Successful conversion of corn stover into microbial lipids at high solids loading by Rhodosporidium toruloides at pilot scale^✩

Abstract

Lignocellulosic biomass (LCB) offers potential feedstocks for biofuels. As it generally involves processes including pretreatment, hydrolysis, and fermentation, LCB-based biorefinery at higher solids loading may improve substrate concentration and reduce operational costs, yet its scaling-up remains a challenge. Here, efficient conversion of corn stover (CS) at high solids loading into microbial lipids was demonstrated in a 1 000 L pilot-scale bioreactor using Rhodosporidium toruloides CGMCC 2.1389. The process employed lab-optimized conditions, including alkaline storage pretreatment of CS (AS-CS) with 6% NaOH for over 60 d at room temperature. The AS-CS (100 kg) was steamed in the 1 000 L bioreactor at 121 ℃ for 1 h at a solids loading of 20%, followed by subsequent removal of alkaline black liquor (ABL) using squeeze technology. The leftover residues were hydrolyzed by enzymes with a total reducing sugar (TRS) recovery of 93.6%. The lipid production in the 1 000 L bioreactor resulted in a lipid titer of 10.6 g/L and a yield of 0.194 g/g (based on consumed TRS). The mass flow analysis suggested that 89.6% of cellulose and 95.5% of hemicelluloses were released to produce lipids with little lignin by-products, avoiding their toxic effects on lipid production. The developed process in this work offers a promising avenue for industrial conversion of LCB into microbial lipids.

Keywords

Microbial lipids / Corn stover / Alkaline storage pretreatment / Pilot-scale / Rhodosporidium toruloides

References

[1] Banerjee, S., Dien, B.S., Eilts, K.K., Sacks, E.J., Singh, V., 2024. Pilot-scale processing of Miscanthus x giganteus for recovery of anthocyanins integrated with production of microbial lipids and lignin-rich residue. Chem. Eng. J. 485, 150117.

[2] Bensah, E.C., Mensah, M., 2013. Chemical pretreatment methods for the production of cellulosic ethanol: Technologies and innovations. Int. J. Chem. Eng. 2013, 719607.

[3] Chen, M., Zhao, J., Xia, L.M., 2009. Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility. Biomass Bioenergy 33, 1381-1385.

[4] Chen, X.X., Yuan, X.C., Chen, S.T., Yu, J.M., Zhai, R., Xu, Z.X., Jin, M.J., 2021. Densifying lignocellulosic biomass with alkaline chemicals (DLC) pretreatment unlocks highly fermentable sugars for bioethanol production from corn stover. Green Chem 23, 4828-4839.

[5] Ching’anda, C., Atehnkeng, J., Bandyopadhyay, R., Callicott, K.A., Orbach, M.J., Mehl, H.L., Cotty, P.J., 2021. Temperature influences on interactions among aflatoxigenic species of Aspergillus section flavi during maize colonization. Front. Fungal Biol. 2, 720276.

[6] Cui, Z.F., Shi, J., Wan, C.X., Li, Y.B., 2012. Comparison of alkaline-and fungi-assisted wet-storage of corn stover. Bioresour. Technol. 109, 98-104.

[7] Deshavath, N.N., Woodruff, W., Eller, F., Susanto, V., Yang, C., Rao, C.V., Singh, V., 2024. Scale-up of microbial lipid and bioethanol production from oilcane. Bioresour. Technol. 399, 130594.

[8] Du, W., Kamal, R., Zhao, Z.K., 2019. Biodiesel. In: Moo-Young, M. (Ed.), Comprehensive Biotechnology, vol. 3. Elsevier, Pergamon, Oxford, pp. 66-78.

[9] Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., Smith, F., 1951. A colorimetric method for the determination of sugars. Nature 168, 167.

[10] Galbe, M., Zacchi, G., 2007. Pretreatment of lignocellulosic materials for efficient bioethanol production. Adv. Biochem. Eng. Biotechnol. 108, 41-65.

[11] Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10-18.

[12] Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust, T.D., 2007. Biomass recalcitrance: Engineering plants and enzymes for biofuels production. Science 315, 804-807.

[13] Huang, Q.T., Wang, Q., Gong, Z.W., Jin, G.J., Shen, H.W., Xiao, S., Xie, H.B., Ye, S.H., Wang, J.H., Zhao, Z.K., 2013. Effects of selected ionic liquids on lipid production by the oleaginous yeast rhodosporidium toruloides. Bioresour. Technol. 130, 339-344.

