CN109517853B - 一种利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法 - Google Patents
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Abstract
本发明公开一种利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法，属于利用微藻生产生物柴油技术领域。本发明所述方法为：将核桃壳提取液作为微藻培养的基础培养基，添加NaNO3，调节pH、分装、灭菌；将微藻接入培养基中，通入CO2，同时添加黄腐酸进行培养；利用紫外分光光度计测定培养条件下微藻的生物量；微藻生长到达稳定期后，利用有机溶剂提取微藻细胞内的油脂。本发明方法提高了微藻中油脂的含量，不仅可以解决核桃壳的处理问题，同时为微藻培养提供了一种新的思路，成功实现了“变废为宝”，节约了生产成本。
Description
技术领域
本发明涉及一种利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法，属于利用微藻生产生物柴油技术领域。
背景技术
生物柴油由于其燃烧性高、无污染、可再生等特点，是传统化石燃料理想的替代能源。作为生产生物柴油的原料，相比众多非粮生物，微藻具有生长速率快，生长周期短，光合作用效率高，而且可以利用微生物发酵技术，在光反应器中高密度培养等优势。一些微藻因其含油量高、易于培养、单位面积产量大等优点，而被称为新一代生物柴油原。但由于微藻生长繁殖条件要求高，前期投资较大，同比造价上还高于化石燃料，所以急需提高其油脂产率。
目前，提高微藻油脂含量的传统方法有：优化发酵条件和基因工程法。对微藻的发酵条件进行改良，可以很大程度的提高微藻的油脂含量；微藻中，油脂的积累涉及到脂类合成和代谢等多方面问题，所以基因工程法仅用单一的一种特异性基因，不能显著的提高油脂含量。
核桃壳是核桃取仁后的坚硬外壳，核桃壳目前被用作农户燃料或者直接丢弃，利用度较低且污染环境。在环境治理方面，核桃壳被用于重金属离子的吸附和活性炭的制备，但以上仅从物理的角度对核桃壳加以利用，至今，尚未有人把核桃壳内的化学物质进行利用。现代研究发现核桃壳含有酚酸类、黄酮类、苷类等多种活性物质，具有抗氧化、抗菌等作用。
发明内容
本发明的目的在于提供一种利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法，利用核桃壳提取液作为基础培养基，通入CO2，进行微藻培养，在黄腐酸的诱导下，微藻的油脂含量和生物量均有提高；本发明不仅可以解决核桃壳的处理问题，还可以提高微藻的油脂含量和生物量，成功实现了“变废为宝”，节约了生产成本；该方法操作简单易行，具体包括以下步骤：
（1）配制培养基：将核桃壳提取液作为微藻培养的基础培养基，添加NaNO3，调节pH、分装、灭菌；
（2）微藻培养：将微藻接入步骤（1）中的培养基中，通入CO2，同时添加黄腐酸进行培养；
（3）生物量和油脂含量的测定：利用紫外分光光度计测定培养条件下微藻的生物量；微藻生长到达稳定期后，利用有机溶剂提取微藻细胞内的油脂。
优选的，本发明步骤（1）中配制培养基的具体方法为：将核桃壳粉碎，煮沸4~4.5h作为基础培养基，NaNO3的添加量为1.1~1.2g/L，pH调节在6.8-7.0之间，灭菌条件为121℃、20 min。
优选的，本发明步骤（2）中微藻的初始接种量为0.38~0.42g/L，CO2的通入量为11~13%，培养基中黄腐酸的浓度为20~80mg/L。
优选的，本发明微藻的培养条件是：光照强度为6000~7000lux，通气量为0.5~0.6L/min，培养温度为24~26℃。
本发明所述微藻生物量浓度的测定方法为常规方法，具体如下：根据藻细胞与吸光度A750值在一定的范围内成线性关系，得出单针藻Monoraphidiumsp.QLZ-3在750nm处的干重与吸光度的关系如下：
Y=0.3226X-0.0025 R 2 =0.9997
Y：生物量（g/L）；X：吸光度值（A750）
测定藻液的A750值，带入上述公式，得出微藻的生物量。
本发明所述油脂的提取方法具体为：将培养至稳定期的微藻，3500r/min离心5min，弃上清，用蒸馏水洗涤沉淀2次，3500r/min离心5min，取沉淀，-80℃冻干；取藻粉，混合2倍质量的石英砂研磨，用氯仿/甲醇（2:1，v/v）提取藻细胞内的油脂，重复提取3次，合并提取液，40℃下烘干称重。
本发明的有益效果：
（1）本发明利用黄腐酸提高了核桃壳提取液中微藻油脂含量和生物量，将核桃壳的处理与微藻培养结合起来，节约了资源，降低了微藻培养的生产成本，并且提高了资源的利用率，变废为宝。
（2）本发明利用核桃壳提取液作为微藻生长的基础培养基，添加不同浓度的黄腐酸，微藻的油脂含量和生物量均不同程度的增长；且当黄腐酸浓度为40mg/L时，微藻的油脂含量和生物量均达到最高，为58.76%和1.48g/L，分别是对照组的1.19和2.82倍。
（3）本发明以核桃壳提取液作为基础培养基培养微藻，实现了废弃物的资源化利用，为核桃壳的处理提供了一种新的方式，同时为微藻的培养提供了一种新的策略。
附图说明
图1黄腐酸不同浓度下微藻的生物量；
图2黄腐酸不同浓度下微藻的油脂含量。
具体实施方式
下面结合具体实施方式对本发明进行详细的说明。
