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Title: Microbubble mediated sequential saccharification, inactivation, and aerobic fermentation with in situ selective product removal
Author: Mulakhudair, Ali R.
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Microbubble mediated technologies are employed for pretreatment steps due to the characteristics of the gas-liquid interface. Traditionally, pretreatment processes are energy intensive operations and use hazardous chemicals such as sulfuric acid and hydrochloric acid, which need to be removed from the pretreatment slurry before feeding it to the fermentation process. Alternative approaches to microbubbles for pretreatment, however, have significant challenges. For example, conventional bubbles are several orders of magnitude larger than the bubble exit pore and therefore have less direct contact with the biomass or delivery the ozone efficiently to the pretreatment slurry. Consequently, these concerns have been addressed in this research, and microbubble-microbe synergy and Ozonolysis-microbe synergy for biomass pretreatment with the developing of microbubbles driven systems, were used to facilitate microbubble generation suitable for pretreatment processes. The first approach was achieved by exploiting the synergy between microbubble-microbe to pretreat lignocellulosic biomass and glucose was the target product. The effects of microbubbles, microbe and the synergy between them on morphology, functional groups and glucose yield were investigated. It was found that free radicals around the gas-liquid interface of the microbubble can readily attack and degrade lignocellulosic biomass, rendering it more amenable to digestion. The combination of microbubbles and Pseudomonas putida—a robust delignification and cellulolytic microbe, further improved biomass degradation and consequently, increased glucose production from wheat straw in comparison to solo pretreatment of the biomass with microbubbles and Pseudomonas putida respectively. In addition, it was found that the highest glucose achieved was 0.27 mg/ml. The second was conducted by exploiting ozonolysis-microbe synergy to pretreat lignocellulosic biomass and glucose was also the target product. The effects of ozonation at various pHs and ozone concentrations, biological pretreatment by Pseudomonas putida and the synergy between them on morphology, functional groups and glucose yield were explored. Ozone is a strong oxidative agent that reacts with lignin by attacking the carbon-carbon double bonds, while P. putida preferentially hydrolyses the exposed cellulolytic parts of the biomass to simple sugars. It was found that both lignin and cellulose contents were reduced under this pretreatment with relatively high glucose recovery. The highest glucose concentration reached was 1.1 mg/ml after 24 hr ozonation at 8.86 mg/L ozone and pH 3 with 50 % reduction in the biological pretreatment duration but crucially, increasing microbial biomass. Using the synergetic approach for the biomass pretreatment is promising approach but leaves the pretreatment slurry contaminated with the cellulolytic microbe, Pseudomonas putida, which needs to be inactivated or removed before feeding the pretreatment slurry into the fermenter. The ability of carbon dioxide enriched microbubbles to inactivate Pseudomonas putida was subsequently investigated. Many drawbacks of the traditional sterilization methods were avoided by using carbon dioxide enriched microbubbles, such as high energy consumption and using toxic and corrosive reagents. It was found that 2-Log reduction in the bacterial population after 90 min was achieved using carbon dioxide enriched microbubbles. Further reductions were achieved by adding additives such as ethanol and acetic acid and the highest reduction performed was 3.5 Log with 10 % ethanol, while a 2.5-Log reduction was achieved with 0.5 % acetic acid. These reductions in the bacterial population were concurrent with changing cells shape from rod cells to coccus shape with cell damage such as lesions and cells death. Subsequently, aerobic fermentation with glucose as a carbon source proceeded with Zymomonas mobilis ZM4 as the microbial fermentation agent. Acetaldehyde has drawn the attention in this research because it is an important chemical, and it can be used in many processes such as plastic manufacturing and fuels production such as ethanol and butanol. Several attempts to produce acetaldehyde from Zymomonas mobilis or genetically modified microbes contained some genes from Zymomonas are reported, but the inhibition of microbial growth by the accumulated acetaldehyde was the main challenge to keep its continuous production. This challenge has been addressed in this study and microbubbles generated by fluidic oscillation were used to remove both acetaldehyde and carbon dioxide from the fermentation broth. Additionally, the oxygenation concurrent with the stripping process by microbubbles efficiently maintained the oxygen concentration in the fermentation broth above the critical oxygen concentration, leading to stable aerobic conditions. The results show that 45 % yield of ethanol and 1 % yield of acetaldehyde with 110 % yield of microbial biomass in comparison with 70 %, 0.5 % and 90 % yield for ethanol, acetaldehyde, and biomass respectively in the initially sparged group were achieved. Also, acetaldehyde was removed from the fermentation broth with 99 % efficiency. Acetaldehyde production in the fermentation was enhanced by selecting the mutant cells with attenuated or modified alcohol dehydrogenase activity using increasing concentrations of allyl alcohol. The results show that 17-fold increase was achieved in the mutant strain in comparison with the wild strain. In addition, the mutant strain produced 90 % less ethanol than the wild VI strain. Also, the acetaldehyde removal efficiency was 88.5 % in comparison with 42 % efficiency achieved with the fine bubbles (bigger bubbles). Additionally, biomass yield produced by the mutant strain was less by a half than the yield produced by the wild strain. To enhance the biomass yield of the mutant strain, different techniques were used to grow this bacterium aerobically, but maintaining sufficient oxygen concentration was challenging in the bacterial propagation stage. Oxygen is the limiting factor in the aerobically grown bacterial cultures, but similarly, the impact of mixing can be critical. The results show that the oxygen uptake rate and mass transfer coefficient are substantial increased using microbubbles technology and there were 41-fold and 150-fold increase in the oxygen uptake rate and mass transfer coefficient respectively in the microbubbles-dosed culture in comparison with the shaking flask culture. This technology can also achieve a proper mixing. Regarding the biomass yield, the mutant strain of Zymomonas mobilis shows an increased yield using the shaking flask (around 100 % and 133 % increases) in comparison with other (microbubbles-dosed and stationary respectively) techniques, while the wild strain produces more biomass in the microbubble-based technique (around 50 % and 100 %) than other (shaking flask and stationary respectively) techniques. In addition, a propagation unit was designed and simulated to grow the mutant strain aerobically in the propagation stage before using this grown biomass as an inoculum to the fermentation process. Fundamentally, the results obtained in this study are achieved in a laminar flow with several orders of magnitude lower energy density than conventional benchmarks, which are a highly turbulent flow.
Supervisor: Zimmerman, William B. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available