Saltworks
Introduction
Microalgal oil is a viable alternative for replacing conventional crude and vegetable oil (Chisti, 2007, Schenk et al., 2008, Griffiths & Harrison, 2009, Rodolfi et al., 2009). To evaluate a microalgal species for its prospective use in aquaculture, whether for biodiesel production, pharmaceuticals or as animal feed in aquaculture facilities, two important characteristics need to be considered. Firstly, growth rate, in terms of cell numbers or biomass and secondly, biochemical composition (Borowitzka, 1992, de Castro Araujo &
Garcia, 2005). The first characteristic is discussed in this chapter; the second in chapter 4. For biodiesel production high biomass and lipid productivity are key desirable characteristics needed in a species (Chisti, 2007, Griffiths &
Harrison, 2009). Another desirable characteristic is the ability to tolerate and grow in extreme environments (Borowitzka, 1992). This is of fundamental importance to the success of any mass microalgal culturing operation. Rapid growth leads to high biomass production. The cost of harvesting microalgal biomass is directly related to harvesting effort. The higher the algal biomass per volume, the less effort is needed to harvest. Another advantage that fast growing species have over slow growing ones is the reduction in contamination risk by their ability to out-compete slower growing species. The chemical composition, e.g. lipids, and the growth rate of microalgae are influenced by environmental conditions, such as light (Morton et al., 1992), temperature (Renaud et al., 1995, Renaud et al., 2002), nutrients (Lynn et al., 2000, Liu et al., 2008, Converti et al., 2009) and salinity (Elenkov et al., 1996, Takagi et al., 2006, Rao et al., 2007). All these factors enhance or reduce the growth rate and lipid production, therefore, a trade-off is needed for optimal production.
41 Halophilic microalgae require high salt concentrations for optimum growth whereas halotolerant microalgae have a response mechanism that permits their existence in a saline medium (Rao et al., 2007). Both groups produce metabolites that help protect them from salt injury and maintain an osmotic balance between the cell and its surrounding environment (Rao et al., 2007).
The genus Dunaliella, is well known for its adaptability to hypersaline environments (Jahnke & White, 2003, Garcia et al., 2007, Rao et al., 2007, Kaçka & Dönmez, 2008) and also serves as a useful model for comparison.
It is advantageous to use indigenous isolates for biodiesel production as they are more tolerant of local conditions (Chu et al., 1996). The effect salinity has on the growth rate of ten local microalgae species, Amphora coffeaeformis var. coffeaeformis (NMMU 0008), Chlamydomonas sp. (NMMU 0037), Dunaliella tertiolecta (NMMU 0020), Navicula gregaria (NMMU 0001), Navicula salinicola (NMMU 0006), Navicula sp. (NMMU 0024), Nitzschia closterium (NMMU 0002), Nitzschia hungarica (NMMU 0004), Tetraselmis sp.
(NMMU 0018), and Thalassiosira sp. (NMMU 0003) isolated from the Swartkops Saltworks were therefore investigated.
Materials and Methods
Study site
The Swartkops Saltworks are located on the south east coast of South Africa just outside of Port Elizabeth. Nutrient rich water is pumped into the evaporation pans from the adjacent Swartkops estuary, the primary source of saline water for the solar saltworks (Du Toit, 2001). The high levels of nutrients promote both micro- and macroalgae production. The organic matter and debris that results from this high productivity, combines with the salt when it crystallize and produce a salt that is black and dirty looking. This pose a serious problem to the quality of salt produced (Du Toit, 2001).
42 Water temperature at the pump house (Figure 3.1) or inlet to the Swartkops saltworks ranges from 12°C to 25°C and for the crystalliser ponds ranges from 15°C to 30°C (Du Toit, 2001). The pH fluctuates between of 7.0 to 8.5 and is relatively stable (Du Toit, 2001). The salinity changes rapidly from one pond to the next, especially between ponds 5, 6 and 7 (Du Toit, 2001). The mean pond depth ranges between 25 cm and 120 cm for ponds 3 and 4, respectively (Du Toit, 2001). Du Toit (2001) reported high ammonium and nitrate concentrations as high as 12.8 µM and 33.0 µM, respectively for the Swartkops saltworks. Phosphate levels were lower at 4.4 µM (Du Toit, 2001).
