培养基是影响赤藓糖醇生产技术水平的重要因素。该研究旨在基于培养基的优化,完成发酵工艺的优化,以实现赤藓糖醇生产技术水平的提高。通过培养基消耗分析技术了解脂耶氏酵母在发酵过程中对氮、磷、硫的需求评估,优化了磷源和氮源添加方案,基于优化方案开发了批次补糖发酵工艺。研究结果表明,氮源和磷源是影响赤藓糖醇发酵的关键因素,磷源浓度主要影响细胞干重, KH2PO4最优添加质量浓度为0.5 g/L;不同氮源对解脂耶氏酵母生长速率和细胞活力的影响是有差异的,酵母浸粉是决定性因素,最优的氮源组合为4 g/L酵母浸粉FM936和1 g/L小麦蛋白胨FP220。在50 L发酵罐上确定了批次补糖工艺,转化率和生产强度分别达到61.71%、2.41 g/(L·h),较优化前分别提高16.06%、42.01%,单罐产量提高了28.75%。研究结果对于降低赤藓糖醇规模化生产成本具有重要意义。
The culture medium is a crucial factor affecting the technological level of erythritol production.This study aimed to enhance erythritol production by optimizing the fermentation process through medium refinement.The nutrient requirements of Yarrowia lipolytica for nitrogen, phosphorus, and sulfur during fermentation was evaluated via spent media analysis.Then the phosphorus source and the combination of nitrogen sources were optimized, leading to the development of a batch supplementation process.Research findings highlighted that phosphorus and nitrogen source were critical determinations in erythritol fermentation.An inadequate concentration of the phosphorus source primarily impacted dry cell weight, with an optimal KH2PO4 concentration identified at 0.5 g/L.Different nitrogen sources exerted distinct effects on growth rate and cell viability, with yeast extract being the decisive factor.The optimal nitrogen source combination comprised 4 g/L yeast extract FM936 and 1 g/L wheat peptone FP220.The batch sugar supplementation process was established using a 50 L fermentation tank, achieving a conversion rate and production intensity of 61.71% and 2.41 g/(L·h) respectively, which were 16.06% and 42.01% higher than those before optimization. The yield per tank increased by 28.75%.This research holds significant implications for reducing the costs associated with large-scale erythritol production.
[1] 邱学良. 产赤藓糖醇亚罗解脂酵母的耐热机制分析及组合策略改造[D]. 江南大学, 2020.
QIU X L. Thermotolerance mechanism analysis and combination strategy transformation of erythritol producing Yarrowia lipolytica[D]. Jiangnan University, 2020.
[2] WANG N, CHI P, ZOU Y W, et al. Metabolic engineering of Yarrowia lipolytica for thermoresistance and enhanced erythritol productivity[J]. Biotechnology for Biofuels, 2020, 13:176.
[3] YANG S L, PAN X W, YOU J J, et al. Systematic metabolic engineering of Yarrowia lipolytica for the enhanced production of erythritol[J]. Bioresource Technology, 2024, 391:129918.
[4] KNEŽEVIĆ K, DAZA-SERNA L, MACH-AIGNER A R, et al. Investigation of ion-exchange membranes and erythritol concentration for the desalination of erythritol culture broth by electrodialysis[J]. Chemical Engineering and Processing-Process Intensification, 2023, 192:109494.
[5] SUWANAPETCH C, VANICHSRIRATANA W. Media optimization for erythritol production by Moniliella sp. BCC25224[J]. Sugar Tech, 2023, 25(1):257-261.
[6] SAVERGAVE L S, GADRE R V, VAIDYA B K, et al. Strain improvement and statistical media optimization for enhanced erythritol production with minimal by-products from Candida magnoliae mutant R23[J]. Biochemical Engineering Journal, 2011, 55(2):92-100.
[7] TOMASZEWSKA L, RYWINSKA A, MUSIAL I, et al. Effect of vitamins source on erythritol biosynthesis by Yarrowia lipolytica Wratislavia K1[J]. Current Opinion in Biotechnology, 2011, 22: S94-S95.
[8] 裴疆森, 黄玲, 张露, 等. 解脂亚罗酵母赤藓糖醇发酵过程的研究[J]. 食品与发酵工业, 2015, 41(4):28-33.
