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J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC CONSTRUCTION OF VERTICAL TUBULAR PHOTOBIOREACTOR FOR MICROALGAE CULTIVATION G.RAMANATHAN1*, K.RAJARATHINAM 2, M.BOOTHAPANDI3, D.ABIRAMI1, G.GANESAMOORTHY3 AND DURAIPANDI3 1 Research Department of Microbiology, 2 Research Department of Botany, and 3 Department of BioEngineering, V.H.N.S.N.College, Virudhunagar-626 001, Tamilnadu, India. *Email: san_than2002@yahoo.co.in ABSTRACT Mass cultivation of microalgae is gaining importance nowadays, because of its bioprospecting property. In this study laboratory scale tubular photobioreactor was constructed for marine microalgae cultivation using cheapest materials such as wooden sheets, wooden reapers, aquarium light, glass tube, and UPVC, PVC couplings. The two marine microalgae Nanochloropsis occulata and Chaetoceros calcitrans were studied. Effect of growth medium, pH was determined in normal culture technique and mass cultivation was carried out in tubular photobioreactor. The growth curve of marine microalgae with various inoculation volume and days of incubation were also determined. F2 medium and pH 8 is suitable for the growth of the two marine microalgae. The biomass production is reached maximum level in 8 days of cultivation at 1.5 OD of inoculum volume. The biomass productivity is higher in tubular photobioreactor than the normal culture system. Keywords: Tubular photobioreactor, Biomass, pH, Nanochloropsis sp. and Chaetoceros sp. Marine microalgae are the fastest growing INTRODUCTION Microalgae are eukaryotic photosynthetic microorganisms, which are used to produce highly valuable compounds. organism, that are less than 2mm in diameter floating in the upper 200 M of the ocean where sunlight is available for J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC photosynthesis. Microalgae are sunlight Closed systems generally have higher driven cell factories that convert Co2 to production rates than open system. Major potential biofuels, foods, feeds and high limitations in open system include poor light value bioactive metabolites (Spolaore et utilization by the cells evaporative losses al.2006). It also produces carotenoids, which diffusion of log to the atmospheres and are requirement used as colouring agents in of large areas of land. neutraceuticals, pharmaceuticals, cosmetics, Furthermore contamination by predators and foods, scavenger and/or quencher of reactive other oxygen species (ROS) and an enhancer for restricted the commercial production of antibody production (Singh et al.2005). microalgae. The production growing heterotrophs have microalgal In comparison with open culture byproducts requires large quantities of algal system a closed photobioreactor is easy to biomass. Biomass is one of the better control sources of energy and hence large scale parameters and can achieve high growth biomass production getting significance. rates (Pulz, 2001; Sierra et al. 2008). A Large scale production of biomass energy photobioreactor could contribute to sustainable development conditions through its design the use of clear on several fronts, environmentally, socially glass tubing for efficient and volumetric and cultivation distribution of light; efficient delivery of techniques are used for mass production of light from the source to the algae. In microalgae. We can cultivate microalgae in addition bioreactor based photosynthetic open system and also in closed system. microalgae cultures were being considered economically. of fast Several with regard to maximizes environmental the growth J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC as a part of the closed ecological support one of the most suitable types for mass system (Li J et al.2003). Fully closed cultivation of algae because they have large photobioreactors provide opportunities for illumination surface (Chisti, 2006). Aeration monoseptic culture of a greater variety of and mixing of the cultures in tubular algae than in open system. Higher biomass photobioreactors are usually done by air- of microalgae productivity is obtained in pump closed photobioreactor cultivation system al.2001). and contamination is also easily prevented. photobioreactors Photobioreactor is a bioreactor which incorporates some type of light source. or air-lift systems Furthermore, are (Traviso long et tubular characterized by gradients of oxygen and Co 2 transfer along the tubes. Virtually any translucent container could be The aim of this work was to called a photobioreactor; however the term construct the vertical tubular bioreactor at is more commonly used to define as a closed low cost and to determine the suitable system. Algae can also be grown in a growth medium, optimum pH, optimum photobioreactor. In photobioreactor algal inoculum level for the mass cultivation of culture can be illuminated by artificial light, marine microalgae. solar light or by both. Some of