Abstract: Salinity and temperature are two of the major factors controlling the growth rate of microalgae. In this study, the effect of salinity and temperature on the growth of marine microalgae; an unidentified Chlorella sp. and Chaetoceros calcitrans were investigated to optimize the microalgal biomass production. These species were cultured at different salinities (20, 25 and 30‰) and temperatures (20, 25 and 30°C). C. calcitrans and Chlorella sp. had significantly higher (p<0.05) growth rate when cultured at salinities of 30 and 25‰, respectively. In terms of temperature, the highest (p<0.05) growth rate was observed in C. calcitrans and Chlorella sp. cultivated at temperatures of 30 and 25°C, respectively. This study indicated that C. calcitrans was suitable to marine condition, whereas Chlorella sp. showed optimum growth at lower salinity and temperature.
INTRODUCTION
Marine microalgae are an excellent source of protein, lipid, carbohydrate and vitamins, i.e., A, B, C, E, folic acid and pantothenic acid (Becker, 2007). Their high nutritional values can provide a high quality nutritional package for different stages of aquaculture animals (Banerjee et al., 2010; Khatoon et al., 2009, 2012). Microalgae that have been found to have good nutritional properties includes Chaetoceros calcitrans (a diatom) and a green alga; Chlorella sp. (Natrah et al., 2007; Goh Jr. et al., 2009; Goh et al., 2010). Optimizing the algae growth rates by manipulating the environmental parameters could become an effective approach in increasing microalgal biomass production (Mata et al., 2010). In fact, marine microalgae have been projected as potential species for various industrial applications due to their fast growth rate and valuable chemical contents (Singh and Gu, 2010).
Salinity and temperature have been shown to induce the characteristic of the nutritional properties in microalgae (Hemaiswarya et al., 2011). Moreover, different culture environment such as seasonal fluctuation, effect of low and high temperature and the species origin can also cause variable growth rates of microalgae (Oliveira et al., 1999; Thompson et al., 1992; Banerjee et al., 2011). Previous study on the arctic sea diatom Chaetoceros spp. showed that the species tolerate low salinity (Zhang et al., 1999).
Variations in salinity also influence several biochemical and physiological mechanisms such as lipid production and growth which are essential in marine organisms (Fava and Martini, 1988). Although temperature is the easiest factor that can be controlled in the practical operation of microalgae cultivation, it is a sensitive factor for algae growth and metabolic processes. Different temperatures have been associated with various quality and composition of microalgae.
There are limited studies on the effect of various salinities and temperatures on phytoplankton especially involving marine Chlorella sp. and C. calcitrans. Thus, a study on the effect of different salinities and temperatures on the growth of two marine microalgae C. calcitrans and Chlorella sp. was carried out in order to contribute to better understanding on this matter.
MATERIALS AND METHODS
Algae cultures: Chaetoceros calcitrans (UPMC-A0010) and Chlorella sp. (UPMC-A0013) were obtained from the Laboratory of Marine Biotechnology, University Putra Malaysia (UPM). Stock cultures were maintained regularly on both liquid and agar plates of Conway medium (Walne, 1966).
Experimental design: Five-day old culture of Chlorella sp. and C. calcitrans (104 cells mL-1) were used as inocula. Ten percent (30 mL v/v) of inocula were inoculated into 500 mL Erlenmeyer flask containing 300 mL of fresh Conway medium with salinities of 20, 25 and 30‰. All treatments were carried out in triplicates. Salinity lower than 30‰ was adjusted by diluting the seawater using Milli-Q water. The cultures were sparged with filter sterilized air at 0.1 min (v/v) and continuously illuminated by fluorescent lamp at a light intensity 120 μmol-2 sec-1, with temperature 25±1°C. The experiment was carried out for 14 days.
The analyses of different growth temperatures were carried out by cultivation of 104 cells mL-1 inoculum in 500 mL Erlenmeyer flask containing 300 mL of fresh Conway medium with salinity 30±2‰. The replicates were cultured in incubators with temperatures of 20, 25 and 30°C under continuous illumination at 600-800 lux light density and sparged with filter-sterilized air by using air pump without additional carbon dioxide.
