EFFECT OF DIFFERENT WAVELENGTHS OF LIGHT ON THE GROWTH OF CHLORELLA PYRENOIDOSA

EFFECT OF DIFFERENT WAVELENGTHS OF LIGHT ON THE GROWTH OF CHLORELLA PYRENOIDOSA

Mounica Asuthkar, Yamini Gunti, Ramgopal Rao S, C S Rao and Rajasri Yadavalli *

Department of Biotechnology, Sreenidhi Institute of Science and Technology, Yamnampet, Ghatkesar, Hyderabad-501301 Telangana, India.

ABSTRACT: Chlorella pyrenoidosa is a fresh water microalgae used as substrate for production of value added products like biodiesel. In this investigation C.pyrenoidosa was grown for ten days in an internally illuminated bubble column photobioreactor at:  450 lux green light (495–570 nm), 480 lux red light (620–750 nm), 1663 lux blue light (450–495 nm), 1986 luxwhite light (380 to 750 nm).The results after a period of ten days of growth, showed that C.pyrenoidosa has grown better under blue light when compared to the other types of light. C.pyrenoidosa biomass produced 33.8% of lipid contentwhen grown under blue light, indicating its potential as alternative source of biodiesel.

Photobioreactor, Biomass,

Red light, Blue light, Green light, White light, Lipid content.

INTRODUCTION: The world has been facing energy crisis in the last decade, in conjunction with irreversible reduction of traditional material of fossil fuels, unsustainable use of energy, accumulation of greenhouse gases in the atmosphere and global warming problem. With the necessary need to decrease carbon production and the decreasing reserves of crude oil, liquid fuels derived from plant material also called biofuels appear to be an attractive substitute source of energy. Microalgae are single-celled organisms that turn solar energy into various forms of chemical energy such as lipids, proteins and carbohydrates. Algae production is being investigated because of the potential to obtain pharmaceuticals, pigments, carbohydrates, fats and other chemicals, used widely in industry ¹.

In addition, algae can be used for wastewater treatment ² and their ability to produce oxygen from CO₂ also contributes to the preservation of the ecological balance.

Microalgae can be produced in photobioreactors (PBR). The most important factor in the design of PBR is the lighting system ³. Most of the energy used in a photobioreactor is for lighting ⁴. To have widespread commercial use, photobioreactors are required to be cheap, durable, and reliable and have an efficient light source.

In order to cultivate microalgae commercially, a durable, reliable, low cost and high efficiency light source is required. Nowadays, among the variety of light sources, light-emitting diodes (LEDs) are the only ones that can achieve this criteria. LEDs are light and small enough to fit into almost any type of photobioreactor and their advantages such as lower heat generation, longer life-expectancy, higher conversion efficiency and others has made them commercially viable ⁵. For the growth of the different kinds of algae, different nutrients in the growth medium and different wavelengths may be necessary.

In this study all environmental conditions, except for the wavelength of light and light intensity, were kept constant when growing Chlorella pyrenoidosa in internally illuminated bubble column photobioreactor. In the experiments red, blue, green and white LED lights were used as illumination sources. The results after a period of ten days of growth showed the effect of the four illumination sources relative to cell mass and biochemical composition of algal biomass.

MATERIALS AND METHODS:

1. pyrenoidosa stock culture was prepared using BG11 medium in photoautotrophic conditions under incessant illumination in Borosil flasks (150 ml). Each liter of the BG11 medium contained NaNO₃-1.5g, K₂HPO₄-0.04g, MgSO₄.7H₂O-0.075g, CaCl₂.2H2O-0.036g, Citricacid-0.006g, NaCO₃-0.02g,H₃BO₃-0.00286g, MnCl₂.4H₂O-0.00181g,ZnSO₄.7H2O-0.00022g,Na₂MoO₄•2H₂O-0.00039g,CuSO₄.5H₂O-0.00008g,Co(NO₃)₂•6H₂O-0.00005g, (NH₄)₆Mo₇O₂₄.4H₂O-0.003g, Na₂EDTA-0.00001g.

