The Chlorella saccharophila (freshwater) and Tetraselmissuecica (marine) microalgae were investigated for their potential as feedstock for the production of biodiesel.  Assessment was based on their ability to produce biomass and lipid accumulation.  Varying concentrations of CO₂ (3, 6 and 9%) were used at 24 h light exposure.  The results indicated that T. suecica produced higher cell yields compared to the C. saccharophila under all parameters tested.  Statistical analysis indicated that the biomass yields achieved using CO₂ at varying concentrations were significantly different from one another.  However, varying CO₂ concentrations over the range of 3 to 9% did not significantly affect the oil yields for both species over the elapsed time.  Thus, from an economic stand point it is much more suitable to use CO₂ at a concentration of 3% as opposed to higher concentrations.  The use of NaHCO₃ as a carbon source on the biomass and oil yields were also evaluated using the same experimental parameters.  Results indicated that the CO₂ carbon source resulted in higher biomass and oil yields for the marine microalgae species when compared to NaHCO₃ carbon source.  However, the Chlorella saccharophila species resulted in higher biomass and lipid yields using the sodium bicarbonate carbon source.  This suggests that different species have a preferred carbon source.  Some are better at the uptake of one source over the other.  Lower oil yields were achieved using the 3% CO₂ as the carbon source compared to NaHCO₃ for the Chlorella saccharophilaspecies.  The Tetraselmissuecicaspecies resulted in a slight increase in oil yield using 3% CO₂ as opposed to NaHCO₃.  The optimal growth conditions for Chlorella saccharophilaare the combination of nutrients, with 24 h light exposure and NaHCO₃ as a carbon source and those for Tetraselmissuecicaare the ammonium nitrate, the 24 h light exposure and 3% CO₂ as a carbon source.

The demonising fossil fuel reserves and the environmental concerns associated with burning fossil fuels have accelerated the need for a renewable energy source that is environmentally friendly.  Increased carbon dioxide emissions have been correlated with the amount of fossil fuel being burnt [1].  Biofuels such as biodiesel and bioethanol are promising substitution for petroleum fuel source [2].  Numerous feedstocks can be used as biomass for biofuel generation which include food waste, agricultural waste, municipal waste and both edible and nonedible oilseeds [3].  Currently, the best crops for biofuel production are oilseeds, but they are considered a food source for many people around the world [2,3].  However, microalgae have been noted to store oil that is 10 folds higher than the leading plant crop [4]. 

Microalgae are abundant microorganisms in nature, able to convert carbon dioxide into biomass which can then be used for biodiesel production via a transesterification reaction process [5].  Microalgae as an alternatefuel source is ideal because of their high growth rates and their ability to store higher lipids than the leading crop plants [6].  In addition, the waste that is generated after oil extraction can be used for other value added products, such as animal feed, organic fertilizers, and other biofuel products such as methane and ethanol via fermentation [7].  The amount of lipids stored in the microalgal cells can be manipulated by changing the environmental parameters such as temperature, pH, nutrient source, carbon source and light duration [8-10]. 

A computer was used to operate and control the various components of the open pond system and record the various measurements.  The light intensity was measured using a Quantum Sensor, SQ-316 Series (Apogee, Logan, Utah, United States of America). The pH was measured using pH electrodes (EW-59001-65, Cole Parmer, Montreal, Quebec, Canada). The temperature was measured using thermocouples (WD-08541-12, Nova-Tech International, Houston, Texas, United States of America). A basic computer program (BASIC Stamp Editor v2.5) allowed the configuration of the operating frequency and duration of the light, aeration unit and collection system. The computer was connected to a data coordinator (cDAQ-9178, National Instruments) which had 24 digital output ports and 24 digital input ports. The digital output ports were connected to electronic circuits which were responsible for the lighting, cooling and collection systems.

Microalgae

One freshwater microalgae (Chlorella saccharophila) and one marine (Tetraselmissuecica)were selected based on their ability to yield high biomass and store lipids [13]. 

The freshwater strain Chlorella saccharophilawas selected for study because of its high lipid content (45%).  This strain is capable of achieving a biomass yield of 3.88 g/L, which is not the highest among the freshwater species, but can however be offset by the fact that it achieves the highest lipid content.  This results in a lipid yield of 1.75 g/L.  The highest biomass yielding algae Scenedesmusobliqus of 4.34 g/L only achieves a lipid content of 38%, which intern results in a lipid yield of 1.69 g/L. Chlorella saccharophilais a green unicellular microalga belonging to the Chlorella genus [14].  The cells have an average size of 7.3 μm[15]. The cells contain a single chloroplast enclosed in a spherical or subspherical form.  These cells reproduce asexually through production of non-motile autospores[16].  This species is able to use glucose [17],bicarbonateandcarbondioxideasthecarbon source for growth [18]. The optimal temperature and pH for growth are 20-24°C and 7.5-8, respectively.