Figure 3.1. The layout of the Swartkops Saltworks.
Microalgae isolation and preparation
The selected species from the Swartkops Saltworks were isolated and described by Sonnekus (2007). After initial growth in multi-well plates the cultures were transferred to and kept in sterile 50 ml Erlenmeyer flasks sealed with cottonwool. They were kept at 18 ± 0.5 °C, in a light cabinet using
cool-43 white lamps at about 80 µmol photons m-2s-1 and a 12:12 h light–dark cycle (Sonnekus, 2007). Stock cultures were established in natural seawater (GF/C filtered), at a salinity of 35 psu, with the nutrients added at the f/2 concentration (Guillard & Ryther, 1962). Silica was excluded from the growth medium for Dunaliella tertiolecta, Chlamydomonas sp. and Tetraselmis sp.
Growth Rate Experiments
In order to evaluate growth at various salinities (17, 35, 52 and 70 psu), the microalgal species were cultured in f/2 medium made up with natural seawater (35 psu) adjusted to a salinity of 17 psu, by diluting natural seawater with distilled water. Salinities of 52 and 70 psu were made by adding appropriate amounts of salt (NaCl) sourced from the Swartkops Saltworks to the natural seawater. All tested species were first acclimated to the new salinity a week prior to the growth experiments. These served as a stock solution form which the experiments were inoculated. Cultures were grown in quintuplicate 250 ml Erlenmeyer flasks containing 150 ml of GF/C filtered medium. The medium and the glassware were sterilised in an autoclave (20 min at 121°C) to prevent growth of unwanted species. After autoclaving and allowing the flasks to stand overnight, they were inoculated with the target species and incubated at 22 ± 0.5°C temperature under 81 ± 2 µmol s–1 m–2 light intensity and 14:10 h light-dark cycle for 10 days. Every day, the cultures where shaken by hand and a 1 ml aliquot was removed and preserved with a drop of formaldehyde. Benthic diatom samples were sonicated for 30 seconds to separate the clumped cells from each other.
The algal cell density was measured daily by counting using an improved Neubauer haemocytometer and a compound light microscope. From changes in the cell density, specific growth rate (µ; d-1) was calculated using equation 1 (Garcia et al., 2007) below
44 µ = lnC1 - lnC0
t1 - t0 (1)
where C0 and C1 (cells ml-1) are cell density values at the beginning (t0) and the end (t1) of a selected time interval between inoculation and maximum cell density, respectively. The cell productivity (cells ml-1 d-1) was calculated using equation 2 below
Productivity = C1 – C0
t1-t0 (2)
where C0 and C1 are cell densities at the beginning (t0) and the end (t1) of a selected time interval between inoculation and maximum cell density, respectively.
Statistical analysis
Data were tested for normality (Shapiro-Wilk W test) and differences in specific growth rate, cell productivity and cell density were tested with the Mann-Whitney U test, using STATISTICA version 8.0 (2007). All p-statistics are reported as P - 2sided. Principal component analysis (PCA) was used (CANOCO, Ter Braak 1987) to determine the relationship between growth and salinity. PCA linear scaling was done on inter-species correlations and species scores were divided by standard deviation with no transformation of species data. No centering and standardization were used either for samples or species. No species-weights and sample-weights were specified.
45 Results
Growth rates and cell productivity for seven members of the Bacillariophyceae and three Chlorophyta species grown at different salinities are shown in Table 3.1 and Table 3.2 respectively. It was observed that the growth rates for all productivity were found for N. hungarica at salinities of 17 psu and 35 psu (U
= 4; z = 1.67; p = 0.09; n=5,5).
