CN114736937A - 一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法 - Google Patents
一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法 Download PDFInfo
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Abstract
本发明属微藻培养技术领域,为解决目前微藻生产中,无法同时提高生物量以及油脂含量的问题,提供一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,斜生栅藻接种于加有水杨酸溶液的BG11液体培养基中,培养基中水杨酸的浓度为20‑40mg/L;培养至微藻稳定期;培养好的藻液用有机溶剂提取藻细胞中的油脂。无需添加碳源,节约成本。水杨酸可以人工合成,无需从植物体内提炼,且对环境无污染。没有损耗微藻生物量,大幅度提高了斜生栅藻油脂含量及产率,油脂含量最大提高到48%接近微藻自身干重的一半,油脂产率超过20mg/L·d‑1。
Description
技术领域
本发明属于微藻培养技术领域,具体涉及一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法。
背景技术
据《BP世界能源统计年鉴》2021统计,化石能源仍是2020年能源消费领域的绝对霸主,占比高达83.1%。化石能源的使用造成CO2等大量温室气体排放,从而导致全球气温升高、冰川融化、物种灭绝等一系列重大灾难。同时,石油价格一方面不断攀升,另一方面其资源具有有限性,寻找一种可以替代石油能源的新能源已经迫在眉睫。
生物质能是唯一可以替代石油能源的无污染能源。生物质能通过植物的光合作用可以再生,可转化为常规的固态、液态和气态燃料,取之不尽、用之不竭,同时也是唯一一种可再生的碳源。生物能源通过自主生产燃料,可以抑制进口石油价格,并减少进口石油费用,使更多的资金能用于改善人民生活和经济的持续发展,从根本上解决能源危机和粮食危机。
由于原料的限制,生物质能源在过去的几十年内并未被大众广泛的应用。如今,作为第三代生物柴油的原料来源—微藻,因其有许多不可替代的优点逐渐进入大众视野,得到重视并进行研究开发。其优点有:(1)来源广、易于培养、周期短;(2)与其他的油料作物相比,微藻的油脂含量及生物质产能都比较高;(3)以微藻为原料制得的生物柴油不含硫,具有碳中性的特点;(4)微藻具有顽强的生命力,在恶劣的环境下也可以生存;(5)微藻能够降解污水中的有机物并回收利用氮磷及吸附去除重金属离子。
国内外有研究通过对微藻施加浓度、种类不同的植物激素,从而改变微藻的性能,提高微藻自身的抗性,诱导微藻产出目标物,如虾青素、叶黄素、DHA等;提高微藻生物量、油脂含量;增强微藻对温度、光强、pH等环境因素的抗性等。国内外学者围绕微藻细胞高密度培养和油脂积累特性方面进行了大量研究,表明营养条件和培养模式对微藻生物量和油脂含量有显著影响。尤其在不利的营养条件(如缺氮、高光、高盐)胁迫下,藻细胞中的碳流会倾向高能产物储存的方向流动从而提高油脂含量,但是胁迫条件下往往会造成微藻生物量的锐减。因此,选择一种能够同时提高生物量及油脂含量的方法是目前的重要任务。
水杨酸是植物体内的一种内源性植物激素,参与调节植物体的种子萌发、细胞生长、光合作用、固氮和衰老等生理过程,同时诱导植物对非生物胁迫(低温、盐度和重金属等)产生抗性,对植物体产生保护作用。此外,水杨酸也可以通过调整氮代谢、氨基酸代谢、糖酵解、磷酸戊糖途径和脂肪酸代谢等相关酶的转录,促进碳水化合物和蛋白降解,使碳流更多地分配到脂肪酸合成途径,从而促进微藻脂质积累。目前,水杨酸的应用主要在于医药、化妆品、食品等领域,但是,将水杨酸应用于微藻培养,以促进油脂积累的生物质能源方面应用的研究尚未报道。
发明内容
本发明为了解决目前微藻培养过程中生物量以及油脂含量无法兼顾的问题,提供了一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法。在不损耗生物量的同时诱导微藻提高油脂含量,提高微藻油脂产率,最终实现生物柴油的优质优量生产。
本发明由如下技术方案实现的:一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,斜生栅藻接种于加有水杨酸溶液的BG11液体培养基中,培养基中水杨酸的浓度为20-40mg/L;培养至微藻稳定期,收集藻种,制成藻粉,提取油脂。
