Introduction
In the past few decades the increase in crude oil prices, the unsustainable use of crude oil and global warming (Gavrilescu & Chisti, 2005, Chisti, 2007, Rittmann, 2008) have placed a renewed urgency on the development of biodiesel as an alternative to petrodiesel. According to some analysts, crude oil reserves will be depleted before 2060 (Rodolfi et al., 2009) if we do not break our dependence on oil. Cheap, renewable, carbon neutral fuel source such as biodiesel can lessen our dependence on crude oil. Biodiesel, or fatty acid methyl esters, is produced by a process known as the transesterification of the triglycerides in vegetable oil - such as sunflower, rapeseed and palm oil – with methanol (Ma & Hanna, 1999, Fukuda et al., 2001, Miao & Wu, 2006). Oil derived from microalgae, as with vegetable oil, is a good source of suitable triglycerides (Demirbas, 2008). Chisti (2007) stated that microalgae biodiesel has the potential to completely displace petro-diesel and meet the global demand for transport fuels. Thus the use of microalgae for biodiesel production is widely regarded as the most efficient way of generating biofuels (Schenk et al., 2008). Marine microalgae are the most suitable of the all microalgae as they produce the very long chain polyunsaturated fatty acids (Harwood & Guschina, 2009) needed for biodiesel production. Currently no biodiesel is produced from microalgae (Chisti, 2007).
The production of microalgal-biodiesel is technically feasible but not yet economically viable (Chisti, 2008). Ratledge and Cohen (2008) however are very pessimistic about the future of biodiesel from microalgae or from any other oleaginous micro-organism.
The choice of biomass used for the production of biodiesel is of great importance.
The use of conventional oil-crops as a bio-oil source will have a negative impact on world food prices and availability (Chisti, 2008, Griffiths & Harrison, In press)
67 as the demand for certain oil-crops increases. On the other hand, the use of microalgae as a source of bio-oil will have a less profound impact on world food prices. Microalgae are also more efficient in the bioconversion of solar energy to biomass (Schenk et al., 2008). Other benefits that microalgae have over conventional oil-crops are that they can be grown in areas unsuitable for conventional crops (Chisti, 2007, Schenk et al., 2008) and can be grown in hypersaline environments. The use of microalgal biomass is not limited to bio-fuel production, but also has the potential to be a source of food for humans, feed for livestock, can be used in aquaculture and the production of chemicals (Spolaore et al., 2006). For these various applications an understanding of the biochemical composition is needed as well as how this changes under different environmental conditions.
Because of the simple, single cell structure of most microalgae, accompanied by fast growth and high oil production, microalgae have the potential to outcompete conventional crops (Chisti, 2007, 2008). Some species of microalgae regularly achieve 50% to 60% of dry weight as lipid (Sheehan et al., 1998) with a mean lipid content of 23 % for nutrient replete conditions (calculated from Griffiths &
Harrison, In press). Lipid content is extremely variable between species and there is even variation between strains of the same species, values ranging between 1 % and 85 % of dry weight have been reported in the literature (Spoehr
& Milner, 1949, Borowitzka, 1988, Chisti, 2007, Rodolfi et al., 2009).
Temperature, irradiance and nutrient availability are key factors influencing lipid production and composition (Guschina & Harwood, 2006, Hu et al., 2008, Converti et al., 2009, Rodolfi et al., 2009) and, to a lesser degree, salinity (Takagi et al., 2006). Conventional biodiesel crops such as soybean and oil palm produces less than 5 % oil in biomass. If, for example, oil palm is to be used, then 61% of all existing all agricultural cropping land needs to be dedicated to oil palm production. This will leave insufficient land for the production of food and other crops. This scenario is, however, different for microalgae. In tropical regions microalgae biomass production can be in the region of 1.535 kg m-3 d-1 (Chisti, 2008). If an average oil content of 30% dry weight in the biomass is
68 assumed then only 3 % of the cropping area (Chisti, 2008) is needed for biodiesel production, leaving more land for the production of food and other crops. Even when microalgal biomass contains only 15 % oil by dry weigh, biodiesel production from microalgae will be still economically feasible and competitive with conventional oil-crops (Chisti, 2008).
