Name: Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 11 min 8 s
Description: Learn to construct and use a Bubble Column Photobioreactor for microalgae cultivation and neutral lipid assays. This cost-effective system supports biofuel research with customizable designs.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Support for this research was provided by USDA National Institute of Food and Agriculture Hatch Project ALA0HIGGINS and the Auburn University Offices of the Provost, the Vice President for Research, and the Samuel Ginn College of Engineering. Support was also provided by NSF grant CBET-1438211.

<ol><li>Prandini, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancement+of+nutrient+removal+from+swine+wastewater+digestate+coupled+to+biogas+purification+by+microalgae+Scenedesmus+spp%5BTitle%5D)+AND+%22Bioresource+Technology%22%5BJournal%5D)">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/pubmed?term=((Phycoremediation+of+dairy+and+winery+wastewater+using+Diplosphaera+sp.+MM1%5BTitle%5D)+AND+%22Journal+of+Applied+Phycology%22%5BJournal%5D)">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/pubmed?term=((Dairy+Wastewaters+for+Algae+Cultivation%2C+Polyhydroxyalkanote+Reactor+Effluent+Versus+Anaerobic+Digester+Effluent%5BTitle%5D)+AND+%22BioEnergy+Research%22%5BJournal%5D)">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/pubmed?term=((Cultivation+of+a+native+alga+for+biomass+and+biofuel+accumulation+in+coal+bed+methane+production+water%5BTitle%5D)+AND+%22Algal+Research%22%5BJournal%5D)">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/pubmed?term=((Oil+accumulation+mechanisms+of+the+oleaginous+microalga+Chlorella+protothecoides+revealed+through+its+genome%2C+transcriptomes%2C+and+proteomes%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">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/pubmed?term=((Combined+nitrogen+limitation+and+hydrogen+peroxide+treatment+enhances+neutral+lipid+accumulation+in+the+marine+diatom+Phaeodactylum+tricornutum%5BTitle%5D)+AND+%22Bioresource+Technology%22%5BJournal%5D)">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/pubmed?term=((Biofuels+from+microalgae--A+review+of+technologies+for+production%2C+processing%2C+and+extractions+of+biofuels+and+co-products%5BTitle%5D)+AND+%22Renewable+Sustainable+Energy+Reviews%22%5BJournal%5D)">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/pubmed?term=((Microalgae+-+Novel+highly+efficient+starch+producers%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">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/pubmed?term=((Utilization+of+Volatile+Fatty+Acids+from+Microalgae+for+the+Production+of+High+Added+Value+Compounds%5BTitle%5D)+AND+%22Fermentation%22%5BJournal%5D)">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/pubmed?term=((Bioprocess+engineering+of+microalgae+to+produce+a+variety+of+consumer+products%5BTitle%5D)+AND+%22Renewable+and+Sustainable+Energy+Reviews%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+new+method+for+genetic+transformation+of+the+green+alga+Chlorella+ellipsoidea%5BTitle%5D)+AND+%22Molecular+biotechnology%22%5BJournal%5D)">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/pubmed?term=((Mutate+Chlorella+sp.+by+nuclear+irradiation+to+fix+high+concentrations+of+CO2%5BTitle%5D)+AND+%22Bioresource+Technology%22%5BJournal%5D)">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/pubmed?term=((Techno-economic+analysis+of+autotrophic+microalgae+for+fuel+production%5BTitle%5D)+AND+%22Applied+Energy%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+Scale+in+a+Photosynthetic+Reactor+System+for+Algal+Remediation+of+Wastewater%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Cofactor+symbiosis+for+enhanced+algal+growth%2C+biofuel+production%2C+and+wastewater+treatment%5BTitle%5D)+AND+%22Algal+Research%22%5BJournal%5D)">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/pubmed?term=((Algal-bacterial+synergy+in+treatment+of+winery+wastewater%5BTitle%5D)+AND+%22Nature+Clean+Water%22%5BJournal%5D)">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/pubmed?term=((Impact+of+thiamine+metabolites+and+spent+medium+from+Chlorella+sorokiniana+on+metabolism+in+the+green+algae+Auxenochlorella+prototheciodes%5BTitle%5D)+AND+%22Algal+Research%22%5BJournal%5D)">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>Lépinay, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+insight+on+interactions+between+bacteria+and+the+marine+diatom+Haslea+ostrearia%3A+Algal+growth+and+metabolomic+fingerprinting%5BTitle%5D)+AND+%22Algal+Research%22%5BJournal%5D)">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/pubmed?term=((Growth+of+three+microalgae+strains+and+nutrient+removal+from+an+agro-zootechnical+digestate%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">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/pubmed?term=((Azospirillum+brasilense+Increases+CO2+Fixation+on+Microalgae+Scenedesmus+obliquus%2C+Chlorella+vulgaris%2C+and+Chlamydomonas+reinhardtii+Cultured+on+High+CO2+Concentrations%5BTitle%5D)+AND+%22Microbial+Ecology%22%5BJournal%5D)">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/pubmed?term=((Effects+of+Escherichia+coli+on+mixotrophic+growth+of+Chlorella+minutissima+and+production+of+biofuel+precursors%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Microplate+assay+for+quantitation+of+neutral+lipids+in+extracts+from+microalgae%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((The+impact+of+elevated+CO2+concentration+on+the+quality+of+algal+starch+as+a+potential+biofuel+feedstock%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">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/pubmed?term=((A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Informatics+for+improved+algal+taxonomic+classification+and+research%3A+A+case+study+of+UTEX+2341%5BTitle%5D)+AND+%22Algal+Research%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGarrett%2C+RH+%22Biochemistry%22">Biochemistry</a>. , 5th Edition, Cengage Learning. 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/pubmed?term=((Chlorella+sorokiniana+UTEX+2805%2C+a+heat+and+intense%2C+sunlight-tolerant+microalga+with+potential+for+removing+ammonium+from+wastewater%5BTitle%5D)+AND+%22Bioresource+Technology%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWang%2C+Q+%22Annual+International+Meeting+of+the+ASABE%22">Annual International Meeting of the ASABE</a>. , American Society of Agricultural and Biological Engineers. Detroit, MI. (2018).</li>
</ol>

