Name: cultivation of green microalgae in bubble column photobioreactors and an assay for neutral lipids
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids at JoVE.com

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。

1. 气泡柱光生物反应器的设置
<ol><li>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。<ol>
<li>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。</li>
		<li>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 "...

<ol>
	<li>Prandini, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+nutrient+removal+from+swine+wastewater+digestate+coupled+to+biogas+purification+by+microalgae+Scenedesmus+spp.">Enhancement of nutrient removal from swine wastewater digestate coupled to biogas purification by microalgae Scenedesmus spp.</a> Bioresource Technology. , 67-75 (2016).</li><li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycoremediation+of+dairy+and+winery+wastewater+using+Diplosphaera+sp.+MM1.">Phycoremediation of dairy and winery wastewater using Diplosphaera sp. MM1.</a> Journal of Applied Phycology. 28 (6), 3331-3341 (2016).</li><li>Passero, M., Cragin, B., Coats, E. R., McDonald, A. G., Feris, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dairy+Wastewaters+for+Algae+Cultivation,+Polyhydroxyalkanote+Reactor+Effluent+Versus+Anaerobic+Digester+Effluent.">Dairy Wastewaters for Algae Cultivation, Polyhydroxyalkanote Reactor Effluent Versus Anaerobic Digester Effluent.</a> BioEnergy Research. 8 (4), 1647-1660 (2015).</li><li>Hodgskiss, L. H., Nagy, J., Barnhart, E. P., Cunningham, A. B., Fields, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+a+native+alga+for+biomass+and+biofuel+accumulation+in+coal+bed+methane+production+water.">Cultivation of a native alga for biomass and biofuel accumulation in coal bed methane production water.</a> Algal Research. 19, 63-68 (2016).</li><li>Gao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil+accumulation+mechanisms+of+the+oleaginous+microalga+Chlorella+protothecoides+revealed+through+its+genome,+transcriptomes,+and+proteomes.">Oil accumulation mechanisms of the oleaginous microalga Chlorella protothecoides revealed through its genome, transcriptomes, and proteomes.</a> BMC Genomics. 15, (2014).</li><li>Burch, A. R., Franz, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+nitrogen+limitation+and+hydrogen+peroxide+treatment+enhances+neutral+lipid+accumulation+in+the+marine+diatom+Phaeodactylum+tricornutum.">Combined nitrogen limitation and hydrogen peroxide treatment enhances neutral lipid accumulation in the marine diatom Phaeodactylum tricornutum.</a> Bioresource Technology. 219, 559-565 (2016).</li><li>Brennan, L., Owende, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofuels+from+microalgae--A+review+of+technologies+for+production,+processing,+and+extractions+of+biofuels+and+co-products.">Biofuels from microalgae--A review of technologies for production, processing, and extractions of biofuels and co-products.</a> Renewable Sustainable Energy Reviews. 14 (2), 557-577 (2009).</li><li>Branyikova, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microalgae+-+Novel+highly+efficient+starch+producers.">Microalgae - Novel highly efficient starch producers.</a> Biotechnology and Bioengineering. 108 (4), 766-776 (2010).</li><li>Chalima, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Volatile+Fatty+Acids+from+Microalgae+for+the+Production+of+High+Added+Value+Compounds.">Utilization of Volatile Fatty Acids from Microalgae for the Production of High Added Value Compounds.</a> Fermentation. 3 (4), (2017).</li><li>Harun, R., Singh, M., Forde, G. M., Danquah, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocess+engineering+of+microalgae+to+produce+a+variety+of+consumer+products.">Bioprocess engineering of microalgae to produce a variety of consumer products.</a> Renewable and Sustainable Energy Reviews. 14 (3), 1037-1047 (2010).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+method+for+genetic+transformation+of+the+green+alga+Chlorella+ellipsoidea.">Development of a new method for genetic transformation of the green alga Chlorella ellipsoidea.</a> Molecular biotechnology. 54 (2), 211-219 (2013).</li><li>Cheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutate+Chlorella+sp.+by+nuclear+irradiation+to+fix+high+concentrations+of+CO2.">Mutate Chlorella sp. by nuclear irradiation to fix high concentrations of CO2.</a> Bioresource Technology. 136, 496-501 (2013).</li><li>Davis, R., Aden, A., Pienkos, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techno-economic+analysis+of+autotrophic+microalgae+for+fuel+production.">Techno-economic analysis of autotrophic microalgae for fuel production.</a> Applied Energy. 88 (10), 3524-3531 (2011).