CN102851211B - 微拟球藻培养基配方及三级培养方法 - Google Patents
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Abstract
本发明提供了一种微拟球藻培养基配方及三级培养方法，配方如下：NaNO3 7.5×10-2g，KH2PO4 1×10-2g，NaHCO3 5×10-1g，柠檬酸铁(FeC6H5O7.5H2O)2×10-4g，VB12 5×10-7g，VB1 1×10-4g，VH 5×10-7g，九二0植物生长刺激素(即赤霉素)5国际单位，人尿2mL，消毒海水1000mL。实验结果表明，使用优化培养基并采用三级培养微拟球藻具有生长速度快、产量高、成本低、易于操作、不易污染的优点。
Description
技术领域
本发明属于海洋微藻养殖领域，尤其涉及一种微拟球藻培养基配方及三级培养方法。
背景技术
微拟球藻(Nannochloropsis oculata)又称微绿球藻，是一种广泛分布的海水藻类，直径为2-4微米，细胞球形，淡绿色，富含不饱和脂肪酸及其它营养元素，其中EPA含量占脂肪酸总量的30％。EPA能降低胆固醇和甘油三酯的含量，促进体内饱和脂肪酸代谢，从而降低血液粘稠度，增进血液循环，提高组织供氧而消除疲劳，并防止脂肪在血管壁的沉积，预防动脉粥样硬化的形成和发展、预防脑血栓、脑溢血、高血压等心脑血管疾病。EPA在减轻炎症方面也起着重要的作用。另外，微拟球藻油脂含量占干重的68％以上，是提炼生物柴油的理想微藻。
目前，微拟球藻规模化培养存在的主要问题是生长缓慢，生物量低，易污染，特别是梅雨季节，不利于大规模培养。以前使用的微拟球藻培养基没有加入碳酸氢钠，但我的实验表明，微拟球藻除了能利用空气中游离的CO2，还能有效地利用培养液中的HCO3-作为碳源。大量实验表明：添加0.5g/L的碳酸氢钠能能很好地促进微拟球藻的生长。另外，我还在培养基中补充了柠檬酸铁、维生素H、赤霉素和人尿，能将产量提高54％。
总之，使用优化培养基并采用三级培养微拟球藻方法具有生长速度快、产量高、成本低、不易污染的优势。
发明内容
为了克服目前技术的不足，本发明提供了一种微拟球藻配方及三级培养方法，具体技术方案如下：
一种微拟球藻培养基，配方如下：NaNO3 7.5×10-2g，KH2PO4 1×10-2g，NaHCO3 5×10-1g，柠檬酸铁(FeC6H5O7.5H2O)2×10-4g，VB12 5×10-7g，VB1 1×10-4g，VH 5×10-7g，九二0植物生长刺激素(即赤霉素)5国际单位，人尿2mL，消毒海水1000mL。
优选地，还包括微量元素0.55～2.25ml/L，所述微量元素配方为：磷酸二氢钠32～40g/L、硫酸铜1.2～3.2g/L、硫酸锌4.2～6.2g/L、氯化钴4～6g/L、氯化锰30～45g/L加水定容至1L。
优选地，所述微量元素溶液中加入120～140g/L的磷酸二氢钠溶液。
优选地，所述微量元素溶液中加入乙二胺四乙酸二钠3～10g/L。
应用本发明所提供的培养基对微拟球藻进行三级培养，包括如下步骤：1)一级培养容器为5000mL三角烧瓶，烧瓶用洗液洗涤、晾干、120℃恒温消毒；培养用的海水比重1.015～1.018，盐度30～32，经过经脱脂棉过滤、煮沸、冷却后加入营养盐；室内培养，室温20-26℃，室内自然光+辅助日光灯管光源照射，光强3000-10000Lx，光暗周期＝14L:10D，指数增长期藻种接种，接种密度300-350×104cell/mL，不充气。培养期间每天摇瓶6次。密度达3000×104cell/mL，即可转入二级培养。2)二级培养容器为50L白塑料桶，白塑料桶刷干净、漂白粉消毒后用消毒水冲干净备用。二级培养用的海水经漂白粉消毒、硫代硫酸钠处理后加入营养盐；将一级培养的藻液按1∶10的比例接种到白桶。室内培养，室温20-26℃，室内自然光+辅助日光灯管光源照射，光强3000-10000Lx，光暗周期＝14L:10D，充气培养，充气量以能将藻细胞冲起悬浮即可。3)三级培养容器为水泥池。面积20-30m2，池深50cm，水深20-30cm，内贴白色瓷砖，三级培养用海水经漂白粉消毒、硫代硫酸钠处理。将二级培养的藻液作藻种转入水泥池培养，室外培养，自然光照射，充气，接种密度200-300×104cell/mL，培养至密度达2000-3000×104cell/mL即可收获。
优选地，包括在步骤2)中充气培养时同时施加臭氧。
优选地，臭氧的浓度为0.5微克/升空气～0.8微克/升空气。
有益效果：
实验结果表明，使用优化培养基并采用三级培养微拟球藻具有生长速度快、产量高、成本低、易于操作、不易污染的优点。
具体实施方式
培养基配制方法
1柠檬酸铁较难溶解，可加少量自来水在火炉上微热至80-90℃，并不断搅拌至全部融化；
2赤霉素为白色结晶粉末，不易溶解于水，可先用酒精充分溶解(一般每g用50mL酒精或60度白酒溶解1小时以上)，再按所需浓度兑水稀释。
3加营养盐的过程中需不断搅水，而且各种营养盐按配方中提供的顺序加入，以免发生化学反应；
4各种营养盐可先配成浓度较高的母液，再依次添加；