The authors have nothing to disclose.

The most important consideration when culturing algae is an understanding of the specific needs of the organism or group of organisms. The algae cultivation system described here can be used to culture a wide range of algae but the specific abiotic factors (temperature, media, pH, light intensity, CO2 level, aeration rate) need to be adjusted to the needs of the organism. Note the parameters described here were used for the cultivation of Chlorella and Auxenochlorella. These organisms are of ...

The application of microalgae in engineering and biotechnology has attracted great interest in recent years. Microalgae are being studied for use in wastewater treatment1,2,3,4, biofuel production5,6,7,8, and the production of nutraceuticals and other high value products9,10. Algae are also being genetically modified at greater rates in an effort to improve their fitness for specific engineering applications11,12. Consequently, there is great interest in experimentation with industrially relevant organisms in controlled settings. The purpose of this method is to communicate an effective approach to culture microalgae in a controlled laboratory environment, and to measure the growth and neutral lipid content of that algae. Improving growth rates and neutral lipid content of microalgae have been identified as two key bottlenecks toward commercialization of algal biofuels13.
A wide range of approaches have been used to culture algae for experimental purposes. In general, these approaches can be divided between large-scale outdoor cultivation and small-scale indoor cultivation. Outdoor cultivation in photobioreactors and open ponds is appropriate for experimentation aimed at scaling up processes that have already been proven at laboratory scale (e.g., to test scale-up of a new high-lipid strain of algae)14. However, indoor small-scale cultivation is appropriate when developing new or improved algae strains or performing experiments aimed at understanding biological mechanisms. In these latter cases, a high-degree of experimental control is required to tease out subtle changes in biological behavior. To that end, axenic cultures are often required in order to minimize the complex biotic factors associated with other organisms (e.g. bacteria, other algae) that inevitably grow in large-scale outdoor systems. Even when studying interactions among algae and other organisms, we have found that use of highly-controlled experimental conditions is helpful when examining molecular exchange among organisms15,16,17.
Within the category of small-scale indoor algae cultivation, a range of approaches have been used. Perhaps the most common approach is to grow algae in Erlenmeyer flasks on a shaker table beneath a light bank18,19. Exchange of oxygen and CO2 takes place by passive diffusion through a foam plug in the top of the flask. Some researchers have improved this set-up through active aeration of the flasks20. Another approach is to cultivate algae in bottles, mixed by stir bar and active aeration. Despite their simplicity, we have found that the use of flasks and bottles often leads to inconsistent results among biological replicates. Presumably this is due to position effects - different positions receive different amounts of light, which also affect internal reactor temperatures. Daily rotation of reactors to new positions can help but does not alleviate the problem because certain stages of algae growth (e.g., early exponential) are more sensitive to positional effects than others (e.g., log phase).
On the opposite side of the spectrum of technological sophistication are fully-controlled commercial photobioreactors. These systems continuously monitor and adjust conditions in the reactor to optimize algae growth. They have programmable lighting, real-time temperature control, and pH control. Unfortunately, they are expensive and typically cost several thousand dollars per reactor. Most scientific and engineering journals require biological replication of results, necessitating the purchase of multiple bioreactors. We present here a bubble column reactor system that bridges the divide between the simple (flask) and sophisticated (fully-controlled bioreactor) approaches for lab-scale algae cultivation. Bubble columns use rising gas bubbles to facilitate gas exchange and mix the reactor. This approach provides some degree of control over the lighting and temperature but does so in a way that is cost-effective. Moreover, we have found this system to yield highly consistent results among biological replicates, reducing the required number of biological replicates needed in order to obtain statistically significant results when compared to the flask or bottle approach. We have also used this system to successfully cultivate mixtures of algae and bacteria21. In addition to algae cultivation, we outline a procedure for measuring the neutral lipid content in the cultured algae. The latter method has been published elsewhere22, but we include the procedure here to provide step-by-step instructions for how to employ it successfully.

