New Experimental and Modeling Appraches to Study the Dynamics of Xylose Fermentation with Sheffersomyces stipitis
Kim, Min Hea
Type of DegreeDissertation
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Ethanol production from lignocellulosic hydrolysates in an economically feasible process requires complete utilization of both glucose and xylose, the main components of cellulose and hemicellulose. Scheffersomyces stipitis (formerly known as Pichia stipitis) has promising potential for converting lignocellulosic biomass into ethanol since it can ferment both hexose and pentose sugars under microaerophillic conditions (a native yeast strain best capable of utilizing xylose to ethanol). However, this strain has several challenges for lignocellulosic ethanol production from xylose as it has a slower sugar consumption rate than S. cerevisiae (hexose fermenting microorganism) and requires oxygen for both growth and maximal ethanol production. In addition, diauxic kinetic is a practical problem associated with mixed sugar utilization by native strains of S. stipitis and even with engineered strains of S. cerevisiae (Kuyper et al. 2005). Although successful cycles of metabolic engineering have improved xylose utilization in recombinant strain of S. cerevisiae, the ethanol production from xylose is still inferior to those of xylose fermentation by native strain of S. stipitis (Jeffries and Jin, 2004; Jin et al. 2004). Since S. stipitis is a respiratory yeast strain, the xylose fermentation performance depends significantly on the oxygenation level of the culture. High aeration rate results in fast cell growth and acetic acid production, while very low aeration (oxygen-limited) often results in xylitol production, both at the expense of reduced ethanol production. Only optimized microaerobic condi-tion promotes ethanol production by maintaining cell viability and NAD+/NADH balance. Hence, it is critical to determine the optimal oxygen utilization rate (OUR) for ethanol production by S. stipitis. In order to quantitatively study the effect of OUR on the fermentation performance, accurate control of OUR is essential. Several studies have been reported on the optimum oxygenation conditions for ethanol fermentation by S. stipitis (Silva et al., 2012; Slininger et al., 2014; Su et al., 2014; Unrean and Nguyen, 2012). Among these studies, most of the experiments were carried out using batch cultures grown in flasks where the Oxygen Transfer Rate (OTR) and/or Dissolved Oxygen (DO) were not effectively controlled. Different OTR levels have been tested simply by changing volume of media, airflow rate, and agitation speed. However, our re-search shows that the inaccuracy and inconsistency of controlling OTR/OUR is problematic in these studies. In order to quantify the metabolic mechanism of OUR on xylose fermentation kinetics by S. stipitis, we have developed experimental protocol and equipment to carry out both single culture and co-culture systems under controlled chemostat. The xylose fermentation kinetics of S. stipitis data collected from single culture experiment was used to design the co-culture experiment to optimize ethanol yield. For the co-culture system, we have developed a novel co-culture bioreactor for the efficient and simultaneous conversion of mixed glucose and xylose to ethanol by S. cerevisiae and S. stipitis, which offers a promising alternative to overcome the difficulties of the existing co-culture systems and to study the dynamic properties of co-culture strains. In addition to this, we used a mathematical modeling tool to describe the kinetics of both single culture and co-culture systems, as well as to validate our results. Also, Principal Component Analysis (PCA) was utilized to enable the extraction of correlations between different cellular physiology with respect to carbon uptake and OUR which helped us to understand and quantify the metabolic mechanism of OUR in continuous fermentation.