the MATERIALS AND METHODS photobioreactors include bubble column (Degen et al. 2001), airlift column (Kaewpintong et al.2007), stirred tank Tubular Photobioreactor Construction and Fabrication (Petkov, 2000) and tubular (Hall et al.2003) The fabrication of photobioreactor photobioreactors.Tubular photobioreactor is was start by drilling a 45 mm hole in the J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC wooden sheet of 0.25″ thickness around the screws (2.50″). The center wooden reaper edges. There are about six holes were made. was used to fix the fluorescent light (40 1.25″ couplings (MTAPL) fixed in the each watts) along the sides by using 1″ plastic hole from the bottom side. Six numbers of brackets. couplings (FTAPL) screwed into the bottom couplings by turning the coupling clock wise. The six hollow glass tube (75 cm X 45 mm) which is bottom closed (Flat bottom) were inserted into the UPVC couplings in a specific position. To avoid shacking of glass tube aquarium sealant was spread on sides of the coupling if necessary. The top edge of all the six hollow glass tube were coupled by 1.25″ UPVC slip and thread adaptor. The four corners and 15 cm of six PVC tubes were fixed with the six PVC threaded plugs and inserted to the holes of the top wooden sheets. This assembly was considered as the top part of the photobioreactor. The air pumps, O2 and Co 2 supplied nutrient inlets were to be provided through the top assemblage for the aeration aquarium air pump were used for Co 2 supply. Collection and maintenance of marine microalgae also the centers of the bottom wooden sheets screwed with wooden reapers (30″) for the purpose of mechanical support. The same size of the wooden sheets which is in the bottom was also been fixed at the top. Both the bottom and top wooden sheets were linked by 30″ wooden reapers by using Two different groups of marine microalgae such as Nanochloropsis occulata and Chaetoceros calcitrans were obtained from Central Marine Fisheries Research Institute (CMFRI), Tuticorin, India. For the cultivation filter sterilized seawater was used along with the required nutrients. J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC About 10 – 20% of the inoculum in the ml conical flask (containing three different growing phase was transferred to the culture medium) as separate manner. The algal flask and those were placed under the white cultures were incubated at 25°C±1°C under fluorescent light (1000 lux) up to 4-5 days 1.2±0.2 klux light intensity with proper for attaining log phase. The time required aeration for 3 weeks. After incubation, the for the maximum cell growth varies growth of microalgae and the total biomass depending on the species, almost most of the were estimated. Among the three, good species require two weeks for completion of yielding medium was selected for the further the growth. The flagellates can be kept for study. two months in their stationary phase in the stock culture room under the controlled light Biomass estimation Biomass concentration (gl-1) was and temperature condition. calculated by measuring dry weight. For dry Cultivation of marine microalgae in different growth medium Marine weight measurement, the microalgal cells were harvested after 21 days of incubation as by centrifugation at 5000 rpm for 15 min. at Nanochloropsis occulata and Chaetoceros room temperature and the cells were washed calcitrans were grown in three different with distilled water. Then the cell was culture media such as F2 medium, MN III placed petri plate and oven dried at 40°C for medium and ASN III medium depending 4 to 6 hours. The dried biomass was cooled upon the requirement of micro and macro and weighed. The difference between the nutrients. initial and final weight were taken and the The microalgae three such different marine microalgal samples were inoculated in 250 J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC biomass weight was calculated. The dry in constructed tubular bioreactor. Inoculum weights were expressed in g/l. concentration is also very important in Effect of pH on marine microalgal biomass growth studies and hence four inoculum concentrations were used for the cultivation in F 2 medium of both marine microalgae Nanochloropsis The effect of pH on growth of the occulata and Chaetoceros calcitrans. The marine microalgae was studied using F2 four inoculums concentration used for the medium with the pH range of 6, 7, 8, 9, and study were OD620 - 0.05, 0.10, 0.15, 0.20 10. The experiments were carried out in respectively. Then the photobioreactor was conical flask containing 100 ml of F2 kept in 25°C±1°C under 1.2±0.2 klux light medium. The pH was adjusted with the help intensity with proper aeration. Growth curve of 8 M NaOH and 1M HCl solution before for two microalgae was also studied. autoclaving. All the flasks were incubated for 3 weeks at 25°C±1°C under 1.2±0.2 klux RESULTS AND DISCUSSION light intensity with