Saccharomyces cerevisiae strain comparison in glucose-xylose fermentations on defined substrates and in high-gravity SSCF: convergence in strain performance despite differences in genetic and evolutionary engineering history

Background: The most advanced strains of xylose-fermenting Saccharomyces cerevisiae still utilize xylose far less efficiently than glucose, despite the extensive metabolic and evolutionary engineering applied in their development. Systematic comparison of strains across literature is difficult due to widely varying conditions used for determining key physiological parameters. Here, we evaluate an industrial and a laboratory S. cerevisiae strain, which has the assimilation of xylose via xylitol in common, but differ fundamentally in the history of their adaptive laboratory evolution development, and in the cofactor specificity of the xylose reductase (XR) and xylitol dehydrogenase (XDH). Results: In xylose and mixed glucose-xylose shaken bottle fermentations, with and without addition of inhibitorrich wheat straw hydrolyzate, the specific xylose uptake rate of KE6-12. A (0.27-1.08 g g(CDW)(-1) h(-1)) was 1.1 to twofold higher than that of IBB10B05 (0.10-0.82 g g(CDW)(-1) h(-1)). KE6-12. A further showed a 1.1 to ninefold higher glycerol yield (0.08-0.15 g g(-1)) than IBB10B05 (0.01-0.09 g g(-1)). However, the ethanol yield (0.30-0.40 g g(-1)), xylitol yield (0.080.26 g g(-1)), and maximum specific growth rate (0.04-0.27 h(-1)) were in close range for both strains. The robustness of flocculating variants of KE6-12. A (KE-Flow) and IBB10B05 (B-Flow) was analyzed in high-gravity simultaneous saccharification and co-fermentation. As in shaken bottles, KE-Flow showed faster xylose conversion and higher glycerol formation than B-Flow, but final ethanol titres (61 g L-1) and cell viability were again comparable for both strains. Conclusions: Individual specific traits, elicited by the engineering strategy, can affect global physiological parameters of S. cerevisiae in different and, sometimes, unpredictable ways. The industrial strain background and prolonged evolution history in KE6-12. A improved the specific xylose uptake rate more substantially than the superior XR, XDH, and xylulokinase activities were able to elicit in IBB10B05. Use of an engineered XR/XDH pathway in IBB10B05 resulted in a lower glycerol rather than a lower xylitol yield. However, the strain development programs were remarkably convergent in terms of the achieved overall strain performance. This highlights the importance of comparative strain evaluation to advance the engineering strategies for next-generation S. cerevisiae strain development

Role of enzyme immobilization in the formulation of enzymes for single use

Immobilized enzymes find application in diverse areas of biosciences as biofuels, biosensors and biocatalysis. Development of a immobilization for an enzyme implies a careful selection and optimization of different parameters that have a critical influence on the final properties, e.g. activity, stability [1,2]. Enzyme stabilization via immobilization is a common target and it is achievable when properly performed [2]. There is the general idea that the use of enzyme in free soluble form is associated to single use due to the simplicity and significant lower cost, whereas the use of immobilized preparations is generally associated to facilitate retention, recycling or continuous use. Nevertheless, there are some constraints where immobilization is a fundamental tool to enable the single use of the enzyme under conditions otherwise not-applicable. Among others, these conditions involve the preservation of enzyme activity during extraction, purification, storage and transport, stabilization under condition of use and easy separation from the medium where it is applied. This communication presents some examples of immobilization strategies and benefits for the application of oxidative enzymes. Glucose oxidase and D-amino acid oxidase were immobilized into porous materials made from natural organic polymers or synthetic organic polymers [3,4]. Under the conditions of use, that involves diluted application and existence of gas-liquid interfaces, the free enzymes have half-lifetime lower than 5 min whereas proper immobilized preparations within porous materials did not show decrease of activity during a time span of observation of 40 h. The storage of the enzyme formulation was also facilitated, immobilized enzymes were stable under simple storage conditions whereas free enzymes required prevention microbiological contamination and tuning of buffer conditions to avoid protein precipitation. Among different strategies, the use of reversible immobilization was evaluated to enable the reuse of support and facilitate an immobilization on/off character. The development reversible one step purification-immobilization simplified the production and application of the immobilizate format compared with the soluble counterpart [3]. Finally, the immobilization was used as a compartmentalization strategy, two enzymes (oxidase-catalase, oxidase-peroxidase) or a pair of oxidase - oxygen probe can be used when they are confined into solid materials compared to incompatible conditions of use in soluble format [5]. Co-joined design of polymeric materials and immobilization strategies is recognized as important for the efficient cost-effective formulation of enzyme immobilizates. [1] Bolivar, J.M., Eisl, I., Nidetzky, B.(2016) Catalysis Today, 259, pp. 66-80. [2] Rodrigues, R.C., Ortiz, C., Berenguer-Murcia, A., Torres, R., Fernández-Lafuente, R. (2013) Chemical Society Reviews, 42 (15), pp. 6290-6307. [3] Wiesbauer, J., Bolivar, J.M., Mueller, M., Schiller, M., Nidetzky, B. (2011) ChemCatChem, 3 (8), pp. 1299-1303. [5] Bolivar, J.M., Consolati, T., Mayr, T., Nidetzky, B. (2013) Biotechnology and Bioengineering, 110 (8), pp. 2086-2095. [4] Bolivar, J.M., Consolati, T., Mayr, T., Nidetzky, B. (2013) Trends in Biotechnology, 31 (3), pp. 194-20

Oscillatory Flow Bioreactor (OFB) Applied in Enzymatic Hydrolysis at High Solid Loadings

Within this study, an enzymatic hydrolysis process using α-cellulosic feedstock was performed in a specially designed plug-flow reactor, referred to as an Oscillatory Flow Bioreactor (OFB). The aims of this approach were to achieve intensification in terms of realising a more energy- and resource-efficient enzymatic hydrolysis, as well as to set the basis for continuous processes in such a reactor. The OFB performance was evaluated for high solid loadings of up to 15 %, and compared to the performance of a Stirred Tank Reactor (STR). Experimental results of the OFB operating at an oscillation frequency of 2 Hz and an oscillation amplitude of 10 mm exhibit better conversion efficiencies (+ 6.7 %) than the STR after 24 h, while requiring only 7 % of the STR power density (W m–3). Therefore, the OFB enables efficient, uniform mixing at lower power densities than STRs for applications with high solid loadings. This work is licensed under a Creative Commons Attribution 4.0 International License