气泡柱光生物反应器中绿色微藻的培养及中性脂质的测定
Summary
在这里, 我们提出了一个协议, 以构建实验室规模的气泡柱光生物反应器, 并将其用于培养微藻。为测定培养生长速率和中性脂质含量提供了一种方法。
Abstract
人们对研究微藻用于生产生物燃料、高价值产品和处理废物等工程应用非常感兴趣。由于大多数新的研究工作都是从实验室规模开始的, 因此需要有成本效益高的方法, 以可重复的方式培养微藻。在这里, 我们交流了在实验室规模的光生物反应器中培养微藻的有效方法, 并测量了该藻类的生长和中性脂质含量。还包括关于如何建立光生物反应器系统的说明。虽然该生物是小球藻和auxeno·bella 的种类, 但该系统可以适应培养各种微藻, 包括藻类与非藻类物种的共培养。股票文化首先是在瓶子中生长, 以产生光比生物反应器系统的接种剂。藻类接种物被浓缩并转移到光生物反应器中, 以便在批量生产模式下进行培养。每天收集样本以获取光学密度读数。在批处理培养结束时, 细胞通过离心机收获, 清洗, 并冻干, 以获得最终的干重浓度。最后的干重浓度用于建立光学密度和干重浓度之间的相关性。随后采用改进的 folch 法从冻干生物质中提取总脂质, 并采用微板法检测其中性脂质含量。此检测方法以前已发布, 但此处包含了协议步骤, 以突出显示经常发生错误的过程中的关键步骤。这里描述的生物反应器系统填补了一个小众之间简单的烧瓶种植和完全控制的商业生物反应器。即使每次治疗只有3-4 个生物复制物, 我们培养藻类的方法也会导致生长和脂质检测的标准偏差很严格。
Introduction
近年来, 微藻在工程和生物技术中的应用引起了极大的兴趣。微藻正在研究中, 用于废水处理1、2、3、4、生物燃料生产5、6、7、8和生产保健品和其他高价值产品9,10。藻类也在以更高的速度进行转基因, 以提高其对特定工程应用的适应性11,12。因此, 人们非常希望在受控环境中试验与工业有关的生物体。该方法的目的是传达一种在可控实验室环境中培养微藻的有效方法, 并测量微藻的生长和中性脂质含量。提高微藻的生长速度和中性脂质含量已被确定为藻类生物燃料商业化的两个关键瓶颈13。
为了实验目的, 人们已经使用了广泛的方法来培养藻类。一般来说, 这些方法可以分为大规模室外种植和小规模室内种植。光生物反应器和开放池塘的室外种植适合于旨在扩大已经在实验室规模上证明的工艺的实验 (例如,测试扩大新的高脂藻类毒株)14。然而, 在开发新的或改良的藻类菌株或进行旨在了解生物机制的实验时, 室内小规模种植是适当的。在后一种情况下, 需要高度的实验控制来梳理生物行为的微妙变化。为此, 通常需要进行空气酸培养, 以尽量减少与其他生物 (如细菌、其他藻类) 有关的复杂生物因素, 这些生物不可避免地生长在大规模的室外系统中。即使在研究藻类和其他生物之间的相互作用时, 我们发现, 在研究15、16、17生物体之间的分子交换时, 使用高度控制的实验条件是有帮助的。
在小型室内藻类养殖类别中, 采用了一系列办法。也许最常见的方法是在 18、19 的灯光库下面的振动台上种植 erlenmeyer 烧瓶中的藻类。氧气和二氧化碳的交换是通过烧瓶顶部的泡沫塞通过被动扩散进行的。一些研究人员通过对烧瓶20 进行主动曝气, 改进了这种设置。另一种方法是在瓶子中培养藻类, 混合搅拌棒和主动曝气。尽管它们很简单, 但我们发现, 烧瓶和瓶子的使用往往会导致生物复制之间的结果不一致。这大概是由于位置效应造成的–不同的位置接收不同数量的光线, 这也会影响内部反应堆的温度。反应堆每天轮换到新的位置可以帮助但不能缓解这一问题, 因为藻类生长的某些阶段 (例如,早期指数) 比其他阶段 (如日志阶段) 对位置效应更敏感。
在技术成熟度的对立面是完全控制的商业光生物反应器。这些系统持续监测和调整反应堆的条件, 以优化藻类的生长。它们具有可编程照明、实时温度控制和 ph 控制。不幸的是, 它们很贵, 每个反应堆通常需要几千元。大多数科学和工程期刊都需要对结果进行生物复制, 因此需要购买多个生物反应器。我们在这里介绍了一个气泡柱反应器系统, 它弥合了实验室规模藻类养殖的简单 (烧瓶) 和复杂 (完全控制的生物反应器) 方法之间的鸿沟。气泡柱使用上升的气泡来促进气体交换和混合反应堆。这种方法提供了对照明和温度的一定程度的控制, 但这样做的方式具有成本效益。此外, 我们发现, 与烧瓶或瓶法相比, 该系统在生物复制中产生了高度一致的结果, 减少了获得统计意义结果所需的生物复制所需的数量。我们还利用该系统成功地培育了藻类和细菌的混合物21。除了藻类养殖外, 我们还概述了测量养殖藻类中性脂质含量的方法。后一种方法已在其他地方发布 22, 但我们在这里包括了一个过程, 以提供如何成功使用它的分步指导。
Protocol
1. 气泡柱光生物反应器的设置 从1升玻璃瓶和杂交管附带的塑料盖中构建一套通风盖 (有关原理图和照片, 请参见图 1 )。为加湿器、混合疏水阀、每个空气提升光反应器和每个瓶反应器建造盖子。 在盖子上钻孔: 生物反应器和加湿器盖需要2个孔;混合疏水阀需要3个孔。 将一个-“o 形圈在一个-8” 面板安装 luer 管接头的螺纹上滑动, 并将其滑入在盖子上钻的 “…
Representative Results
此过程在 od 550 nm 处生成藻类光学密度数据的时间过程 (图 4 a)。光学密度和干重浓度数据可以相互关联 (图 4b)。这是通过在冷冻干燥步骤后首先计算最终的干重藻类浓度来实现的。其次, 培养系列稀释的光学密度 (在取样的最后一天进行) 与实际干重浓度可以相互关联。对于低细胞浓度, 可以使用线性相关性, 而对于较高的细胞浓…
Discussion
在养殖藻类时, 最重要的考虑因素是了解生物体或生物群的具体需求。这里描述的藻类养殖系统可以用来培养广泛的藻类, 但具体的非生物因素 (温度、介质、ph 值、光强、二氧化碳水平、曝气率) 需要根据生物体的需要进行调整。请注意, 此处描述的参数用于培养小球藻和auxenooclella。这些生物具有工业意义, 因为它们能耐受高营养、光和温度水平27。然而, 通…
