高级检索

  • ISSN 1006-3080
  • CN 31-1691/TQ

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

刘畅, 陈阳, 田锡炜, 庄英萍, 储炬, 王泽建. 烷烃为底物合成槐糖脂发酵过程供氧控制优化[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20210221001
引用本文: 刘畅, 陈阳, 田锡炜, 庄英萍, 储炬, 王泽建. 烷烃为底物合成槐糖脂发酵过程供氧控制优化[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20210221001
LIU Chang, CHEN Yang, TIAN Xiwei, ZHUANG Yingping, CHU Ju, WANG Zejian. Optimization of Oxygen Supply Control During the Fermentation of Sophorolipids Synthesis from Alkane as Substrate[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20210221001
Citation: LIU Chang, CHEN Yang, TIAN Xiwei, ZHUANG Yingping, CHU Ju, WANG Zejian. Optimization of Oxygen Supply Control During the Fermentation of Sophorolipids Synthesis from Alkane as Substrate[J]. Journal of East China University of Science and Technology. 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
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

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

  • 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。

     

  • 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

    AbbreviationsEnglish names
    GlcGlucose
    G6PGlucose-6-phosphate
    G1PGlucose-1-phosphate
    F6PFructose-6-phosphate
    R5PRibose-5- phosphate
    GAPGlyceraldehydes-3-phosphate
    PyrPyruvate
    LacLactic acid
    AcCoAAcetyl coenzyme A
    CitCitrate
    ICitIsocitrate
    SucCoASuccinyl coenzyme A
    SucSuccinate
    MalMalate
    OAAOxaloacetate
    UDPGUDP-glucose
    HexHexadecane
    Hexol1-Hexadecanol
    HexalHexadecanal
    PAPalmitic acid
    HPAHydroxylated palmitic acid
    NAA-SLNon-acetylated acidic sophorolipid
    MAA-SLMono-acetylated acidic sophorolipid
    BAA-SLDi-acetylated acidic sophorolipid
    NADPHNicotinamide adenine dinucleotide phosphate
    NADHNicotinamide adenine dinucleotide
    FADH2Flavine adenine dinucleotide, reduced
    O2Oxygen
    CO2Carbondioxide
    下载: 导出CSV

    表  2  代谢通量方程式

    Table  2.   Metabolic flux equation

    No.Reaction equation
    r1Glc + ATP → G6P
    r2G6P → F6P
    r3G6P → R5P + 2 · NADPH + CO2
    r43 · R5P → 2 · F6P + GAP
    r5F6P + ATP→2 · GAP
    r6GAP → Pyr + 2 · ATP + NADH
    r7Pyr → Pyr.m
    r8Pyr.m → AcCoA.m + NADH + CO2
    r9AcCoA.m + OAA.m → Cit.m
    r10Cit.m →ICit
    r11ICit → SucCoA + 2 · NADH + 2 · CO2
    r12SucCoA → Suc +ATP
    r13ICit + AcCoA.m → Mal.m + Suc
    r14Suc → Mal.m + FADH2
    r15Mal.m → OAA.m + NADH
    r16OAA.m → Pyr.m + ATP + CO2
    r17Pyr + NADH → Lac
    r18Cit.m → Cit
    r19Cit +ATP → AcCoA + OAA
    r20OAA + NADH → Mal
    r21Mal → Mal.m
    r22G6P → G1P
    r23G1P → UDPG
    r24Hex + O2 + 2 · NADPH → Hexol
    r25Hexol + O2 → Hexal
    r26Hexal → PA + 2 · NADH
    r27PA + O2 + 2 · NADPH → HPA
    r28HPA + 2 · UDPG → NAA-SL
    r29NAA-SL + AcCoA → MAA-SL
    r30MAA-SL+ AcCoA→ BAA-SL
    r31PA → 8 · AcCoA + 7 · FADH2 + 7 · NADH
    r32NADH + 0.5 · O2 → 2.5 · ATP
    r33FADH2 + 0.5 · O2 → 1.5 · ATP
    r34O2.ex → O2
    r35CO2 → CO2.ex
    r36Glc.ex → Glc
    r37Hex.ex → Hex
    r38NAA-SL → NAA-SL.ex
    r39MAA-SL → MAA-SL.ex
    r40BAA-SL → BAA-SL.ex
    r41Cit → Cit.ex
    r42Pyr → Pyr.ex
    r43Suc → Suc.ex
    r44Lac → 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
    下载: 导出CSV

