CULTURE OF Chlorella ellipsoidea IN DIFFERENT CULTURE MEDIA

An experiment of algal culture was conducted in natural light and temperature conditions at a balcony of a room at the 2nd floor of Fisheries Faculty Building facing the north. The experiment was done to evaluate the growth of Chlorella ellipsoidea in four different media, viz, medium I (inorganic), medium II (organic, whole pulse powder extract), medium III (organic, whole lentil powder extract) and medium IV (organic, whole gram powder extract) under natural environment conditions during January-June, 2015. Growth rates of the algal species in four different media were found not significantly different. The alga, C. ellipsoidea attained maximum cell density of 28.89×106 cell ml-1 in the 15th day in medium I, of 30.69×106 cell ml-1 in the 13th day in medium II, of 26.18×106 cell ml-1 in the 15th day in medium III and of 21.12×106 cell ml-1 in the 13th day in medium IV. The ranges of air temperature, water temperature and light intensity were 21°C to 38°C, 23°C to 36°C and 2.28×103to 9.60×103 Lux respectively during the culture period. The average sunshine period was 5.87±2.82 hrs. Total alkalinity, free CO2, pH , NO3-N and PO4-P of algal culture media I, II, III and IV were 128, 540, 554 and 322 mgL-1; 32, 162, 102, 70 mgL-1; 7.4, 8, 7.9 and 7.9; 180, 36.6, 62.4 and 150 mgL-1, and 25.2, 48.2, 42.4 and 45.6 mgL-1, respectively. According to ANOVA of cell densities of cultures of C. ellipsoidea under treatments are not significantly different (F=1.441077). It is clear that differences between them are not significant i.e. mean algal cell densities are more or less same as differences between treatments are less than 20%.


Introduction
Following global fears of an uncontrollable human population boom during the late 1940s and the early 1950s, Chlorella was seen as a new and promising primary food source and as a possible solution to the then-current world hunger crisis.Many people during that time thought hunger would be an overwhelming problem and saw Chlorella as a way to end the crisis by providing large amounts of high-quality food for a relatively low cost (Belasco, 1997).
Microalgae play an important role in aquaculture as live food for larval stage of many species of crustacean and fish as well as for all stages of bivalves and as food for the zooplankton.However, microalgae are eventually fed to late larval and juvenile fish and crustaceans in hatcheries (Renaud et al., 1991).Addition of various microalgae to the water during early first feeding of marine fish larvae frequently has resulted in improved growth and survival (Howell, 1979).It is observed that nursery cultivation of bivalve molluses required a consistent supply of suitable algae culture to maintain growth (Claus, 1981).Actually, successful fish culture practices primarily depend on the maintenance of healthy aquatic environment and the production of sufficient fish food organisms.
Microalgae are an essential component of the diet of marine bivalve mollusk (e.g.Oysters, Clams, Scallop and Mussels), the larvae of some marine gastropods (abalone), larva of salt water shrimp, fish species (e.g.Tilapia, Silver carp, Milk fish) and zooplankton.To some extent, microalgae are also used for rearing the larvae of freshwater prawn and larvae of some marine fish like sea bass (Fujimura and Okamoto, 1972).
Microalgae are used as a human food source for over-populated countries (Barlew, 1953) and for space travel (Haldane, 1951).There are several suggested advantages of microalgae.If algae are grown under suitable environment conditions, the protein yield from it may be quite better (Spoehr and Milner, 1949).In China and Japan, Int.J. Agril.Res.Innov.& Tech.7 (1): 51-57, June, 2017 seaweeds and certain other algae Porphyra, Ulva, Alaria and Chlorella are most commonly used.These not only consist of an important ingredient of soups but also are used for flavoring meat.Different vitamins such as B, C, Folic acid and Niacin are also found in them (Kumar and Singh, 1976).In Japan, Korea, Myanmar, China, America, Mexico and many countries of Europe the marine algae are very popular food, especially it is used as salad.Algae are also used as poultry food in different farms and it can be suggested the addition of algae in animal feeding (Becker, 1994).
It has been demonstrated that algae can also be used as fertilizer.The blue-green algae, which are rich in nitrogen and phosphorous, are excellent fertilizer.A suitably, blended mixture of seaweed and cyanophycean manures (i.e.bloom of Microcystis sp.) may serve as an ideal fertilizer and this can relieve the acute shortage of fertilizers in developing countries in tropical region; bottom-mud of dried up ponds is regularly used as manure in crop cultivation, the manurical value is mostly due to the high content of blue-green algae (Kumar and Singh, 1976).
Chlorella is a genus of single-cell green algae of the phylum of Chlorophyta.Its shape is spherical, about 2 to 10 micron in diameter, and has no flagella.The green photosynthetic pigments chlorophyll-a, chlorophyll-b are found in chloroplast of Chlorella.Requiring only carbon dioxide, water, sunlight, and a small amount of minerals to reproduce, it multiplies rapidly through photosynthesis.So, considering very high importance of culture of microalgae, C. ellipsoidea, a very important microalgae, has been selected for culture in different media to evaluate the best culture media.