1. pyrenoidosa cultures were grown in 200 ml flasks in a shaker fixed at 100 rpm, by aseptically transferring an inoculum of 50 ml seed culture (10 day old) into freshly sterilized BG11 media (100 ml) exposed to incandescent light. A 10% v/v culture was used as inoculums.

Design of the Photobioreactor:

Fig. 1 illustrates the design and dimensions of the photo bioreactor assembled with two acrylic columns, each with an external diameter of 0.165 m and the inner diameter 0.155 m.  The height of the acrylic column was 0.43 m. The aspect ratio of the present bubble column reactor is 2.77.In general aspect ratio of a bubble column reactor ranges from 2-3. Four glass housings were installed within the column, each housing with 0.02 m diameter and a height of 0.38 m. Algal culture as grown in the inside column to a final volume of 4.5 l provided with an illuminated surface area of 0.1219 m². Ambient air was filtered (0.2 µm pore size) and bubbled along with pure CO₂ through a porous plate sparger located just above the bottom of the inside cylinder. The gas flow rate of CO₂ and air was adjusted at 1% and both were mixed in a gas mixing chamber prior to entering the Bioreactor.

To compensate evaporation losses, a constant volume of culture was maintained by replenishing the nutrient rich medium (3 liters).  The pH was monitored continuously using pH probe and controlled manually. Temperature generated due to sparging and metabolic reactions inside the reactor was controlled using the coolant that is pumped outside chamber through a recirculation process. The coolant (water) was supplied from overhead tank into the external column.

FIG 1: EXPERIMENTAL SETUP OF PHOTO BIOREACTOR WITH MIXING CHAMBER [INSIDE THE BOX: TRIANGULAR ARRANGEMENTS OF THE LIGHTS FOR BETTER PENETRATION OF LIGHT ON ALL SIDES INSIDE THE REACTOR].

Red, Green, Blue and White LEDs in the form of strips were attached to an 18 cm stick and soldered for connections. These LEDs on a solid support were connected to an adaptor through wires for power supply. Light intensity of green light (495–570 nm) was measured to be 450 lux. Intensity of red light (620–750 nm) was measured to be 480 lux. Intensity of blue light (450–495 nm) was measured to be 1663 lux .Intensity of white light (380 to 750 nm) was measured to be 1986 lux. The initial OD of all the samples in four different wavelengths on first day of inoculation was 0.04. After 10days of cultivation of algal culture in BG11 medium, the media was taken out from the photobioreactor and centrifuged at 5000 rpm for about 10mins and the pellet was collected. The dry algal biomass was analyzed for its biochemical composition.

Estimation of Lipid, Carbohydrate and Protein: The carbohydrate content was analyzed based on the procedure published by earlier scientists⁶. In short, 0.1 g dried algal pellet was acidified by adding 20 ml 2.5 M HCl. The acidified solution was then hydrolyzed at 100°C for 30 min and neutralized to pH 7. The volume was adjusted to 100 ml. The filtered sample was subjected to 3,5-dinitrosalicylic acid (DNS) assay ⁷.

Lipid extraction was performed in a way that was similar to the procedures reported by earlier studies ⁸. Briefly, 1.0 g dried cells were ground in amortar and pestle. The dried powder is then transferred to a 50 ml glass centrifuge tube. Chloroform was then added to the tube to make the chloroform/ methanol (2:1, v/v). The tube was vortexed for 5 min and was allowed to stand for 24 h. After that, the tube was centrifuged at 4,000g for 15 min to remove the algal solids. The supernatant was collected and the solvent was vaporized using Rotovap at 65⁰C. Oil left in the flask without solvent was weighed to calculate lipid content.

