Abstract: A saline water micro algae was isolated from Tiab estuary. The Al-25 strain was identified to be as genus Chlorella. The maximum number of viable cells was 50/8 x 104 CFU mL-1 with 852 mg L-1 of DCW and the maximum specific growth rate and biomass productivity were estimated to be 72 h and 852 mg L-1, respectively. Crude protein content of Chlorella Al-25 was about 55-58%.
INTRODUCTION
The green algae of the genus Chlorella (Beijerinck, 1890) belongs to the family of Chlorellaceae (Hoek et al., 1995). Species of this genus are widespread in fresh water and in the sea, air and soil. In asexual reproduction stage this micro algae are produce none motile autospore. It is becoming increasingly evident that the development of low-cost, high-quality protein feed is crucial for the future success of aquaculture industry (Rumsey, 1981). A Single Cell Protein (SCP), microalgae have been used as essential food for the larval stages of fish and shellfish (Benemann, 1992) and yeast and bacteria have been considered as algal substitute for several species of filter feeders (Epifanio, 1979a, b; Kim et al., 1998). Chlorella are also used for microbial protein production and as protein rich food for sewage oxidation (Kessler, 1982). The Chlorella vulgaris has ability to produce carotenoid of lutein which is very important in human serum and food as well (Li et al., 2001). Chlorella species are very simple unicellular algae and also the members of this genus are easy to cultivate and wildly used in various physiological studies. The identification of Chlorella species is difficult because morphological and physiological characteristics normally changed with the environmental conditions. However the Chlorella cells do not exhibit characteristics that differentiate them from the morphological properties which are typically the basis of the classical taxonomy treatment of other algae (Shihira and Krauss, 1965). Although the traditional taxonomic characteristics of Chlorella sp. indicate that morphological, biochemical and physiological properties are used in its identification, the cells size and shape are variable and largely depend on varying nutrition and environmental factors (Fott and Novakova, 1969). The objective of this study was to isolate, identify and purify Chlorella sp. from Tiab estuary in Iran.
MATERIALS AND METHODS
This study was carried out at the University of Alzahra and A.C.E.C.R., Water sampling was done from spring of 2004 to winter of 2005. Water samples as an isolating source of micro algae were collected from Hormozgan province, Tiab estuary in Persian golf of Iran. Micro algae were newly isolated from water samples by the methods described by Watanabe et al. (1992) and APHA/AWWA/WPCH (1989).
Chlorella identification: Micro algae were identified as described by Carmelo (1997) and Kessler and Huss, (1992).
Morphological observations: Algal samples of each Chlorella species were observed with a zeiss microscope equipped with Normarski interference optics and the number of endospores and the size of 100 cells of each isolate were measured with a micrometer eyepiece. The observations were made at different stages of the life cycle, either on cells in exponential phase or on cells in late stationary phase of growth.
Culture conditions: Table 1 shows the details of culture conditions. Two identified the micro algae were isolated from different regions at first the ability of these strains to grow on different media has been evaluated. Table 2-5 shows the details of components of these media.
| Table 1: | Details of cultural conditions |
| Table 2: | Specific culture media for isolation of Chlorella strain Al-25 |
| 9 cc from A solution and 1cc from B solution add to 1 L of culture medium | |
| Table 3: | B medium |
| Table 4: | C medium |
| Note: 1 cc of every stock solution of C medium add to 1 L of sea water | |
The micro algae culture experiments were conducted to determined the culture conditions of the isolates. The algal strains were grown in 250 mL Erlenmeyer flasks contains 25 mL of modified Sato medium (Richmond, 1983) with 3 g L-1 NaNO3 as a nitrogen source and a pH adjusted to 8.1 by adding NaHCO3. The initial pH of the medium was 7.8. Cultures were incubated for 5 days at 26 to 28°C under a 12 h light: 12 h dark period using cool white fluorescent light and also very mild aeration is provided. Table 1 shows the details of cultural conditions. For the salt tolerance tests the algae were grown at different concentrations of NaCl (2, 4, 6 and 8%).
| Table 5: | D medium (9), basis of mineral materials solution |
Assay: The algal growth and Cell density was assessed by measuring the absorbance at 660nm using a Spectrophotometer (LI-250 CECIL, Inc.,UK).
Protein extraction: Total protein were exterated using a Lowry method as described by Lowry et al. (1951) and also micro kjeldahl method (APHA, 1985). The concentration of total protein was determined by measuring the absorbance at 660 and 730 nm using a spectrophotometer (Hellebust and Craigie, 1973).
RESULTS AND DISCUSSION
Isolation of micro algae: A new saline water micro algae was isolated which could grow well in modified Sato media. It is a single cell green algae named to Chlorella strain Al-25 and the cell size is about 5 microns referred to Fig. 1. The Al-25 strain was identified as genus Chlorella according to the Beijerinck 1890. Chlorella Al-25 was cultured under 12 light:12 h dark period using cool white fluorescent light (3000-3300 LUX) at 26°C to 28°C. Growth characteristics including: Total chlorophyll and maximum cell concentration measured by using Haemocytometer lam method. Present results indicated that Chlorella Al-25 were grown in different salt concentration medium in this study. The maximum dry weight biomass was obtained with 852 mg/L/dw of biomass after 72 h cultivation. Growth characteristics were evaluated by the following two characteristics the linear growth rate and the maximum cell concentration. Figure 2 shows the growth of the Chlorella Al-25 strain. Growth curve was produce for Chlorella strain Al-25, growing in modified Sato broth using Optical Density (OD), measurements at 660 nm.
| Fig. 1: | Chlorella sp. |
| Fig. 2: | Chlorella growth curve |
The results from the growth curves of the Chlorella strain Al-25 tested in sato broth indicate that maximum of algae cells concentration was after 72 h cultivation. The growth rate was remarkably low after 48 h cultivation with maximum cells concentration with 188 mg/L/dw of biomass and reached to the stationary phase after 72 h. Chlorella strain Al-25 was cultured in sato medium for single cell protein production. Chlorella strain Al-25 cultured in sato broth under shaker-grown condition.
The total protein was extracted as described by Lowry et al. (1951) and micro kjeldahl. The results of total protein extraction shows that Chlorella strain Al-25 produce 3/15 mg mL-1 of medium. The crude protein content of Chlorella strain Al-25 was about 55-57 %. This protein content was higher than yeast cells (50/5%) (Kobayashi and Kurata, 1978), Cellulomonas sp. (44%) and was lower than most photosynthetic (Thanikachalam and Rangarjan, 1986). These results show that Chlorella Al-25 is a promising strain to cultivate for protein production.