CN114934081B - 一种微生物混菌发酵提高油脂收率的方法 - Google Patents
一种微生物混菌发酵提高油脂收率的方法 Download PDFInfo
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Abstract
本发明涉及一种微生物混菌发酵提高油脂收率的方法,分别活化圆红冬孢酵母和小球藻;先将活化小球藻接种至发酵培养基中培养,之后接种活化圆红冬孢酵母,进行混菌发酵。本发明通过运用混菌发酵的手段,微藻为酵母生长提供O2,并且吸收利用酵母生产的CO2和有机酸,缓解了发酵后期pH较低对于油脂生产的抑制。
Description
技术领域
本发明属于微生物发酵领域,具体涉及一种微生物混菌发酵提高油脂收率的方法。
背景技术
随着世界人口的不断增加,人们对于食物和能源的需求日益增长,这一问题加剧了全球气候变化等环境问题。而微藻潜力巨大,代谢多种多样,因此在可持续生产增值产品方面具有巨大的潜力。微藻可以利用光合作用进行碳固定,这有助于减少CO2的排放。在自然系统中,微藻能够与异养微生物结合在一起(主要是细菌和酵母),异养微生物利用藻类产生的O2来氧化有机底物和产生CO2。这种藻类和异养微生物之间的有益协同具有巨大的研究前景。添加微藻后,酵母纯培养物的溶解氧显著增加。因此发现微藻的产氧有利于共培养异养微生物的生物量和脂质产量。产油酵母有能力将各种碳饲料转化为复杂的次生代谢产物,是生物燃料前体的生产者。微藻在生长过程中吸收无机碳(CO2)、氮(NH4 +、NO3 ﹣、NO2)和磷(PO4 3-),产生含氧(O2)碳水化合物、脂质和蛋白质。微藻释放的氧气被细菌用来分解废水中的有机物。这些细菌释放二氧化碳和一些促进微藻生长的生长促进剂。在光的生态系统中,光合微生物和异养生物之间的主要相互作用之一是CO2和O2之间的良性气体交换循环。在共培养体系中,pH的调节至关重要。微藻和酵母之间存在气体的相互供应,酵母的快速生长产生大量的二氧化碳,发酵液pH快速下降,较低的pH影响了酵母生长。而微藻光合作用产生氧气,氧气使得发酵液pH维持在一个较高的水平。因此共培养体系的pH可以在气体交换中维持平衡,菌株的生长性能得到提高。
廉价底物糖蜜的开发和应用也有巨大的前景,糖蜜又称为糖浆,是制糖工业将压榨出的甘蔗汁液或者甜菜、柑橘、玉米糖汁液,经加热、中和、沉淀、过滤、浓缩、结晶等工序制糖后所剩下的副产物,主要成分为糖类,糖蜜中富含大量还原糖,以及蔗糖,并且含有丰富的矿物质,所以糖蜜可作为碳源应用在发酵领域中。糖蜜中的主要成分为糖类,糖蜜含蔗糖约24%-36%,其他糖约12-24%;以及矿物质含量较高约8%-10%,并且含有少量金属离子,和生物素等营养成分,所以糖蜜可作为碳源及氮源应用于发酵领域中。
发明内容
本发明的目的在于解决圆红冬孢酵母发酵后期pH较低抑制发酵和油脂产量不高的问题,提供一种微生物混菌发酵提高油脂收率的方法。
为实现上述技术目的,本发明采用如下方案:
一种微生物混菌发酵提高油脂收率的方法,包括:
分别活化圆红冬孢酵母和小球藻;
先将活化小球藻接种至发酵培养基中培养,之后接种活化圆红冬孢酵母,进行混菌发酵。
作为一种优选的实施方式,发酵温度为25~30℃;优选30℃。
作为一种优选的实施方式,活化圆红冬孢酵母和活化小球藻的接种比例为1:1~1:3;优选1:2。
作为一种优选的实施方式,所述圆红冬孢酵母为圆红冬孢酵母(Rhodosporidiumtoruloides)Z11,小球藻为小球藻(Chlorella sp)GY-H4。圆红冬孢酵母是性状优良的产油酵母。酵母是目前研究较多的产油微生物,大多数酵母的脂肪酸组成相似,分布较为单一,以C16和C18系脂肪酸为主,软脂酸和硬脂酸是主要的饱和脂肪酸,油酸含量一般比较丰富,亚油酸含量较少,有些酵母还含有棕榈油酸。某些产油酵母虽然也能合成多不饱和脂肪酸,但其含量远低于霉菌。由于酵母的脂肪酸组成与植物油脂相似且酵母能够利用多种碳源合成油脂,因而是制备生物柴油的理想原料。常见的产油酵母有浅白色隐球酵母、弯隐球酵母、斯达氏油脂酵母、茁芽丝孢酵母、产油油脂酵母、胶粘红酵母等。