    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

    BatchSLs Titer/(g·L−1Glc consumption/gAlk consumption/gSLs Productivity/(g·L−1·h−1a)YSLs/substrateb)YSLs/Glcb)YSLs/Alkb)
    High oxygen79.1267.9179.80.830.440.741.10
    Low oxygen55.0278.190.90.570.370.491.51
    a) SLs Productivity = SLs titer/Fermentation time
    b) Ym (SLs)/Substrate consumption
    下载: 导出CSV

    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)GlucoseAlkaneSLsO2CO2Carbon recovery/%a)
    High oxygen0.218±0.0030.162±0.0020.0738±0.0012.807±0.0081.573±0.005100.9±0.5
    Low oxygen0.221±0.0020.0687±0.0010.0413±0.0011.329±0.0060.985±0.00495.1±0.2
    a) Carbon recovery=[(qSLs×32+qCO2×1)/( qglucose×6+qalkane×16)]×100%
    下载: 导出CSV

    表  5  优化后发酵过程关键指标参数

    Table  5.   Key parameters of different process was optimized

    BatchSLs Titer/(g·L−1Glc consumption/gAlk consumption/gSLs Productivity/(g·L−1·h−1YSLs/substrateYSLs/GlcYSLs/Alk
    Optimization83.8299.3182.20.870.430.701.15
    下载: 导出CSV
  • [1] JEZIERSKA S, CLAUS S, VAN BOGAERT I. Yeast glycolipid biosurfactants[J]. Febs Letters, 2018, 592 (8): 1312-1329. doi: 10.1002/1873-3468.12888
    [2] SHAH M, SIVAPRAGASAM M, et al. Production of sophorolipids by Starmerella bombicola yeast using new hydrophobic substrates[J]. Biochemical Engineering Journal, 2017, 127: 60-67. doi: 10.1016/j.bej.2017.08.005
    [3] HU Y, JU L. Purification of lactonic sophorolipids by crystallization[J]. Journal of Biotechnology, 2001, 87 (3): 263-272. doi: 10.1016/S0168-1656(01)00248-6
    [4] CHEN Y, LIN Y M, TIAN X W, et al. Real-time dynamic analysis with low-field nuclear magnetic resonance of residual oil and sophorolipids concentrations in the fermentation process of Starmerella bombicola[J]. Journal of Microbiological Methods, 2019, 157: 9-15. doi: 10.1016/j.mimet.2018.12.007
    [5] SAERENS K, SAEY L. One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola[J]. Biotechnology and Bioengineering, 2011, 108 (12): 2923-2931. doi: 10.1002/bit.23248
    [6] 杨帆. 两种槐糖脂表面特性和生物活性初步研究[D]. 沈阳: 辽宁大学, 2011.
    [7] BACCILE N, BANAT M, et al. Development of a cradle-to-grave approach for acetylated acidic sophorolipid biosurfactants[J]. Acs Sustainable Chemistry & Engineering, 2017, 5(1): 1186-1198.
    [8] CERESA C, RINALDI M, et al. Inhibitory effects of lipopeptides and glycolipids on C. albicans-Staphylococcus spp. dual-species biofilms[J]. Frontiers in Microbiology, 2021: 11. doi: 10.3389/fmicb.2020.545654
    [9] FENG L Y, JIANG X P, HUANG Y N, et al. Petroleum ydrocarbon-contaminated soil bioremediation assisted by isolated bacterial consortium and sophorolipid[J]. Environmental Pollution, 2021, 273: 116476. doi: 10.1016/j.envpol.2021.116476
    [10] YING G G. Fate, behavior and effects of surfactants and their degradation products in the environment[J]. Environment International, 2006, 32(3): 417-431. doi: 10.1016/j.envint.2005.07.004
    [11] PAREKH V J, PANDIT A B. Optimization of fermentative production of sophorolipid biosurfactant by Starmerella bombicola NRRL Y-17069 using response surface methodology[J]. International Journal of Pharmacy and Biological Sciences, 2011, 1 (3): 103-106.
    [12] BAJAJ V, TILAV A. Enhanced production of bioactive sophorolipids by Starmerella bombicola NRRL Y-17069 by design of experiment approach with successive purification and characterization[J]. Journal of Oleo Science, 2012, 61(7): 377. doi: 10.5650/jos.61.377
    [13] MADDIKERI G L, GOGATE P R, PANDIT A B. Improved synthesis of sophorolipids from waste cooking oil using fed batch approach in the presence of ultrasound[J]. Chemical Engineering Journal, 2015, 263: 479-487. doi: 10.1016/j.cej.2014.11.010
