ISSN Original Article Comparative study on growth of Skeletonema costatum: amicroalga as live feed for aquaculture importance

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1 Available online at International Journal of Research in Fisheries and Aquaculture Universal Research Publications. All rights reserved ISSN Original Article Comparative study on growth of Skeletonema costatum: amicroalga as live feed for aquaculture importance Mayavan Veeramuthu Rajeswari*and Thangavel Balasubramanian Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai , Tamilnadu, India. *Corresponding Author Received 30 June 2014; accepted 10 July 2014 Abstract A microalga, Skeletonema costatum isolated from Vellar estuary, southeast coast of India were cultured under 28 ± 1 o C and salinity 28 PSU with 50 µmol m -2 s -1 of light using different culture media such as TMRL medium (Tang Kang Marine Research Lab. Medium), TMRL medium with urea, Miquel s medium and Conway of Walne s medium. The results evidenced that maximum cell numbers in exponential phase were noticed in Conway medium (600 cells/ml) followed by Miquel s medium (500 cells/ml), TMRL medium (470 cells/ml) and TMRL medium with urea (450 cells/ml) and chlorophyll a content was about 3.38, 1.25, 1.73 and 1.64 µg/ml respectively. The results of the present study concluded that the Conway medium was found to be the most suitable culture medium for the enhancement algal biomass and also recommended for the mass culture of S. costatum as live feed for larval rearing in hatcheries Universal Research Publications. All rights reserved Key words: Microalga, Culture medium, Skeletonema costatum, Vellar estuary, Live feed. 1. Introduction Planktonic micro algae, as primary producers, are the biological starting points of the aquatic food-chain and also help in the energy flow (1). It is therefore logical that the management of micro-algal populations and their productivity is an integral part of any aquaculture operation. Since aquaculture is one of the most rapidly growing industries in the field of food production, culture of micro algae in the hatcheries has become an essential prerequisite for the rearing of economically important cultivable organisms. The success of any hatchery system (for crustaceans, oysters, sea cucumbers or fishes) entirely depends on the availability of the suitable micro algae for raising them in culture so as to be fed to the larvae of culturable shell or finfish species (2). The microalgae used as feed in hatcheries vary in size, environmental requirements, growth rate, and nutritional value (3). When selecting a species for culture, it is important to take all of these parameters into consideration. Most hatcheries grow a variety of species that serve different needs throughout the production cycle with respect to size, production cycle with respect to size, digestibility, culture characteristics, and nutritional value(4). Microalgal culture is the most expensive and technically challenging aspect of all hatchery operations. The cost of producing microalgal feed ranges from $100 to $400 per dry kilogram ($45 to $180 per pound) of microalgal biomass(5). Microalgal cultivation technology encompassing its biomass and products have received much attention in the last 5-6 decades owing to their potential as possible commodities and numerous other industrial applications (6-9). Numerous studies have shown that nutrients affect the growth of marine phytoplankton, and nitrogen and phosphorus concentrations are especially relevant (10-14). For culturing of micro-algae, either diatoms or other nanoplankters in the laboratory, various culture media are being used depending on the type of organism to be cultured and its growth characteristics. Miquel (1982) introduced a very effective culture medium for diatoms and nannoplankters (15). Although various other media have been used for the microalgal culture, Walneand TMRL mediumis being used efficiently for maintaining stock culture in the laboratory and also for mass culture. However, the techniques of culturing microalgae involve a clear understanding of their nutritional requirements. Although various studies have been carried out on effect of temperature (16) and ph (17) on growth of marine diatom Skeletonema costatum, an experimental study on effect of various culture media on growth of microalga S. costatum conducted in order to find out a suitable culture medium for 117