5三级培养各阶段营养盐配方和浓度相同。
一种微拟球藻培养基，其特征在于该微拟球藻培养基配方如下：NaNO3 7.5×10-2g，KH2PO41×10-2g，NaHCO3 5×10-1g，柠檬酸铁(FeC6H5O7.5H2O)2×10-4g，VB12 5×10-7g，VB1 1×10-4g，VH 5×10-7g，九二0植物生长刺激素(即赤霉素)5国际单位，人尿2mL，消毒海水1000mL。
优选地，还包括微量元素0.55～2.25ml/L，所述微量元素配方为：磷酸二氢钠32～40g/L、硫酸铜1.2～3.2g/L、硫酸锌4.2～6.2g/L、氯化钴4～6g/L、氯化锰30～45g/L加水定容至1L。
优选地，所述微量元素溶液中加入120～140g/L的磷酸二氢钠溶液。
优选地，所述微量元素溶液中加入乙二胺四乙酸二钠3～10g/L。
微拟球藻三级培养方法
1一级培养
一级培养容器为5000mL三角烧瓶，烧瓶用洗液洗涤、晾干、120℃恒温消毒；培养用的海水比重1.015，盐度30，经过经脱脂棉过滤、煮沸、冷却后加入营养盐；室内培养，室温20-26℃，室内自然光+辅助日光灯管光源照射，光强3000-10000Lx，光暗周期＝14L:10D，指数增长期藻种接种，接种密度300-350×104cell/mL，不充气。培养期间每天摇瓶6次。密度达3000×104cell/mL，即可转入二级培养。
2二级培养
二级培养容器为50L白塑料桶，白塑料桶刷干净、漂白粉消毒后用消毒水冲干净备用。二级培养用的海水经漂白粉消毒、硫代硫酸钠处理后加入营养盐；将一级培养的藻液按1∶10的比例接种到白桶。室内培养，室温20-26℃，室内自然光+辅助日光灯管光源照射，光强3000-10000Lx，光暗周期＝14L:10D，充气培养，充气量以能将藻细胞冲起悬浮即可。
3三级培养
三级培养容器为水泥池。面积20-30m2，池深50cm，水深20-30cm，内贴白色瓷砖，三级培养用水经漂白粉消毒、硫代硫酸钠处理。将二级培养的藻液作藻种转入水泥池培养，室外培养，自然光照射，充气，接种密度200-300×104cell/mL，培养至密度达2000-3000×104cell/mL即可收获。
优选地，包括在步骤2)中充气培养时同时施加臭氧。
优选地，臭氧的浓度为0.5微克/升空气～0.8微克/升空气。
上述所有的区间都可以实现，不止端点和中点能实现，在此特别说明。
最后应说明的是：以上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：其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分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实施例技术方案的精神和范围。
Claims (1)
1.培养微拟球藻的三级培养方法，其特征在于包括如下步骤：
配置一种微拟球藻培养基，该微拟球藻培养基配方如下：NaNO37.5×10-2g，KH2PO41×10-2g，NaHCO35×10-1g，柠檬酸铁2×10-4g，VB125×10-7g，VB11×10-4g，VH5×10-7g，九二0植物生长刺激素5国际单位，人尿2mL，消毒海水1000mL；
还包括微量元素0.55～2.25ml/L，所述微量元素配方为：磷酸二氢钠32～40g/L、硫酸铜1.2～3.2g/L、硫酸锌4.2～6.2g/L、氯化钴4～6g/L、氯化锰30～45g/L加水定容至1L；
所述微量元素溶液中加入乙二胺四乙酸二钠3～10g/L；
微拟球藻的三级培养步骤为：
1)一级培养容器为5000mL三角烧瓶，烧瓶用洗液洗涤、晾干、120℃恒温消毒；培养用的海水比重1.015～1.018，盐度30～32，经过经脱脂棉过滤、煮沸、冷却后加入营养盐；室内培养，室温20-26℃，室内自然光+辅助日光灯管光源照射，光强3000-10000Lx，光暗周期＝14L：10D，指数增长期藻种接种，接种密度300-350×104cell/mL，不充气；培养期间每天摇动6次，密度达3000×104cell/mL，即转入二级培养；
2)二级培养容器为50L白塑料桶，白塑料桶刷干净、漂白粉消毒后用消毒水冲干净备用；二级培养用的海水经漂白粉消毒、硫代硫酸钠处理后加入营养盐；将一级培养的藻液按1∶10的比例接种到白桶；室内培养，室温20-26℃，室内自然光+辅助日光灯管光源照射，光强3000-10000Lx，光暗周期＝14L：10D，充气培养，充气量以能将藻细胞冲起悬浮即可；
3)三级培养容器为水泥池；面积20-30m2，池深50cm，水深20-30cm，内贴白色瓷砖，三级培养用水经漂白粉消毒、硫代硫酸钠处理；将二级培养的藻液作藻种转入水泥池培养，室外培养，自然光照射，充气，接种密度200-300×104cell/mL，培养至密度达2000-3000×104cell/mL即可收获；
在步骤2)中充气培养时同时施加臭氧，臭氧的浓度为0.5微克/升空气～0.8微克/升空气。
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