    [14] WANG H, KAUR G, TO M H, et al. Efficient in-situ separation design for long-term sophorolipids fermentation with high productivity[J]. Journal of Cleaner Production, 2019, 246: 118995.
    [15] 刘新歌, 马晓静, 何洪波. 槐糖脂合成的廉价底物替代研究进展[J]. 生物加工过程, 2017, 15(3): 59-68. doi: 10.3969/j.issn.1672-3678.2017.03.010
    [16] PEDRO JIMENZE -PEALVER, DAVEREY A, et al. Use of wastes for sophorolipids production as a transition to circular economy: State of the art and perspectives[J]. Reviews in Environmental Science and Bio/Technology, 2019, 18(1).
    [17] 彭怀丽, 李红娜, 张丽. 石油污染土壤中正十六烷降解菌的效果研究[J]. 农业资源与环境学报, 2017, 34(3): 257-265.
    [18] 张施阳, 朱瑞利, 李辉. H2O2供氧条件下Burkholderia cepacia好氧降解三氯乙烯和苯酚的共代谢机理[J]. 华东理工大学学报(自然科学版), 2015, 41(1): 48-53, 71. doi: 10.3969/j.issn.1006-3080.2015.01.008
    [19] 陈玉琳, 刘秋, 于基成. 海洋石油降解菌AH07的分离鉴定及其石油降解性能分析[J]. 微生物学杂志, 2020, 40(01): 32-36. doi: 10.3969/j.issn.1005-7021.2020.01.004
    [20] GUILMANOV V. BALLISTRERI A. Oxygen transfer rate and sophorose lipid production by Candid bombicola[J]. Biotechnology and Bioengineering. 77(5): 489-494.
    [21] 滕丽丽. 含透明颤菌血红蛋白基因重组菌的构建及其对菌株发酵产槐糖脂的影响[D]. 青岛科技大学, 2019.
    [22] HUANG F C, PETER A, SCHWAB W. Expression and characterization of CYP52 genes involved in the biosynthesis of sophorolipid and alkane metabolism from Starmerella bombicola[J]. Applied and Environmental Microbiology, 2014, 80(2): 766-776. doi: 10.1128/AEM.02886-13
    [23] LI J S, HUI L, LI W W, et al. Identification and characterization of a flavin-containing monooxygenase MoA and its function in a specific sophorolipid molecule metabolism in Starmerella bombicola[J]. Applied Microbiology & Biotechnology, 2016, 100(3): 1307-1318.
    [24] 苏怡. 催化裂化汽油烯烃组成对其辛烷值影响的气相色谱分析研究[D]. 兰州理工大学, 2020.
    [25] CHEN Y, TIAN X W, LI Q H, et al. Target-site directed rational high-throughput screening system for high sophorolipids production by Candida bombicola[J]. Bioresource Technology, 2020, 315: 123586.
    [26] LI Y, QIN J, WU H, et al. In vitro inhibitory effect of lysionotin on the activity of cytochrome P450 enzymes[J]. Pharmaceutical Biology, 2020, 58(1): 695-700. doi: 10.1080/13880209.2020.1787468
    [27] 赵仕兰. 高等植物长链脂肪醇氧化酶初步研究[D]. 上海交通大学, 2009.
    [28] WANG T, LIU T, WANG Z, et al. A rapid and accurate quantification method for real-time dynamic analysis of cellular lipids during microalgal fermentation processes in Chlorella protothecoides with low field nuclear magnetic resonance. Journal of Microbiological Methods, 2016, 124: 13-20.
    [29] YANG L, LI Y, ZHANG X, et al. Metabolic profiling and flux distributions reveal a key role of acetyl-CoA in sophorolipid synthesis by Candida bombicola[J]. Biochemical Engineering Journal, 2019, 145: 74-82. doi: 10.1016/j.bej.2019.02.013
    [30] SAERENS K M J, VAN B I N A. Characterization of sophorolipid biosynthetic enzymes from Starmerella bombicola[J]. Fems Yeast Research, 2015(7): 7.
    [31] JEZIERSKA S, CLAUS S, et al. Redirecting the lipid metabolism of the yeast Starmerella bombicola from glycolipid to fatty acid production[J]. Journal of Industrial Microbiology and Biotechnology, 2019, 46(9): 1-10.
    [32] SAERENS K M J, SAEV L, SOETAERT W. One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola[J]. Biotechnology and Bioengineering, 2011, 108(12): 2923-2931. doi: 10.1002/bit.23248
  • 加载中
图(5) / 表(5)
计量
  • 文章访问数:  414
  • HTML全文浏览量:  109
  • PDF下载量:  2
  • 被引次数: 0
出版历程
  • 收稿日期:  2021-02-21
  • 网络出版日期:  2021-05-07

目录

    /

    返回文章
    返回