Effect of cell immobilization and pH on Scheffersomyces stipitis growth and fermentation capacity in rich and inhibitory media

Abstract

Background

A wide range of value-added products can potentially be produced by bioprocessing hardwood spent sulfite liquors (HSSLs) that are by-products of pulp and paper industry with a high pentose sugar content. However, besides sugars, HSSLs contain considerable amounts of sulfonated lignin derivatives and acetic acid that inhibit the metabolic activity of most microorganisms. Scheffersomyces stipitis is a yeast with high capacity to ferment the pentose sugar xylose under appropriate microaerophilic conditions but it has limited tolerance to HSSL inhibitors. In the present study, cultivations of suspended and immobilized S. stipitis were compared in terms of growth capacity and by-product formation using rich medium and HSSL to investigate whether the immobilization of cells in calcium alginate beads could be a protection against inhibitors while favoring the presence of microaerophilic conditions.

Results

Whereas cell immobilization clearly favored the fermentative metabolism in rich medium, pH control was found to play a more important role than cell immobilization on the ethanol production efficiency from bio-detoxified HSSL (bdHSSL), leading to an improvement of 1.3-fold on the maximum ethanol productivity than using suspended cells. When immobilization and pH control were applied simultaneously, the ethanol yield improved by 1.3-fold with unchanged productivity, reaching 0.26 g ethanol.(g glucose + xylose)⁻¹. Analysis of the immobilized beads inside revealed that the cells had grown in the opposite direction of the cortex.

Conclusions

Immobilization and pH control at 5.5, when applied simultaneously, have a positive impact on the fermentative metabolism of S. stipitis, improving the ethanol production efficiency. For the first time light microscopic analysis of the beads suggested that the nutrient and mass transfer limitations played a more important role in the fermentation than a possible protective role against inhibitors.

Keywords

Scheffersomyces stipitis / Hardwood spent sulfite liquor / Cell immobilization / Light microscopy / Ca alginate beads / Xylose fermentation / Stress tolerance

References

[1.] Nigam JN. Ethanol production from hardwood spent sulfite liquor using an adapted strain of Pichia stipitis. Journal of Industrial Microbiology & Biotechnology, 2001, 26(3): 145-150.

[2.] Pereira SR, Portugal-Nunes DJ, Evtuguin DV, Serafim LS Xavier AMRB. Advances in ethanol production from hardwood spent sulphite liquors. Process Biochemistry, 2013, 48(2): 272-282. http://dx.doi.org/10.1016/j.procbio.2012.12.004

[3.] Jeffries TW, Grigoriev IV, Grimwood J, Laplaza JM, Aerts A, Salamov A, Schmutz J, Lindquist E, Dehal P, Shapiro H, Jin YS, Passoth V, Richardson PM. Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis. Nature Biotechnology, 2007, 25(3): 319-326.

[4.] Pereira SR, Ivanuša Š, Evtuguin DV, Serafim LS, Xavier AMRB. Biological treatment of eucalypt spent sulphite liquors: a way to boost the production of second generation bioethanol. Bioresource Technology, 2012, 103(1): 131-135. doi:10.1016/j.biortech.2011.09.095

[5.] Xavier AMRB, Correia MF, Pereira SR, Evtuguin DV. Second-generation bioethanol from eucalypt sulphite spent liquor. Bioresource Technology, 2010, 101(8): 2755-2761. doi:10.1016/j.biortech.2009.11.092

[6.] Parajó JC, Domínguez H, Domínguez J. Biotechnological production of xylitol. Part 3: operation in culture media made from lignocellulose hydrolysates. Bioresource Technology, 1998, 66(1): 25-40.

[7.] Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates I: inhibition and detoxification. Bioresource Technology, 2000, 74: 17-24.

[8.] Marques AP, Evtuguin DV SM, Amado FML, Prates A. Structure of lignosulphonates from acidic magnesium-based sulphite pulping of Eucalyptus globulus. Journal of Wood Chemistry and Technology, 2009, 29: 337-357.

