Development of Discontinous Filtration System for Enhancing Biomass Production and Co 2 Fixation from Chlorella vulgaris Buitenzorg

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1 International Journal on Algae, 2012, 14(4): Development of Discontinous Filtration System for Enhancing Biomass Production and Co 2 Fixation from Chlorella vulgaris Buitenzorg M. DIANURSANTI 1, NASIKIN & A. WIJANARKO 2 1 Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia 2 Kampus Universitas Indonesia, Depok 1642, Indonesia danti@che.ui.ac.id anondho@eng.ui.ac.id ABSTRACT Currently, microalgae such Chlorella vulgaris is using as a raw material for biofuels. Chlorella vulgaris has ability to absorb CO 2 and metabolized to produce lipids, proteins and carbohydrates in its cell body. Chlorella culture was cultivated in mid-scale bubble column photobioreactor which was sparged by air which contains 5% CO 2. It s biomass production rate could be keep constant hight by adjusting its culture cell density. These arrangements will be implemented through a discontinuous filtration system thathas been proven to increase production of Ch. vulgaris biomass about two times more than cultivation without filtration system. The results also have shown that the average rate of CO 2 fixation obtained at the filtration system was larger. Discontinuous filtration system is very potential to be developed as a method for the production of biomass. This discontinuous filtration system resulted an accumulation of lipids around 36.7% in its internal cell. KEYWORDS: Chlorella vulgaris, biomass production, discontinuous filtration, CO 2 fixation, lipid accumulation. INTRODUCTION Microalgae is a photosynthetic microorganism that is rich in essential substance such lipid, protein, carotene< carbohydrate, pigment, have an ability to elimenate the greenhouse gas, CO 2. Chlorella have a potential purpose for health supplements potential, cosmetics and pharmaceuticals, animal feed, chemical Originally published in Algologia, 2012, 22(4), pp ISSN Begell House Inc.,

2 M. Dianursanti, Nasikin & A. Wijanarko products and fuel stock (Ohtaguchi & Wijanarko, 2002; Becker, 2004; Wijanarko et al., 2004; 2006a, b). Light intensity factor was important for its photosynthetically growth to convert light energy using CO 2 as corbon source to make various biological compounds. At higher light intensity, specific growth rate and biomass production of this photosynthetic microorganism will be increase to the point of optimum (Hirata et al., 1996). Effect of illumination on the ability of biomass production and CO 2 fixation of photosynthetic organisms depends on the quality of light (in this case the magnitude of the intensity of light as well as how long the daily exposure time) (Wirosaputro, 2002). Previous research resuits show that at optimal illumination settings, Chlorella sp. production was increased significantly. Ch. vulgaris biomass production can be increased up to 60% by the method of lightening alteration that was conducted in a lab-scale photobioreactor (750 dm 3 ) (Wijanarko et al., 2006b). However, this method will require enormous energy for lightening and its application in large scale production will be difficult. Adjusting its culture cells density relatively constant through discontinuous filtration system may become a solution. This treatment will trap the most of biomass to keep its culture cell density relatively constant at its growth phase. This cells trapping method make phenomenon of self-shading effect that often occurs in photosynthetically bacterial cultivation can be overcome. Thus, a given light intensity can be sufficient for its cellular growth, and it is unnecessary to altered light intensity during cell growth. MATERIALS AND METHODS Microalgal cultures, medium and chemical Chorella vulgaris was supplied from Fresh Water Fishery Research Center, Depok, Indonesia. Cell of algae was cultured in Benneck medium contain of 100 mg MgSO 4 7H 2 O, 200 mg KH 2 PO 4, 500 mg NaNO 3, 3-5 mg FeCl 3 per liter. This strain is cultivated in a single 18 dm 3 bubble column photobioreactor at light intensity of 5.0 Klx and sparged by air which contains 5% CO 2. Experimental system with photobioreactor In this study, bioreactor is a flat plate reactor which is not too thin and sparged with an aerator through the gas distributor. The reactor type was used for increasing its cellular growth such large lighting surface area, efficient mass transfer rate, high biomass yield, and also easy to operate and sterilize. Photobioreactor scheme is shown in Figure 1. Existence of suction pump and 358

