grown in a reactor and half the reactor contents are harvested once a limiting nutrient is depleted; the reactor is then replenished with the same amount of fresh medium, initiating a new cycle. Nutrient depletion is sensed by control parameters, such as dissolved oxygen and carbon dioxide evolution rate. Thereby, cycling is not dictated by a pre-set cycle time but rather by cell growth itself – hence the name “self-cycling” fermentation. A direct result of implementing this feedback control system is great stability, even when encountering perturbations and nutrient heterogeneity. In addition, by eliminating the lag and stationary phases, SCF presents greatly improved productivity compared to conventional batch reactor (BR) operation. Due to the nutrient cycle, synchrony is observed in many SCF studies, a characteristic feature of SCF. <br>
In this work, the feasibility and impact of SCF operation was investigated for one yeast and two bacteria: Saccharomyces cerevisiae, Escherichia coli, and Methylotuvimicrobium buryatense 5GB1C. <br>
In the first study, the effects of SCF operation on S. cerevisiae engineered to overproduce shikimic acid – a valuable compound that can be used as precursor to many aromatic compounds – were assessed. Yield, volumetric productivity, and specific productivity of shikimic acid were all found to be greatly improved compared to BR operation (4-fold, 4-fold, and 3-fold greater, respectively). Global gene regulation patterns, elucidated through transcriptomic analysis, provided insights into the regulatory mechanisms leading to these significant improvements. They also led to the first demonstration of synchrony in SCF from a gene regulation perspective. <br>
In the second study, SCF long and short cycle schemes implemented for Escherichia coli and Saccharomyces cerevisiae cultures were investigated to uncover patterns in glucose consumption, carbon dioxide evolution rate, and cell replication during SCF operation. SCF Short cycles significantly improved biomass productivity compared to long cycles and helped identified the relation between doubling time and SCF cycle time. Stemming from these results and previous SCF articles, three trends in the co-occurrence of characteristic events during SCF cycles were identified and summarized: 1) three key events of SCF (i.e., the depletion of a plateau of the limiting nutrient, the completion of synchronized cell replication, and characteristic points of control parameters) occur concomitantly; 2) cell replication ends prior to the concurrence of the other two events; and 3) the limiting nutrient is depleted or reaches a plateau later than the joint occurrence of the other two events. This work uncovers the potential of SCF as a research tool to explore microbial physiological properties (e.g., nutrient uptake, proliferation, and respiration intensity) and highlights the enhanced performance of the short cycle scheme. Moreover, a novel description of SCF was established thoroughly, together with the revealed key trends providing a solid framework for further SCF development and applications.<br>
In the third study, SCF and fed-batch strategies were successfully implemented to cultivate Methylotuvimicrobium buryatense 5GB1C, a methanotrophic bacterium, using methanol as the carbon source. A new control parameter, culture reflectance, was used to establish stable SCF operation, leading to a 3-fold or 10-fold increase in volumetric biomass productivity (depending on the SCF scheme implemented) as compared to BR. On the other hand, the fed-batch operation, when compared to BR, resulted in a 26-fold improvement in biomass density. These results provide an important initiative for exploring methanotroph-mediated methanol bioconversion.<br>
Overall, the present work broadens our understanding of SCF operation, its properties, and its effects on cells. In terms of novelty, these works include: 1) the first characterization of SCF at a transcriptomic level; 2) the first study distinguishing the impacts of short and long SCF cycle schemes on cell cultures; 3) the first in-depth survey of SCF characteristic events and corresponding trends; 4) a new definition of SCF; 5) the first study that incorporated culture reflectance as control parameter leading to stable SCF operation; and 6) the implementation of fed-batch and SCF schemes in cultures of methanotrophic bacteria using methanol as carbon source. These advances in knowledge will help researchers and bioprocessing engineers adopt and adapt this advanced semi-continuous operation.</p>