[9.] Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K, Zacchi G. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme and Microbial Technology, 1999, 24: 151-159.

[10.] Palmqvist E, Grage H, Meinander NQ, Hahn-Hägerdal B. Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnology and Bioengineering, 1999, 63: 46-55.

[11.] Palmqvist E, Almeida JS, Hahn-Hägerdal B. Influence of furfural on anaerobic glycolytic kinetics of Saccharomyces cerevisiae in batch culture. Biotechnology and Bioengineering, 1999, 62(4): 447-454.

[12.] Ding MZ, Wang X, Yang Y, Yuan YJ. Metabolomic study of interactive effects of phenol, furfural, and acetic acid on Saccharomyces cerevisiae. OMICS, 2011, 15(10): 647-653.

[13.] Sanchez OJ, Cardona CA. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresource Technology, 2008, 99(13): 5270-5295.

[14.] Passoth V, Cohn M, Schafer B, Hahn-Hägerdal B, Klinner U. Analysis of the hypoxia-induced ADH2 promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast. Yeast, 2003, 20: 39-51.

[15.] Klinner U, Fluthgraf S, Freese S, Passoth V. Aerobic induction of respirofermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis. Applied Microbiology and Biotechnology, 2005, 67: 247-253.

[16.] Huang C-F, Lin T-H, Guo G-L, Hwang W-S. Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis. Bioresource Technology, 2009, 100(17): 3914-3920.

[17.] Helle SS, Murray A, Lam J, Cameron DR, Duff SJB. Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor. Bioresource Technology, 2004, 92: 163-171.

[18.] Palmqvist E, Hahn-Hägerdal B, Szengyel Z, Zacchi G, Rèczey K. Simultaneous detoxification and enzyme production of hemicellulose hydrolysates obtained after steam pretreatment. Enzyme and Microbial Technology, 1997, 20: 286-293.

[19.] Jönsson LJ, Palmqvist E, Nilvebrant NO, Hahn-Hägerdal B. Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor. Applied Microbiology and Biotechnology, 1998, 49: 691-697.

[20.] Oliveira MED, Vaughan BE, Rykiel EJ Jr. Ethanol as fuel: energy, carbon dioxide balances and ecological footprint. Biosci, 2005, 55(7): 593-602.

[21.] Desimone MF, Degrossi J, D’Aquino M, Diaz LE. Ethanol tolerance in free and sol–gel immobilised Saccharomyces cerevisiae. Biotechnology Letters, 2002, 24: 1557-1559.

[22.] Pourbafrani M, Talebnia F, Niklasson C, Taherzadeh MJ. Protective effect of encapsulation in fermentation of limonene-contained media and orange peel hydrolyzate. International Journal of Molecular Sciences, 2007, 8: 777-787.

[23.] Nikolić S, Mojović L, Rakin M, Pejin D. Bioethanol production from corn meal by simultaneous enzymatic saccharification and fermentation with immobilized cells of Saccharomyces cerevisiae var. ellipsoideus. Fuel, 2009, 88(9): 1602-1607. http://dx.doi.org/10.1016/j.fuel.2008.12.019

[24.] Kourkoutas Y, Bekatrou A, Banat IM, Marchant R, Koutinas AA. Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiology, 2004, 21: 277-397.

[25.] Zhang W, Mou H, Cui H, Zhang Y. Isolation of xylose fermentation strains for ethanol production and xylose fermentation research. Chemical and Biochemical Engineering Quarterly, 2014, 28(1): 117-124.

[26.] Varanasi P, Singh P, Auer M, Adams PD, Simmons BA, Singh S. Survey of renewable chemicals produced from lignocellulosic biomass during ionic liquid pretreatment. Biotechnology for Biofuels, 2013, 6(14): 1-9.

[27.] Sugiura S, Oda T, Izumida Y, Aoyagi Y, Satake M, Ochiai A, Ohkohchi N, Nakajima M. Size control of calcium alginate beads containing living cells using micro-nozzle array. Biomaterials, 2005, 26: 3327-3331.