The marine microalgae strain Tetraselmissuecicawas selected for this study because of its high biomass yield of 4.48 g/L and comparatively high lipid content.  This species achieves a lipid content of 23% which is not the highest among the other species but can, however, be offset by the fact that it achieves the highest biomass yield.  This results in a lipid yield of 1.03 g/L, while the Chaetocerosmuelleri, with the highest lipid content of 34%, only achieves a biomass yield of 0.98 g/L, which results in a lipid yield of 0.33 g/L.  Tetraselmissuecicagrows as single cells.  They are motile and can be compressed or curved, but they are never twisted [19].  The cells are spherical or elliptic with a length of 35 μm and a width of 14 μm.  This species is able to use both sodium bicarbonate [20] and carbon dioxide [21] as the carbon source for growth. The optimal temperature and pH for growth are 18-24°C and 7-9, respectively [22]. 

Experimental Design

Two set of experiments were carried out. In the first set of experiments, the selected freshwater (Chlorella saccharophila) and marine (Tetraselmissuecica) microalgae species were grown in an open pond system using NaHCO₃ as a carbon source.  The sodium bicarbonate (NaHCO₃) was administered at a concentration of 1300 mg/L.  Ammonium nitrate was used for the marine microalgae and a combination of nutrients (ammonium nitrate, ammonium sulfate and ammonium phosphate) was used for the freshwater algae as sources of nitrogen at the optimum light exposure (24 h) as recommended by [13].  The light intensity was kept at 480 μmol/m² s¹ and the nitrogen content, pH and temperature were kept constant at 70 mg/L, 8.3-8.9 and 22°C, respectively.  In the second set of experiments the effects of carbon dioxide concentration on the algae biomass and oil content were evaluated. Carbon dioxide was administered at concentrations of 3, 6 and 9% (v/v in air).  The algae wereexposed to full light exposure (24 hand the light intensity was kept at 480 μmol/m² s¹).  A combination of nutrients (ammonium nitrate, ammonium phosphate and ammonium sulfate) was used as nutrient for the freshwater microalgae and ammonium nitrate was used for the marine microalgae.  The nitrogen content, pH and temperature were the same as in the first set of experiment.  The best results obtained with CO₂ were compared with those obtained with NaHCO₃. 

Preparation of Liquid Medium for Inoculum Growth

The freshwater microalgae medium was prepared on algal proteose medium (ATCC Catalog Medium No. 847, American Type Culture Collection, Manassas, Virginia, United States of America) and was made up by adding 1 g of proteose peptone (Difco 0120) to 1 L of Bristols solution (Table 1). Bristols solution was prepared by adding the following amounts from the prepared stock solutions: 10mL of NaNO₃, 10 mL of CaCl₂, 10 mL of MgSO₄ 7H₂O, 10 mL of K₂HPO₄, 10 mL of KH₂PO₄, 10 mL ofNaCl,0.05 mLofFeCl₃and940 mL of distilled water.  The stock solutions were prepared as follows: 10 g of NaNO₃ in 400 mL of distilled water, 1g of CaCl₂ in 400 mL of distilled water, 3 g of MgSO₄ 7H₂O in 400 mL of distilled water, 3 g of K₂HPO₄ in 400 mL of distilled water, 7 g of KH₂PO₄ in 400 mL of distilled water and 1 g of NaCl in 400 mL of distilled water. 

Preparation of Solid Medium for Inoculum Growth

The inoculum for Tetraselmissuecica microalga was prepared by taking 5 mL of the liquid sample (UTEX LB 2286, Cedarlane, Burlington, Ontario, Canada)and adding it to 125 mL Erlenmeyer flask containing 25 mL of F/2 liquid media and then left to grow at room temperature for 2 weeks at a photocycle of 14 h light and 10 h dark.  The mixture was then transferred to a 500 mL Erlenmeyer flask containing 250 mL of F/2 liquid media and was left to grow for 2 weeks at a photocycle of 14 h light and 10 h dark.  Finally, the media was transferred from the 500 mL flask into a 30 L bioreactor containing 25 L of F/2 liquid media and left to grow for 2 additional weeks at a cycle of 14 h light and 10 hour dark.

Preparation of Algae Production Media

The freshwater production medium is a modification of the Fitzgerlad medium [24].  The preparation of the stock solutions for this media is shown in Table 3.  The medium was made up by the addition of 1 mL of each of the stock solutions A, B, C and D to 1 L distilled water (Table 4).  

A modification of the F/2 media [23] was used as the production medium for the marine microalga.  The medium was modified by eliminating the addition of sodium nitrate.  The medium consists primarily of autoclaved ocean water (Halifax Waterfront, Halifax, Nova Scotia, Canada). Table 5 shows the elemental analysis of the components present in the marine water which was performed at the Mineral Engineering Center of Dalhousie University.