本发明实施例中微藻生物量浓度的测定方法为常规方法，具体如下：根据藻细胞与吸光度A750值在一定的范围内成线性关系，得出单针藻Monoraphidium sp. QLZ-3在750nm处的干重与吸光度的关系如下：
Y=0.3226X-0.0025 R 2 =0.9997
Y：生物量（g/L）；X：吸光度值（A750）
测定藻液的A750值，带入上述公式，得出微藻的生物量。
油脂的提取方法具体为：将培养至稳定期的微藻，3500r/min离心5min，弃上清，用蒸馏水洗涤沉淀2次，3500r/min离心5min，取沉淀，-80℃冻干；取藻粉，混合2倍质量的石英砂研磨，用氯仿/甲醇（2:1，v/v）提取藻细胞内的油脂，重复提取3次，合并提取液，40℃下烘干称重。
实施例1
将核桃壳煮沸4h作为基础培养基，外源添加NaNO3（1.1g/L），pH调节在6.8-7.0之间，灭菌条件为121℃、20min。微藻培养温度24~26℃，初始接种量为0.38g/L，气体流速为0.6L/min，CO2体积分数为11%，添加浓度为20mg/L的黄腐酸；每天定时取样，利用紫外分光光度计测得生物量，培养结束后利用有机溶剂提取油脂。
结果：当核桃壳提取液中黄腐酸浓度为20mg/L时，微藻的油脂含量为56.74%，生物量最高达到1.28g/L。
实施例2
将核桃壳煮沸4.5h作为基础培养基，外源添加NaNO3（1.2g/L），pH调节在6.8-7.0之间，灭菌条件为121℃、20min。微藻培养温度24~26℃，初始接种量为0.42g/L，气体流速为0.6L/min，CO2体积分数为11%，添加浓度为40mg/L的黄腐酸；每天定时取样，利用紫外分光光度计测得生物量，培养结束后利用有机溶剂提取油脂。
结果：当核桃壳提取液中黄腐酸浓度为40mg/L时，微藻的油脂含量为58.76%，生物量最高达到1.48g/L。
实施例3
将核桃壳煮沸4 h作为基础培养基，外源添加NaNO3（1.1g/L），pH调节在6.8-7.0之间，灭菌条件为121℃、20 min。微藻培养温度24~26℃，初始接种量为0.38g/L，气体流速为0.5L/min，CO2体积分数为13%，添加浓度为60mg/L的黄腐酸；每天定时取样，利用紫外分光光度计测得生物量，培养结束后利用有机溶剂提取油脂。
结果：当核桃壳提取液中黄腐酸浓度为60mg/L时，微藻的油脂含量为54.00 %，生物量最高达到1.36g/L。
实施例4
将核桃壳煮沸4h作为基础培养基，外源添加NaNO3（1.1g/L），pH调节在6.8-7.0之间，灭菌条件为121℃、20 min。微藻培养温度24~26℃，初始接种量为0.4g/L，气体流速为0.6L/min，CO2体积分数为12%，添加浓度为80mg/L的黄腐酸；每天定时取样，利用紫外分光光度计测得生物量，培养结束后利用有机溶剂提取油脂。
结果：当核桃壳提取液中黄腐酸浓度为80mg/L时，微藻的油脂含量为52.12%，生物量最高达到1.38g/L。
对比实例
将核桃壳煮沸4 h作为基础培养基，外源添加NaNO3（1.2g/L），pH调节在6.8-7.0之间，灭菌条件为121℃、20 min；微藻培养温度24~26℃，初始接种量为0.42g/L，气体流速为0.5L/min，CO2体积分数为12%；每天定时取样，利用紫外分光光度计测得生物量，培养结束后利用有机溶剂提取油脂。
结果：当核桃壳提取液中黄腐酸浓度为0mg/L时，微藻的油脂含量为49.54%，生物量最高达到1.18g/L。
利用紫外分光光度计测定对比实例和实施例1-4中的生物量，见图1。
利用有机溶剂提取对比实例和实施例1-4中微藻的油脂，见图2。
结果表明：本发明中，以核桃壳提取液为基础培养基，外源添加NaNO3（1.2g/L），同时通入13%的CO2，随着黄腐酸浓度的增加，微藻中的油脂含量先增加后减少，且当黄腐酸浓度为40mg/L时，微藻的油脂含量和生物量达到最大，为58.76%和1.48g/L，分别是对比实例的1.19和2.82倍。
Claims (3)
1.一种利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法，其特征在于：具体包括以下步骤：
（1）配制培养基：将核桃壳提取液作为微藻培养的基础培养基，添加NaNO3，调节pH、分装、灭菌；
（2）微藻培养：将微藻接入步骤（1）中的培养基中，通入CO2，同时添加黄腐酸进行培养；
（3）生物量和油脂含量的测定：利用紫外分光光度计测定培养条件下微藻的生物量；微藻生长到达稳定期后，利用有机溶剂提取微藻细胞内的油脂；
步骤（1）中配制培养基的具体方法为：将核桃壳粉碎，煮沸4~4.5h作为基础培养基，NaNO3的添加量为1.1~1.2g/L，pH调节在6.8-7.0之间，灭菌条件为121℃、20 min。
2.根据权利要求1所述利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法，其特征在于：步骤（2）中微藻的初始接种量为0.38~0.42g/L，CO2的通入量为11~13%，培养基中黄腐酸的浓度为20~80 mg/L。
3.根据权利要求1所述利用黄腐酸提高核桃壳提取液中微藻油脂含量的方法，其特征在于：微藻的培养条件是：光照强度为6000~7000lux，通气量为0.5~0.6L/min，培养温度为24~26℃。
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