作为一种优选的实施方式,发酵培养基碳氮比为200~300;优选300。
作为一种优选的实施方式,将活化小球藻接种至发酵培养基中培养48-72h后接种活化圆红冬孢酵母;优选72h。
作为一种优选的实施方式,所述圆红冬孢酵母选用圆红冬孢酵母(Rhodosporidium toruloides)Z11,保藏编号为CCTCC NO:M 2021226;所述小球藻为小球藻(Chlorella sp)GY-H4。
作为一种优选的实施方式,发酵培养为振荡培养,优选转速110rpm。
作为一种优选的实施方式,发酵培养阶段,光照时间为12:12。
酵母在发酵过程中经常出现氧气供应不足、pH值变化剧烈、底物供应不足等问题。酵母生长过程中需要大量氧气,酵母通过代谢途径(如TCA循环)产生大量的二氧化碳,从而降低发酵过程中的pH值。在混合培养中,微藻可以作为需氧酵母的氧气发生器,而酵母生长反过来提供二氧化碳,以促进微藻的生长。这种良性的气体循环还可以缓解O2/CO2限制导致的生长限制。此外,微藻还能代谢发酵液中的一些有机酸,从而调节pH值,尤其是在发酵后期。在共培养体系中,细胞裂解后可获得金属和氨基酸等营养元素(图1)。
此外,廉价底物的开发也有很良好的应用。该体系的底物葡萄糖可以用廉价底物糖蜜替代。使用糖蜜作为底物发酵生物量可达到31.9g/L。
与现有技术相比,本发明的技术优势如下:
(1)本发明通过运用混菌发酵的手段,微藻为酵母生长提供O2,并且吸收利用酵母生产的CO2和有机酸,缓解了发酵后期pH较低对于油脂生产的抑制。
(2)本发明通过发酵条件优化,大幅度提高了油脂含量,油脂含量超过70%,同时不饱和脂肪酸含量增加,在30℃条件下发酵首次出现纯培养中未出现的不饱和脂肪酸亚麻酸,亚麻酸为高附加值不饱和脂肪酸,是人体必需脂肪酸之一,对人体有诸多生理功能,如降血脂、防血栓等。除作保健品外,也是重要的化工原料,增加了下游产物附加值。
(3)该发明成果将丰富和发展混菌发酵理论和实践,实现油脂试生产的绿色和高效合成;同时也为生物发酵法提产量提供新的思路和借鉴,在理论和实际应用方面均具有重要价值。
附图说明
图1为混菌发酵原理图。
图2为酵母、微藻分别发酵产油脂和混菌发酵产油脂对比。
图3为发酵培养基C/N比的优化。
图4为酵母和微藻最佳接种比例的优化。
图5为酵母和微藻最佳接种时间的优化。
图6为酵母和微藻混菌发酵最佳温度的优化。
图7为YC培养基和糖蜜培养基中生物量(A)、pH值(B)和脂肪酸组成(C)的比较。
具体实施方式
实施例中,圆红冬孢酵母分类命名为圆红冬孢酵母(Rhodosporidiumtoruloides)Z11,已于申请人的在先专利申请CN 113308387A中公开,保藏编号为CCTCCNO:M 2021226。
小球藻分类命名为小球藻(Chlorella sp)GY-H4,购自上海光语生物科技有限公司。
酵母活化培养基配方为:蛋白胨20g/L,葡萄糖20g/L,酵母粉10g/L,溶剂为水。
小球藻活化培养基配方为:硝酸钠1.4-1.6g/L,磷酸氢二钾0.03-0.05g/L,七水硫酸镁0.07-0.08g/L,七水氯化钙0.03-0.04g/L,碳酸钠0.01-0.03g/L,柠檬酸0.005-0.007g/L,柠檬酸铁0.03-0.05g/L,溶剂为水。
实施例1圆红冬孢酵母和小球藻发酵产油脂
(1)将圆红冬孢酵母和小球藻以接种量5%v/v分别接种到YPD培养基和小球藻BG11培养基中活化,25℃、110rpm分别活化24h和72小时;
(2)将圆红冬孢酵母和小球藻以总接种量20%v/v,1:1接种到酵母发酵培养基中,25℃、110rpm发酵120h;
(3)同时圆红冬孢酵母以10%v/v接种量接入酵母发酵培养基,30℃、110rpm发酵120h;小球藻以10%v/v接种量接入酵母发酵培养基,25℃、110rpm发酵120h。
酵母发酵培养基成分为:葡萄糖70g/L,酵母粉0.7g/L,硫酸铵0.1g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,溶剂为水。