3 Development of Discontinous Filtration System filter container with ultra filter which has pore size between 0.01 to 0.01 µm are used for absorbing excess microalgae product. This ultra filter has been widely used to filter impurities contained in water at water treatment processes. Experimental design of discontinuous filtration system In this discontinuous system, similar to batch cultivation system, culture medium was illuminated at 5.0 Klx that was an optimum illuminating condition for cellular culture optical density (OD 600 nm ) of and sparged by flowing air contain 5% CO 2. Cellular measurement was taken every 6 hours for 204 hours. At the OD 600 nm tend of 0.600, cellular culture was harvested. Harvesting is done by sucking cellular culture around a half volume of bioreactor into the water filter housing. Furthermore, un-harvested cellular culture will be added with reentered supernatant medium. Cellular culture density at OD 600 nm reached is quite dense and cellular growth tended in stationary phase is a reason why this harvesting process is done. Culture filtration around a half volume of bioreactor and re-enter supernatant medium made remaining cellular density is about an it also make required cultivation time to reach cellular culture optical denstty of is not too long, so it make every harvesting process can be done in a short period of time. Calculation of microalgal growth rate Biomass measurement is carried out using visible light spectrophotometer with a wavelength of 600 nm (OD 600 nm ). Cellular specific growth rate µ, that is logarithmically proportional to incident biomass density X and was calculated by following logarithmic equation: µ = 1 X dx dt (1) Calculation of CO 2 fixation rate The amount of CO 2 concentration was measured by using gas chromatography. Calculation of percentage amount of fixated CO 2 was done by following equation (Hirata et al., 1996; Wijanarko et al., 2006a, b). dy Y CO2 Y = CO2in Y Y CO 2in CO2out x100% (2) 359

4 M. Dianursanti, Nasikin & A. Wijanarko Lipid extraction The determination of lipid content in inner cellular sample using the method of Bligh-Dyer: 1 ml samples of Chlorella mixed and extracted into the solution (mixture of methanol and chloroform with ratio of 2:1) and its supernatant is then carried out and centrifuged for 10 minutes. Furthermore, sediment yield which was dominated by lipids are dried and weighed. DISCUSSION In this research, culture of Ch. vulgaris is cultivated by discontinuous filtration system and unused filtration system. Of these methods then compared the growth rate and CO 2 fixation ability, as well as the lipid content. Initial OD 600 nm is 0.2 and illuminated by 5.0 Klx light intensity and aerated by flowed air which contains 5% v/v CO 2. A growth result of both systems is shown in Figure 2. It can be seen that in beginning interval, cellular growth in both treatments have similar trend. However, after 50 hours cultivation, cell growth began to show a different trend. This indicates cellular self shading phenomenon as consequence of rapid increasing biomass density in medium culture in unused filtration system (Falkowsky & Owens, 1980; Wijanarko & Dianursanti, 2009). In discontinuous filtration system, it shown that total cellular biomass density, which is defined as cellular culture density and ultra filter trapped cellular density, was increasing to 16.4x10-1 g/dm 3. However in unused filtration system, its total cellular density tended an increasing to 8.04x10-1 g/dm 3 and it means that presence of discontinuous filtration system was increase cellular growth around two times. Refer to growth result, after 170 hours cultivation, cellular growth in unused filtration system had entered the stationary phase, while in discontinuous filtration system, cell growth is still in exponential growth phase. It is caused of a periodically discontinuous trapping of the cells from its cellular culture, marking remained culture, cell density in state of obtaining sufficient light. In this discontinuous filtration system, re-entered supernatant medium to remained cellular culture density makes the whole cells in culture media still get adequate lighting and avoid self-shading effects. These results shown a similar pattern with previous results, which was concluded that cellular cultivation process using filtration system will improve its cellular growth for getting its more biomass yield (Wijanarko et al., 2008; Wijanarko & Dianursanti, 2009). Furthermore, in discontinuous filtration reactor, the addition of re-entered supernatant medium, resulted periodically growth rate increasing..therefore, it 360