[14] Huang, Q.T., Kamal, R., Shen, H.W., Lu, H.B., Song, J.Y., Chu, Y.D., Xue, C., Zhao, Z.K., 2022. Pilot-scale conversion of corn stover into lipids by the red yeast rhodosporidium toruloides. J. Environ. Chem. Eng. 10, 108858.

[15] Isroi, M.R., Syamsiah, S., Niklasson, C., Cahyanto, M.N., Lundquist, K., Taherzadeh, M.J., 2011. Biological pretreatment of lignocelluloses with white-rot fungi and its applications: A review. BioResources 6, 5224-5259.

[16] Jarvis, M., 2003. Cellulose stacks up. Nature 426, 611-612.

[17] Jin, M.J., Bothfeld, W., Austin, S., Sato, T.K., La Reau, A., Li, H.B., Foston, M., Gunawan, C., LeDuc, R.D., Quensen, J.F., McGee, M., Uppugundla, N., Higbee, A., Ranatunga, R., Donald, C.W., Bone, G., Ragauskas, A.J., Tiedje, J.M., Noguera, D.R., Dale, B.E., Balan, V., 2013. Effect of storage conditions on the stability and fermentability of enzymatic lignocellulosic hydrolysate. Bioresour. Technol. 147, 212-220.

[18] Jönsson, L.J., Martín, C., 2016. Pretreatment of lignocellulose: Formation of inhibitory by-products and strategies for minimizing their effects. Bioresour. Technol. 199, 103-112.

[19] Jørgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction of lignocellulose at high-solids concentrations. Biotechnol. Bioeng. 96, 862-870.

[20] Kamal, R., Huang, Q.T., Lu, H.B., Shah, A.M., Zhang, J.L., Zhao, Z.K., 2024. High solids loading conversion of corn stover for lipid production by rhodosporidium toruloides. Ind. Crops Prod. 209, 117997.

[21] Kim, J.S., Lee, Y.Y., Kim, T.H., 2016. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour. Technol. 199, 42-48.

[22] Kim, J.Y., Heo, S., Choi, J.W., 2018. Effects of phenolic hydroxyl functionality on lignin pyrolysis over zeolite catalyst. Fuel 232, 81-89.

[23] Lefyedi, M.L., Taylor, J.R.N., 2006. Effect of dilute alkaline steeping on the microbial contamination, toxicity and diastatic power of sorghum malt. J. Inst. Brew. 112, 108-116.

[24] Li, Q., Kamal, R., Chu, Y.D., Wang, Q., Yu, X., Huang, Q.T., 2020. Automated pressurized liquid extraction of microbial lipids from oleaginous yeasts. Appl. Biochem. Biotechnol. 192, 283-295.

[25] Li, H.X., Zhai, R., Chen, X.X., Jiang, X.X., Li, C., Deng, Q.F., Xu, Z.X., Jin, M.J., 2024. Modeling and design of fed-batch strategies for achieving 255 g/L sugar concentration from high-solid enzymatic hydrolysis of pretreated corn stover. Chem. Eng. J. 486, 150268.

[26] Lu, X.H., Li, F., Li, Y.M., Fang, S., Zhou, X., Zhao, Z.Z., Liu, P., 2022. Components analysis of recycled alkali black liquor combined with corn straw under ozone pretreatment. BioEnergy Res 15, 1204-1212.

[27] Masri, M.A., Garbe, D., Mehlmer, N., Brück, T.B., 2019. A sustainable, high-performance process for the economic production of waste-free microbial oils that can replace plant-based equivalents. Energy Environ. Sci. 12, 2717-2732.

[28] Menon, V., Rao, M.L., 2012. Trends in bioconversion of lignocellulose: Biofuels, platform chemicals and biorefinery concept. Prog. Energy Combust. Sci. 38, 522-550.

[29] Modenbach, A.A., Nokes, S.E., 2012. The use of high-solids loadings in biomass pretreatment: A review. Biotechnol. Bioeng. 109, 1430-1442.

[30] Modenbach, A.A., Nokes, S.E., 2014. Effects of sodium hydroxide pretreatment on structural components of biomass. Trans. ASABE 57, 1187-1198.

[31] Nishiyama, Y., Sugiyama, J., Chanzy, H., Langan, P., 2003. Crystal structure and hydrogen bonding system in cellulose i 𝛼 from synchrotron X-ray and neutron fiber diffraction. J. Am. Chem. Soc. 125, 14300-14306.