Highest yield for Nitzschia hungarica (4.60 ± 0.82 ×104 cells ml-1) was obtained at a salinity of 17 psu and the lowest (3.30 ± 0.84 ×104 cells ml-1) at a salinity of 35 psu. No significant difference (U = 6; z = 1.25; p =0.22; n = 5,5) in yield was found between salinities of 17 and 35 psu at the end of the cultivation period (Figure 3.2). Nitzschia hungarica cultures died at salinities of 52 psu and greater.
The maximum specific growth rate for Amphora coffeaeformis was highest at a salinity of 35 psu and significantly lower (p <0.05) at salinities of 52 psu and greater (Table 3.1). The slight increase in specific growth rate from 0.89 ± 0.09 d-1 to 0.93 ± 0.05 d-1, at salinities of 17 and 35 psu respectively, was not significant ( U = 7; z = -1.04; p = 0.30; n = 5,5). Maximum productivity was found at a salinity of 17 psu and decreased with increased salinity (70 psu).
Significant differences in final cell productivity were found for all tested salinities (p < 0.05). The decrease in cell density for A. coffeaeformis in relation to salinity is seen in Figure 3.3. Maximum cell density (7.18 ± 1.16 × 105 cells mL-1) occurred 6 days after inoculation at 17 psu salinity and was followed by a steady decline in cell numbers (Figure 3.3). The highest concentration of cells obtained at 70 psu salinity (0.74 ± 0.10 × 105 cells mL-1) was almost ten times lower than that found at a salinity of 17 psu (7.18 ± 1.16
46
× 105 cells mL-1). Significant differences in cell density were found at the end of the cultivation period for all tested salinities (p < 0.05).
0 2 4 6 8 10
Time (days) 0
1 2 3 4 5 6
Cell density (× 104 ml-1 )
17 ‰ 35 ‰
Figure 3.2. Cell density over time at 17 and 35 psu for Nitzschia hungarica. Bars = +1 S.D.
47
0 2 4 6 8 10
Time (days) 0
2 4 6 8 10
Cell density (× 105 ml-1 )
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.3. Cell density over time at 17, 35, 52 and 70 psu for Amphora coffeaeformis var.
coffeaeformis. Bars = +1 S.D.
Navicula gregaria attained the highest growth rate (0.92 ± 0.09 d-1) at a salinity of 35 psu with no significant difference in growth rate (U = 3; z = -1.88;
p = 0.05, n= 5,5) between 17 psu and 35 psu. However, these growth rates were significantly higher (p < 0.05) than at 52 % (0.63 ± 0.05 d-1) and 70 psu (0.47 ± 0.03 d-1). Highest productivity (Table 3.1) for N. gregaria was found at 17 psu salinity and decreased with increased salinity. Significant differences (p < 0.01) in cell productivity for N. gregaria were found between all tested salinities, except between 17 psu and 35 psu. The cell density was affected by salinity (Figure 3.4). N. gregaria attained the highest yield at 17 psu (9.47 ± 1.11 × 105 cells mL-1) and the lowest concentrations were at 70 psu (4.62 ± 0.41 × 105 cells mL-1). A longer lag phase at a salinity of 70 psu was observed for N. gregaria.
48
0 2 4 6 8 10
Time (days) 0
2 4 6 8 10 12
Cell density (× 105 ml-1)
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.4. Cell density over time at 17, 35, 52 and 70 psu for Navicula gregaria. Bars = +1 S.D.
Maximum growth rate and productivity (Table 3.1) for Navicula salinicola was obtained at a salinity of 17 psu and decreased as salinity increased. No significant difference (U = 8; z = 0.83; p = 0.42; n = 5,5) in specific growth rate was found between salinities of 35 and 52 psu. There was no significant difference in productivity between 17 psu and 35 psu salinity. Yield was clearly affected by salinity (Figure 3.5). After 10 days of cultivation the highest yield (3.65 ± 0.81 × 106 cells mL-1) for N. salinicola was obtained at a salinity of 35 psu and the lowest yield (0.10 ± 0.02 × 106 cells mL-1) was found at a salinity of 70 psu. Significant differences in yield were found at the end of the cultivation period for all tested salinities (p < 0.05), with 17 psu being the exception (U = 8; z = -0.83; p = 0.42; n = 5,5).