Analytical methods: Biomass estimation and determination of cell number. One milliliter aliquots of cultures were filtered on fiberglass Whatman GF/F filters using a vacuum pump and washed with a solution of ammonium formate (0.5 M) to remove silicate residue. The filters were dried at 100°C for 4 h to volatilize the ammonium formate. The dry weights of algae biomass were determined gravimetrically and the growths were expressed in terms of dry weight (g DW L-1). Meanwhile, the cell number was determined and counted using haemocytometer.
Growth analysis: Growth was expressed in terms of growth rates using the following equation:
where, F1 is the biomass at time of harvest (t1) and F0 is biomass at time of zero (t0) (Guillard, 1973).
Statistical analysis: Statistical package for social sciences (SPSS 15.0) were applied for one way ANOVA tests in the evaluation of differences in the mean values. Significance was tested at 95% level.
RESULTS
Effect of salinity on growth: The best growth of C. calcitrans occurred at salinity of 30‰, with the highest growth rate of 0.28±0.03 μ day-1 (p<0.05) and maximum cell count of 2.7x106 cells mL-1 on the 12th day of the culture period (Fig. 1 and 2a). Meanwhile, Chlorella sp. reached the maximum cell count of 3.1 x 106 cells mL-1 on 12th day in culture grown at salinity of 25‰ with a growth rate of 0.37±0.01 μ day-1 (Fig. 1 and 2b, p<0.05). The maximum biomass (p<0.05) obtained from C. calcitrans and Chlorella sp. were 1.52±0.2 and 1.34±0.4 g DW L-1, respectively (Fig. 3).
Effect of temperature on growth: In C. calcitrans, the highest growth rate was observed in cultures at 30°C (0.27±0.02 μ day-1) with maximum cell count (p<0.05) of 2.1x106 cells mL-1 on day 10 (Fig. 4, 5a). However, there were no significant changes (p>0.05) in growth rates in cultures grown at 20 and 25°C. Meanwhile, Chlorella sp. showed the best growth at 25°C with the highest growth rate of 0.35±0.04 μ day-1 and cell count of 2.9x106 cells mL-1 on day 10th (Fig. 4 and 5b, p<0.05). Chaetoceros calcitrans and Chlorella sp. reached the maximum biomass (p<0.05) of 1.44±0.5 and 1.42±0.3 g DW L-1, respectively (Fig. 6).
| Fig. 1: | Growth rates of Chaetoceros calcitrans and Chlorella sp. in response to different salinities, Vertical bars are means±SE (n = 3). Means with different letters are significantly different at p<0.05 |
| Fig. 2(a-b): | Changes of mean densities (n =3) of (a) Chaetoceros calcitrans and (b) Chlorella sp. under different salinities during the culture period |
| Fig. 3: | Biomass for Chaetoceros calcitrans and Chlorella sp. under different salinities, Vertical bars are means±SE (n = 3). Means with different letters are significantly different at p<0.05 |
| Fig. 4: | Growth rates of Chaetoceros calcitrans and Chlorella sp. in response to different growth temperatures, Vertical bars are means±SE (n = 3). Means with different letters are significantly different at p<0.05 |
| Fig. 5(a-b): | Changes in mean cell densities (n = 3) of (a) Chaetoceros calcitrans and (b) Chlorella sp. under different temperatures during the culture period |
| Fig. 6: | Biomass of Chaetoceros calcitrans and Chlorella sp. at different temperatures, Vertical bars are means±SE (n = 3). Means with different letters are significantly different at p<0.05 |
DISCUSSION
Microalgae growth is highly dependent on the environmental conditions where the variables that affect the growth rates are different from one species to another. However, the most studied variables are salinity, pH, temperature, light as well as the nutrition (Banerjee et al., 2011; Liang et al., 2009; Raghavan et al., 2008; Ji and Sherrell, 2008). In the present study, C. calcitrans and Chlorella sp. showed that the growth rate increased with increasing salinity. In the present study, C. calcitrans and Chlorella sp. reached the highest growth rates of 0.28±0.03 and 0.37±0.01 μ day-1, respectively at the optimum salinity. In fact, the biomass production of C. calcitrans and Chlorella sp. achieved in this study was higher than the values reported in previous studies (<1.0 g DW L-1) (Araujo and Garcia, 2005).