Protein estimation is done using Lowry’s method ⁹. Briefly, 0.1 g algal powder is taken and 2ml of 5%TCA is added. The mixture is centrifuged at 2000rpm for 10min.Solution containing pellet is dissolved in 1.5ml of 0.1N NaOH. From the above prepared solution 0.2 ml is taken into 20ml test tube and 5ml of alkaline copper solution is added. After incubation at room temperature for 15min, 0.5ml of Folin-Ciocalten reagent is added. Again the mixture is incubated for 30min and OD values were taken at 540nm.

The specific growth rate (μ) of C.pyrenoidosa under green, red, blue and white lights was calculated using the formula

μ = (ln X₂ - lnX₁)/Δt,

Where X₂ is the final cell concentration, X₁ is the initial cell concentration and Δt is the time required for the increase in concentration from X₁ to X₂.

The doubling time (t_d) of C.pyrenoidosa was also calculated for the four different wavelengths of lights using the equation t_d= 0.693 / µ.

RESULTS AND DISCUSSION:

FIG 2: INTERNALLY ILLUMINATED BUBBLE COLUMN PHOTOBIOREACTOR WITH DIFFERENT WAVELENGTHS OF LIGHT

Fig.2 shows the set up of different wavelengths of light in internally illuminate bubble column photobioreactor. The effect of four different LED lights on the growth of Chlorella pyrenoidosa was observed for 10 days. There was a significant difference in the growth of algae when they were grown in four different lights (Fig. 3). The growth of C.pyrenoidosa growth increased till 8th day in all the wavelengths and after that growth became constant as microalgae could have entered the stationary phase either due to unavailability of nutrient medium or substrate inhibition.

The maximum growth of C.pyrenoidosa was observed in blue light (Fig. 3). The growth of C.pyrenoidosa in red light was less when compared to other three lights. This was in accordance with previous studies which reported that growth of microalgae is less in red light when compared to blue light. The energy content of blue light (400–480 nm) is greater than that of red light (620–750 nm) ¹⁰.

In addition to differences in the energy intensity, specific components of light are known to impact the cellular regulatory processes including: chlorophyll synthesis, photo damage repair, and cell division. For example, blue light was shown to be essential for the division of Chlamydomonas reinhardtii cells ¹¹. Evidence for the influence of blue light on short-term growth rate is equivocal ¹². Previous studies ¹³ also reported that the blue LED allows the light intensity to penetration deep into the batch culture thus enhancing the doubling of microalgae and cell density.

FIG. 3: CHLORELLA PYRENOIDOSA GROWTH CURVE IN DIFFERENT WAVELENGTHS OF LIGHT

TABLE 1: SPECIFIC GROWTH RATE AND DOUBLING TIME OF C.PYRENOIDOSA IN VARIOUS WAVELENGTHS OF LIGHT

From Table 1, specific growth rate value was observed to be maximum for blue light (0.506 day^-1) and minimum value for red light (0.215 day^-1) whereas for green and white light it was the same. Doubling time for C.pyrenoidosa in the presence of red light was slower that is 77.35 hours while in the presence of  blue light it was rapid (32.64 hours). In the presence of green and white light C.pyrenoidosa took 69.12 hours to get doubled.

TABLE 2: BIOCHEMICAL ANALYSIS FOR C.PYRENOIDOSA UNDER DIFFERENT WAVELENGTHS OF LIGHT

The biochemical analysis was performed on the dry biomass obtained from photobioreactor after 10 days of cultivation. From Table 2 it was found that the protein and carbohydrate content was high in algal biomass grown under blue light when compared with other wavelengths of light. Lipid productivity is a product of lipid content and dry cell weight of algal biomass, which in turn decides its suitability as alternative source of biodiesel. As the dry cell weight of C.pyrenoidosa in other light intensities was less when compared with blue light, the lipid analysis was performed only for the algal biomass grown in blue light. It was found that the lipid content was 3.38mg/g in that biomass sample.