5 Development of Discontinous Filtration System can be said that the discontinuous filtration system could make its cellular growth rate remains high and make its cultivation time longer. Bicarbonate ion concentration [HCO - 3 ] trend was shown in Figure 3. This figure showed the amount diluted CO 2 which was available and ready to consume for micro algae s metabolism. These ions were calculated from ph measurement using combination of Henry s Law and the Hendersen-Hasselbalch s equation (Wijanarko et al., 2004, 2006a, b). It can be seen that the concentration of bicarbonate ions from unused filtration system is higher than the discontinuous filtration. During cultivation growth, increasing of biomass make an increasing of culture medium ph and it tend by increasing OH - ions that was produced by its photosynthesis and incorporated by CO 2 which was contained in aerated air to make bicarbonate ion. Furthermore, increasing biomass yield indirectly make an increasing of bicarbonate ion in culture medium (Kaplan et al., 1980). Meanwhile, in discontinuous systems, the process of culture cells entrapped before re-entered its supernatant yield into the bioreactor, making a part of dissociated CO 2 from bicarbonate ion entrapped with cellular filter residue and also gone to atmosphere and furthermore, contained bicarbonate ion in the culture medium becomes down after each cellular harvesting. Higher cellular growths in discontinuous filtration system, is due to its CO 2 fixation was higher than the other system. Increasing of dissolved CO 2 in the cellular culture medium directly make increasing of [HCO - 3 ] that can be easily consumed by micro algae. Discontinuous filtration system tends an increasing of CO 2 fixation ability about 62.3% comparing to another treatment result. This escalation of CO 2 fixation ability obtained that discontinuous filtration method is potential to be utilized for reducing greenhouse gas, CO 2. TABLE. Lipid content of cultivation Type of system Lipid content (% weight) Unused filtration 35.8 Discontinuous filtration 36.7 Results of cultivation for 204 hours is then carried out the analysis of lipid content. The essence of this test is to prove that the cultivation methods used do not cause disturbances in the lipid content of cells. Lipid content analysis results can be seen in the following Table. 361

6 International Journal on Algae, 2012, 14(4): Figure 1. Experimental equipment 362

7 Development of Discontinous Filtration System Figure 2. Total biomass concentration and cellular specific growth of Chlorella vulgaris: A un-used filtration system; B discontinuous filtration system 363

8 M. Dianursanti, Nasikin & A. Wijanarko Figure 3. Incident bicarbonate ion concentration [HCO 3 - ] and incident fixated CO 2 concentration of Chlorella vulgaris: A unsed filtration system; B filtration system 364

9 International Journal on Algae, 2012, 14(4): From above results, it can be seen that inner cellular lipid content values obtained from both methods of cultivation did not change significantly. This result shows that discontinuous filtration method potential to be developed cause of it did not change of its cellular content and sustainable production of microalgae Ch. vulgaris was proven. Finally, its lipid content results make this microalgae become a promising potential to be utilized as raw material for the manufacture of alternative fuel. CONCLUSIONS Chlorella vulgaris Buitenzorg cultivation using discontinuous filtration system has succeeded in increasing growth rate around two times and also its CO 2 fixation ability. Furthermore, discontinuous filtration system is very potential as an alternative method for global climate change problem solving. In addition, based on Ch. vulgaris lipid content analysis, this discontinuous system is potential for producing high economically value biomass of algal oil as an alternative fuel. ACKNOWLEDGMENT The author would like to thanks Irfan Pratama, Tangguh Wijoseno, Faris Najmuddin for their contribution in the experimental works. REFERENCES Becker, W Microlgae in human and animal nutrition. Pp in: Handbook of microalgae culture. Blackwell, Oxford. Falkowsky, P.G. & T.G. Owens Light-Shade Adaptation. Plant Physiol. 66: Hirata, S., M. Hayashitani & S. Tone Characterization of Chlorella cell cultures in batch and continuous operations under a photoautotrophic condition. J. Chem. Eng. Jap. 29(6) Kaplan, A., M.R. Badger & J.A. Bery Photosynthesis and the intracellular inorganic carbon pool in the bluegreen alga Anabaena variabilis: Response to external CO 2 concentration. Planta 149: Ohtaguchi, K. & A. Wijanarko Elevation of the efficiency of cyanobacterial carbon dioxide removal by monoethanolamine solution. Technology 8: Wijanarko, A. & Dianursanti PPFD alteration and cellular filtration for large scale biomass production of Chlorella vulgaris Buitenzorg. J. Biosci. and Bioeng. 108:

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