2 mass culture of this species for aquaculture importance. 2. Materials and Methods 2.1. Collection and isolation of S. costatum Microalgal samples were collected from marine zone of the Vellar estuary (11 29 N; E), southeast coast of India using a phytoplankton net (No.32 with the mesh size of 48 µm) operated from a boat. The collected plankton samples were immediately brought to the laboratory and subjected to curious examination under binocular microscope and the micro alga S. costatum was isolated using Agar plating method. Briefly, 1.5 g of agar was added to 1 liter of natural sea-water (28 PSU) and sterilized by autoclaving for 15 minutes, poured in sterilized petri-dishes and incubated at 25 o C for 24 h. The microalga, S. costatum was picked up and streaked on surface of the agar plates and was incubated in an incubation chamber for 8 days with 25 µmol m -2 s -1 oflight at 25 C. After incubation, grown colonies were removed by inoculation loop and transferred tothe culture tubes. Further, cultures from the culture tubes were transferred to 250 ml Erlenmeyer flasks in order to maintain stock culture. Axenic culture of S. costatum was raised by treating with gentamycin (400 µg/ml) and choloromphenical (200 µg/ml) to avoid bacterial growth was used as an inoculum for the growth experiments in four different medium such as (i) TMRL medium (Tung Kang Marine Research Lab) (ii) TMRL medium with urea, (iii) Miquel s medium and (iv) Conway or Walne s medium. The filtered and aged seawater was used for preparation of the medium. All the glassware used for the culture experiments were cleaned and aseptically sterilized. Fig.1(a) Cell growth and Chlorophyll a content of S. costatum grown on TMRL medium. Fig.1(c) Cell growth and Chlorophyll a content of S. costatum grown on Miquel s medium. Fig.1(b) Cell growth and Chlorophyll a content of S. costatum grown on TMRL medium with urea. Fig.1(d) Cell growth and Chlorophyll a content of S. costatum grown on Conway medium Composition of the culture medium used (All the chemicals were kept separately in 10 ml reagent bottles and 1 ml of each was added to 1 liter of sterilized seawater) 118

3 TMRL medium Potassium nitrate : 10 g /100 ml of distilled water Sodium orthophosphate : 1 g /100 ml of distilled water Ferric chloride : 0.3 g /100 ml of distilled water Sodium silicate : 0.1 g/100 ml of distilled water TMRL medium with urea Potassium nitrate : 10 g /100 ml of distilled water Sodium orthophosphate : 1 g /100 ml of distilled water Ferric chloride : 0.3 g /100 ml of distilled water Sodium silicate : 0.1 g/100 ml of distilled water Urea : 0.1 g/100 ml of distilled water Miquel s medium A. Potassium nitrate : 20.2 g /100 ml distilled water B. Sodium orthophosphate : 4 g Calcium chloride : 2 g Ferric chloride : 2 g in 2 mlof HClin 100 ml of distilled water. (0.55 ml of A and 0.5 ml of B was added to one liter of sterilized seawater) Conway or Walne s medium A. Potassium nitrate : 100 g Sodium orthophosphate : 20 g EDTA (NA) : 45 g Boric acid : 33.4g and Ferric chloride : 0.36 g in 1 liter of distilled water. B. Zinc chloride : 4.3 g Cobalt chloride : 4.0 g Copper sulphate : 4.0 gand Ammonium molybdate : 1.8 g in 1 liter of distilled water. C. Vitamin B (Thiamine) : 200 mg in 100 ml of distilled water Vitamin B (Cyanocobalamine) mg in 100 ml of distilled water. (A, B and C were prepared and kept separately in different reagent bottles. Then, 1 ml of A, 0.5 ml of B and 0.1 ml of C were added to 1 liter of sterilized seawater.) 2.3. Culture of Skeletonema costatum finfish and shellfish larvae feed on the minute plant A total of twenty test tubes for each medium components of their choice which are really available in the (15mL) were prepared and sterilized. Further, 1 ml of S. aquatic system. But in the hatchery system, it is essential to costatum inoculum was added to each tube with the initial provide food which is acceptable to the larvae of the culture concentration of 1 x 10 4 cells/ml. The tubes were kept for 10 animal for their growth and further development. In the early days in artificial light (50 µmol m -2 s -1 ) under 12 h light and critical stages of rearing the larvae in hatcheries, some dark cycles at 28±1 o C and all the experiments were phytoflagellates (species of Isochrysis, Pavlova, Dicrateria, conducted in duplicate Estimation of cell growth and chlorophyll a Chromulina and Tetraselmis) and some nannoplankters (species of Chlorella and Synechocystis) were used as basic Cell growth was estimated from the aliquots food. For feeding the post larvae of crustaceans and spat withdrawn at every 2 days