Disclosures
The authors have nothing to disclose.
Acknowledgements
美国农业部国家粮食和农业舱口项目 ala0higins 和奥本大学院长办公室、研究副总裁和 samuel ginn 工程学院为这项研究提供了支持。nsf 赠款 cbet-1438211 也提供了支助。
Materials
| Supplies for airlift photobioreactor setup | |||
| 1 L Pyrex bottles | Corning | 16157-191 | For bottle reactors, humidifiers |
| 1/2" hose clamp | Home Depot | UC953A | or equivalent |
| 1/4" female luer to barb | Nordson biomedical | Nordson FTLL360-6005 | 1/4" ID, PP |
| 1/4" ID, 3/8" OD autoclaveable PVC tubing | Thermo-Nalgene | 63013-244 | 50' |
| 1/4" in O-rings | Grainger | 1REC5 | #010 Medium Hard Silicone O-Ring, 0.239" I.D., 0.379"O.D. |
| 1/8" Female luer to barb | Nordson biomedical | FTLL230-6005 | |
| 1/8" ID, 1/4" OD autoclaveable PVC tubing | Thermo-Nalgene | 63013-608 | 250' |
| 1/8" male spinning luer to barb | Nordson biomedical | MLRL013-6005 | |
| 1/8" multiport barb | Nordson biomedical | 4PLL230-6005 | 1/8" multiport barb |
| 1/8" NPT to barb | Nordson biomedical | 18230-6005 | 1/8" 200 series barb |
| 1/8" panel mount luer | Nordson biomedical | Nordson MLRLB230-6005 | 1/8", PP |
| 10 gallon fish tank | Walmart | 802262 | Can hold up to 8 bioreactors depending on layout |
| 100-1000 ccm flow meter | Dwyer | RMA-13-SSV | For bottle reactors |
| 2 ft fluorescent light bank | Agrobrite | FLT24 T5 | |
| 200-2500 ccm flow meter | Dwyer | RMA-14-SSV | For air regulation upstream of humidifier |
| 250 mL Pyrex bottles | Corning | 16157-136 | For gas mixing after humidifier |
| 50-500 ccm flow meter | Dwyer | RMA-12-SSV | For hybridization tube reactors |
| 5-50 ccm flow meter | Dwyer | RMA-151-SSV | For CO2 flow rate control |
| Air filters 0.2 µm | Whatman/ Fisher | 09-745-1A | Polyvent, 28 mm, 0.2 µm, PTFE, 50 pack |
| Check valves | VWR | 89094-714 | |
| Corning lids for pyrex bottles | VWR | 89000-233 | 10 GL45 lids |
| Female luer endcap | Nordson biomedical | Nordson FTLLP-6005 | Female stable PP |
| Hybridization tubes | Corning | 32645-030 | 35×300 mm, pack of 2 |
| Light timer | Walmart | 556393626 | |
| Locknuts | Nordson biomedical | Nordson LNS-3 | 1/4", red nylon |
| Low profile magnetic stirrer | VWR | 10153-690 | Low profile magnetic stirrer |
| Male luer endcap | Nordson biomedical | Nordson LP4-6005 | Male plug PP |
| Spinning luer lock ring | Nordson biomedical | Nordson FSLLR-6005 | |
| Stir bars – long | VWR | 58949-040 | 38.1 mm, for bottle reactors |
| Stir bars – medium | VWR | 58949-034 | 25 mm, for hyridization tubes |
| Supplies and reagents for culturing algae | |||
| 0.2 µm filters | VWR | 28145-491 | 13 mm, PTFE, for filtering spent media from daily culture sampling |