</li><li>Sales, C. M., Au, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Scale+in+a+Photosynthetic+Reactor+System+for+Algal+Remediation+of+Wastewater.">Comparison of Scale in a Photosynthetic Reactor System for Algal Remediation of Wastewater.</a> Journal of Visualized Experiments. (121), e55256 (2017).</li><li>Higgins, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofactor+symbiosis+for+enhanced+algal+growth,+biofuel+production,+and+wastewater+treatment.">Cofactor symbiosis for enhanced algal growth, biofuel production, and wastewater treatment.</a> Algal Research. 17, 308-315 (2016).</li><li>Higgins, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algal-bacterial+synergy+in+treatment+of+winery+wastewater.">Algal-bacterial synergy in treatment of winery wastewater.</a> Nature Clean Water. 1 (6), (2017).</li><li>Higgins, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+thiamine+metabolites+and+spent+medium+from+Chlorella+sorokiniana+on+metabolism+in+the+green+algae+Auxenochlorella+prototheciodes.">Impact of thiamine metabolites and spent medium from Chlorella sorokiniana on metabolism in the green algae Auxenochlorella prototheciodes.</a> Algal Research. 33, 197-208 (2018).</li><li>L&#233;pinay, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+insight+on+interactions+between+bacteria+and+the+marine+diatom+Haslea+ostrearia:+Algal+growth+and+metabolomic+fingerprinting.">First insight on interactions between bacteria and the marine diatom Haslea ostrearia: Algal growth and metabolomic fingerprinting.</a> Algal Research. 31, 395-405 (2018).</li><li>Franchino, M., Comino, E., Bona, F., Riggio, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+three+microalgae+strains+and+nutrient+removal+from+an+agro-zootechnical+digestate.">Growth of three microalgae strains and nutrient removal from an agro-zootechnical digestate.</a> Chemosphere. 92 (6), 738-744 (2013).</li><li>Choix, F. J., Lopez-Cisneros, C. G., Mendez-Acosta, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azospirillum+brasilense+Increases+CO2+Fixation+on+Microalgae+Scenedesmus+obliquus,+Chlorella+vulgaris,+and+Chlamydomonas+reinhardtii+Cultured+on+High+CO2+Concentrations.">Azospirillum brasilense Increases CO2 Fixation on Microalgae Scenedesmus obliquus, Chlorella vulgaris, and Chlamydomonas reinhardtii Cultured on High CO2 Concentrations.</a> Microbial Ecology. 76 (2), 430-442 (2018).</li><li>Higgins, B., VanderGheynst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Escherichia+coli+on+mixotrophic+growth+of+Chlorella+minutissima+and+production+of+biofuel+precursors.">Effects of Escherichia coli on mixotrophic growth of Chlorella minutissima and production of biofuel precursors.</a> PLoS One. 9 (5), e96807 (2014).</li><li>Higgins, B., Thornton-Dunwoody, A., Labavitch, J. M., VanderGheynst, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplate+assay+for+quantitation+of+neutral+lipids+in+extracts+from+microalgae.">Microplate assay for quantitation of neutral lipids in extracts from microalgae.</a> Analytical Biochemistry. 465, 81-89 (2014).</li><li>Tanadul, O. U., Vandergheynst, J. S., Beckles, D. M., Powell, A. L., Labavitch, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+elevated+CO2+concentration+on+the+quality+of+algal+starch+as+a+potential+biofuel+feedstock.">The impact of elevated CO2 concentration on the quality of algal starch as a potential biofuel feedstock.</a> Biotechnology and Bioengineering. 111 (7), 1323-1331 (2014).</li><li>Folch, J., Lees, M., Sloane Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> Journal of Biological Chemistry. 226 (1), 497-509 (1957).</li><li>Higgins, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Informatics+for+improved+algal+taxonomic+classification+and+research:+A+case+study+of+UTEX+2341.">Informatics for improved algal taxonomic classification and research: A case study of UTEX 2341.</a> Algal Research. 12, 545-549 (2015).</li><li>Garrett, R. H., Grisham, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. , 578-730 (2012).</li><li>de-Bashan, L. E., Trejo, A., Huss, V. A. R., Hernandez, J. -. P., Bashan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorella+sorokiniana+UTEX+2805,+a+heat+and+intense,+sunlight-tolerant+microalga+with+potential+for+removing+ammonium+from+wastewater.">Chlorella sorokiniana UTEX 2805, a heat and intense, sunlight-tolerant microalga with potential for removing ammonium from wastewater.</a> Bioresource Technology. 99 (11), 4980-4989 (2008).</li><li>Wang, Q., Higgins, B., Ji, H., Zhao, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annual International Meeting of the ASABE. , (2018).</li></ol>

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...

