The culture of the of green alga ChloreJJa ellipsoidea was conducted under .... nonh at 2nct floor of the Fisheries Faculty Building, Bangladesh Agricultural ...
Bangladesh J Fish. Res., 9(2), 2005: 185-190
©BFRI
Culture of green algae Chlorella ellipsoidea in inexpensive media M. Shahidur Rahman*, M. Anwar Hossain, S. Fatema and M. Amzad Hossain1 Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 1 Dept. of Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh * Corresponding author
Abstract The culture of the of green alga ChloreJJa ellipsoidea was conducted under namral conditions at the same place simultaneously in five different media, viz., medium-! (inorganic medium), medium-H (powdered whole-pulse medium), medium-HI (medium of pulse bran), medium-IV (mixed medium = 50% inorganic medium + 50% wholepulse powder medium), medium-V (mixed medium = 50% inorganic medium + 50% pulse bran medium). The culture was done in 500 ml conical flask. Growth rates of C. ellipsoidea in five different media were different and reached maximum ceH densities of 0.63 x 106 cells ml·1 in 8 days in medium-1, 4.02 x 106 ceU ml· 1 in 10 days in medi.um-H, 3.62 X 106 ceHs ml·1 in 9 days in medium-HI, 4.38 x l0 6 ceHs ml·1 in ll days in mediumIV and 4.36 x 106 ceHs ml" 1 in ll days in medium V. The range of air temperature was 20 to 33°C and that of culmre media was 24 to 32°C and light intensity was 2000 to 7000 lux during the culture period. The inexpensive culmre media were found to be significantly useful for algal culture. Key words :Algae culture, Chiorella ellipsoidea, Pulse bran, Light intensity
Introduction Algae are very important in various ways. People of some countries such as China and Japan, have long been using seaweeds and certain other algae as a source of food. Some of the more commonly used algae are Porphyra~ Ulvay Alariay Chlorellay Chondrus, etc. The brown algae Ascophyllumy Laminaria and Fucus are used as stock feed for sheep and catde in maritime districts of Ireland and Scotland. Planktonic algae form the major food of protozoans, crustaceans and fishes. Marine flageHal(es and other microalgae are cultivated for rearing the larvae of marine fishes, especiaHy sheHfish (Kumar and Singh 1976). Algae are also used as fertilizer. The blue-green algae, which are rich in nitrogen and phosphorus, are exceHem fertilizer (Kumar and Singh 1976). The algae Chlorella grows very fast under a variety of conditions and i.ts photosynthetic pigments and reserve products are similar to those of higher plants. Biochemical analysis of its cells has revealed that they may be very rich in proteins (about 50%), fats (about 20%), carbohydrates (about 20%), amino acids, vitamins and minerals. Some algae produce antibacterial substances effective against a number of
M.S. Rahman er al.
pathogenic bacteria. ChloreHin obtained from Chlorella was the first such substance. In view of this many laboratory and pilot plant studies, on the feasibility of growing Chlorella in mass cultures for use as human food or animal feed, have been conducted i.n Japan, Germany, the USA and Israel (Kumar and Singh 1976). From a modest beginning with Chiorella tablets in Japan in late fifties, new endeavors have emerged as specialized industries the world over, aiming to produce health food, food additives, animal feed, biofertilizers and an assortment of natural products (Vonshak and Rishmend 1988). Culture of Chlorella sp. in media of powdered whole pulse and pulse bran is very inexpensive and simple. Main aim of the experiment was to develop inexpensive culture medium for large scale production of algae. In brief, the objectives of the present study were to prepare inexpensive culture media using pulse bran and whole pulse powder and to compare the results of algal cultures in inexpensive organic media with those of inorganic medium. Materials and methods The seeds of Chlorella ellipsoidea was collected from pond water. Phytoplankton seeds were cultured in test tubes in sterilized medium i.n which inoculation was given using medicinal injection syringe from centrifuged pond water after examining under a microscope. Then through microscopic examination of the repeated cultures in the test tubes, seeds of C ellipsoidea were collected for the present experiment. Preparation of inorganic medium was done according to Stanier et al. (1971) with slight modification.