49
0 2 4 6 8 10
Time (days) 0
1 2 3 4 5
Cell density (× 106 ml-1 )
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.5. Cell density over time at 17, 35, 52 and 70 psu for Navicula salinicola. Bars = +1 S.D.
The highest productivity for Navicula sp. was obtained at a salinity of 35 psu and the lowest was at a salinity of 70 psu. Productivity at 52 psu was significantly different (U = 0; z = 2.50; p = 0.007; n = 5,5) from that at 70 psu.
The highest specific growth rate was attained (0.57 ± 0.07 d-1) at a salinity of 35 psu with no significant differences (p > 0.05) between salinities of 17 psu and 52 psu. Navicula sp. attained the highest cell density at a salinity of 35 psu (10.66 ± 1.23 × 106 cells mL-1) and the lowest concentrations at 70 psu (2.32 ± 0.26 × 106 cells mL-1). Significant differences in cell density were found at the end of the cultivation period for all tested salinities (p < 0.05), except between 17 psu and 52 psu salinities (Figure 3.6).
50
0 2 4 6 8 10
Time (days) 0
4 8 12 16
Cell density (× 105 ml-1)
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.6. Cell density over time at 17, 35, 52 and 70 psu for Navicula sp. Bars = +1 S.D.
Nitzschia closterium attained maximum productivity and specific growth rate (Table 3.1) at a salinity of 35 psu. Both the productivity and specific growth rate obtained at 35 psu were significantly higher (p <0.05) compared to salinities of 17, 52 and 70 psu. Although the specific growth rates at 17 psu and 70 psu salinities are similar (Table 3.1), 0.65 ± 0.08 d-1 and 0.68 ± 0.01 d
-1 respectively, the productivity at 17 psu salinity was significantly higher (U = 11; z = 0.20; p = 0.84; n=5, 5) when compared to 70 psu. The specific growth rate at a salinity of 52 psu was significantly higher (p < 0.05) than that attained at salinities of 17 and 70 psu. At a salinity of 35 psu and 8 days of growth, the yield, peaked at 9.72 ± 0.31 × 105 cells mL-1. A similar maximum (Figure 3.7) was reached 2 days later but at a lower salinity (17 psu). At a salinity of 70 psu N. closterium exhibited a longer lag phase, approximately 2 days longer, compared to lower salinities (Figure 3.6). Maximum yield at 52 psu (8.21 ± 0.82 × 105 cells mL-1) and 70 psu (6.20 ± 0.54 × 105 cells mL-1) salinity were reached after 8 and 10 days, respectively, after initial inoculation of the culture medium.
51
0 2 4 6 8 10
Time (days) 0
2 4 6 8 10 12
Cell density (× 105 ml-1)
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.7. Cell density over time at 17, 35, 52 and 70 psu for Nitzschia closterium. Bars = +1 S.D.
The increase in salinity, from 17 psu to 70 psu, significantly decreased (U = 0;
z = 2.50; p = 0.007; n = 5,5) the specific growth rate of Thalassiosira sp. from 0.44 ± 0.03 d-1 to 0.17 ± 0.05 d-1. Productivity was the highest at a salinity of 17 psu followed by 70 psu. The former had a productivity of 1.57 ± 0.29 × 104 cells mL-1 d-1 and the latter 0.19 ± 0.09 × 104 cells mL-1 d-1. A Mann-Whitney U test revealed significant differences (p < 0.05), comparing all salinities with the exception of comparing 52 psu to 70 psu. This held true for both specific growth rate and productivity. It was observed that the growth of Thalassiosira sp. was influenced by the salinity of the culture medium. The growth was lowest at salinities of 52 and 70 psu and highest at a salinity of 17 psu, in which the attained maximum yield was 1.38 ± 0.28 × 105 cells mL-1. Significant differences in yield were found at the end of the cultivation period between low and high salinities (p < 0.05), with the exception of 35 psu (Figure 3.8).