PEI J S, HUANG L, ZHANG L, et al. Erythritol production with Yarrowia lipolitica[J]. Food and Fermentation Industries, 2015, 41(4):28-33.
[9] TACHIBANA S, WATANABE K, KONISHI M. Estimating effects of yeast extract compositions on Escherichia coli growth by a metabolomics approach[J]. Journal of Bioscience and Bioengineering, 2019, 128(4):468-474.
[10] BAPAT P M, BHARTIYA S, VENKATESH K V, et al. Structured kinetic model to represent the utilization of multiple substrates in complex media during rifamycin B fermentation[J]. Biotechnology and Bioengineering, 2006, 93(4):779-790.
[11] RICHARDSON J, SHAH B, BONDARENKO P V, et al. Metabolomics analysis of soy hydrolysates for the identification of productivity markers of mammalian cells for manufacturing therapeutic proteins[J]. Biotechnology Progress, 2015, 31(2):522-531.
[12] DODIA H, SUNDER A V, BORKAR Y, et al. Precision fermentation with mass spectrometry-based spent media analysis[J]. Biotechnology and Bioengineering, 2023, 120(10):2809-2826.
[13] DODIA H, MISHRA V, NAKRANI P, et al. Dynamic flux balance analysis of high cell density fed-batch culture of Escherichia coli BL21 (DE3) with mass spectrometry-based spent media analysis[J]. Biotechnology and Bioengineering, 2024, 121(4):1393-1405.
[14] XU Y C, YANG H R, BRENNAN C S, et al. Cellular mechanism for the improvement of multiple stress tolerance in brewer’s yeast by potassium ion supplementation[J]. International Journal of Food Science & Technology, 2020, 55(6):2419-2427.
[15] ARAOZ M, GRILLO-PUERTAS M, DE MORENO DE LEBLANC A, et al. Inorganic phosphate modifies stationary phase fitness and metabolic pathways in Lactiplantibacillus paraplantarum CRL 1905[J]. Frontiers in Microbiology, 2024, 15:1343541.
[16] 汪文丽, 崔树茂, 唐鑫, 等. 两歧双歧杆菌F35氮源利用的差异性解析[J]. 食品与发酵工业, 2021, 47(19):21-28.
WANG W L, CUI S M, TANG X, et al. Various nitrogen source utilization in Bifidobacterium bifidum F35[J]. Food and Fermentation Industries, 2021, 47(19):21-28.
[17] 朱丹凤, 王园园, 崔树茂, 等. 罗伊氏乳杆菌氮源利用的选择性与特征分析[J]. 食品与发酵工业, 2018, 44(11):35-41.
ZHU D F, WANG Y Y, CUI S M, et al. Selectivity and characteristic analysis of nitrogen source utilized by Lactobacillus reuteri[J]. Food and Fermentation Industries, 2018, 44(11):35-41.
[18] JEYA M, LEE K M, TIWARI M K, et al. Isolation of a novel high erythritol-producing Pseudozyma tsukubaensis and scale-up of erythritol fermentation to industrial level[J]. Applied Microbiology and Biotechnology, 2009, 83(2):225-231.
[19] BATISTOTE M, DA CRUZ S H, ERNANDES J R. Altered patterns of maltose and glucose fermentation by brewing and wine yeasts influenced by the complexity of nitrogen source[J]. Journal of the Institute of Brewing, 2006, 112(2):84-91.
[20] KIM J, KIM K H. Effects of minimal media vs. complex media on the metabolite profiles of Escherichia coli and Saccharomyces cerevisiae[J]. Process Biochemistry, 2017, 57:64-71.
[21] YANG H R, ZONG X Y, CUI C, et al. Peptide (Lys-Leu) and amino acids (Lys and Leu) supplementations improve physiological activity and fermentation performance of brewer’s yeast during very high-gravity (VHG) wort fermentation[J]. Biotechnology and Applied Biochemistry, 2018, 65(4):630-638.
[22] RZECHONEK D A, DOBROWOLSKI A, RYMOWICZ W, et al. Recent advances in biological production of erythritol[J]. Critical Reviews in Biotechnology, 2018, 38(4):620-633.
[23] KHATAPE A B, DASTAGER S G, RANGASWAMY V. An overview of erythritol production by yeast strains[J]. FEMS Microbiology Letters, 2022, 369(1): fnac107.