Preparation ofinexpensive organic media Two inexpensive organic media were prepared using whole pulse powder and pulse bran (powder and bran of Vigna mungo). Both whole-pulse powdered medium and pulse-bran medium were prepared in 36 Htre plastic buckets mixing 250g whole-pulse powder and 250g pulse-bran in 25 Htres tape water individuaHy. After a week one teaspoonful urea was added into each of buckets. After 25 days partiaHy-decomposed whole-pulse powder and pulse-bran mixture with water were filtered through with thin markin doth and solid materials were discarded and then after a week the dear supernatant was siphoned to another bucket and one teaspoonful of Hme (CaO) was added to the extract solution to make it clear and then it was used as culture medium.
Environmental conditions The air temperature, temperature of culture medium (by a maximum-minimum thermometer) and Hght intensity (by a luxmeter) were recorded daily during the culmre period and data of sunshine period and rainfall were collected from Weather Yard of Bangladesh Agricultural University, Mymensingh.
186
Culture of the green alga
Chemical sraws ofthe culwre media Chemical status such as pH, total alkalinity, free C0 2, phosphate-phosphorus and nitrate-nitrogen of the culture media were determined foHowing standard methods in the modern laboratory ofBAU-ODA link programme.
Experimental design Culture was carried om simultaneously under natural conditions at same place in five different media viz. medium-I (inorganic medium), medium-II (medium of wholepulse powder), medium-HI (medium of pulse-bran), medium-IV (mixed medium = 50% inorganic medium+ 50% whole pulse powder medium), medium-V (mixed medium = 50% inorganic medium + 50% pulse-bran medium) in 500 ml conical flask with 4 replications for each of the culture media. Seeds of C ellipsoidea were used from the previous laboratory cultures in conical flask. Two hundred and fifty (250) ml medium was taken in a conical flask and 7 ml algal seed was inoculated. The mouth of the conical flask was covered with mosquito net to avoid insect contamination. The conical flasks were kept in natural environmental conditions in a bakony of a room facing the nonh at 2nct floor of the Fisheries Faculty Building, Bangladesh Agricultural University where sufficient sun-light was available throughout the day. The cultures were performed for a period of 15 days in August'96. Cell densities of the algal cultures were determined under a compound microscope using Sedgewick rafter counting ceH (S-R cell). Results Results of cultures of C ellipsoidea in five different media have been presented in Tables l and 2. Culture of C ellipsoidea in medium-!, started with 0.08 x 106 cell/ml, anained maximum cell density of 0.63x10 6 ceHs/ml in 8 days and average ceH density was 0.41 ± 1.12 x 106 ceUs/ml in which pH, total alkalinity, free C0 2, nitrate-nitrogen and phosphate phosphorus were 7.93, 134 mg/L, 22 mg/L, 34.8 mg/L and 0.958 mg/L, respectively (Tables 1 & 2). Culture of C ellipsoidea in medium-H, reached maximum ceU density of 4.06 x 106 ceHs/ml in 10 days from an initial density of 0.02 x 106 ceUs/ml and average ceH density was 2.28±1.25 x 106 ceHs/ml in which pH, total alkalinity, free C0 2, nitrate-nitrogen and phosphate-phosphorus were 8.24, 582 mg/L, 24 mg/L, 7.2 mg/L and 0.779 mg/L, respectively (Tables 1 & 2). In medium-HI, the initial cell density was 0.09 x 106 ceUs/ml which attained maximum cell density of 3.62 x 106 ceUs/ml in 9 days and average cell density was 2.24± 1.12 x 106 ceUs/ml in which