52
0 2 4 6 8 10
Time (days)Time (hrs) 0
0.4 0.8 1.2 1.6 2
Cell density (× 105 ml-1 )
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.8. Cell density over time at 17, 35, 52 and 70 psu for Thalassiosira sp. Bars = +1 S.D.
Chlamydomonas sp. grew best at a salinity of 17 psu, but showed moderate growth over the entire salinity range tested (Table 3.2). At 70 psu the growth was about 37 % of that at a salinity of 17 psu. Significant differences in productivity were found between all tested salinities (p < 0.05). The specific growth rate decreased from 0.78 ± 0.05 d-1 to 0.29 ± 0.02 d-1 as salinity increased from 17 psu to 70 psu. The decrease in specific growth rate with an increase in salinity was found to be significant at all tested salinities (p < 0.01) (Table 3.2). Chlamydomonas sp. attained a maximum yield at a salinity of 17 psu, 10 days after inoculation. Cell concentration at a salinity of 70 psu reached a maximum concentration of 0.21 ± 0.02 × 106 cells mL-1 5 days after inoculation. Significant differences (p < 0.05) in cell density were found at the end of the cultivation period for all tested salinities (Figure 3.9).
53
0 2 4 6 8 10
Time (days) 0
4 8 12
Cell density (× 106 ml-1)
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.9. Cell density over time at 17, 35, 52 and 70 psu for Chlamydomonas sp. Bars = +1 S.D.
54
Table 3.1 Productivity and specific growth rate for six Bacillariophyceae species isolated from a warm-temperate solar saltworks, grown at four different salinities (data shown as mean ±1 SD, n = 5, except where indicated).
Salinity (psu)
Species (collection number)
Amphora coffeaeformis
Navicula gregaria
Navicula
salinicola Navicula sp. Nitzschia closterium
Nitzschia hungarica
Thalassiosira sp.
(NMMU 0008) (NMMU 0001) (NMMU 0006) (NMMU 0024) (NMMU 0002) (NMMU 0004) (NNMU 0003)
Growth rate, µ (d-1)
17 0.89 ± 0.09a 0.83 ± 0.06a 1.09 ± 0.07a 0.46 ± 0.06 0.65 ± 0.08a 0.31 ± 0.03 0.44 ± 0.03a 35 0.93 ± 0.05b 0.92 ± 0.09b 0.71 ± 0.07ab 0.57 ± 0.07#a 1.19 ± 0.17ab 0.27 ± 0.04 0.33 ± 0.05ab 52 0.47 ± 0.04ab 0.63 ± 0.05abc 0.67 ± 0.04ac 0.46 ± 0.07 1.05 ± 0.02bc CD 0.22 ± 0.05ab 70 0.41 ± 0.02ab 0.47 ± 0.03abc 0.32 ± 0.07abc 0.41 ± 0.04a 0.68 ± 0.01bc CD 0.17 ± 0.05ab
Productivity (× 105 cells mL-1 d-1)
17 1.42 ± 0.24a 1.57 ± 0.22a 5.41 ± 1.05a 0.55 ± 0.10a 1.48 ± 0.08a 5.71 ± 1.43* 1.57 ± 0.29**ab 35 0.83 ± 0.16ab 1.42 ± 0.23b 4.48 ± 0.55b 1.49 ± 0.17#ab 2.28 ± 0.35ab 4.00 ± 1.20* 0.84 ± 0.30**ab 52 0.38 ± 0.04abc 0.94 ± 0.04abc 1.36 ± 0.16abc 0.43 ± 0.06abc 1.66 ± 0.11abc CD 0.35 ± 0.17**ab 70 0.11 ± 0.02abc 0.56 ± 0.05abc 0.12 ± 0.03abc 0.30 ± 0.02abc 0.96 ± 0.02abc CD 0.19 ± 0.09**ab
R2 0.86 0.88 0.86 0.69 0.86 0.99 0.97
* (× 103 cells mL-1 d-1) a, b, c ,d denotes significant differences
** (× 104 cells mL-1 d-1) CD culture died after 24 hrs
# n = 4
55
Table 3.2. Productivity and specific growth rate of three species of warm-temperate solar saltworks microalgae belonging to the Chlorophyta , grown at four different salinities (data shown as mean ±1 SD, n = 5, except where indicated).