Preparation of organic media Collection of pulse powder, lentil powder and gram powder
For preparation of organic medium, pulse powder (Vigna mungo), lentil powder (Lens culinaris) and gram powder (Cicer arientium) were collected from Choto Bazaar, Mymensingh City.
Preparation of inexpensive culture medium using whole pulse powder (Vigna mungo) according to the method of Rahman (2000).
This medium was prepared by mixing 1 kg pulse powder (Vigna mungo) in 30-litre tap water.After a week, 15 g urea was added into mixture.After three weeks, pulse powder mixture was filtered through thin marking cloth to discard solid materials and then after several days the clear supernatant was siphoned to another bucket.After adding lime for making medium clear, pH of the medium increased to about 10.Then to lower the pH to 7, conc.H2SO4was added to the medium at the rate of 0.325 ml per litre and after one week, the medium was ready for use as algal culture medium.
Preparation of inexpensive organic culture medium using whole lentil powder (Lens culinaris) and gram powders (Cicer arientium) are more or less similar to the pulse powder preparation.

Study of the environment factors
Water temperature (°C) Maximum and minimum water temperature data were taken by a maximum-minimum thermometer daily.

Air temperature (°C)
Maximum and minimum air temperature data were taken by a maximum-minimum thermometer daily.

Light
Light intensity (Lux) at the place of algal culture was measured by a digital Lux-Meter (LX-1010B) daily during the culture period.

Sunshine period and rainfall
Data of sunshine period and rainfall were collected from the "Weather Yard" under the Department of Irrigation and Water Management of Bangladesh Agricultural University, Mymensingh.

Determination of chemical status of culture media
pH: pH of the culture media were measured by an electronic digital pH meter (Hanna Instruments Co.) before starting culture experiment.
Total Alkalinity: Total alkalinity of culture media was determined by methyl orange indicator method (APHA, 1971).

Nitrate-nitrogen (NO3-N):
For determining nitrate-nitrogen, samples were filtered through high glass microfiber filter paper with the help of vacuum pressure bottle.Then nitrate-nitrogen of the culture media was determined by a Nitrate Meter (Hanna Instruments Co.).

Phosphate-phosphorus (PO4-P):
For Phosphate-phosphorus determination samples were filtered in the same way of nitrate-nitrogen and Phosphate-phosphorus of the culture media were determined by phosphate Meter (Hanna Instruments Co.).

Estimation of cell density (cells ml -1 ) of Chlorella ellipsoidea culture by a haemacytometer
The calculation of the cell density of algal culture (cells ml -1 ) was done by the following formula (modified from Rahman, 1992) Where, N = No. of plankton cells per ml of culture medium.A = Average no.plankton cells in 0.1 cubic mm × 10 = Average no.plankton cells in 1 cubic mm

Statistical analysis
A computer through the pregame SPSS having three replications did ANOVA of cell densities of C. ellipsoidea, cultured in four different media (four treatments) each.

Results
Table 2. Daily variation of mean cell density of culture of Chlorella ellipsoidea cultured in four different media for a period of 21 days (Treatments 4, Replications 3).Fig. 1.Composition of daily variation of mean cell density (× 10 6 cell ml -1 ) of C. ellipsoidea cultured in medium I (inorganic), medium II (organic, whole pulse powder extract), medium III (organic, whole lentil powder extract) and medium IV (organic, whole gram powder extract).The result of analysis of ANOVA of mean cell densities of C. elliposoidea of three replications of treatment-I (inorganic medium), treatment-II (organic medium, whole pulse powder extract), Treatment-III (organic medium, whole lentil powder extract) and treatment-IV (organic medium, whole gram powder extract) have been presented in Table.

