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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 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.
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....
1. Setup of Bubble Column Photobioreactors
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.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
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 | 35x300 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 | 100x15 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 24x2 | 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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