The specific growth rate of 0.68 ± 0.12 d-1 was obtained for Tetraselmis sp. at a salinity of 35 psu, however this was not significantly different (U = 5; z = -1.46; p
= 0.15; n = 5,5) from the specific growth rate (0.59 ± 0.06 d-1) obtained at a salinity of 17 psu. No significant difference (U = 7; z = 0.61; p = 0.55; n = 5,5) was found between the specific growth rates of the two hypersaline cultures (52
& 70 psu salinity), but the specific growth rate was found to be significantly lower (U = 0; z = 2.50; p = 0.007; n = 5,5) than that of the poly-euhaline cultures (17 &
35 psu salinity). The highest productivity (2.01 ± 0.34 × 105 cells mL-1 d-1) was obtained at a salinity of 35 psu and the lowest (0.52 ± 0.06 × 105 cells mL-1 d-1)
56 was found at a salinity of 70 psu with no significant differences (p > 0.05) between salinities of 17 and 52 psu. The response in cell growth of Tetraselmis sp. grown at different culture medium salinities is illustrated in Figure 3.10. As far as yield is concerned Tetraselmis sp. favours a salinity of 35 psu (Figure 3.10).
Tetraselmis sp., at the end of the cultivation period and at a salinity of 35 psu, attained a yield of 10.87 ± 0.28 × 105 cells mL-1, significantly higher (p ≤ 0.01) compared to salinities of 17 (6.42 ± 1.42 ×105 cells mL-1), 52 (5.97 ± 0.83 ×105 cells mL-1) and 70 psu (4.80 ± 0.54 ×105 cells mL-1). Yield at a salinity of 70 psu was also significantly lower (p ≤ 0.03) than those cultures grown at salinities of 17 and 52 psu.
0 2 4 6 8 10
Time (days) 0
4 8 12 16
Cell density (× 105 ml-1)
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.10. Cell density over time at 17, 35, 52 and 70 psu for Tetraselmis sp. Bars = +1 S.D.
Maximum growth for Dunaliella tertiolecta occurred at a salinity of 35 psu (Table 3.2). Salinity was responsible for significantly reducing specific growth rate from 0.66 ± 0.03 d-1 at 35 psu to 0.59 ± 0.05 d-1 at 70 psu. The highest productivity was obtained at a salinity of 35 psu. No significant differences in cell productivity
57 were found for between the tested salinities (p > 0.05), with the exception of that at 35 psu. Figure 3.11 shows the growth curves of D. tertiolecta cultures measured at various salinities. At a salinity of 35 psu a yield of 1.71 ± 0.08 ×106 cells mL-1, was attained. This was significantly higher (p < 0.01) compared to other tested salinities. No significant differences in yield were found between 17, 52 and 70 psu (p >0.05).
0 2 4 6 8 10
Time (days) 0
0.5 1 1.5 2 2.5
Cell density (× 106 ml-1)
17 ‰ 35 ‰ 52 ‰ 70 ‰
Figure 3.11. Cell density over time at 17, 35, 52 and 70 psu for Dunaliella tertiolecta. Bars = +1 S.D.