pH, total alkalinity, free C0 2, nitrate-nitrogen, phosphate-phosphorus were 8.30, 404 mg/L, 22 mg/L, 6.4 mg/L, and 0.842 mg/L, respectively (Tables 1 & 2). In medium-IV, the culture of C. ellipsoidea started with 0.18 x 106 ceHs/ml, attained a maximum cell density of 4.38 x 106 ceHs/ml in 11 days and average cell density was 3.98 ± 1.48 x 106 ceHs/ml in which pH, total alkalinity, free C0 2, nitrate-nitrogen and phosphate-phosphorus were 8.14·, 404 mg/L, 23 mg/L, 17.5 mg/L and 0.842 mg/L, respectively (Tables 1 & 2). In medium-V, i:he culture started with 0.07 x 106 ceUs/ml, attained a maximum cell density of 4.36 x 106 187
5.4
nil
Rainfall (mm)
nil
nil
nil
10.2
9.8
10.1
27.1 24.2-30
26.6 21.631.5
26.5 25-28
28.8 26-31.5
4 3.5-4.5
26.5 21.4-31.5
3 2.5-3.5
3 2.5-3.5
2.5 2-3
2.07
27.3 23-31.5
1.90
0.37
0.07
1.89
25.5 24-27
1.06
0.41
0.18 3.50
2.92
nil
8.5 nil
nil
nil
9.3
9.6
10.6
nil
8.6
27.5 22-33
9.5 nil
10.4 nil
27.5 22.4-32.6
27.5 22.2-31.8
25.5 24.5-26.5
3.5 2.5-4.5
3.52
4.18
2.38
2.34
27.7 22.3-33
26.3 25.5-27
26 25-27
26 25-27
27.2 22-32.4
26.3 25-27.5
2.75 2-3.5
3.5 2.5-4.5
5.5 4.5-6.5
4.32
4.36
4.36
2.84
2.70
4.08
27.1 21.6-32.5
25.5 24-27
25.5 24-27 27.4 22.8-32
3.5 2.5-4.5
3.5 2.5-4.5
4.31
3.98
3.72
6 4.5-7.5
2.48
3.28
4.38
3.17
3.61
3.62
3.28
2.91
2.10
3.90
3.78
4.02
2.66
2.18
1.87
25.8 24.5-27
1.07
0.48
0.09
0.84
0.45
0.02
Sunshine period (hrs)
Culture medium/ Environmental factors Medium-! (inorganic) Medium-11 (whole pulse extract) Medium-III (pulse bran extract) Medium-IV (mixed, I+ II) Medium-V (mixed, I+ III) Light intensity (xl0 3 lux) Temperature of medium (0C) Air temperature (0C)
nil
9.0
26.9 21.3-32.4
25.5 24.5-26.5
3.25 2.5-4
3.18
3.54
2.02
2.12
nil
9.7
26.9 20.8-33
25.5 24.5-26.5
2.7 2-3.4
2.80
3.44
1.52
1.70
nil
9.3 ±1.3
Mean +S.D. 0.412 ±0.18 2.18 ±1.25 2.24 ±1.12 3.92 ±1.48 2.66 ±1.38 3.59 ±1.05 26.0 ±0.9 27.2 ±0.4
Table 1. Cell densities (x 106 cells/ml) of C. ellipsoidea cultured in 5 different media for a period of 15 days and average environmental conditions with their range during experimental period
Cui ture of the green alga
cells/ml in 11 days and average cell density was 2.66± 1.38 x 106 ceUs/ml in which pH, total alkalinity, free C0 2, nitrate-nitrogen and phosphate-phosphorus were 8.19, 320 mg/L, 22 mg/L, 16.3 mg/L and 0.911 mg/L, respectively (Tables 1 & 2). Table 2. Chemical status of the culture media Culture medium
Medium-I (inorganic) Medium-II (whole pulse extract) Medium-III (pulse bran extract) Medium-IV (mixed, l+Il) Medium-V (mixed, I+III)
pH
Free C0 2 (Mg/L)
7.93 8.24 8.30 8.14 8.19
22 24 22 23 22
Total alkalinity (mg/L) B4 582 404 346 320
N0 3-N (mg/L)
P0 4-P (mg/L)
34.8 7.2 6.4 17.5 16.3
0.958 0.779 0.842 0.864 0.911
The cultures in five different media were done simultaneously under natural conditions at the same place. The range of air temperature was 20 to 30°C, culture medium temperature was 24 to 3L5°C, range oflight intensity was 2000 to 7000 lux and sunshine period was in the range of 5.4 to 10.6 hrs. and there was on rainfall during the culture period (Table 1). Discussion The culture of C ellipsoidea were conducted simultaneously under natural conditions at the same place in 5 different media but growing abilities of the alga in different media were different (F = 8.90**). Growing abilities of the alga in medium-IV, V, II and III were significantly better than in medium-! (t-values are 5.39**, 4.88**, 3.914** and 3.838**), which proves that inexpensive organic culture media are effective and useful for algal culture as the expensive inorganic