<table><tbody><tr><td>&lt;strong&gt;Supplies for airlift photobioreactor setup&lt;/strong&gt;</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&quot; hose clamp</td><td>Home Depot</td><td>UC953A</td><td>or equivalent</td></tr><tr><td>1/4&quot; female luer to barb</td><td>Nordson biomedical</td><td>Nordson FTLL360-6005</td><td>1/4&quot; ID, PP</td></tr><tr><td>1/4&quot; ID, 3/8&quot; OD autoclaveable PVC tubing</td><td>Thermo-Nalgene</td><td>63013-244</td><td>50&#039;</td></tr><tr><td>1/4&quot; in O-rings</td><td>Grainger</td><td>1REC5</td><td>#010 Medium Hard Silicone O-Ring, 0.239&quot; I.D., 0.379&quot;O.D.</td></tr><tr><td>1/8&quot; Female luer to barb</td><td>Nordson biomedical</td><td>FTLL230-6005</td><td></td></tr><tr><td>1/8&quot; ID, 1/4&quot; OD autoclaveable PVC tubing</td><td>Thermo-Nalgene</td><td>63013-608</td><td>250&#039;</td></tr><tr><td>1/8&quot; male spinning luer to barb</td><td>Nordson biomedical</td><td>MLRL013-6005</td><td></td></tr><tr><td>1/8&quot; multiport barb</td><td>Nordson biomedical</td><td>4PLL230-6005</td><td>1/8&quot; multiport barb</td></tr><tr><td>1/8&quot; NPT to barb</td><td>Nordson biomedical</td><td>18230-6005</td><td>1/8&quot; 200 series barb</td></tr><tr><td>1/8&quot; panel mount luer</td><td>Nordson biomedical</td><td>Nordson MLRLB230-6005</td><td>1/8&quot;, 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 &amp;micro;m</td><td>Whatman/ Fisher</td><td>09-745-1A</td><td>Polyvent, 28 mm, 0.2 &amp;micro;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&quot;, 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>&lt;strong&gt;Supplies and reagents for culturing algae&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;micro;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>&lt;strong&gt;Supplies and reagents for lipid extraction and neutral lipid assay&lt;/strong&gt;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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

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

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the treatment of wastes. As most new research efforts begin at laboratory scale, there is a need for cost-effective methods for culturing microalgae in a reproducible manner. Here, we communicate an effective approach to culture microalgae in laboratory-scale photobioreactors, and to measure the growth and neutral lipid content of that algae. Instructions are also included on how to set up the photobioreactor system. Although the example organisms are species of Chlorella and Auxenochlorella, this system can be adapted to cultivate a wide range of microalgae, including co-cultures of algae with non-algae species. Stock cultures are first grown in bottles to produce inoculum for the photobioreactor system. Algae inoculum is concentrated and transferred to photobioreactors for cultivation in batch mode. Samples are collected daily for the optical density readings. At the end of the batch culture, cells are harvested by centrifuge, washed, and freeze dried to obtain a final dry weight concentration. The final dry weight concentration is used to create a correlation between the optical density and the dry weight concentration. A modified Folch method is subsequently used to extract total lipids from the freeze-dried biomass and the extract is assayed for its neutral lipid content using a microplate assay. This assay has been published previously but protocol steps were included here to highlight critical steps in the procedure where errors frequently occur. The bioreactor system described here fills a niche between simple flask cultivation and fully-controlled commercial bioreactors. Even with only 3-4 biological replicates per treatment, our approach to culturing algae leads to tight standard deviations in the growth and lipid assays.

This procedure yields a time course of algal optical density data at OD 550 nm (Figure 4A). The optical density and dry weight concentration data can be correlated (Figure 4B). This is accomplished by first calculating the final dry weight algae concentration after the freeze-drying step. Next, the optical density of the culture serial dilution (performed on the last day of sampling) and the actual dry weight concentrations can b...

Here, we present a protocol to construct lab-scale bubble column photobioreactors and use them to culture microalgae. It also provides a method for the determination of the culture growth rate and neutral lipid content.

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

cultivation-green-microalgae-bubble-column-photobioreactors-an-assay

Research

JoVE Journal

Environment

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

Analysis of Fatty Acid Content and Composition in Microalgae

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

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

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

This protocol demonstrates the use of a phenotype microarray (PM) technology platform to define metabolic requirements of Chlamydomonas reinhardtii, a green microalga, and refine an existing metabolic network model.

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments.

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

The present study highlights the advantages of employing the method developed by Jeffrey and Humphrey for extracting and quantifying fat-soluble pigments from microalgae. This method serves as a valuable tool for assessing the influence of growth factors on chlorophyll production and cellular content in these organisms.

Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae

Learn to construct and use a Bubble Column Photobioreactor for microalgae cultivation and neutral lipid assays. This cost-effective system supports biofuel research with customizable designs.

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

Explore More Videos