Optimization of Oxygen Supply Control During the Fermentation of Sophorolipids Synthesis from Alkane as Substrate
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摘要: 槐糖脂是最具前途的生物表面活性剂之一，广泛应用于化妆品、石油、制药等行业。本研究以正十六烷作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。通过代谢通量计算整合发酵过程关键酶活性分析，发现供氧水平显著影响细胞脂肪酸和羟基脂肪酸的合成，同时对葡萄糖和烷烃的利用程度造成明显的改变。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，因此后续有望成为过程调控的关键参数进一步应用于过程优化。最后，通过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，而且也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: Sophorolipids (SLs) is one of the most promising biosurfactants, which has been widely used in cosmetics, petroleum, pharmaceutical and other industrial fields. In this study, n-hexadecane was used as hydrophobic substrate to investigate the effect of different oxygen supply levels on SLs synthesis. Through the 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 auotient (RQ) could well represent the changes of intracellular metabolic flux, so as to be expected to act as a key process parameter to regulate the cell metabolism in the further. Finally, by providing the appropriate oxygen supply level during the synthesis period of SLs, it can not only enhance the utilization rate of relatively expensive substrate alkane, but also significantly reduce the power input, thus improving the production economy. The results of this study would be easily extended to other hydrophobic substrates such as rapeseed oil and oleic acid for SLs fermentation, and would provide a solid theoretical basis for the development and application of industrial-scale SLs fermentation process control strategies.-
Key words:
- Candida bombicola /
- sophorolipids /
- hexadecane /
- oxygen metabolism /
- metabolic flux
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图 1 以烷烃为底物合成槐糖脂发酵过程参数变化
a—Speed adjustment and OUR changes; b—Cell dry weight changes; c—Sophorolipids production changes; d—CER and RQ changes
Figure 1. Parameter changes in the fermentation process of sophorolipids synthesis using alkanes as substrates
图 2 不同供氧条件下槐糖脂发酵过程的代谢网络模型（自上而下分别为高供氧组和低供氧组）
Figure 2. Metabolic network model of sophorolipids fermentation process under different oxygen conditions. (From top to bottom are the high oxygen group and the low oxygen group)
图 3 不同氧条件槐糖脂发酵过程关键酶活
a—P450 monooxygenase; b—Long-chain fatty alcohol oxidase
Figure 3. Key enzyme activities in the sophorolipids fermentation process under different oxygen conditions
图 4 槐糖脂发酵阶段性调整
a—phased stirring speed and RQ; b—phased productivity of sophorolipids
Figure 4. Phase adjustment of sophorolipids fermentation
图 5 优化后槐糖脂发酵参数
a—Stirring speed, OUR and RQ; b—Sophorolipids titer
Figure 5. Sophorolipids fermentation parameters after optimization
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 English names 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 O2 Oxygen CO2 Carbondioxide 
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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 + 2 · NADPH + CO2 r4 3 · R5P → 2 · F6P + GAP r5 F6P + ATP→2 · GAP r6 GAP → Pyr + 2 · ATP + 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 + 2 · NADH + 2 · CO2 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 + 2 · NADPH → Hexol r25 Hexol + O2 → Hexal r26 Hexal → PA + 2 · NADH r27 PA + O2 + 2 · NADPH → HPA r28 HPA + 2 · UDPG → NAA-SL r29 NAA-SL + AcCoA → MAA-SL r30 MAA-SL+ AcCoA→ BAA-SL r31 PA → 8 · AcCoA + 7 · FADH2 + 7 · NADH r32 NADH + 0.5 · O2 → 2.5 · ATP r33 FADH2 + 0.5 · O2 → 1.5 · ATP 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 注：表中为了区分同一物质在细胞液、线粒体和胞外的不同分布，下标.m或.ex分别表示物质处于线粒体内或胞外
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表 3 不同供氧水平下发酵过程关键指标参数比较
Table 3. Comparison of key parameters under different oxygen supply levels
Batch 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) High oxygen 79.1 267.9 179.8 0.83 0.44 0.74 1.10 Low oxygen 55.0 278.1 90.9 0.57 0.37 0.49 1.51 a) SLs Productivity = SLs titer/Fermentation time
b) Ym (SLs)/Substrate consumption
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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
qp/(mmol·g−1·h−1) Glucose Alkane SLs O2 CO2 Carbon recovery/%a) 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+qCO2×1)/( qglucose×6+qalkane×16)]×100%
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表 5 优化后发酵过程关键指标参数
Table 5. Key parameters of different process was optimized
Batch SLs Titer/（g·L−1） Glc consumption/g Alk consumption/g SLs Productivity/（g·L−1·h−1） YSLs/substrate YSLs/Glc YSLs/Alk Optimization 83.8 299.3 182.2 0.87 0.43 0.70 1.15
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