具体包括如下步骤:
(1)藻种液的制备:将斜生栅藻加入到BG11液体培养基中,在25℃、光强3000lux的恒温培养箱下培养至对数增长期,作为后续实验的藻种液;每日采用恒温摇床150r/min摇2~3次,每次15min,保证微藻均匀分布,避免微藻贴壁生长,将微藻培养至对数增长期,作为后续实验的藻种液;
(2)藻液的培养:步骤(1)所获得的藻种液接种于含有水杨酸溶液的BG11培养基中,控制培养条件为光照强度3000lux,温度25℃,调节初始pH为6.8-7.2,光暗比12h/12h,每日采用恒温摇床150r/min摇2~3次,每次15min,培养至稳定期收获;培养基中水杨酸的浓度为20-40mg/L;
(3)提取油脂:收集步骤(2)中稳定期的藻液,离心分离,采用冷冻干燥机制成干燥的藻粉,用有机溶剂提取藻细胞中的油脂;有机溶剂为氯仿、甲醇和水的混合溶液,混合溶液中氯仿、甲醇、水的体积比为1:1:0.9。
所述斜生栅藻为Scenedesmus obliquus FACHB-12,培养好的藻液中斜生栅藻的细胞密度范围为1.5-1.9g/L,干重范围为0.73-0.76g/L,油脂含量范围为35-49%,油脂产率>20mg/L·d-1。
本发明使用的斜生栅藻Scenedesmus obliquus(FACHB-12),购置于中国科学院武汉水生生物研究所淡水藻种库。
本发明中,所述的水杨酸溶液的浓度范围为20-40mg/L,研究发现在本发明所述范围内水杨酸浓度与油脂产率呈现线性关系,水杨酸浓度越高,油脂产率越高。
本发明中斜生栅藻每日在150r/min,25℃的恒温摇床下摇2-3次,每次10~15min。
所述培养方法下微藻细胞密度范围为1.5-1.9g/L,干重范围为0.73-0.76g/L,油脂含量范围为35-49%,油脂产率超过20mg/L·d-1。
与现有技术相比,本发明的优点是:本发明所述斜生栅藻培养方式为自养培养,无需添加碳源,通过恒温培养箱的鼓风系统鼓入的空气中的CO2提供碳源,实现碳减排、碳中和,节约成本。采用的植物激素水杨酸可以人工合成无需从植物体内提炼方便易得,水杨酸使用浓度低对环境几乎没有影响无污染。利用水杨酸诱导微藻产脂,在提高斜生栅藻生物量的同时大幅度提高了油脂含量及产率,油脂含量最大提高到48%,接近微藻自身干重的一半,油脂产率超过20mg/L·d-1。
本发明在不损耗生物量的前提下提高油脂含量,进而提高油脂产率,旨在增加油脂的产能。以CO2为碳源进行的自养培养,希望通过微藻利用空气中的CO2产出油脂,实现碳减排,减少微藻培养的成本。
附图说明
图1为本发明实施例中斜生栅藻细胞密度变化图;
图2为本发明实施例中斜生栅藻比生长速率变化图;
图3为本发明实施例中斜生栅藻干重对比图;
图4为本发明实施例中斜生栅藻不同时期叶绿素a变化图;
图5为本发明实施例中斜生栅藻油脂含量及产率图;
图6为本发明实施例中水杨酸浓度与油脂产率的线性图;
图7为本发明实施例中水杨酸对斜生栅藻FTIR图;
图8为本发明水杨酸诱导斜生栅藻产脂机制图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明中的一部分实施例而非全部的实施例。基于本发明实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
除非另有定义,所有在此使用的技术和科学术语和本发明所属领域内的技术人员所通常理解的意思相同,在此公开引用及他们引用的材料都将以引用的方式被并入。
本领域技术人员意识到的通过常规实验就能了解到的描述的特定实施方案的等同技术都将包含在本申请中。
下述实施例中的实验方法,如无特殊说明,均为常规方法;下述实施例中所用的仪器设备,如无特殊说明,均为实验室常规仪器设备;下述实施例中所用的实验材料,如无特殊说明,均为由常规生化试剂商店购买得到的。本发明所使用的斜生栅藻(Scenedesmus obliquus,FACHB-12),购置于中国科学院武汉水生生物研究所淡水藻种库,水杨酸(Salicylic acid)购于天津市光复科技发展有限公司,为优级纯。
实施例1:一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,包括如下步骤:
(1)制备藻液:将斜生栅藻扩培在BG11培养基内,在光强3000lux、25℃的恒温培养箱下培养到对数增长期,以作为后续实验激素诱导的藻种液。
(2)微藻培养:向150ml培养基中接种步骤1中10-15%的藻液,并加入20mg/L的水杨酸,培养条件为3000lux、25℃、光暗比=12h:12h,培养至稳定期,隔日测定细胞密度、比生长速率,实验结束时测定干重、油脂含量产率。