实验发现,实施例使用的小球藻产物中未测出油脂,圆红冬孢酵母单发酵和混菌发酵,如图2所示,同等条件下混菌培养相比圆红冬孢酵母发酵的生物量积累具有明显优势,pH的调控也更加明显。通过对于油脂的对比和分析发现,在25℃下混合培养相比30℃单菌发酵仍具有更高的油脂含量。
实施例2混菌发酵
本实施例说明圆红冬孢酵母和小球藻混菌发酵步骤及效果,并对发酵培养基C/N比进行了优化
(1)将圆红冬孢酵母和小球藻以接种量5%v/v分别接种到酵母活化培养基和小球藻活化培养基中活化,25℃、110rpm分别活化24h和72小时;
(2)将圆红冬孢酵母和小球藻以总接种量20%v/v,1:1接种到发酵培养基中,25℃、110rpm发酵120h;
(3)调整碳氮比为100、200、300进行发酵优化,共3组,每组3个平行。
碳氮比为100时,发酵培养基配方为:葡萄糖70g/L,酵母粉0.73g/L,硫酸铵0.1g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,溶剂为水。
碳氮比为200时,发酵培养基配方为:葡萄糖70g/L,酵母粉1.19g/L,硫酸铵0.1g/L,
磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,溶剂为水。
碳氮比为300时,发酵培养基配方为:葡萄糖70g/L,酵母粉2.59g/L,硫酸铵0.1g/L,
磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,溶剂为水。
培养基碳氮比的调整在整个实验工作中是一个非常重要的部分。酵母生产过程中的脂质含量受菌株、碳源、碳氮比、培养基组成和培养条件等因素的影响。确定油脂生产的最佳条件是很重要的。大多数初步研究都采用烧瓶培养来确定最佳条件,包括碳氮比。不同的C/N影响脂质产量,较高的C/N导致较高的脂质含量。因此,氮限制条件常被用于各种产油生物较高的脂类积累。如图3所示,碳氮比为300时,生物量和油脂收率最高,分为别15.8g/L和38.87%,后续实施例均采用碳氮比为300时的发酵培养基进行发酵培养。
实施例3发酵接种比例优化
(1)将圆红冬孢酵母和小球藻以接种量5%v/v接种到活化培养基中,25℃、110rpm分别活化24h和48小时;
(2)将圆红冬孢酵母和小球藻以总接种量20%v/v接种到发酵培养基中,25℃、110rpm发酵120h;圆红冬孢酵母和小球藻接种比例分别为1:1、1:2、1:3。
由于小球藻的生长速度低于酵母的生长速度,因此需要调节接种比例来增强小球藻作为氧气发射器和pH调节的作用。实验结果如图4所示,圆红冬孢酵母和小球藻1:2的接种比例生物量的积累和油脂的含量最高,分别为16.37g/L和42.83%。
实施例4发酵接种时间优化
(1)将圆红冬孢酵母和小球藻以接种量5%v/v接种到活化培养基中,25℃、110rpm分别活化24h和48小时;
(2)将圆红冬孢酵母和小球藻以总接种量20%v/v,比例为1:2接种到发酵培养基中,先接种小球藻,圆红冬孢酵母的接种时间分别为延迟接种24h、48h、72h;发酵条件为25℃、110rpm发酵120h;
小球藻对于培养基的适应时间比酵母要缓慢一些,并且在新的生长环境有生长延滞期,因此延迟接种酵母,为小球藻生长提供更加条件有利于生物量的积累和油脂的生产。结果如图5所示,最佳接种时间为延迟接种72h,生物量和油脂收率分别为20.93g/L和45.62%。
实施例5发酵温度优化
(1)将圆红冬孢酵母和小球藻以接种量5%v/v接种到活化培养基中,25℃、110rpm分别活化24h和48小时;
(2)将圆红冬孢酵母和小球藻以总接种量20%v/v,比例为1:2接种,酵母延迟接种72h到发酵培养基中,110rpm发酵120h,发酵温度分别为20℃、25℃、30℃。
温度对于生长有着至关重要的影响。本申请首次发现不同温度对于混菌体系中油脂组分的影响,在30℃下油脂产物组分中首次出现不饱和脂肪酸亚麻酸,实验结果如图6所示,在30℃的生物量和油脂收率分别为29.25g/L和70.94%。
实施例6廉价底物替代