Prior to the Aquatic Species Program (Sheehan et al., 1998) little work had been done to enhance lipid production in microalgae and most work focussed on nutrient deficiency as the major key factor triggering lipid production. The focus of nutrient deficiency as a trigger was instigated by Spoehr and Milner (1949), who demonstrated that nitrogen starved Chlorella pyrenoidosa was able to accumulate up to 85% of biomass as lipid. Nutrient deficiency is regarded as the most efficient method of increasing lipid content in algae (Rodolfi et al., 2009).
However, Sheehan et al. (1998) came to the conclusion that lipid productivity is not enhanced under nutrient deficient conditions, because the reduction in growth rate and productivity is greater and cannot be offset by the high cellular lipid content attained when the algal cells are nutrient starved. If nutrient deficiency is not really a viable alternative (Sheehan et al., 1998) to enhancing lipid production then other stressors need to be investigated. Various authors have shown that a change in salinity also triggers the production of lipids (Cooksey & Chansang, 1976, Fabregas et al., 1984, Renaud & Parry, 1994, Takagi et al., 2006, Matsubara et al., 2007, Abid et al., 2008).
The Aquatic Species Program isolated and screened more than 3000 strains of organisms for their oil content and ability to grow at extreme temperatures, salinities, and pH (Sheehan et al., 1998). This collection was reduced to about 300 strains with no recommended “biodiesel species” identified. More recently, Rodolfi et al., (2009) recommended two marine microalgal genera, Nannochloropsis and Tetraselmis, as suitable for biodiesel production.
Nannochloropsis was selected on its ability to respond with increased oil production under nitrogen deficient conditions and high constitutional lipid content (about 30%), whereas, Tetraselmis for its robustness and high productivity.
69 Although there is rapidly growing interest in the use of microalgae for biodiesel production, there is little published data on the effect that hyper-salinity has on total lipid produced. Most of the published literature focuses on the effect that lower (< 35 psu) salinities (Fabregas et al., 1984, Renaud & Parry, 1994) have on the production of lipids or how nutrient limitation affects lipid production (Fabregas et al., 1984, Reitan et al., 1994, Chu et al., 1996, de la Peña, 2007, Converti et al., 2009, Widjaja et al., 2009).
A change in total and intracellular composition of lipids in response to environmental salinity has been reported by various authors (Renaud & Parry, 1994, Xu & Beardall, 1997, Abid et al., 2008) and forms the basis for the following hypothesis: an increase in salinity of microalgal cultures will produce more microalgal oil; however, when the salinity optimum for lipid production is exceeded, lipid productivity will be negatively affected. The objective of this study was to investigate the effect that salinity, within the range 17 to 70 psu, has on the total lipid content of ten species of warm-temperate solar saltworks microalgae, grown in 1.5-l laboratory batch culture for a period of 6 days. A second aspect of the present study was to find from the 10 species isolated, the most suitable microalgae for biodiesel production. Results are discussed from a biodiesel point of view and a possible production scenario is suggested.
Materials and Methods
Isolation and preparation of microalgae
Isolation and preparation methods for the ten microalgae species are described in chapter 2.
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Growth of microalgae for lipid experiments
The growth conditions were the same as those described in chapter 2. Species that obtained a specific growth rate of 0.2 d-1 or less were not considered for lipid extraction. Two species, Thalassiosira sp. and Nitzschia hungarica, fell below this threshold and were therefore not selected for the total lipid experiments at salinities of 52 psu and greater. The cultures were grown, for 6 days, in quintuplicate 3 L Erlenmeyer flasks containing 1.5 L of GF/C filtered and autoclaved NSW supplemented with F/2 medium at salinities of 17, 35, 52 and 70 psu. Algae were harvested, by means of filtration (using GF6 filter paper), 6 days after initial inoculation. The filter papers containing the microalgae were freeze dried overnight for lipid extraction the following day.
Extraction of lipids
Total lipids were determined gravimetrically by a modified Bligh and Dyer (1959) method (Takagi et al., 2006). Approximately 100 mg of dried algae (filter paper included) were used for extraction. The ratio of filter to microalgae was known and a control showed that the glass-fibre filter had no effect on the lipid extraction process. The biomass (filter included) was blended with 3 mL chloroform/methanol (2:1 v/v) and the mixture was allowed to stand for two hours at room temperature. The solvent phase was recovered by centrifugation. The same process was repeated three times and the solvents were added together.