Ordination of species indicates their tolerance with regard to salinity, separated on the major ordination axis (x-axis, Figure 3.12) by growth rate and on the y-axis by means of salinity (Figure 3.11 & Figure 3.13). Nitzschia hungarica is separated from the other species based on low specific growth rate at salinities of 35 psu and less. N. hungarica shows stenohaline characteristics, capable of tolerating only slight variations in salt concentrations with a preference towards lower salinities.
58 Thalassiosira sp., Navicula salinicola, Amphora coffeaeformis, Chlamydomonas sp., Tetraselmis sp and Navicula gregaria are grouped together for their preference towards 35 psu salinity. Thalassiosira sp. is separated from N.
gregaria on its preference for lower salinities, whereas N. gregaria prefers higher salinities. These six species display euryhaline characteristics, tolerating a wider range of salinities. D. tertiolecta, N. closterium and Navicula sp (NMMU 0024) were the most tolerant towards high salinity and grew best under hypersaline conditions.
Ordination of growth rates indicates the dissimilarity of salinity treatments, separated on the major ordination axis (x-axis, Figure 3.12) by specific growth rate and on the y-axis by salinity (Figure 3.12 and Figure 3.13). The salinities of 35, 52 and 70 psu are separated from one another due to an overall decrease in specific growth rate with increased salinity. Specific growth rates, in general, were much lower at 70 psu salinity when compared to all other salinities. The ordination clearly shows the effect salinity has on the growth rates of the microalgae.
59
Figure 3.12. Principal Components Analysis (PCA) of species, for specific growth rate, separated on the major ordination axis (x-axis) by specific growth rate and on the y-axis by salinity, (x-axis eigenvalue = 0.948, y-axis eigenvalue = 0.028. Cumulative percentage variance = 94.8 %, Sum of all eigenvalues = 1.000).
0.6 1.2
-0 .4 0 .6
Nav_greg
Nit_clos Tha_sp Nit_hung
Nav_sali Amp_coff
Tet_sp
Dun_tert Nav_sp
Chl_sp
SPECIES
Diatoms Flagellated Green
Stenohaline
Euryhaline
Hyperhaline
60
Figure 3.13. Principal Components Analysis (PCA) of salinities, for specific growth rate, separated on the major ordination axis (x-axis) by specific growth rate and on the y-axis by salinity, (x-axis eigenvalue = 0.948, y-axis eigenvalue = 0.028. Cumulative percentage variance = 94.8 %, Sum of all eigenvalues = 1.000).
0.5 1.3
-0.40.6
SAMPLES
17 ppt 35 ppt 52 ppt 70 ppt
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Discussion
Measuring the growth rates of microalgal populations is an informative way to determine the effect salinity has on the growth or state of a microalgal culture (Affan et al., 2007). By understanding the behaviour of a species under a set of known conditions, reproducible high-density mass cultures can be maintained (Affan et al., 2007). Difficulty arises however when trying to compare the results obtained in this study with those published in the literature as culture conditions differ from study to study. Most of published literature on growth rates focuses on nutrient limitation, especially nitrogen, followed by phosphate and also silica deficiency for diatoms. The effect of temperature and light intensity on growth is also well studied in the literature when compared to the amount of information available on the effect of salinity. The literature that is available on the effect of salinity concentrates on the 5 to 35 psu salinity range and ignores the higher salinity values.
Marine diatoms are known to have a broad tolerance to salinity (Williams, 1964) and this was found in the present study (figure 3.12). The experimental results agree with much of the published literature on marine and estuarine diatoms.
Measured growth rates (µ, d-1) ranged between 1.19 ± 0.17 d-1 for Nitzschia closterium, and 0.17 ± 0.05 d-1, Thalassiosira sp. Such values are in the range of those reported by other authors, Renaud et al. (1999), Jahnke and White (2003) and Griffiths and Harrison (2009). However, they are lower than those reported for temperate diatoms (range 1.68 – 3.83 d-1) reported by Chen and Durbin (1994).
At a salinity of 17 psu, three species, Navicula gregaria, Navicula salinicola and
At a salinity of 17 psu, three species, Navicula gregaria, Navicula salinicola and