(626d) Efficient Bioconversion of Glucose/Xylose Mixtures for Ethanol Production Using a Novel Co-Culture System

With the inevitable depletion of the world’s conventional oil and gas supply, there has been an increasing worldwide interest in alternative sources of energy. In recent years, growing attention has been devoted to the conversion of lignocellulosic biomass into fuel ethanol, which offers great environmental and potential economic benefits over fossil fuel. The cellulosic biomass is cost-effective compared to low-cost petroleum; the cellulosic biomass is widely available at $40 per dry ton, and this price is equivalent to petroleum at $17.5 per barrel on both a mass and energy basis (Lynd et al., 1999). This potentially abundant lignocellulosic biomass contains fermentable sugars, mainly composed of glucose and xylose. It is worth noting that ethanol production from lignocellulosic hydrolysates in an economically feasible process requires complete utilization of both glucose and xylose. Specifically, the efficient utilization of xylose offers an opportunity to reduce the cost of producing ethanol by 25% (Aristidou and Penttila, 2000). The conventional yeast strain, Saccharomyces cerevisiae, is the most frequently used microorganism for ethanol production and it has both high ethanol tolerance and high fermentation yield and rate, but its inability to ferment xylose limits its use in ethanol production with lignocellulosic biomass. Although successful cycles of metabolic engineering have improved xylose utilization in recombinant S. cerevisiae, the ethanol production from xylose is still low (Madhavan et al., 2009; Bengtsson et al., 2009; Matsushika et al., 2008). The xylose-fermenting yeast, Pichia stipitis shows the highest native capacity for xylose fermentation of any known microbes and can convert both glucose and xylose into ethanol. However, the ethanol productivity of this strain is at least five to ten-folds lower than that obtained with S. cerevisiae during glucose fermentation (Alfenore et al., 2004; Agbogbo et al., 2006). Furthermore, P. stipitis shows a diauxic growth on the mixed substrates and has low ethanol tolerance, which inhibits its metabolism when ethanol concentration reaches above 30 g/L.

To achieve efficient utilization of glucose/xylose mixtures for ethanol production, most existing research has focused on developing a single recombinant strain which can ferment the mixed sugars derived from lignocelluloses to produce ethanol. In parallel with the ongoing development of the genetic recombinant strategy, co-culture strategy has been recognized as a promising and cost-effective way to co-ferment a mixture of glucose and xylose for ethanol production. However, efficient ethanol production by conventional co-culture systems faces several difficulties and the major ones are the competition for dissolved oxygen between the two microbes, the diauxic behavior of xylose-fermenting strain (i.e., they cannot use xylose in the presence of glucose) and their low ethanol tolerance (Gutierrez-River et al., 2012; Chandel et al., 2011; Li et al., 2011). In addition, very limited research has been done to investigate the dynamic properties of co-culture systems. Since the interactions between two microorganisms (glucose-fermenting and xylose-fermenting strains) are complex due to different assimilation pathways and regulatory mechanisms, it is important to develop new approaches to help understand and explore the dynamic behaviors of the co-culture system.

In this study, we designed and customized a novel bioreactor with an innovative fermentation scheme to offer a promising alternative of overcoming the difficulties of the existing co-culture systems. With this innovative fermentation scheme, the simultaneous complete utilization of both glucose and xylose was achieved. In addition, both diauxic phenomenon and oxygen deficiency of P. stipitis, were circumvented by providing the optimal dissolved oxygen required for efficient xylose fermentation. Also, the pseudo-continuous fermentation, i.e. continuous fermentation with cell retention, enhanced ethanol tolerance of both strains significantly by allowing the cells to adapt to a high ethanol concentration and environmental pressure. Specifically, the ethanol tolerance of P. stipitis (strain from pseudo-continuous fermentation) had increased 3.5-fold compared with unadapted strain of P. stipitis in the presence of 60g/L of ethanol. Furthermore, the dynamic properties of co-culture system were studied under pseudo-continuous fermentation to identify the optimal co-culture conditions by manipulating oxygen transfer rate (mmolg^-1hr^-1), substrate feed rate (ghr^-1), and substrate feed ratio (glucose/xylose ratio).

Reference

Agbogbo FK, Coward-Kelly G, Torry-Smith M, Wenger KS. (2006). Fermentation of glucose/xylose mixtures using Pichia stipitis. Process Biochem, 41, 2333-2336.

Alfenore S, Cameleyre X, Benbadis L, Bideaux, C, Uribelarrea JL, Goma G, Molina-Jouve C, Guillouet SE. (2004). Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process. Appl Microbiol Biotechnol, 63, 537-542.

Aristidou A, Penttila M. (2000). Metabolic engineering applications to renewable resource utilization, Curr Opin Biotechnol, 11, 187-198.

Bengttson O, Hahn-Hagerdal B, Gorwa-Grauslund ME. (2009). Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae. Biotechnol Biofuels, 2, 9.

Chandel AK, Singh OV, Narasy ML, and Rao V. (2011). Bioconversion of Saccharum spontaneum (wild sugarcane) hemicellulosic hydrolysate into ethanol by mono and co-cultures of Pichia stipitis NCIM3498 and thermotolerant Saccharomyces cerevisiae-VS₃, New Biotechnol, 28, 593-599

Gutierrez-Rivera B, Waliszewski-Kubiak K, Carvajal-Zarrabal O, and Aguilar-Uscanba MG. (2012). Conversion efficiency of glucose/xylose mixtures for ethanol production using Saccharomyces cerevisiae ITV01 and Pichia stipitis NRRL Y-7124. J Chem Technol Biotechnol, 87, 263-270.

Li Y, Park J, Shiroma R, and Tokuyasu K. (2011). Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae prior to xylose fermentation, J Biosci Bioeng, 111, 682-686.

Lynd LR, Wyman CE, and Gerngross TU. (1999). Biocommodity Engineering. Biotechnol Prog, 15, 777-793.

Madhavan  A, Tamalampudi S, Srivastava A, Fukuda H, Bisaria VS, and Kondo A. (2009). Alcholic fermentation of xylose and mixed sugars using recombinant Saccharomyces cerevisiae engineered for xylose utilization. Appl Microbiol Biotechnol, 82, 1037-1047

Matasushika A and Sawayama S. (2008). Efficient bioethanol production from xylose by recombinant Saccharomyces cerevisiae requires high activity of xylose reductase and moderate xylulokinase activity. J Biosci Bioeng, 106, 306-309.