The sample was then filtered (GF6) and the remaining biomass rinsed with 3mL chloroform/methanol (2:1). After extraction, methanol and a 1 % NaCl solution were added to adjust the ratio of methanol, chloroform and water to 2:2:1 (v/v/v).
The methanol/water layer was removed, the chloroform evaporated, and the sample freeze-dried overnight and weighed as the total lipid.
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Statistical analysis
Data were tested for normality (Shapiro-Wilk W test). Differences in total lipid were tested with the Mann-Whitney U test, using STATISTICA version 8.0 (2007). All p-statistics were reported as P - 2sided. Principal components analysis (PCA) was used (CANOCO, Ter Braak 1987) to determine the relationship between different species over all salinities. 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 for either samples or species. No species-weights and sample-weights were specified.
Results
The trends in total lipid content of the algae with regard to salinity are shown in Table 4.1. The diatom with the highest percentage lipid content was Navicula salinicola, followed by Navicula sp. and Amphora coffeaeformis. For the flagellated greens, Chlamydomonas sp. attained the highest total lipid content with 22.64 ± 1.19 % of DW at a salinity of 17 psu. However, this was not significantly (p < 0.05) higher than the lipid values measured for Tetraselmis sp.
and Dunaliella tertiolecta. Lipid content for D. tertiolecta ranged from 11.64 ± 2.43 to 17.03 ± 4.37 % DW, for 17 and 70 psu, respectively. A Mann-Whitney U Test indicated no significant increase (U = 4; z = -1.77; n = 5,5; p = 0.09) in lipid content for D. tertiolecta with increased salinity. Seven of the ten species showed no significant (p < 0.05) increases in total lipid with an increase in salinity from 17 to 35 psu. Two of the species, Nitzschia closterium and Navicula salinicola, showed significantly (p < 0.03) lower lipid content at 17 psu than at 70 psu.
Chlamydomonas sp. had a significantly lower (U = 0; z = 2.61; n = 5,5; p = 0.007) lipid content) than at 35 psu. Lipid content for N. salinicola, Navicula sp. and Chlamydomonas sp. were significantly higher (p < 0.03 for all three species) at
72 35 psu than at 52 psu salinity. A. coffeaeformis and N. closterium had significantly higher lipid content at 35 psu salinity than at 70 psu.
Table 4.1 Total lipid content (% DW) for ten species of microalgae isolated from a warm-temperate solar saltworks and grown at four different salinities (data shown as mean ± SD, n = 5, except where indicated).
Salinity (psu)
Species Collection
number 17 35 52 70
Amphoracoffeaeformis (NMMU 0008) 5.10 ± 1.46 6.90 ± 1.63 7.82 ± 3.95 3.52 ± 1.54 Navicula gregaria (NMMU 0001) 6.48 ± 2.09 7.68 ± 2.27 6.89 ± 5.20 4.51 ± 2.75 Navicula. salinicola (NMMU 0006) 6.68 ± 1.18 10.86 ± 4.59a 5.29 ± 1.72a 2.78 ± 0.36a
Navicula sp. (NMMU 0024) 5.60 ± 4.61 8.91 ± 1.14b 2.97 ± 1.47b - Nitzschia. closterium (NMMU 0002) 6.80 ± 1.37 7.13 ± 1.17 6.38 ± 2.57c 4.24 ± 1.27c Nitzschia. hungarica (NMMU 0004) 7.57 ± 2.42 5.80 ± 0.88 - -
Thalassiosira sp. (NMMU 0003) 5.71 ± 2.68 6.33 ± 2.03 - - Chlamydomonas sp. (NMMU 0037) 22.64 ± 1.19d 7.71 ± 2.46d 3.10 ± 1.56d - Dunaliella tertiolecta (NMMU 0020) 11.64 ± 2.43 13.87 ± 1.88 15.17 ± 3.72 17.03 ± 4.37
Tetraselmis sp. (NMMU 0018) 17.45 ± 7.90 12.80 ± 1.58 14.07 ± 2.82 14.20 ± 2.36
a, b, c ,d
denotes significant differences
Ordination of the lipid content (Figure 4.1) indicates the preferred salinities for lipid production for each species, separated on the x-axis by total lipid produced and on the y-axis by salinity. N. hungarica and Thalassiosira sp. are grouped together, as both species produced a similar percentage of total lipid at salinities of 17 and 35 psu with no lipid produced at salinities of 52 and 70 psu. Navicula sp. is separated from N. hungarica and Thalassiosira sp. because it produced a