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。

<table>
	<tbody>
		<tr>
			<td>Supplies for airlift photobioreactor setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 L Pyrex bottles</td>
			<td>Corning</td>
			<td>16157-191</td>
			<td>For bottle reactors, humidifiers</td>
		</tr>
		<tr>
			<td>1/2&#34; hose clamp</td>
			<td>Home Depot</td>
			<td>UC953A</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>1/4&#34; female luer to barb</td>
			<td>Nordson biomedical</td>
			<td>Nordson FTLL360-6005</td>
			<td>1/4&#34; ID, PP</td>
		</tr>
		<tr>
			<td>1/4&#34; ID, 3/8&#34; OD autoclaveable PVC tubing</td>
			<td>Thermo-Nalgene</td>
			<td>63013-244</td>
			<td>50&#39;</td>
		</tr>
		<tr>
			<td>1/4&#34; in O-rings</td>
			<td>Grainger</td>
			<td>1REC5</td>
			<td>#010 Medium Hard Silicone O-Ring, 0.239&#34; I.D., 0.379&#34;O.D.</td>
		</tr>
		<tr>
			<td>1/8&#34; Female luer to barb</td>
			<td>Nordson biomedical</td>
			<td>FTLL230-6005</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8&#34; ID, 1/4&#34; OD autoclaveable PVC tubing</td>
			<td>Thermo-Nalgene</td>
			<td>63013-608</td>
			<td>250&#39;</td>
		</tr>
		<tr>
			<td>1/8&#34; male spinning luer to barb</td>
			<td>Nordson biomedical</td>
			<td>MLRL013-6005</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8&#34; multiport barb</td>
			<td>Nordson biomedical</td>
			<td>4PLL230-6005</td>
			<td>1/8&#34; multiport barb</td>
		</tr>
		<tr>
			<td>1/8&#34; NPT to barb</td>
			<td>Nordson biomedical</td>
			<td>18230-6005</td>
			<td>1/8&#34; 200 series barb</td>
		</tr>
		<tr>
			<td>1/8&#34; panel mount luer</td>
			<td>Nordson biomedical</td>
			<td>Nordson MLRLB230-6005</td>
			<td>1/8&#34;, PP</td>
		</tr>
		<tr>
			<td>10 gallon fish tank</td>
			<td>Walmart</td>
			<td>802262</td>
			<td>Can hold up to 8 bioreactors depending on layout</td>
		</tr>
		<tr>
			<td>100-1000 ccm flow meter</td>
			<td>Dwyer</td>
			<td>RMA-13-SSV</td>
			<td>For bottle reactors</td>
		</tr>
		<tr>
			<td>2 ft fluorescent light bank</td>
			<td>Agrobrite</td>
			<td>FLT24 T5</td>
			<td></td>
		</tr>
		<tr>
			<td>200-2500 ccm flow meter</td>
			<td>Dwyer</td>
			<td>RMA-14-SSV</td>
			<td>For air regulation upstream of humidifier</td>
		</tr>
		<tr>
			<td>250 mL Pyrex bottles</td>
			<td>Corning</td>
			<td>16157-136</td>
			<td>For gas mixing after humidifier</td>
		</tr>
		<tr>
			<td>50-500 ccm flow meter</td>
			<td>Dwyer</td>
			<td>RMA-12-SSV</td>
			<td>For hybridization tube reactors</td>
		</tr>
		<tr>
			<td>5-50 ccm flow meter</td>
			<td>Dwyer</td>
			<td>RMA-151-SSV</td>
			<td>For CO2 flow rate control</td>
		</tr>
		<tr>
			<td>Air filters 0.2 &#181;m</td>
			<td>Whatman/ Fisher</td>
			<td>09-745-1A</td>
			<td>Polyvent, 28 mm, 0.2 &#181;m, PTFE, 50 pack</td>
		</tr>
		<tr>
			<td>Check valves</td>
			<td>VWR</td>
			<td>89094-714</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning lids for pyrex bottles</td>
			<td>VWR</td>
			<td>89000-233</td>
			<td>10 GL45 lids</td>
		</tr>
		<tr>
			<td>Female luer endcap</td>
			<td>Nordson biomedical</td>
			<td>Nordson FTLLP-6005</td>
			<td>Female stable PP</td>
		</tr>
		<tr>
			<td>Hybridization tubes</td>
			<td>Corning</td>
			<td>32645-030</td>
			<td>35x300 mm, pack of 2</td>
		</tr>
		<tr>
			<td>Light timer</td>
			<td>Walmart</td>
			<td>556393626</td>
			<td></td>
		</tr>
		<tr>
			<td>Locknuts</td>
			<td>Nordson biomedical</td>
			<td>Nordson LNS-3</td>
			<td>1/4&#34;, red nylon</td>
		</tr>
		<tr>
			<td>Low profile magnetic stirrer</td>
			<td>VWR</td>
			<td>10153-690</td>
			<td>Low profile magnetic stirrer</td>
		</tr>
		<tr>
			<td>Male luer endcap</td>
			<td>Nordson biomedical</td>
			<td>Nordson LP4-6005</td>
			<td>Male plug PP</td>
		</tr>
		<tr>
			<td>Spinning luer lock ring</td>
			<td>Nordson biomedical</td>
			<td>Nordson FSLLR-6005</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir bars - long</td>
			<td>VWR</td>
			<td>58949-040</td>
			<td>38.1 mm, for bottle reactors</td>
		</tr>
		<tr>
			<td>Stir bars - medium</td>
			<td>VWR</td>
			<td>58949-034</td>
			<td>25 mm, for hyridization tubes</td>