CONCLUSION: Growth of C.pyrenoidosa was more in the presence of blue light compared to green, red and white lights asblue LED allows the light intensity to penetration deep into the batch culturecompared to the other lights. Specific growth rate of C.pyrenoidosa  under  blue light was found to be maximum (0.506 day^-1) and showed a rapid doubling time of 32.64 h. Also the protein and carbohydrate content were found to be more in blue light when compared to other three wavelengths of light.

ACKNOWLEDGEMENTS: We thank the Management of Sreenidhi Institute of Science and Technology (SNIST) and Technical Education Quality Improvement Programme(TEQIP-II) funded by World bank  for their financial support in carrying out this in-house project.

REFERENCES:

1. Hoppe HA, Levring T, Tanaka Y (Eds.): Marine algae in pharmaceutical science. Walter de Gruyter, Berlin 1979; 25-119.
2. Rajasri Y, Goutham Rao H: Two stage treatment of dairy effluent using immobilized Chlorella pyrenoidosa. Journal of Environmental Health Science and Engineering 2013; 11(1):36 doi: 10.1186/2052-336X-11-36.
3. Barta, D J, Tibbitts T W, Bula R J, Morrow R C: Application of light emitting diodes for plant irradiation in space bases. COSPAR Meeting in The Hague, The Netherlands, June 28, 1990.
4. Rajasri Y, Ramgopal Rao S, C.S. Rao: Lipid productivity in Chlorella pyrenoidosa under nitrogen starvation: Energy evaluation of an internally illuminated bubble column photo bioreactor. Asian Jr. of Microbiol. Biotech. Env. Sc. 2014;16 (1) : 245-251
5. Chun-Yen Chen, Kuei-Ling Yeh, Rifka Aisyah, Duu-Jong Lee, Jo-Shu Chang: Cultivation, Photobioreactor Design and Harvesting of Microalgae for Biodiesel Production: A Critical Review . Bioresource Technology 2011; 102 :71–81.
6. Miao X, Wu Q: High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. J Biotechnol 2004; 110:85–93.
7. Miao XL, Wu QY, Wu GF, Zhao NM: Sucrose accumulation in salt-stressed cells of agp gene deletion-mutant in cyanobacterium Synechocystis PCC 6803. FEMS Microbiol Lett. 2003; 218: 71–77.
8. Zhu M, Zhou PP, Yu LJ: Extraction of lipids from Mortierellaalpina and enrichment of arachidonic acid from the fungal lipids. Bioresource Technol .2002; 84:93–95.
9. Lowry Oh, Rosebrough Nj, Farr Al, Randall Rj: Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193(1):265-75.
10. Ankita J, Ruben Michael C, Ganti S Murthy: Effects of Environmental Factors and Nutrient Availability on the Biochemical Composition of Algae for Biofuels Production: A Review. Energies 2013; 6: 4607-4638.
11. Voigt J ,Münzner P: Blue light-induced lethality of a cell wall-deficient mutant of the unicellular green alga Chlamydomonas reinhardtii. Plant Cell Physiol.1994; 35: 99–106.
12. Borodin VB: Effect of red and blue light on acclimation of Chlamydomonasreinhardtii to CO₂-limiting conditions. Rus. J. Plant Physiol.2008; 55: 441–448
13. Madiha Atta, Ani Idris Ataullah Bukhari, Suzana Wahidin: Intensity of Blue LED Light: A Potential Stimulus for Biomass and Lipid Content in Fresh Water Microalgae Chlorella vulgaris. Bioresource Technology 2013; 148: 373–378.
14. Shen Y, Yuan W, Pei Z, Mao E: Culture of microalga Botryococcus in livestock wastewater. Trans ASABE 2008; 51:1395–1400.
    

    ---

How to cite this article:

Asuthkar M, Gunti Y, Rao SR, Rao CS and Yadavalli R: Effect of Different Wavelengths of Light on the Growth of Chlorella Pyrenoidosa. Int J Pharm Sci Res 2016; 7(2): 847-51.doi: 10.13040/IJPSR.0975-8232.7(2).847-51.

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.