interval. Briefly, 0.1 ml of the juveniles of bivalves, planktonic diatoms (species of culture sample from each tube was transferred to a Neubuer Chaetoceros, Skeletonema and Thalassiosira) were raised in haemocytometer (0.1 mm deep) and cell growth in terms of numbers of cells wereenumerated. Five counts were made from each tube to ensure accuracy and mean values were calculated. For estimation of chlorophyll a, 10 mlfrom each the algal laboratories as primary food (1).Of which, Skeletonema costatum finds an important place since itcan be acceptable to many culturable shell and finfish larvae and also various studies made earlier were also concluded that the S. costatum Grev. Cleve as an ideal candidate for live feed culture tube was harvested by centrifugation at 3000 xg for 5 algal species throughout in world. In addition, it is minutes and 1 ml of 90% acetone was added to the pellet comparatively easier to maintain this ubiquitous alga in and kept in refrigerator for overnight in dark. After the culture under varying physico - chemical conditions. complete extraction of pigments, the samples were Although many culture media are being used for the culture centrifuged for 10 min and the supernatant was made up to 10 ml using 90% acetone. Further, the optical density of the in order to find out most suitable medium for extracts was measured at 630, 645 and 660 nm (Hitachi 220) and the concentration of chlorophyll a was then calculated using the extinction co-efficient values (18). 3. Results and discussion In the natural environment, commercially important 119 of microalgae, the present experimental study was conducted S. costatum that would quicken the exponential phase with higher chlorophyll a content. Growth (in terms of cell numbers) and chlorophyll a content of S. costatum recorded in different culture media seems that the growth rate as well as chlorophyll a content

4 (1.73 µg/ml) was comparatively higher in TMRL medium. The growth was increased from initial to the fourth day and started decreasing after sixth day onwards (Fig.1).Similarly, increasing trend was observed in growth rate in TMRL urea medium up to the fourth day (450cells/mL) and decreased from sixth totenth day (Fig.1b). The chlorophyll a content also registered a similar trend with increasing from 0.64 (initial day) to 1.64 µg/ml (fourth day) and decreased from 0.91 to a minimum of µg/ml on tenth day (Fig. 1b).Support to the above fact, Hu et al. (2011) stated that the S. costatum was grew faster initially and the cell density was decreased quickly with time later in growth phase at different nitrate (NaNO 3 ) and phosphate (NaH 2 PO 4 ) levels(19). S. costatum had a greater capacity to take up and reduce NO 3 relative to growth N demands than the flagellated species (20). In addition, Eppleyet al. also suggested that the degree of light/dark differences in nitrogen assimilation differs among species and could be relevant for competition among species for growth-limiting nutrients and hence for species succession (21). The S. costatum diatom assimilated nitrate and ammonium primarily during the day and grew well when irradiance was fairly high (22). The increase in cell numbers and chlorophyll a content was observed in Miquel s medium up to fourth day of culture (500 cells/ml) and decreased gradually with further increase in culture days and was being minimum (280 cells/ml) at tenth day (Fig.1c).The growth rate on Conway medium was also in same trend as noticed earlier and maximum growth (600 cells/ml) was recorded at fourth day and decreased gradually afterwards. Similarly, chlorophyll a content was also increased from initial to fourth day and was being maximum on fourth day (3.38 µg/ml) and decreased to 0.18 µg/ml after sixth day onwards (Fig.1d). The results of the present study indicated that the microalga S. costatum exhibited significant increase in cell growth up to the fourth day and started decrease from the sixth to tenth day. Although similar trend has been reported in all the four different culture medium used, the growth performance in terms of cell number and chlorophyll a content was comparatively higher in Conway medium which contains vitamin B 1 and B 12 and several micro and macro nutrients to enhance the growth of S. costatum. But, Kang et al. concluded that the F/2 (Guillard s) medium was the suitable culture medium for culture of microalgae(23). The cell count was found to be maximum in exponential phase in Conway medium (600 cells/ml), followed by Miquel s medium (500 cells/ml), TMRL Medium (470 cells/ml) and TMRL medium with urea (450 cells/ml) which clearly evidenced that the Convey medium was the most suitable medium for getting more algal biomass in low volume of area which could beuseful in hatcheries for raising mass cultures to use as live feed. 4. Acknowledgement Authors are thankful to the authorities of Annamalai University for providing facilities and also the Ministry of Earth Science (MoES), Govt. of India, New Delhi for the financial support. 