Salinity is the main factor for the process of giving life to plants and can cause retardation of central metabolic activities such as photosynthesis (Liska et al., 2004). Microalgae differ in their adaptability to salinity and based on their tolerance as they are grouped as halophilic and halotolerant (Rao et al., 2007). In natural condition or marine waters, active multiplication normally started at day 5 or 7 of the growth phase. The culture can last for 2 or 3 weeks depending on the microalgae species. In this study, C. calcitrans culture increased progressively from the beginning of the culture period culminating in maximum densities and biomass on day 12 in all treatments. There were significant differences (p<0.05) in the growth rate amongst different salinities indicating that salinity was an important factor to be considered when culturing C. calcitrans on a large scale. Similarly, Chlorella sp. showed similar growth pattern with the highest production on the 12th day. However, the effect of salinity on Chlorella sp. growth was significantly different compared to C. calcitrans as the growth response was slowest at high salinity (Fig. 1). Different algae species response varies to different salinities as have been reported in previous studies (Huang et al., 2011; Hu and Gao, 2006; Takagi et al., 2006; Zhang et al., 1999). Sudhir and Murthy (2004) illustrated that although high salt content influence physiological process in microorganism, each species differs in the growth response to salinity.
Saros and Fritz (2000) showed that diatom physiology can be affected directly or indirectly via interaction with other growth factors such as the ion composition in the saline system. In a few species of diatoms, low salinity can result in decrease of cell dimension (Lynn et al., 2000). However, other diatom species such as Thalassionema eccentrica and Pseudo-nitzschia seriata are able to survive salinity up to 150‰ (Nagasathya and Thajuddin, 2008). Although both marine microalgae are capable of growing and photosynthesizing in salinities ranging from 20 to 30‰, significant decrease (p<0.05) in growth patterns as salinity decreases was observed in C. calcitrans. This trend might be associated with the limitation of nutritional factors in sea water after dilution (Raghavan et al., 2008).
In the present study, both microalgae species were able to tolerate growth temperature range from 20 to 30°C. Chaetoceros calcitrans performed best at 30°C (0.27±0.01 μ day-1) and this finding was within the range (27-30°C) reported by Renaud et al. (2002). At its optimum temperature, the cell density of C. calcitrans observed in this study was significantly higher than the values (1.6x105 cells mL-1) reported by Phatarpekar et al. (2000). Meanwhile, lower growth temperatures reduced the cell density of this diatom. Sheehan (1998) illustrated that increasing the growth temperature induced the cell proliferation, probably due to the changes of cell metabolic activities in response to the environmental stress.
The green microalgae Chlorella sp. growth appeared to be at optimum when cultured at 25°C (0.35 μ day-1). At this temperature, the growth rate was 40% higher than value reported in C. vulgaris (Converti et al., 2009). Thus, this study illustrated that Chlorella sp. proliferated at a maximum rate at low temperature, an observation which was also reported in Chlorella sorokiniana (Franco et al., 2012). Although Chlorella sp. cultured at high temperature (30°C) resulted in significant decrease (p<0.05) in growth rate, the values obtained in this study were two times higher than those reported for C. vulgaris cultured at a similar temperature (Converti et al., 2009). This finding illustrated that tolerance to temperature changes is species specific (Oliveira et al., 1999). The ability of Chlorella sp. and C. calcitrans in adapting to different temperatures indicated that they are versatile candidates for outdoor mass culture.
CONCLUSION
This study illustrated that the salinity of 30‰ and temperature of 25-30°C induced optimum cell proliferation in C. calcitrans. Meanwhile, Chlorella sp. required a lower salinity of 25‰ and temperature of 25°C for optimum growth. It can be concluded that both marine microalgae had different ranges of tolerance and adaptability to environmental changes. In this study, Chlorella sp. and C. calcitrans were demonstrated as fast growing microalgae and they can be considered as suitable species for large outdoor microalgal cultivation.
ACKNOWLEDGMENT
This research was funded by the Ministry of Science, Technology and Innovation Malaysia (MOSTI) through Johor State Biotechnology Satellite Project No., (BSP(J)/BTK/001(4)).