(3)油脂含量:将步骤2培养的微藻收获,真空冷冻干燥制成藻粉。取0.01mg藻粉于干燥洁净的离心管中,向其中加入0.8mL蒸馏水,再加入1mL氯仿和2mL甲醇,振荡2min使其混合均匀后,超声破碎1min。再向溶液中加入1ml氯仿,振荡1min混合均匀,再加入1ml蒸馏水,再振荡1min后在4000rpm的转速下离心5min。离心后,用针筒抽取最下层氯仿相转移到另一干燥洁净恒重的离心管,再向原管中加入2mL氯仿,振荡1min后,在4000rpm的转速下离心5min,将氯仿相转移到上一次的氯仿相中,该操作重复两次。将三次转移的氯仿相在80℃烘干至质量恒定,称重得所提取油脂重量。
结果表明,添加20mg/L水杨酸的实验组,斜生栅藻细胞密度为1.885g/L,干重为0.760g/L,获得的油脂含量及产率分别为33.66%、15.97mg/L·d-1。
实施例2:一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,包括如下步骤:
(1)微藻培养:向150ml培养基中接种步骤1中10-15%的藻液,并加入40mg/L的水杨酸,其他方法同实施例1所述方法。
(2)油脂含量测定方法同实施例1所述方法。
结果表明,添加40mg/L水杨酸的实验组,斜生栅藻细胞密度为1.567g/L,干重为0.738g/L,获得的油脂含量及产率分别为48.49%、22.38mg/L·d-1。
对比例1:微藻培养:向150ml培养基中接种步骤1中10-15%的藻液,未向BG11培养基中添加水杨酸,其他方法同实施例1所述方法。油脂含量测定方法同实施例1所述方法。
结果表明,未添加水杨酸的对照组,斜生栅藻细胞密度为1.402g/L,干重为0.525g/L,获得的油脂含量及产率分别为28.05%、9.20mg/L·d-1。
对比例2:微藻培养:向150ml培养基中接种步骤1中10-15%的藻液,并加入5mg/L的水杨酸,其他方法同实施例1所述方法。油脂含量测定方法同实施例1所述方法。
结果表明,添加5mg/L水杨酸的诱导组,斜生栅藻细胞密度为1.420g/L,干重为0.518g/L,获得的油脂含量及产率分别为37.51%、12.13mg/L·d-1。
对比例3:微藻培养:向150ml培养基中接种步骤1中10-15%的藻液,并加入10mg/L的水杨酸,其他方法同实施例1所述方法。油脂含量测定方法同实施例1所述方法。
结果表明,添加10mg/L水杨酸的诱导组,斜生栅藻细胞密度为1.577g/L,干重为0.635g/L,获得的油脂含量及产率分别为32.70%、13.34mg/L·d-1。
结果分析:表1汇总了不同浓度水杨酸对斜生栅藻细胞密度、干重、油脂含量及产率的影响。
表1 不同浓度水杨酸对斜生栅藻光密度、油脂含量及产率的影响
对比例1中在未添加水杨酸进行刺激斜生栅藻生长的情况下,细胞密度、油脂含量及产率的含量都是最低的,难以达到大规模生产生物油脂的基本要求。根据对比例2、3可以看出,添加低浓度的水杨酸对斜生栅藻有促进生长产脂的效果,细胞密度为1.577g/L,较对比例1提升了约15.8%,干重为0.653g/L,提高了约24.38%,油脂产率高达13.34mg/L·d-1,提高了42.7%。而从实例1、2中,一方面微藻受到植物激素水杨酸的刺激促进了干重的提高,另一方面微藻自身在受到水杨酸诱导后油脂含量提高。在双重作用下,斜生栅藻的油脂产率有大幅度的提升。通过表1可以看出细胞密度、干重最高点出现在水杨酸浓度为20mg/L时,分别为1.885g/L、0.760g/L,较对比例1提高了34.45%、44.76%。油脂含量及产率最高出现在水杨酸浓度在40mg/L时,油脂含量高达48.49%,接近微藻自身重量的一半,油脂产率为22.38mg/L·d-1,是对比例1的2.4倍。
水杨酸加快了斜生栅藻的生长速率见图2,加入水杨酸后对比例2、3和实施例1、2中比生长速率均有不同程度的增加,表明水杨酸能够加快微藻的生长速率,促使微藻提前进入对数生长期,同时,高浓度水杨酸延长了微藻生长周期见图1,提高微藻生物量、油脂积累的周期,从而提高了微藻的干重质量、油脂含量。