5. References 1. C.P. Gopinathan, Micro algae culture as live feed. CMFRI - Winter School Course Manual on Recent advances in breeding and larviculture of marine finfish and shellfish, Central Marine Fishery Research Institute Bulletin, (2009) C.P. Gopinathan, Live-feed culture - Micro Algae, Bullettin. Central Marine Fish Research Institute, 48 (1996) M.M. Helm, N.Bourne and A.Lovatelli, Hatchery culture of bivalves: A Practical Manual, FAO Fisheries Technical Paper Number 471. Rome, Italy. (2004) 200 pp. 4. A.Muller-Feuga, J.Moal and R.Kaas, The Microalgae of Aquaculture. in: J.G. Stottrup and L.A. McEvoy (eds.), Live Feeds in Marine Aquaculture, Blackwell Publishing, Oxford, UK.(2003) pp. 5. G.H. Wikfors, Microalgal culture. R.R. Stickney (ed.), Encyclopedia of Aquaculture. John Wiley & Sons, Inc., New York.(2000) pp. 6. P.Spolaore, C. Joannis-Cassan, E.Duran and A.Isambert, Commercial applications of microalgae, Journal of Bioscience and Bioengenering. 101 (2006) K.H. Cardozo, T.Guaratini, M.P. Barros, V.R. Falcao, A.P. Tonon, N.P.Lopes, S. Campos, M.A.Torres, A.O.Souza, P.Colepicolo and E.Pinto, Metabolites from algae with economical impact, Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 46 (2007) N.T. Eriksen, The technology of microalgal culturing, Biotechnology Letters. 30 (2008) A.Satoh, K.Ichii, M.Matsumoto, C.Kubota, M.Nemoto, M.Tanaka, T.Yoshino, T.Matsunaga and T.Tanaka, A process design and productivity evaluation for oil production by indoor mass cultivation of a marine diatom, Fistulifera sp. JPCCDA0580, Bioresource Technology. 137 (2013) R.Riegman, M. de Boer, L. de SenerpontDomis, Growth of harmful marine algae in multispecies cultures, Journal of Plankton Research. 18(10) (1996) I.J.Hodgkiss, K.C. Ho, Are changes in N: P ratios in coastal waters the key to increased red tide blooms?,hydrobiologia. 352 (1997) R. Riegman, Species composition of harmful algal blooms in relation to macronutrient dynamics. In: Physiological Ecology of Harmful Algal Blooms (Anderson DM, Cembella AD, Hallegraeff GM, eds.), Springer Verlag, Berlin, Germany. (1998) pp. 13. M. Frangopulos, C.Guisande, E. deblas and I.Maneiro, Toxin production and competitive abilities under phosphorus limitation of Alexandrium species, Harmful Algae. 3(2) (2004) Y.Shi, H.Hu and W.Cong, Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandriumtamarense (Gonyaulacales, Dinophyceae), Phycology Research. 53(1) (2005)

5 15. Miquel. De la culture artificiella des diatomes, Comptes Rendus de l'académie des Sciences, Paris.94(1892) D.C.O. Thornton and B.Thake, Effect of temperature on the aggregation of Skeletonema costatum (Bacillariophyceae) and the implication for carbon flux in coastal waters, Marine Ecology Programme Series.174 (1998) E.Granum and S.M.Myklestad, A photobioreactor with ph control: demonstration by growth of the marine diatom Skeletonema costatum,journal of Plankton Research. 24(2002) J.D.H.Strickland and T.R. Parsons, A practical handbook of seawater analysis, Bulletin of Fisheries Research Board of Canada.167 (1972) 310pp. 19. H.Hu, J.Zhang and W.Chen, Competition of bloomforming marine phytoplankton at low nutrient concentrations, Journal of Environmental Science. 23(4) (2011): M.W. Lomas and P.M. Glibert, Comparisons of nitrate uptake, storage, and reduction in marine diatoms and flagellates, Journal of Phycology. 36(5) (2000): R.W. Eppley, and J.L. Coatsworth, Uptake of nitrate and nitrite by Ditylumbrightwellii- kinetics and mechanisms, Journal of Phycology. 4(2) (1968) R.W. Eppley, J.N. Rogers and J.J. McCarthy, Light/dark periodicity in nitrogen assimilation of the marine phytoplanktersskeletonema costatum and Coccolithushuxleyi in N-limited chemostat culture, Journal of Phycology. 7(2) (1971) K.H.Kang, Z.J. Qian, B.Ryu and S. K.Kim, Characterization of growth and protein contents from microalgae Naviculaincerta with the investigation of antioxidant activity of enzymatic hydrolysates,food Science Biotechnology.20(1) (2011) Source of support: Nil; Conflict of interest: None declared 121