Understanding changes in microbial metabolism of Clostridium termitidis under different growth conditions using continuous, real-time monitoring of fermentation end-products
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Date
2019-09-14
Authors
Hossain, Md. Eftekhar
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Abstract
Metabolic shifts are associated with physico-chemical environment changes during the growth phase of many types of cells. An inability to monitor rapid and/or subtle changes in microbial metabolism may sometimes limit our understanding of microbial physiology. The central focus of this thesis was to better understand changes in microbial metabolism induced by changes in growth conditions (carbon-excess, end-product concentration such as acetate, quick removal of product gases), which may be acquired by continuous monitoring. Clostridium termitidis was chosen as the model organism for this work. A mass spectrometry-based Titration and Off-Gas Analysis (TOGA) system was used as an on-line monitoring tool for headspace gases (H2, CO2, and ethanol) analysis. In addition, GC, HPLC, and spectrophotometry methods were also used for other metabolites analysis as off-line techniques. Several key metabolic shifts were recognized during the early stationary growth phase under carbon-excess condition, compared to carbon-sufficient and carbon-limited conditions. The H2 and CO2 production rates decreased significantly, while the specific rates of ethanol and formate synthesis increased significantly. As a result, the substrate specific yields of H2 were gradually decreased from exponential phase to late stationary phase, whereas the ethanol yields were increased. Decreased CO2 synthesis and increased formate synthesis suggested that carbon flux and reducing equivalent shifted from the pyruvate ferredoxin oxidoreductase (PFOR) pathway to the pyruvate formate lyase (PFL) pathway, resulting in a sudden increase of ethanol synthesis. The addition of exogenous acetate during fermentation, resulted in decreased H2 yields and significantly increased ethanol yields. The substrate specific yields of H2 and CO2 also increased significantly in cultures with a high turbulent growth environment, achieved by a high gas sparging rate (HSR) compared to the cultures with a low turbulent growth environment, with a low gas sparging rate (LSR). The growth of C. termitidis under LSR favoured higher ethanol yields, via the PFL pathway, whereas growth of C. termitidis under HSR was unfavorable to ethanol synthesis due to re-direction of carbon and reducing equivalents towards the PFOR pathway. An algorithm was developed to create a model to estimate concentrations of non-volatile fermentation end-products using reaction stoichiometry from real-time, on-line, headspace data for H2, CO2, and ethanol concentrations obtained with the TOGA system, as well as acid/base balance. The proposed algorithm could not only reliably predict acetate, formate, and lactate concentrations, but also was able to tell qualitatively and quantitatively the metabolic process trends of C. termitidis, such as the physiological state (exponential or stationary phase) during which a particular end-product was synthesized, as well as whether the concentration of this end-product decreased or increased as growth conditions changes (metabolic shifts). Thus, real-time, on-line headspace data, together with continuous predicted information were used to detect precise metabolic shifts that resulted in changes in fermentation end-product yields. This integrated process was less cumbersome and offers a number of advantages, such as less labour intensive sampling and minimized contamination threats, which is suitable for industrial scale fermentations.
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Clostridium termitidis, Microbial metabolism, Metabolic shifts, Continuous monitoring, Titration and off-gas analysis (TOGA), Stoichiometric algorithm, Variable substrate load, Headspace gases, End-product induced metabolism