culture medium. Fatema (1996) carried out a similar culture experiment of the green alga, Scenedesmus sp. in five different media, such as inorganic medium, inexpensive organic media of pulse-bran, and whole-pulse powder and mixed media. The results and observations of her experiment are in conformity with those of the present experiment. Luong-Van Thinh (1994) reponed, in a culture of Isochrysisaff. galbana, maximum cal density of 5 to 6 x 106 cells mi- 1 at a temperature of 24°C in an inorganic medium and a1$o observed that population density increased under high light conditions. ManinezJeronimo and Espinosa-Chavez (1994), in an experiment of mass culture of micro algae, Ankistrodesmus falcatus and Scenedesmus incrassatulus at temperature (25 to 27°C) in organic medium, found average cell density of A. falcatus 1.4 x 106 cells/ml and S. incrassatulus 3 x 106 ceHs/ml which are more or less similar to the average cell densities of some cultures of the present experiment. Some of the results of the present experiment are similar to the findings of Vass and Bhanou (1973) who observed cell densities in a culture of Chiorella sp. 2 to 3 x 106 cells/ml at a temperature of 25 to 30°C in a mixed medium (nutrient medium + commercial fenilizer) and pH of this medium 189
M.S. Rahman er al.
was 7 to 8 and Hght intensity was 1000 lux. James et al. (1988) observed, in an experiment of growth of Chlorella sp., the range of ceU density of about 20 x 106 cells/ml 6 to about 80 x 10 ceHs/ml in which pH were 6.0, 6.5, 7.0, 8.0 and 8.5, these results of cell densities are higher than those of the present study. Khan (1996) carried out an experiment on the culture of Fibrocapsa japonica i.n laboratory and found at ma..xi.mum cell density of5.51±0.31 x 104 cells/ml in which temperature was 25°C and pH was 7.8 to 8.5. Maximum cell density was observed on the 14rh day of the culture period. Acknowledgement The present experiment was a trial under a research project no. 98/06/RC financed by ARMP, BARC, IDA Credit No. 2815-BD. References Fatema, S., 1996. Culture of Phytoplankton in Inexpensive Medium. M.S. Thesis, Department of Fisheries Biology and Limnology, Faculty of Fisheries, Bangladesh Agri.cuh:ural University, Mymensiingh. James, C.M., A.M. Al-Khars and P. Chorbani, 1988. pH dependent growth of chloreUa in a continuous culture system. f World Aquat. Soc.~ 19(2): 27-35. Khan, S., 1996. Toxins from Rophidophycean Flagellates. Ph.D. Thesis, United Graduate School of Agricultural Science, Kagoshima University, Japan. 151 p. Kumar, H.D. and H.N. Singh, 1976. A text book on Algae. 2nct ed. Affiliated East-West Press, New Delhi.. 216 p. Luong-Van Thinh, 1994. Potential use of ageing cultures of Isochrysis off galbana (lsochrysis Tahitan, T. Iso.) as starter cultures for live algal food production in tropical aquaculture. f Appl. Phyco17 6: 357-358. Maniinez-Jeronimo, F. and F. Espinosa-Chavez, 1994. A laboratory-scale system for mass culture of freshwater microalgae in polythene bags. f Appl. Phycol.7 6(4) : 423-426. Stanier, R.Y., R. Kunisawa, M. Mandel and G. Cohen-Bazire, 1971. Purification and properties of unicellular blue-green algae (Order: Chroococcales). Bact. Rev.~ 35: 171-205. Vass, K.K. and K.K. Bhanou, 1973. A simple and inexpensive method for the mass culture of Chlorella sp. f Inland Fish. Soc. India, V : Vonshak, A. and A. Richmond, 1988. Mass production of the blue-green alga Spimlina, an overview. Biomass, 15 : 233-247.
(Manuscript received 29 November 2004)
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