		</tr>
		<tr>
			<td>Supplies and reagents for culturing algae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m filters</td>
			<td>VWR</td>
			<td>28145-491</td>
			<td>13 mm, PTFE, for filtering spent media from daily culture sampling</td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>Air-tite</td>
			<td>89215-216</td>
			<td>For filtering spent media from daily culture sampling</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td>Sterile (or equivalent)</td>
		</tr>
		<tr>
			<td>10 mL Serological pipettes</td>
			<td>Greiner Bio-One</td>
			<td>82050-482</td>
			<td>Sterile (or equivalent)</td>
		</tr>
		<tr>
			<td>100 mm plates</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>100x15 mm stackable petri dishes, sterile</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Greiner Bio-One</td>
			<td>82050-276</td>
			<td>Sterile (or equivalent), polypropylene</td>
		</tr>
		<tr>
			<td>2 mL Serological pipette tips</td>
			<td>Greiner Bio-One</td>
			<td>82051-584</td>
			<td>Sterile (or equivalent)</td>
		</tr>
		<tr>
			<td>2 mL tubes</td>
			<td>VWR</td>
			<td>87003-298</td>
			<td>Sterile (or equivalent)</td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Greiner Bio-One</td>
			<td>82050-348</td>
			<td>Sterile (or equivalent), polypropylene</td>
		</tr>
		<tr>
			<td>96 well microplate</td>
			<td>Greiner Bio-One</td>
			<td>89089-578</td>
			<td>Polystyrene with lid, flat bottom</td>
		</tr>
		<tr>
			<td>Inocculating loops</td>
			<td>VWR</td>
			<td>80094-478</td>
			<td>Sterile (or equivalent)</td>
		</tr>
		<tr>
			<td>Liquid carbon dioxide tank and regulator</td>
			<td>Airgas</td>
			<td>CD-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies and reagents for lipid extraction and neutral lipid assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL bead tubes</td>
			<td>VWR</td>
			<td>10158-556</td>
			<td>Polypropylene tube w/ lid</td>
		</tr>
		<tr>
			<td>96 well microplates</td>
			<td>Greiner Bio-One</td>
			<td>82050-774</td>
			<td>Polypropylene, flat bottom</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Walmart</td>
			<td>550646751</td>
			<td>Only use regular bleach, not cleaning bleach</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>BDH</td>
			<td>BDH1109-4LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>BDH</td>
			<td>BDH1115-1LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>BDH</td>
			<td>BDH1133-1LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>BDH20864.400</td>
			<td></td>
		</tr>
		<tr>
			<td>Nile red</td>
			<td>VWR</td>
			<td>TCN0659-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette tips</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BDH</td>
			<td>BDH9286-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vegetable oil</td>
			<td>Walmart</td>
			<td>9276383</td>
			<td>Any vegetable oil should work as long as it is fresh</td>
		</tr>
		<tr>
			<td>Zirconia/ silica beads (0.5 mm diameter)</td>
			<td>Biospec products</td>
			<td>11079105z</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Mettler-Toledo</td>
			<td>XS205DU</td>
			<td>Capable of at least 4 decimal accuracy</td>
		</tr>
		<tr>
			<td>Bead homogenizer</td>
			<td>Omni</td>
			<td>19-040E</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop micro centrifuge</td>
			<td>Thermo</td>
			<td>Heraeus Fresco 21 with 24x2</td>
			<td>Including rotor capable of handling 1.5 and 2 mL tubes</td>
		</tr>
		<tr>
			<td>Dry block heater</td>
			<td>VWR</td>
			<td>75838-282</td>
			<td>Including dry block for a microplate</td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>Labconco</td>
			<td>7670520</td>
			<td>2.5L freeze drying system</td>
		</tr>
		<tr>
			<td>Large benchtop centrifuge</td>
			<td>Thermo</td>
			<td>Heraeus Megafuge 16R Tissue</td>
			<td>Including rotors capable of handling 400 mL bottles, 50 mL tubes, and 15 mL tubes</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M2</td>
			<td>Capable of reading absorbance and fluorescence</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>VWR</td>
			<td>10153-838</td>
			<td></td>
		</tr>
	</tbody>
</table>

