烷烃为底物合成槐糖脂发酵过程供氧控制优化

刘畅; 陈阳; 田锡炜; 庄英萍; 储炬; 王泽建

doi:10.14135/j.cnki.1006-3080.20210221001

烷烃为底物合成槐糖脂发酵过程供氧控制优化

doi: 10.14135/j.cnki.1006-3080.20210221001

华东理工大学生物反应器工程国家重点实验室, 生物工程学院，上海 200237

详细信息

作者简介:
刘畅：刘　畅 (1996—)，女，江苏连云港人，硕士生，研究方向为发酵过程工艺优化。E-mail：15751222520@163.com

通讯作者:
田锡炜，E-mail：tahfy@163.com

中图分类号: Q815
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出版历程
- 收稿日期: 2021-02-21
- 网络出版日期: 2021-05-07
- 刊出日期: 2022-04-22

Optimization of Oxygen Supply Control During the Fermentation of Sophorolipids Synthesis from Alkane as Substrate

State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China

摘要

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（R_Q）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。
- 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 (R_Q) 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.
- 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的发酵过程参数变化

Figure 1. Parameter changes of sophorolipids synthesis using alkanes as substrates during the fermentation process

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图 2 不同供氧条件下槐糖脂发酵过程的代谢网络模型（上下数据分别为高供氧组和低供氧组的代谢通量）

Figure 2. Metabolic network model of sophorolipids fermentation process under different oxygen conditions (The upper and lower data represent the metabolic flux of the high oxygen group and the low oxygen group, respectively)

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图 3 不同供氧条件下槐糖脂发酵过程关键酶活

Figure 3. Key enzyme activities in the sophorolipids fermentation process under different oxygen conditions

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图 4 槐糖脂发酵过程阶段性调整

Figure 4. Phase adjustment of sophorolipids fermentation process

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图 5 优化后槐糖脂发酵参数

Figure 5. Sophorolipids fermentation parameters after optimization

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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

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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 + 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

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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)	Y_{SLs/substrate}^b)	Y_SLs/Glc^b)	Y_SLs/Alk^b)
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

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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

Batch	q/(mmol·g⁻¹·h⁻¹)					Carbon recovery/%^a)
Batch	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=[(q_SLs×32+$q_{{\rm{CO}}_2} $)/( q_glucose×6+q_alkane×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 fermentation process after optimization

SLs titer/（g·L⁻¹）	Glc consumption/g	Alk consumption/g	SLs productivity/（g·L⁻¹·h⁻¹）^a）	Y_{SLs/substrate}^b）	Y_SLs/Glc^b）	Y_SLs/Alk^b）
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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