Cell density in four algal culture media
Cell density of C. ellipsoidea in medium I (inorganic medium) ranged from 2.05 to 28.89 (×10 6 ) cell ml -1 , during the culture period.The average cell density was 16.91±6.98(×10 6 ) cells ml -1 .During the culture period of C. ellipsoidea in medium I, exponential phase was attained up to 15 th day from the starting of culture and after that from stationary phase cell density began to decline toward death phase.
During the culture period, the range of cell density of C. ellipsoidea in medium II (organic, whole pulse powder extract) ranged from 2.05 to 30.69 (×10 6 ) cells ml -1 .The average cell density was 15.35±7.63(×10 6 ) cell ml -1 .During the culture period of C. ellipsoidea in medium II, exponential phase was attained up to 13 th day from the starting of culture and after that from stationary phase cell density began to decline toward death phase.
During the culture period, the range of cell density of C. ellipsoidea in medium III (organic, whole lentil powder extract) ranged from 2.05 to 26.18 (×10 6 ) cell ml -1 .The average cell density was 13.21±6.24(×10 6 ) cell ml -1 .During the culture period of C. ellipsoidea in medium III, exponential phase was attained up to 15 th day from the starting of culture and after that from stationary phase cell density began to decline toward death phase.
Cell density of C. ellipsoidea in medium IV (organic, whole gram powder extract) ranged from 2.05 to 21.12 (×10 6 ) cells ml -1 , during the culture period.The average cell density was 13.68±4.48(×10 6 ) cells ml -1 .During the culture period of C. ellipsoidea in medium IV, exponential phase was attained up to 13 th day from the starting of culture and after that from stationary phase cell density began to decline toward death phase.Jahan (2011), in an experiment of culture of Chlorella sp. in three culture media (pulse bran extract medium, soil extract medium and inorganic medium), observed maximum ell densities in pulse bran extract medium (56.32×10 6 cells ml -1 ), in soil extract medium (102.99×10 6cell ml -1 ) and in inorganic medium (64.23×10 6 cells ml -1 ).These results of cell densities are much higher in comparison to those of the present experiment.Ara (2010), in an experiment of culture of Chlorella sp. in three culture media (treated eutrophic-pond-water medium, inorganic medium and organic medium), found maximum cell densities in eutrophic-pond-water medium (161.69 10 6 cells ml -1 ),in inorganic medium (170.03 10 6 cells ml -1 ), and in organic medium (165.03 10 6 cells ml -1 ).These results of cell densities are much higher in comparison to those of the present experiment.Sultana (2009), in an experiment of culture of Chlorella sp. in three culture media (organic, inorganic and eutrophic pond water media), recorded maximum cell densities in organic medium (35 10 6 cells ml -1 ), in inorganic medium (45.05 10 6 cells ml -1 ) and in eutrophic pond water medium (24.05 10 6 cells ml -1 ).These results are more or less similar to those of the present experiment.Mishu (2008), in an experiment of culture of green alga, Scenedesmus sp. in two culture media, medium I (organic) and medium II (inorganic), observed that the maximum cell densities of 17.03 10 cells ml -1 in 9 days in medium I, maximum densities of 17.41 10 6 cells ml -1 in medium II in 8 days, which are much lower than those of present experiment.Wongsnansilp et al. (2007) did an experiment on the culture of an alga, Chlorosarcinopsis sp.(PSU/CHL20).He found the highest cell density of 14.8 10 6 cells ml -1 in 30 °C on 14 days of culture, which are much lower than those of than the present study.Hossain (1996), in an experiment of algal culture of C. ellipsoidea in five different media, viz, medium I (inorganic medium), medium II (medium of whole pulse powder), medium III (medium of pulsed bran), medium IV (mixed medium = 50% inorganic + 50% whole pulse powder medium) and medium V (mixed medium = 50% inorganic + 50% pulse bran medium), found that the ranges of cell densities were 0.08 to 0.62 (×10 6 ) cells ml -1 in medium I, 0.02 to 4.02 (×10 6 ) cells ml -1 in medium II, 0.18 to 4.38 (×10 6 ) cell ml -1 in medium IV, 0.07 to 4.38 (×10 6 ) cells ml -1 in medium V.These results of cell densities of C. ellipsoidea were much lower than those of the present study.

Statistical analysis
According to ANOVA (Table 5) of cell densities of cultures of C. ellipsoidea under treatments I, II, III and IV, it can be concluded that cell densities under 4 treatments are not significantly different (F=1.4441077), that is the four algal culture media are more or less of similar quality.But the mean amount of cell densities fr0m higher to lower are 30.69±1.73 (×10 6 ) cells ml -1 (medium II, organic, whole-pulse-powder extract), 28.89±1.12(×10 6 ) cells ml -1 (medium I, inorganic), 26.18±0.75(×10 6 ) cells ml -1 (medium IV, organic, whole-gram-powder extract).So, highest suitable medium was medium II (organic, whole-pulse-powder extract) although the differences among media are not significant and medium I and medium II are almost of same quality.
Experimental layoutCulture of C. ellipsoidea was done in four types of medium.Seeds of C. ellipsoidea were collected from previous cultures, which were done by the researcher Professor Md.Shahidur Rahman of the Department of Fisheries Management, BAU, Mymensingh.
-1 N.B.Media were sterilized.The culture was done in natural light and temperature conditions on a steel framed glass shelf in a balcony of a room at 2 nd floor of the Fisheries Faculty Building, BAU, Mymensingh.

Table 4 .
Chemical status of the culture media.

Table 5 .
Analysis of variance (ANOVA) of cell densities of cultures of C. ellipsoidea under treatment I, II, III and IV.