Optimization of Oxygen Supply Control During the Fermentation of Sophorolipids Synthesis from Alkane as Substrate
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摘要: 以正十六烷为疏水底物考察了不同供氧水平对槐糖脂合成代谢的影响。通过对发酵过程中的代谢通量分布和关键酶活性的整合分析,发现供氧水平显著影响细胞脂肪酸和羟基脂肪酸的合成,同时显著影响葡萄糖和烷烃的利用程度。在线生理参数呼吸商(RQ)能够很好地表征胞内代谢通量的变化,后续有望成为过程调控的关键参数并可以进一步应用于过程优化。最后,在槐糖脂合成期提供合适的供氧水平,不但能够有效提升相对昂贵底物烷烃的利用率,而且能够显著降低功率输入,从而在两个方面提高生产经济性。本文研究结果同样适用于其他菜籽油、油酸等疏水性底物的槐糖脂发酵过程,并为工业规模槐糖脂发酵过程控制策略的开发和应用提供坚实的理论基础。
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关键词:
- Candida bombicola /
- 槐糖脂 /
- 正十六烷烃 /
- 氧代谢 /
- 代谢通量
Abstract: Sophorolipid (SL) is one of the most promising biosurfactants widely used in cosmetics, petroleum, pharmaceutical and other industrial fields. In this study, n-hexadecane was used as a hydrophobic substrate to investigate the effect of different oxygen supply levels on SLs synthesis. Through integrated analyses of metabolic flux distribution and key enzyme activities during the fermentation process, it was found that the oxygen supply level significantly affected the syntheses of fatty acids and hydroxyl fatty acids, and, simultaneously, caused significant changes in the utilization of glucose and alkanes. The on-line physiological parameter respiratory quotient (RQ) well dictated the changes of intracellular metabolic flux, which could act as a key process parameter to regulate cell metabolism. Finally, by enabling an appropriate oxygen supply level during the synthesis period of SLs, the utilization rate of the relatively expensive alkane substrate could be optimized, and the related power input reduced, thus improving the production economy. The results of this study could be extended to the study of other hydrophobic substrates such as rapeseed oil and oleic acid for SLs fermentation, and provided a theoretical basis for the control of industrial-scale SLs fermentation process.-
Key words:
- Candida bombicola /
- sophorolipids /
- hexadecane /
- oxygen metabolism /
- metabolic flux
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表 1 代谢网络中相关代谢物对照表
Table 1. Comparison table of related compounds in metabolic network
Abbreviations Name Glc Glucose G6P Glucose-6-phosphate G1P Glucose-1-phosphate F6P Fructose-6-phosphate R5P Ribose-5- phosphate GAP Glyceraldehydes-3-phosphate Pyr Pyruvate Lac Lactic acid AcCoA Acetyl coenzyme A Cit Citrate ICit Isocitrate SucCoA Succinyl coenzyme A SUC Succinate MAL Malate OAA Oxaloacetate UDPG UDP-glucose Hex Hexadecane Hexol 1-Hexadecanol Hexal Hexadecanal PA Palmitic acid HPA Hydroxylated palmitic acid NAA-SL Non-acetylated acidic sophorolipid MAA-SL Mono-acetylated acidic sophorolipid BAA-SL Di-acetylated acidic sophorolipid NADPH Nicotinamide adenine dinucleotide phosphate NADH Nicotinamide adenine dinucleotide FADH2 Flavine adenine dinucleotide, reduced 表 2 代谢通量方程式
Table 2. Metabolic flux equation
No. Reaction equation r1 Glc + ATP → G6P r2 G6P → F6P r3 G6P → R5P + 2NADPH + CO2 r4 3R5P → 2F6P + GAP r5 F6P + ATP→2GAP r6 GAP → Pyr + 2ATP + NADH r7 Pyr → Pyr.m r8 Pyr.m → AcCoA.m + NADH + CO2 r9 AcCoA.m + OAA.m → Cit.m r10 Cit.m →ICit r11 ICit → SucCoA + 2NADH + 2CO2 r12 SucCoA → Suc +ATP r13 ICit + AcCoA.m → Mal.m + Suc r14 Suc → Mal.m + FADH2 r15 Mal.m → OAA.m + NADH r16 OAA.m → Pyr.m + ATP + CO2 r17 Pyr + NADH → Lac r18 Cit.m → Cit r19 Cit +ATP → AcCoA + OAA r20 OAA + NADH → Mal r21 Mal → Mal.m r22 G6P → G1P r23 G1P → UDPG r24 Hex + O2 + 2NADPH → Hexol r25 Hexol + O2 → Hexal r26 Hexal → PA + 2NADH r27 PA + O2 + 2NADPH → HPA r28 HPA + 2UDPG → NAA-SL r29 NAA-SL + AcCoA → MAA-SL r30 MAA-SL+ AcCoA→ BAA-SL r31 PA → 8AcCoA + 7FADH2 + 7NADH r32 NADH + 0.5O2 → 2.5ATP r33 FADH2 + 0.5O2 → 1.5ATP r34 O2.ex → O2 r35 CO2 → CO2.ex r36 Glc.ex → Glc r37 Hex.ex → Hex r38 NAA-SL → NAA-SL.ex r39 MAA-SL → MAA-SL.ex r40 BAA-SL → BAA-SL.ex r41 Cit → Cit.ex r42 Pyr → Pyr.ex r43 Suc → Suc.ex r44 Lac → Lac.ex The subscripts .m and .ex indicate that the substances are inside the mitochondria or outside the cell, expectively 表 3 不同供氧水平下发酵过程关键参数比较
Table 3. Comparison of key parameters under different oxygen supply levels during the fermentation process
Batch SLs titer/(g·L−1) SLs consumption/g Glc consumption/g Alk consumption/g SLs productivity/(g·L−1·h−1)a) YSLs/substrateb) YSLs/Glcb) YSLs/Alkb) High oxygen 79.1 198.2 267.9 179.8 0.83 0.44 0.74 1.10 Low oxygen 55.0 137.3 278.1 90.9 0.57 0.37 0.49 1.51 a) SLs productivity = SLs titer/Fermentation time;b) Substrate consumption ratio 表 4 不同供氧水平下菌株72~96 h的比速率和碳回收率
Table 4. Specific rate and carbon recovery rate of strains under different oxygen levels at 72—96 h
Batch q/(mmol·g−1·h−1) Carbon recovery/%a) Glucose Alkane SLs O2 CO2 High oxygen 0.218±0.003 0.162±0.002 0.0738±0.001 2.807±0.008 1.573±0.005 100.9±0.5 Low oxygen 0.221±0.002 0.0687±0.001 0.0413±0.001 1.329±0.006 0.985±0.004 95.1±0.2 a) Carbon recovery=[(qSLs×32+$q_{{\rm{CO}}_2} $)/( qglucose×6+qalkane×16)]×100% 表 5 优化后发酵过程关键指标参数
Table 5. Key parameters of fermentation process after optimization
SLs titer/(g·L−1) Glc consumption/g Alk consumption/g SLs productivity/(g·L−1·h−1)a) YSLs/substrateb) YSLs/Glcb) YSLs/Alkb) 83.8 299.3 182.2 0.87 0.43 0.70 1.15 a) SLs productivity = SLs titer/Fermentation time;b) Substrate consumption ratio -
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