Gas profiling in quasi-closed pressure regulated anaerobic fermentation systems
Creators: name: Rotbart, Asaf
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320602
subject: Medical biotechnology diagnostics (incl. biosensors)
classificationCode: 400399
subject: Biomedical engineering not elsewhere classified
classificationCode: 410304
subject: Environmental biotechnology diagnostics (incl. biosensors)

description: Fermentation of organic materials by microorganisms is an essential component in a variety of medical, industrial and agricultural applications. Many of these fermentations take place in quasi-closed pressure regulated anaerobic fermentation systems and involve the production of different gases. These gases are highly indicative as they are identifiable with biological processes and different bacteria species. Profiling gas components in such systems can assist with their microbial activities analysis, diagnosis and monitoring. However, methods for gas profiling in such fermentation systems lack real-time, accurate, simple, portable and cost-effective gas profiling technologies for continuously measuring gases in both anaerobic headspaces and in liquid media.<br><br>The aim of this PhD research is to enhance the understanding, diagnosis and monitoring of these systems and their associated applications using gas components. This was specifically achieved by resolving the limitations and inadequacies of gas profiling in quasi-closed pressure regulated anaerobic fermentation systems.<br><br>Firstly, the author of this thesis thoroughly reviewed the methods utilized for accurate profiling of gas components. Specifically, he focused on profiling intestinal gases produced in-vitro during fecal incubation. Secondly, the author investigated the calculation methods for profiling the production of these gases and their kinetics. Finally, the author explored gas profiling in both liquid and gas phases for in-situ monitoring of anaerobic digestion fermentation systems.<br><br>The first stage involved addressing limitations of profiling intestinal gases produced by incubation of fecal matters in-vitro. The past available technologies for sensing colonic gases in-vitro were either bulky, expensive, offline or included only limited number of gas types. In addition, the gut environment in-vitro is generally simulated with N2 as an inert gas where the supplementation of important fermentation gases, such as CO2 and H2, was not understood.<br><br>As such, the author developed a low-cost, portable and real-time gas sensing technology for monitoring CO2, CH4, H2, H2S and NOx simultaneously in the anaerobic headspace of fecal fermentation systems in-vitro. The author demonstrated the performance of the new technology on healthy human fecal samples and validated the new technology for both accuracy and reproducibility.<br><br>The author also explored the impact of the initial headspace environment composition on the fermentation gas profiles. It was found that supplying the reactor with CO2 enhanced CH4 and H2 production and inhibited H2S production. Furthermore, it was shown that fecal incubation together with high fermentable fibre could suppress H2S production. Finally, the author found that healthy human fecal samples did not produce NOx spontaneously.<br><br>In the second stage, the author investigated the calculation methods for profiling the production of gases and their kinetics in quasi-closed pressure regulated anaerobic fermentation systems. Surprisingly, the author discovered that there was no existing standardized or comprehensive method for such calculations. Therefore, the author developed a rigorous gas fermentation model and a novel mass-flow equation for accurately profiling the produced gases and introduced these into the literature. This new model was designed to match the commonly used commercial fermentation systems, making the new technology readily available for many applications and studies. The author demonstrated the performance of the new model for human fecal sample incubation using the in-vitro technology developed in the first stage and validated its accuracy. Moreover, the author found that the contribution of newly introduced components in the mass-flow equation exceeded 9.1% of the overall gas profile.<br><br>In the final stage, the author researched the monitoring capability of anaerobic digestion processes using in-situ measurements of gas components in both liquid and gas phases. As an integral part of the microbial activity of anaerobic digestion processes, gas components have the potential of providing the necessary information for monitoring such processes effectively. However, current technologies for gas sensing in liquid-phase have been inadequate. Previously, Real-time profiling of gas components in both phases simultaneously has not been thoroughly studied due to lack of the required technology. This has possibly hindered important insights about the system’s health.<br><br>In order to conduct this research, the author developed a novel, relatively simple, low-cost technique for measuring gas components in both phases simultaneously. Using this technique, dissolved gases were measured in-situ using membrane protected gas sensors which, in comparison to other approaches, eliminated many complications, delays or sample contamination. The author demonstrated the performances of the new technology on a series of anaerobic digestion batch experiments and confirmed its accuracy, longevity and reproducibly.<br><br>Utilizing the new technique, the author identified patterns and signatures that were associated with process imbalances but not clearly observed in commonly used indicators such as volatile acids and pH. The author also explored the impact of inoculum age on the process and showed that, relative to freshly collected inoculum, processes using aged inoculum had a higher potential to enter imbalanced states and failure.<br><br>It is the position of this author that the insights and technological advances achieved in this PhD research have contributed significantly to the advancement of the field of anaerobic fermentation. In particular, this was achieved by creating new, simple, accurate and reliable technologies, while adding significantly to the knowledge of quasi-closed, pressure regulated anaerobic fermentation systems and their applications.