73 small amount of lipid at 52 psu salinity. Chlamydomonas sp., N. hungarica, Thalassiosira sp. and Navicula sp. did not yield any lipid at 70 psu salinity and therefore they are separated from the rest. Navicula salinicola, Nitzschia closterium, Navicula gregaria, A. coffeaeformis and Tetraselmis sp. are grouped together as these five species showed optimal lipid productivity at salinities of 35 and 52 psu. N. salinicola attained maximum lipid at 35 psu salinity. The rest of the species within the 35 – 52 psu optimal salinity group showed a higher salinity preference, with the diatom A. coffeaeformis achieving its highest percentage lipid content at a salinity of 52 psu. Tetraselmis sp. also showed a preference towards hypersaline conditions for optimal lipid production. The two green flagellates, Tetraselmis sp. and Dunaliella tertiolecta, are separated from the other species based on higher lipid productivity at a salinity of 70 psu. D.
tertiolecta had the greatest lipid productivity of all species at a salinity of 70 psu.
In Figure 4.2 there is large dissimilarity in lipid production at a salinity of 17 psu, at higher salinities this dissimilarity is more focused. The dissimilarity at 17 psu salinity can be attributed to the large variation in observed total lipid content for each of the different species. Eight of the ten species had similar lipid content at 17 psu. The other two species had significantly higher lipid content and therefore contribute to the large variation observed in lipid content at 17 psu, of which Chlamydomonas sp. was the main contributor. Variation in total lipid content was less at salinities of 35, 52 and 70 psu. At high salinities there is a lower lipid content in the algae. However a Kruskal-Wallis test shows that there is no significant : H = 3; α = 7.59; n = 168; p = 0.055) difference between the different salinity groups.
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Figure 4.1 Principal Components Analysis (PCA) of species, for total lipid content (x-axis eigenvalue = 0.792, y-axis eigenvalue = 0.119. Cumulative percentage variance of species data
= 91.1. Sum of all eigenvalues = 1.000).
0.4 1.0
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T e t _ s p D u n
_ t e r t N a v
_ s p C h l
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SPECIES
Diatoms Flagellated green
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Figure 4.2 Principal Components Analysis (PCA) of samples indicates the total lipid content with regard to salinity, separated on the major axis (x-axis,) by total amount (% DW) of lipid and on the y-axis by means of salinity (x-axis eigenvalue = 0.792, y-axis eigenvalue = 0.119. Cumulative percentage variance of species data = 91.1. Sum of all eigenvalues = 1.000).
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Discussion
The first part of the hypothesis, an increase in salinity of microalgal cultures will produce more microalgal oil; can be rejected for all species examined. Three species, Navicula gregaria, Nitzschia closterium and Thalassiosira sp. conform to the second part of the hypothesis, when the salinity optimum for lipid production is exceeded, lipid productivity will be negatively affected. This study found no consistent pattern for the production of lipid as influenced by salinity. This same observation was made with regards to temperature. In general, maximum lipid content coincides with optimal range in temperature (Opute, 1974, Renaud et al., 1995). Renaud and Parry (1994) found that there was an optimal salinity for the production of lipids and this varied from species to species. This optimal salinity range can be used to increase the lipid content of microalgae. For example, growing Chlamydomonas sp. at a salinity of 17 psu will result in higher lipid content when compared to 35 psu and above. Dunaliella tertiolecta on the other hand will result in more lipid at higher salinities than lower salinities. Thus, we recommend a salinity of 70 psu for maximum lipid yield for Dunaliella tertiolecta.
The increase in lipid for Dunaliella tertiolecta with increased salinity might be due to salt tolerant character of Dunaliella. All other species showed a decrease in lipid content with increased salinity. Consequently, salinities of 17 and 35 psu are considered to be appropriate to achieve high lipid yield.