将廉价底物糖蜜进行简单稀释处理,作为培养基的碳源。发酵培养基配方为:稀释糖蜜70g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,溶剂为水。使用糖蜜作为底物发酵生物量可达到31.9g/L(图7)。
与现有技术相比,该技术更加绿色创新,利用混菌发酵的优势,缓解圆红冬孢酵母在发酵后期pH较低抑制油脂生产的问题。同时,大幅度提高油脂的生产,不饱和脂肪酸含量大大增加,出现高附加值产物如亚麻酸等,是一个新型高效绿色的油脂生产手段。
Claims (1)
1.一种微生物混菌发酵提高油脂收率的方法,其特征在于,包括:
将圆红冬孢酵母和小球藻以接种量5% v/v接种到活化培养基中,25 ℃、110 rpm分别活化24 h和48小时;
酵母活化培养基配方为:蛋白胨20g/L,葡萄糖 20g/L,酵母粉 10g/L,溶剂为水;
小球藻活化培养基配方为:硝酸钠1.4-1.6 g/L,磷酸氢二钾0.03-0.05 g/L,七水硫酸镁0.07-0.08 g/L,七水氯化钙0.03-0.04 g/L,碳酸钠0.01-0.03 g/L,柠檬酸0.005-0.007g/L,柠檬酸铁0.03-0.05 g/L,溶剂为水;
将活化小球藻接种至发酵培养基中培养48-72h后接种活化圆红冬孢酵母,在30℃下混菌发酵;活化圆红冬孢酵母和活化小球藻的接种比例为1:2;
发酵培养基配方为:葡萄糖70g/L,酵母粉2.59g/L,硫酸铵0.1g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5 g/L,溶剂为水;
所述圆红冬孢酵母选用圆红冬孢酵母(Rhodosporidium toruloides)Z11,保藏编号为CCTCC NO:M 2021226;所述小球藻为小球藻(Chlorella sp)GY-H4。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102080119A (zh) * | 2009-11-26 | 2011-06-01 | 北京化工大学 | 混合培养酵母和藻类生产油脂的方法 |
CN105441524A (zh) * | 2016-01-27 | 2016-03-30 | 扬州大学 | 一种通过共培养提高以蔗糖为碳源下微藻油脂产率的方法 |
CN106010993A (zh) * | 2016-06-12 | 2016-10-12 | 福建师范大学 | 生产亚麻酸的圆红冬孢酵母及其制备方法 |
CN108587919A (zh) * | 2018-05-31 | 2018-09-28 | 江南大学 | 一种通过共培养小球藻和粘红酵母生产生物油脂的方法 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102080119A (zh) * | 2009-11-26 | 2011-06-01 | 北京化工大学 | 混合培养酵母和藻类生产油脂的方法 |
CN105441524A (zh) * | 2016-01-27 | 2016-03-30 | 扬州大学 | 一种通过共培养提高以蔗糖为碳源下微藻油脂产率的方法 |
CN106010993A (zh) * | 2016-06-12 | 2016-10-12 | 福建师范大学 | 生产亚麻酸的圆红冬孢酵母及其制备方法 |
CN108587919A (zh) * | 2018-05-31 | 2018-09-28 | 江南大学 | 一种通过共培养小球藻和粘红酵母生产生物油脂的方法 |
Non-Patent Citations (1)
Title |
---|
Jiayin Ling等.Lipid production by a mixed culture of oleaginous yeast and microalga from distillery and domestic mixed wastewater.Bioresource Technology.2014,第173卷132-139. * |
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