| 1 mL syringes | Air-tite | 89215-216 | For filtering spent media from daily culture sampling |
| 1.5 mL tubes | VWR | 87003-294 | Sterile (or equivalent) |
| 10 mL Serological pipettes | Greiner Bio-One | 82050-482 | Sterile (or equivalent) |
| 100 mm plates | VWR | 25384-342 | 100×15 mm stackable petri dishes, sterile |
| 15 mL tubes | Greiner Bio-One | 82050-276 | Sterile (or equivalent), polypropylene |
| 2 mL Serological pipette tips | Greiner Bio-One | 82051-584 | Sterile (or equivalent) |
| 2 mL tubes | VWR | 87003-298 | Sterile (or equivalent) |
| 50 mL tubes | Greiner Bio-One | 82050-348 | Sterile (or equivalent), polypropylene |
| 96 well microplate | Greiner Bio-One | 89089-578 | Polystyrene with lid, flat bottom |
| Inocculating loops | VWR | 80094-478 | Sterile (or equivalent) |
| Liquid carbon dioxide tank and regulator | Airgas | CD-50 | |
| Supplies and reagents for lipid extraction and neutral lipid assay | |||
| 2 mL bead tubes | VWR | 10158-556 | Polypropylene tube w/ lid |
| 96 well microplates | Greiner Bio-One | 82050-774 | Polypropylene, flat bottom |
| Bleach | Walmart | 550646751 | Only use regular bleach, not cleaning bleach |
| Chloroform | BDH | BDH1109-4LG | |
| Dimethyl sulfoxide | BDH | BDH1115-1LP | |
| Isopropyl alcohol | BDH | BDH1133-1LP | |
| Methanol | BDH | BDH20864.400 | |
| Nile red | VWR | TCN0659-5G | |
| Pasteur pipette tips | VWR | 14673-010 | |
| Sodium chloride | BDH | BDH9286-500G | |
| Vegetable oil | Walmart | 9276383 | Any vegetable oil should work as long as it is fresh |
| Zirconia/ silica beads (0.5 mm diameter) | Biospec products | 11079105z | |
| Equipment | |||
| Analytical balance | Mettler-Toledo | XS205DU | Capable of at least 4 decimal accuracy |
| Bead homogenizer | Omni | 19-040E | |
| Benchtop micro centrifuge | Thermo | Heraeus Fresco 21 with 24×2 | Including rotor capable of handling 1.5 and 2 mL tubes |
| Dry block heater | VWR | 75838-282 | Including dry block for a microplate |
| Freeze dryer | Labconco | 7670520 | 2.5L freeze drying system |
| Large benchtop centrifuge | Thermo | Heraeus Megafuge 16R Tissue | Including rotors capable of handling 400 mL bottles, 50 mL tubes, and 15 mL tubes |
| Microplate reader | Molecular Devices | SpectraMax M2 | Capable of reading absorbance and fluorescence |
| Vortex mixer | VWR | 10153-838 |
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Cite This Article
Wang, Q., Peng, H., Higgins, B. T. Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids. J. Vis. Exp. (143), e59106, doi:10.3791/59106 (2019).