All diatom species responded to salinity over the experimental range 17 to 70 psu and attained similar lipid values. Diatoms in general contain more lipid than other algae (Brown et al., 1997), however, the diatoms in the present study attained lower average total lipid contents than the flagellated green algae. Lipid values ranged from 2.97 to 10.86 % DW. These values are lower than those reported by Griffiths and Harrison (2009) Amphora coffeaeformis attained maximum lipid content (7.82 ± 3.95) at a salinity of 52 psu. This is a 53 % improvement on the total lipid (5.10 ± 1.46) content at a salinity of 17 psu. A. coffeaeformis was the only diatom species that showed the ability to increase its lipid content at higher
77 salinities, although there was no increase above a salinity of 52 psu. De la Peña (2007) reported on the effects of nutrient media on the lipid composition of a tropical isolate of an Amphora species. Lipid content ranged from 26.4 to 81.5 % DW. These values are several orders of magnitude higher than those reported in the present study. This is a good indication that lipid content may be enhanced by nutrient stress rather than by salinity.
Five of the six diatoms species in this study, namely Navicula gregaria, Nitzschia closterium, Thalassiosira sp., Navicula salinicola and Navicula sp. showed a maximum total lipid content at 35 psu salinity. Work done in the mid 1990’s by Renaud et al. (1994, 1994, 1995) on the effect that temperature and salinity had on the biochemical composition of tropical diatom isolates to be used as potential feed in aquaculture, reported higher lipid contents for Nitzschia closterium and Nitzschia paleacea. N. paleacea showed a decrease in lipid content at high salinity with maximum lipid content obtained at a salinity of 10 psu. To my knowledge, the present study is the first to report on the lipid content of Nitzschia hungarica.
All three Navicula species showed maximum total lipid content at a salinity of 35 psu. Of all the diatoms tested, N. salinicola showed the greatest variation in lipid content over the range of salinities used, with both the highest and lowest lipid contents. At a salinity of 52 psu N. gregaria was the most productive of the three Navicula species, producing 30 and 132 % more lipid than N. salinicola and Navicula sp., respectively. Renaud et al. (1994) report a 24.2 % total lipid content for a marine Navicula species. Three freshwater Navicula species attained an average lipid content of 24 to 51 % of DW (Griffiths & Harrison, 2009), for nutrient replete and deficient conditions. Both the freshwater and marine isolates of Renaud et al. (1994) will be better candidates for the production of biodiesel.
However, for the tested Navicula species nutrient limitation will yield in more lipid rather than salinity.
Lipid content of nutrient-replete green algae, those that have been investigated, ranges from 13 to 31 % DW with a mean of 23 % DW (Griffiths & Harrison,
78 2009). The lipid values obtained for Chlamydomonas sp., Dunaliella tertiolecta and Tetraselmis sp. in the present study are well within the range reported in literature for nutrient-replete conditions (Maddux & Jones, 1964, Chisti, 2007, Patil et al., 2007, Griffiths & Harrison, 2009). On the other hand, the lipid content reported in the literature for green algae under nutrient-depleted conditions are a magnitude or two higher (Griffiths & Harrison, 2009) than those reported in the present study. Nutrient-depleted conditions will result in more lipid yield than increased salinity conditions.
Chlamydomonas sp. and Navicula sp. showed moderate growth rates (Table 3.2) at salinities of 52 psu and greater. However, after 6 days of growth, their growth was very slow at 70 psu salinity and subsequent testing for lipid content was halted due to insufficient biomass. The low specific growth and biomass yield of Thalassiosira sp., Nitzschia hungarica, Navicula sp. and Chlamydomonas sp. at high salinities automatically excluded them as potential candidates for biodiesel production when grown at high salinities. The PCA ordination (Figure 4.1) illustrates this by separating Thalassiosira sp., Nitzschia hungarica, Navicula sp.
and Chlamydomonas sp from the other species.
Chisti (2007) reported a 23 % by dry weight oil content for Dunaliella primolecta.
Takagi et al. (2006) reported higher lipid values for the genus Dunaliella in their study on the effect salt has on the intracellular accumulation of lipid. They
Takagi et al. (2006) reported higher lipid values for the genus Dunaliella in their study on the effect salt has on the intracellular accumulation of lipid. They