水杨酸能够提高微藻中叶绿素a的含量(如图4),提高了微藻光合作用中生产叶绿素a某些酶的活性,例如光合碳同化相关酶,同时提高了光合电子传递效率(ETR)及PSⅡ调节性能量耗散(NPQ),YNPQ,从而增加了叶绿素a的含量,从而提高光合作用的效率,增加生物量。藻类在各种胁迫条件下都可能利用其色素沉着系统作为防御机制,因此在营养物质匮乏的稳定期,尤其是在缺氮的条件下,细胞内源性含氮化合物含量降低,高浓度的SA遏制微藻对光的捕获和吸收,减少了叶绿素a的合成,从而导致碳水化合物和蛋白质代谢途径受到抑制,反而增加了细胞中油脂合成途径,促进碳流向油脂方面转化,积累脂质。
从图6可以看出,在本实验浓度范围内,添加水杨酸的浓度与油脂产率呈正相关,即水杨酸浓度越高,对微藻的油脂产率越高。因此,为显著提高斜生栅藻的油脂产率,优选20-40mg/L的水杨酸浓度可以获得最高的油脂含量及产率。在此条件下油脂含量范围为33-49%,油脂产率16-23 mg/L·d-1。
根据图7,SA诱导后细胞官能团—酯基、羧基、磷酸基团等都发生了变化,糖类、蛋白质等物质峰的减弱、消失和脂类物质峰的增加、加强都表明SA对斜生栅藻油脂提升具有积极作用。
图8为水杨酸刺激斜生栅藻产脂机制,多数绿藻的储藏物质是淀粉,在叶绿体中以淀粉粒形式存在,而在缺乏养分等逆境条件下会积累大量的油脂。一方面,光合作用所固定的过量的碳进入磷酸已糖库,以淀粉形式储存在叶绿体内,为后续碳流向脂类转化奠定基础,另一方面,还可通过糖酵解途径(EMP)生成丙酮酸,经丙酮酸脱氢酶催化直接得到乙酰辅酶a。在营养物质充足的情况下,SA提高了微藻中叶绿素的含量,进而促进微藻的光合作用,从而大量合成生物量,积累了淀粉等储藏物质,产生二羟基丙酮磷酸,在甘油-3-磷酸脱氢酶的作用下合成甘油-3-磷酸,经过卡尔文循环、糖酵解生成脂肪酸的前驱物质-乙酰辅酶a,再经过一系列反应生成脂肪酸。在稳定期营养物质缺乏,高浓度SA抑制叶绿素合成,碳水化合物和氮代谢途径受到抑制,反而增加了油脂合成途径,促进淀粉酶活性上升,淀粉降解为葡萄糖单体,葡萄糖进入EMP途径生成乙酰辅酶a合成脂肪酸。此外,SA的酸性和氧化性胁迫微藻,造成脂质过氧化,激发微藻合成磷脂的能力以抵抗不利环境,进而提高微藻自身的抗逆性,加速脂肪酸的正向合成。
因此,通过表1及图1-8中,斜生栅藻的细胞密度、比生长速率、干重等指标以及斜生栅藻产脂机制的讨论结果来看,这些结果为水杨酸诱导斜生栅藻生产油脂提供了有利的数据及理论支持。可以看出,在自养条件下,添加植物激素水杨酸能够促进斜生栅藻生长,诱导其油脂含量及产率的提高,降低成本提高生物油脂的产量。水杨酸投加范围为20-40mg/L之间,获得最高的生物量和油脂产率。由此可见,利用植物激素水杨酸诱导斜生栅藻提高油脂含量及产率是一种可行的、经济的、有效的方法,满足制备生物柴油的基本需求。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,其特征在于:斜生栅藻接种于加有水杨酸溶液的BG11液体培养基中,培养基中水杨酸的浓度为20-40mg/L;培养至微藻稳定期,收集藻种,制成藻粉,提取油脂。
2.根据权利要求1所述的一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,其特征在于:具体包括如下步骤:
(1)藻种液的制备:将斜生栅藻加入到BG11液体培养基中,在25℃、光强3000lux的恒温培养箱下培养至对数增长期,作为后续实验的藻种液;每日采用恒温摇床150r/min摇2~3次,每次15min;
(2)藻液的培养:步骤(1)所获得的藻种液接种于含有水杨酸溶液的BG11培养基中,控制培养条件为光照强度3000lux,温度25℃,调节初始pH为6.8-7.2,光暗比12h/12h,每日采用恒温摇床150r/min摇2~3次,每次15min,培养至稳定期收获;培养基中水杨酸的浓度为20-40mg/L;
(3)提取油脂:收集步骤(2)中稳定期的藻液,离心分离,采用冷冻干燥机制成干燥的藻粉,用有机溶剂提取藻细胞中的油脂;有机溶剂为氯仿、甲醇和水的混合溶液,混合溶液中氯仿、甲醇、水的体积比为1:1:0.9。
3.根据权利要求1或2所述的一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,其特征在于:所述斜生栅藻为Scenedesmus obliquus FACHB-12,培养好的藻液中斜生栅藻的细胞密度范围为1.5-1.9g/L,干重范围为0.73-0.76g/L,油脂含量范围为35-49%,油脂产率>20mg/L·d-1。
4.根据权利要求1所述的一种利用植物激素水杨酸促进斜生栅藻生长产脂的方法,其特征在于:所述培养基中水杨酸的浓度为40mg/L。
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