cultivation of green microalgae in bubble column photobioreactors and an assay for neutral lipids

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。

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...

Watch this Scientific Journal Video about Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids at JoVE.com

Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

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。

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

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...

该协议为构建和使用气泡柱光生物反应器系统以种植微藻提供了说明，对于任何研究藻类生物燃料、废水处理或藻类生物学的人，都应感兴趣。我们已经找到了尖顶反应堆系统，以产生非常有信誉的结果。反应堆系统也可定制到大量的藻类，成本远远低于许多商业产品。演示这个程序的将是我实验室的研究生王启辰。要开始设置气泡柱光生物反应器，请从一升玻璃瓶的塑料盖和杂交管中构建一套通风盖，如文本协议所述。将 1/4 英寸 O 环滑过 1/8 英寸面板安装装置的胎面，然后滑入盖中钻出的 1/4 英寸孔中。将第二个 1/4 英寸滑过螺纹，使盖子夹在两个 O 环之间。然后将锁紧螺母滑到螺纹上，然后拧紧其以将面板安装诱饵固定到位。现在捕捉锁定环暴露在外的男性诱惑，从盖子投影。对盖子上的每个孔重复此过程。对于将在气泡柱和瓶子反应器上使用的盖子，将 1/8 英寸母诱饵连接到 1 1/2 英寸 1/8 英寸 ID PVC 管。将这些连接到盖子上每个暴露的男性诱饵配件上。将止回阀连接到 1/8 英寸部件之一的自由端。然后连接一个男性的诱惑到巴布配件的第二块1/8英寸的管道从盖子投射。单击旋转锁环到位，将 0.2 微米空气滤清器固定到位。空气递送系统、鱼缸、搅拌板和灯光的组装，如文字协议描述。通过使用真空泵去除超藻，集中已结的微藻存量。在每个瓶子中留下少于100毫升的介质，但避免去除已落的藻类。将藻浆悬浮并转移到无菌的50毫升离心管中。将1000次G离心5分钟，进一步浓缩藻类。在生物安全柜中，去除足够的超高能，为12个光生物反应器实现约80毫升藻类浓缩物的总体积。避免吸尘颗粒。将藻类浓缩物转移到消毒容器中。现在，在每个光生物反应器中加入六毫升藻浆，并加入一个10毫升血清学移液器。旋转生物反应器，将藻类混合到介质中。使用血清移液器从每个生物反应器中抽出两毫升样品，并转移到两毫升管中。每 24 小时收集两毫升样品，以监测培养进度。使用测试条检查样品 ph，并根据需要调整反应器。拧紧生物反应器盖，将所有生物反应器放入鱼缸水浴中。将气化、二氧化碳和照明调整到适合物种的水平。采样后每天旋转生物反应器位置。将每个培养样品的 200 微升在三孔中，用于 96 孔微孔板的孔。然后在550纳米和680纳米时测量光学密度。用一个研究生圆柱器测量每个生物反应器的固定量藻类培养量，并在离心瓶中转移。将培养培养 4，696 次 G 离心五分钟。小心地吸出超吸器，将其丢弃。将颗粒转移到标有 50 毫升的管中。用蒸馏水冲洗离心瓶，然后将内装物转移到50毫升管中。确保总体积不超过 45 毫升。如文本协议所述，清洗藻类颗粒后，丢弃超微。然后，将7.5毫升蒸馏水加入每50毫升管中。现在，涡流50毫升管，并转移藻浆到预重15毫升管。用额外的蒸馏水冲洗50毫升管，将液体转移到15毫升管中，使这些管子中的总体积控制在12毫升以下。离心15毫升管并像之前一样丢弃超高管后，在零下80摄氏度下用颗粒冷冻管至少30分钟，准备冷冻干燥。