[32] Parsons, S., Abeln, F., McManus, M.C., Chuck, C.J., 2019. Techno-economic analysis (TEA) of microbial oil production from waste resources as part of a biorefinery concept: Assessment at multiple scales under uncertainty. J. Chem. Technol. Biotechnol. 94, 701-711.

[33] Patel, A., Arora, N., Mehtani, J., Pruthi, V., Pruthi, P.A., 2017. Assessment of fuel properties on the basis of fatty acid profiles of oleaginous yeast for potential biodiesel production. Renew. Sustain. Energy Rev. 77, 604-616.

[34] Robles-Iglesias, R., Naveira-Pazos, C., Fernández-Blanco, C., Veiga, M.C., Kennes, C., 2023. Factors affecting the optimisation and scale-up of lipid accumulation in oleaginous yeasts for sustainable biofuels production. Renew. Sustain. Energy Rev. 171, 113043.

[35] Scheller, H.V., Ulvskov, P., 2010. Hemicelluloses. Annu. Rev. Plant Biol. 61, 263-289.

[36] Soccol, C.R., Dalmas Neto, C.J., Soccol, V.T., Sydney, E.B., da Costa, E.S.F., Medeiros, A.B.P., de Souza Vandenberghe, L.P., 2017. Pilot scale biodiesel production from microbial oil of rhodosporidium toruloides DEBB 5533 using sugarcane juice: Performance in diesel engine and preliminary economic study. Bioresour. Technol. 223, 259-268.

[37] Tan, L., Zhong, J., Jin, Y.L., Sun, Z.Y., Tang, Y.Q., Kida, K., 2020. Production of bioethanol from unwashed-pretreated rapeseed straw at high solid loading. Bioresour. Technol. 303, 122949.

[38] Wang, W., Wang, X.M., Zhang, Y., Yu, Q., Tan, X.S., Zhuang, X.S., Yuan, Z.H., 2020. Effect of sodium hydroxide pretreatment on physicochemical changes and enzymatic hydrolysis of herbaceous and woody lignocelluloses. Ind. Crops Prod. 145, 112145.

[39] Weinberg, Z.G., Ashbell, G., 2003. Engineering aspects of ensiling. Biochem. Eng. J. 13, 181-188.

[40] Xiao, L.P., Song, G.Y., Sun, R.C., 2017. Effect of hydrothermal processing on hemicellulose structure. In: Hydrothermal Processing in Biorefineries. Springer International Publishing, Cham, pp. 45-94.

[41] Yang, X.B., Jin, G.J., Gong, Z.W., Shen, H.W., Bai, F.W., Zhao, Z.K., 2014. Recycling biodiesel-derived glycerol by the oleaginous yeast rhodosporidium toruloides Y4 through the two-stage lipid production process. Biochem. Eng. J. 91, 86-91.

[42] Yu, Y., Xu, Z.X., Chen, S.T., Jin, M.J., 2020. Microbial lipid production from dilute acid and dilute alkali pretreated corn stover via Trichosporon dermatis. Bioresour. Technol. 295, 122253.

[43] Yu, Y., Yu, J.M., Wang, Z., Yuan, X.C., Chen, X.X., Zhai, R., Xu, Z.X., Jin, M.J., 2021. Development of DLC and DLCA pretreatments with alkalis on rice straw for high titer microbial lipid production. Ind. Crops Prod. 172, 114086.

[44] Yu, Y., Sha, Y.Y., Yu, J.M., Zhou, L.L., Chen, X.X., Zhai, R., Xu, Z.X., Jin, M.J., 2022. DLC(sa) and DLCA(sa) pretreatments boost the efficiency of microbial lipid production from rice straw via Trichosporon dermatis. Fuel 309, 122117.

[45] Zhang, Y., Kamal, R., Li, Q., Yu, X., Wang, Q., Zhao, Z.K., 2022. Comparative fatty acid compositional profiles of rhodotorula toruloides haploid and diploid strains under various storage conditions. Fermentation 8, 467.

[46] Zhang, B., Liu, X.C., Bao, J., 2023. High solids loading pretreatment: The core of lignocellulose biorefinery as an industrial technology: An overview. Bioresour. Technol. 369, 128334.

[47] Zhang, J.L., Lyu, L.T., Liang, S.Y., Zhao, Z.K., 2025. Yeast lipid technology for biomass refinery. Sci. Sin. Chim. 55, 28-36.