Novel LED-Based Light Source for Cultivation of Phototrophic Organisms in a Stirred-Tank Bioreactor

Martin Grolms1, Claudia M. Hther1, Sebastian Kleebank1, Rick Bradley2, Tian Ong3, Janice OBrien3, Jean-Franois Hamel3
1

DASGIP AG, Jlich, Germany; 2DASGIP BioTools LLC, Shrewsbury, MA; 3MIT, Chemical Engineering Department, Cambridge, MA

Introduction Algae seem to be suited for regenerative energy applications due to their photosynthetic efficiency and high yield in biomass. This biomass can be used for the industrial production of biofuels (1). The challenge in this field is that only a few micro-algae species offer suitable high yields in the desired lipids. Both known and newly discovered algae have to be investigated with regard to their growth parameters (2). Thus, the conditions under which Tetraselmis sp. and Dunaliella tertiolecta provide high lipid yields for biofuel production were investigated. Materials and Methods Experimental Strains Tetraselmis sp. and Dunaliella tertiolecta were provided by Provasoli-Guillard National Center for Culture of Marine Phytoplankton and Bigelow Laboratory for Ocean Sciences. The 10 m x 14 m phytoplankton Tetraselmis sp. and the 9 - 11 m long, motile, unicellular, and rod to ovoid-shaped Dunaliella tertiolecta are of special interest for biodiesel production due to their high lipid levels. Cultivation Medium Two sets of 900 mL medium PM0021 (35 g/L Instant Ocean, 5 mL/L ProLine F/2 Part A and 3 mL/L ProLine F/2 Part B) were inoculated with 100 mL Tetraselmis sp. (optical density at = 600 nm (OD600) 0.004) and 100 mL Dunaliella tertiolecta (OD600 0.070), respectively, resulting in working volumes of 1 L each. Photobioreactors All relevant process parameters were controlled in DASGIP PhotoBioreactors (PBR), in particular different light spectra and light intensities. Spectral composition could be controlled online via the DASGIP Control 4.0 Software by adjusting three groups of light spectra. The unique internal arrangement of the DASGIP light emitting diode (LED) illuminators guarantees most effective and consistent light supply of the culture. Spectral light intensity and profiles of each spectrum were controlled individually. Through these means, the PBR enabled the customization of the growth conditions in phototrophic cultivations.

Diagram 2: Cultivation of Dunaliella tertiolecta using the PhotoBioreactor: Dissoved Oxygen Concentration [% v/v], Inlet Gas Flow Rate [sL/h] and Inlet Gas Oxygen Concentration [% v/v] depending on Light Intensity [mol photons/s].

Results and Discussion The decrease of the inlet gas flow rate from 5.8 sL/h to 3.2 sL/h, between 96 h and 144 h, with 0 % inlet gas oxygen concentration could be an indication for a reduced oxygen production rate of the organisms. This suggests that illumination with a constant intensity may harm the cells. For that reason, after 172 h the illumination profile was changed during the run into a day/night cycle profile. Nighttime spanned for 11 h and daytime lasted 13 h, in which the intensity was increased using the cosine mode to simulate sunrise and sunset. It was observed that the oxygen uptake of the cells increased during the night cycle, whereas the oxygen production increased during the daytime cycles, reaching a maximum production rate at maximum illumination intensities. At the beginning of the cultivation, a constant inlet gas flow rate of 3 sL/h was applied and only the inlet gas oxygen concentration was used as a controller output for DO control. After 75 h, the DO controller was changed and the inlet gas flow rate was added to the DO controller output. After changing the illumination profile to day/night cycles, it could be observed that the oxygen production was dependent on the illumination intensities of the cells. Nevertheless, the dissolved oxygen concentration could not be maintained at 20 % DO. After 240 h, the DO controller was modified again, using the scripting option of the software, resulting in a constant dissolved oxygen concentration of 20 % within both day and night cycles.

Figure 1: DASGIP PhotoBioreactor DR03 with integrated LED Illuminators. In this study a working volume of 1.0 L was used.

The DASGIP benchtop vessels are each fitted with an overhead drive and four illuminators. These illuminators are placed inside each bioreactor and serve optimized light spectra with defined wavelengths to meet the specific photosynthesis requirements. Based on their specific wavelengths, the LEDs are combined into three groups with defined spectral ranges: A (453 nm), B (572 nm, 625 nm, 640 nm), and C (660 nm, 780 nm). Day/night phases of solar output are programmed using the undulated Cosine mode. Start time was 5:30 a.m. and the highest light intensity was achieved at 12:00 p.m. Cultivation Parameters Table 1 shows the process parameters controlled with the DASGIP Control Software, such as temperature, agitation rate, pH, dissolved oxygen (DO) concentration and aeration rate. O2 production and CO2 consumption were determined by using DASGIP OffGas Analyser with integrated BlueSens sensors.
Diagram 1: Light spectra of LEDs are optimized to meet various photosynthesis requirements with 453 nm, 572 nm, 625 nm, 640 nm, 660 nm and 780 nm.

Diagram 3: Cultivation of Tetraselmis sp. and Dunaliella tertiolecta using the DASGIP PhotoBioreactor DR03: Viable cell concentration by cultivation time.

After changing the illumination cycle to a day/night cycle profile, it could be observed that the viable cell concentration of Tetraselmis sp. started to increase. This suggests that constant illumination could harm the cells. Overall, it is apparent that, in the DASGIP PhotoBioreactors, it was possible to grow the algae organisms - Tetraselmis sp. and Dunaliella tertiolecta - up to viable cell numbers of 1.5 106 cells/mL and 2.6 106 cells/ mL, respectively. Conclusion The results revealed that Tetraselmis sp. and Dunaliella tertiolecta are promising candidates for the production of biofuels, due to their high lipid level and because of their good culturability in photobioreactors. Improved growth was obtained by imitating a day/night cycle, through slowly increasing and decreasing the light intensity, ratherthan immediately switching the lights on or off. In order for biofuels production from algae feedstock to become feasible in the alternative energy market, further research must be conducted to improve the competitiveness of this option and to increase and optimize production quantities. References 1. Singh MK and Dhar DW (2011): Microalgae as Second Generation Biofuel. A Review. Agronomy Sust. Developm. DOI:10.1007/s13593-011-0018-0 2. Araujo GS, Matos LJ, Goncalves LR, Fernandes FA and Farias WR (2011): Bioprospecting for Oil Producing Microalgae Strains: Evaluation of Oil and Biomass Production for Ten Microalgae Strains. Bioresour Technol. 102(8): 5248-50

Table 1: Starting parameters for the cultivation of Tetraselmis sp. and Dunaliella tertiolecta using the DASGIP

Experimental strains pH Aeration rate [sL/h] Inlet gas setting [% v/v] Temperature [C] Light intensity [mol photons /s]

Agitation rate [rpm]

Tetraselmis sp. 8.0 2 O2 = 21 and CO2 = 0.1 30 A= 0.50 B= 0.15 C= 0.35 115

Dunaliella tertiolecta 8.0 3 O2 = 21 and CO2 = 0.0 20 A= 0.50 B= 0.15 C= 0.35 115