Microalgae Culture Using the DASGIP® PBR4 Module for Illumination with a New BrunswickTM CelliGen® 310 Stirred-tank Bioreactor

The number of bioprocess applications for microalgae has increased in recent years, particularly in the fi eld of biofuel production. The combination of the New Brunswick CelliGen 310 stirred-tank bioreactor and the DASGIP LED Illumination System creates a bioreactor setup which is capable of supporting high density microalgal growth. Using the stand-alone Eppendorf DASGIP PBR4 Module, LED illumination spectra and intensities can be controlled for optimal support of all types of chlorophylls and carotenoids. For this study in which high density culture of up to 1.5 x 107 cells/mL was achieved, the unicellular freshwater alga, Dunaliella tertiolecta, was used. Figure 1: Equipment used to assemble the bioreactor – CelliGen 310 (left) and DASGIP PBR4 Module with LED Illumination Devices (right) APPLICATION NOTE No. 332 I Page 2 CelliGen 310 stirred-tank bioreactor with the addition of the DASGIP LED Illumination system without the use of a DASGIP photobioreactor (Figure 1). This bioreactor setup is able to generate high-density microalgae cultures which can be used for strain engineering and other research and development endeavors. The stand-alone DASGIP PBR4 Module for precise control of illumination settings allows growing plant cell suspensions, green or brown algae as well as phototrophic bacteria like cyanobacteria or green sulfur bacteria on variable lighting conditions. The DASGIP LED Illumination Devices were designed to provide the most eff ective light supply for the highest photosynthesis and growth rates (Figure 3). Up to 4 devices per bioreactor can be installed using the industry-standard PG13.5 headplate port and serve optimized light spectra with defi ned wavelengths to meet the specifi c photosynthesis requirements. The spectral composition of the LEDs supports all types of chlorophylls and carotenoids (Figure 3). Materials and Methods Consumable Materials Table 1 details the consumable reagents and materials that were used in this study. Algae Inoculum Culture D. tertiolecta (Figure 4) was cultured in vent cap fl at bottom Erlenmeyer fl asks (VWR®, USA) in a New Brunswick Innova® 44R shaker outfi tted with the programmable photosynthetic light bank (Eppendorf, Inc., USA). Sterile Instant Ocean (United Pet Group, Inc., USA) was used as a base to which f2 media supplements were added at the concentrations recommended by the kit protocol (ProvasoliGuillard National Center for Marine Algae and Microbiota, USA). Instant Ocean with f2 supplements is referred to as f2 medium – so named because it represents a reducedTable 1: Materials, media and cells Material Supplier Order no. Dunaliella tertiolecta Provasoli-Guillard National Center for Marine Algae and Microbiota CCMP362 f/2 culture medium supplements Provasoli-Guillard National Center for Marine Algae and Microbiota f/2 medium TAP growth medium Gibco® A1379801 1x Dulbecco’s Phosphate buffered saline Life Technologies® 14190-144 Instant Ocean® United Pet Group, Inc. SS15-10 250 mL PC vent cap Erlenmeyer flask VWR® 89095-266 Figure 3: Available wavelengths of LED lights are illustrated – light spectra of the LEDs are optimized to meet various photosynthesis requirements with 453, 572, 625, 640, 660 and 780 nm available Figure 2: Growth of two algae strains using a DASGIP PhotoBioreactor combined with the PBR4 LED Illumination System Figure 4: D. tertiolecta grown in bioreactor conditions – scale bar represents 100 μm, photographed at 100x using an Olympus® IX51 inverted microscope (OLYMPUS, Japan) equipped with an Infi nity2 CCD camera (Lumenera®, Canada) APPLICATION NOTE No. 332 I Page 3 strength version of the original “f medium.”[4] While f2 medium provides trace metals, a nitrogen and phosphorous source, and vitamins, Tris acetate phosphate (TAP) medium provides optimal salinity and buff er conditions for algae growth. Robust microalgal growth was observed using a combination of f2 and TAP, formulated as follows: 1 part f/2 medium and 9 parts TAP medium (f2:TAP). Cultures were inoculated at an approximate density of 1 x 106 cells/mL and maintained at 23 °C with a shaking speed of 100 rpm in constant light conditions. Culture density was monitored using a Vi-CELL® automated cell counter (Vi-CELL XR; Beckman Coulter, Inc., USA #731050). Bioreactor culture Log phase D. tertiolecta were used to inoculate a CelliGen 310 1 L glass water-jacketed vessel (Eppendorf, Inc., USA). The vessel was outfi tted with a standard gelfi lled pH sensor, a pitched-blade impeller and three DASGIP LED Illumination Devices (DASGIP GmbH, Germany). An inoculation density of 1 x 106 cells/mL in a fi nal working volume of 1.8 L of the f2:TAP combination described above was used. The vessel was fi lled to maximum working volume of 1.8 L to allow as much LED light into the culture medium as possible. Since algal cultures tend to become more alkaline over time, CO2 gas was used to maintain pH without base addition. When CO2 was directly sparged into the culture, the pH dropped too rapidly and resulted in a reduction in viability (data not shown); therefore, pH control was accomplished using the overlay option installed on the CelliGen 310 controller (Eppendorf, Inc., USA). The pH control was cascaded to overlay CO2 fl ow. A pH deadband of 0.2 was used. The DASGIP PBR4 Module was used to control LED light delivery to the growing culture. This module allows individual control of three independent wavelength channels emitting light of 6 diff erent wavelengths, as shown in Table 2. For green algae, LEDs in the red, orange and blue ranges were used which is appropriate for an organism containing chlorophyll A and B (Table 2). Setpoints for the LED illumination sticks are shown in Table 3. Table 2: Wavelengths of PBR4 channels Channel Description Available wavelengths (nm) A Bright red, near infared 660, 780 B Green, yellow, orange 572, 625, 640