在每含有20毫克冷冻干藻的两毫升管中加入1.5毫升的福尔奇溶剂。将大约0.5毫升的氧化硅珠倒入每个管中，直到液位达到2毫升。在珠磨机中，以6.5米/秒的速度将藻类样品均匀化20秒。将管子转移到冰上30秒冷却样品。然后，再重复五次，以完全提取脂质。通过含有不锈钢丝网盘的五毫升注射器过滤均质，将珠子应变，在 15 毫升管中收集滤盐。用1.5毫升的耳蜗溶剂清洗珠子，必要时用西环将液体推穿。重复此洗涤 2 次，并收集 15 毫升管中的所有滤液，产生约 6 毫升的最终体积。将1.2毫升0.9%氯化钠溶液加入15毫升管中的福尔奇提取物中，并加入涡流以混合。将15毫升管以6000倍G离心5分钟。使用 15 毫升管侧的线记录底部氯仿相容量到最接近的 0.1 毫升。然后使用玻璃牧场移液器将底部相转移到玻璃邪恶。要进行中性脂质测定，用甲醇稀释脂质提取物和植物油标准三倍。对于每稀释，样品添加80微升到96孔聚丙烯微孔板四甲酸酯。对于溶剂空白，在四甲酸酯中应用80微升的福尔奇溶剂。对于标准，添加10、30、60、90和120微升稀释植物油标准也四分之一。将微孔板放在烟气罩中，在 55 摄氏度的预热干块加热器上，20 至 30 分钟，直到所有溶剂蒸发。从加热块上取下微孔板，让它冷却到室温。然后，将30微升异丙醇添加到每个井中，然后通过上下移液混合。确保所有移液器通道混合溶液并重新产生脂质，从而产生均匀的绿色液体。现在，每毫升加入200微升，每毫升1微克，每井有一个尼罗河红色溶液，然后上下管10次混合。在室温下孵育5分钟后，在每井中加入20微升50%漂白剂溶液，并上下管5次，以混合良好。在室温下，让板孵育30分钟。30分钟后，在530纳米的激发下，每5至10分钟读取一次样品中的荧光，575纳米发射，自动切断设置为570纳米，直到藻类样本的信号稳定下来。此过程可生成 OD 550 纳米的 algel 光学密度数据的时间过程。控制培养在新鲜的 NH4 介质上生长。治疗之一，是共同培养辅助球菌原糖二化物和在新鲜与生俱来的NH4培养基上生长的亚佐斯皮里鲁姆胸罩。治疗二，是一种在先天NH4介质上生长的azinic A.原型二化物，每毫升50毫克的AA，完全抑制藻类生长。治疗三，是阿齐尼奇 A.原型二，生长在从巴西利安花花的花介质上。生长曲线显示辅助球菌原二化物在120小时进入晚期对数生长。此处显示的是 550 纳米的光学密度与最终干重浓度之间的核心曲线，使用第二阶多项式拟合。最后，可将着色应用于时间过程光学密度数据，获得干重生长曲线。这里也使用相同的治疗方法，只不过小球菌是培养的，而不是辅助球菌原糖二氧化硅。中性脂质测定中获得的干重中性脂质与相应薄层色谱板上的三糖醇含量相关。与A.原型二剂不同，IAA治疗没有抑制C.Sorkiniana的生长。在提取藻类脂质后，可以分析剩余的细胞托盘的淀粉和细胞壁含量，从而提供细胞内储能产品的详细图片。这里介绍的方法使阿尔格尔生物燃料和废水处理领域的新发现得以实现。具体来说，这个系统被用来更好地了解细菌如何影响凝胶的生长。

Research

JoVE Journal

Environment

Analysis of Fatty Acid Content and Composition in Microalgae

脂肪酸组分和含量微藻分析

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

科研

环境科学

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

重金属和微藻的产其他无机污染物的量化

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

一种用于检测汞和镉的厌氧生物传感器检测方法

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

爬行动物皮肤脂类的化学分离、定量和分离

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...