proper aeration. After The mass cultivation of marine incubation, the growth of microalgae and the microalgae require a appropriate culture total biomass were estimated. system with consistent cultivation parameters which will be suitable for Mass cultivation and growth studies producing algae based products which has Marine microalgae such as pharmaceutical, biotechnological and Nanochloropsis occulata and Chaetoceros marketed commercially. Most of the large calcitrans were grown in the selected culture scale cultivation of microalgae has been medium (F2 medium, pH-8) for mass success with open system of cultivation. cultivation. The experiment was carried out J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC However major short coming at the open was calculated, which was higher in F2 culture system contamination with bacteria medium than ASN III and MN III medium. and other contaminants as well as changes in So F2 medium was preferred for further local climatic conditions, cost of the land experimental and water. Further perspective of mass microalgae Chaetoceros calcitrans have culture of microalgae will required closed 4.6g/l of dried biomass and Nanochloropsis system because of the algae and algal occulata contain lower amount (i.e.) 3.4g/l products must be grown free of potential of contaminants such as heavy metals and (Figure.1).The present study found that the microorganisms. Continuous culture and significant effect of pH on biomass yield of good control over the growth environment marine microalgae.When compared to all result in a consistent product quality and pH ranges pH 8 yield higher amount of highest operating cell density. In this present marine investigation Chaetoceros a small scale tubular dried analysis. biomass microalgal Among in F2 biomass calcitrans these medium (Figure.2). could produce photobioreactor with the capacity of 4L was higher amount of dried biomass (4.9g/l) constructed using low cost material and has compared with Nanochloropsis occulata been used for the mass cultivation of marine (4.0g/l) at pH 8. Dayananda et al. (2005) microalgae. reported the effect of media and culture After 21 days of incubation the conical flasks have the greenish bloom of algal biomass. The amount of total biomass conditions on growth and production of Botrycoccus braunii. biomass J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC Figure 1. Total biomass of two marine microalgae in different growth medium Figure 2. Effect of various pH range on growth of two marine microalgae in F2medium The array of long and low diameter they have been major advantages in polythene tubing designed in a clearly operation of closed tubular bioreactor for deficient scaling up of the original pilot Dunaliella scale device enable the accumulation of new Wiffels (2003). Cultivation of microalgae in algae and algal products. In recent years suitable inoculum concentration is necessary salina culture, Hejazi and J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC which can effectively shorten the lag phase inoculum volume and 8 days of cultivation of cell growth and allowed to go into period were maintained to determine the logarithmic phase very earlier. OD620-0.15 optimum requirements for getting higher inoculum produces maximum amount of biomass of both microalgae in closed tubular biomass at the 12th day of cultivation. photobioreactor. Yue-Hui Zhu (2008) was Chaetoceros produce reported that inoculum volume of 0.15 for 8.82g/l dried biomass (Figure.3) compared Dunaliella salina. Continuous cultivation with Nanochloropsis occulata (Figure.4). was used to study the growth curve of the The of microalgae. Both Chaetoceros calcitrans cultivation, will determine the sufficient and Nanochloropsis occulata reached steady biomass of any microalgal culture system. In state from 7th day of cultivation in this photobioreactor. calcitrans inoculum present volume study the could and four days different Dried Biomass (g/L) Figure 3. Growth of Chaetoceros Calcitrans at different volume of inoculum 10 9 8 7 6 5 4 3 2 1 0 0.5 1 1.5 2 1 3 5 7 9 11 Time of cultivation (Days ) 13 15 J. Algal Biomass Utln. 2011, 2 (2): 41– 52 TUBULAR PBR FOR MICROALGAE CULTIVATION © PHYCO SPECTRUM INC Figure 4. Growth of Nanochloropsis occulata at different volume of inoculum photoinhibition. Major advantage of tubular CONCLUSION Compare with other culturing system a tubular illuminating outdoor photobioreactor surface cultures area fairly as s suitable good large for biomass productivity relatively chief. The closed photobioreactor provided opportunity for monoseptic cultures of great variety of algae then is possible in open system. The closed photobioreactor will also over come the major impact of solar radiation- photobioreactor would be acclimated to high light and therefore the negative effect of photoinhibition would be minimal in such system. 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