Experimental Protocol

To each compartment in the open pond system a total of 4.75 L of freshwater production media was added.  The amount of nutrient was added to the production medium.  This solution was enriched with the desired carbon source (1.3 g/L of sodium bicarbonate, 3% CO₂, 6% CO₂ or 9% CO₂).  To this, 250 mL of Chlorella saccharophila inoculum was added to each compartment.  The cells were exposed to 24 h light and left to grow for 10 days.  Every otherday, 100 mL sample was taken for experimental analyses.  The samples were analyzed for pH and biomass yield.  At the end of the run the biomass was harvested from the liquid media using a Sorvall T1 Centrifuge (Thermo Scientific, Marietta, Ohio, United States of America). The supernatant from the centrifuge tubes was decanted and the cells were collected for biomass yield and oil content analyses.  The marine medium was used with marine algae and the same procedure was followed using the ammonium nitrate nutrient system.

Microalgae Biomass Determination

Microalgae Biomass Using CO₂ as a Carbon Source

Effect of Microalgae Type

The effects of microalgae type on the cell yield are shown in Figure 4.  Tetraselmissuecica achieved higher cell yields than the Chlorella saccharophila at all carbon dioxide concentrations.  Tetraselmissuecica resulted in cell numbers of 1.148x10⁶, 2.213x10⁶ and 1.930x10⁶ cells/mL while Chlorella saccharophila resulted in cell numbers of 0.181x10⁶, 0.553x10⁶ and 1.019x10⁶ cells/mL at the CO₂ concentrations of3%, 6% and 9%, respectively. 

Chinnasamy et al. [31] reported a biomass productivity of 23 mg/L/d for Chlorella saccharophila.  Herrera-Valencia et al. [27] achieved biomass productivity of 154.3 mg/L/dfor Chlorella saccharophila.  Hempel et al. [32] noted a biomass productivity of 310 mg/L/d for the freshwater species Chlorella saccharophila.  Bondioli et al. [28] noted a biomass productivity of 237 mg/L/d for Tetraselmissuecica.  Moheimani [33] achieved a biomass productivity of 320 mg/L/d for Tetraselmissuecica. 

These findings agree with the findings of this study in which case the biomass yield and productivity obtained for the Tetraselmissuecica are higher than those obtained from the Chlorella saccharophila.  In this study the dry cell yield and biomass productivity for the Chlorella saccharophilaranged from 280.5 mg/L to 1579.45 mg/L and 28 mg/L/d to 154 mg/L/d, while those for Tetraselmissuecica species 1779.4-2991.5 mg/L and 177.9-299.2 mg/L/d, respectively.  These values are within the reported range in the literature. 

Effect of Carbon Dioxide Concentration

The effect of carbon dioxide concentration on the biomass yield of the marine and freshwater microalgae is illustrated in Figure 5.  As the carbon dioxide concentration increased from 3% to 9%, a gradual increase in cell yield for Chlorella saccharophila was observed.However, an increase in CO₂ concentration from 3 to 6% resulted in increased yield for the Tetraselmissuecica species, but a further increase to 9% resulted in decreased cell yields. 

Yue and Chen [39] reported that the freshwater microalgae Chlorella species resulted in an increase of 200% in algal growth rate when 1% CO₂ was used in the medium as opposed to the ambient air.  They also noted that higher concentrations of CO₂ resulted in a decline in algal growth as a result of increased acidity.  Nakano et al. [40]reported that the microalgae species Euglena gracilis exposed to varying concentrations of CO₂ (5-45%) grew best at 5% CO₂ while concentrations greater than 45% inhibited growth. Maeda et al. [41] noted that Chlorella sp. microalgae was capable of growing at 100% CO₂ concentrations, but the highest growth rate was achieved at 10% CO₂ concentration.  Hanagata et al. [42] reported that the algae Scenedesmus sp. can grow at 80% CO₂ concentration, but the maximum growth was achieved at 10-20% CO₂ concentration.  Studies performed by Ho et al. [43] and Jacob-Lopes et al. [44] found that excess CO₂ absorbed in the media has a negative effect on the microalgae growth. 

Kaewkannetra et al. [34] noted that an increase in CO₂ concentration from 5% to 15% resulted in an increase in biomass yield from 1.7 g/L to 2.3 g/L.  However, further increase in CO₂ concentration resulted in a decreased yield.  Yue and Chen [39] noted that the microalgae species Chlorella grown in CO₂ concentration over the range of 0.035-70% and noted the highest cell concentration of 5.9 g/L at 10%CO₂ concentration.  Ho et al. [43] and de Morris and Costa [45] noted that an optimum CO₂ concentration for the species Scenedesmus exists between 10% and 15%. 

Goswami et al. [35] noted that the microalgae Selenastrum sp. resulted in biomass productivity of 0.667, 0.889, 0.797 and 0.778 mg/L/d at carbon dioxide concentrations of 4,400, 5,200, 7,500 and 8,200 ppm, respectively.  Devgoswami et al. [46] noted that upon an increase in CO₂ concentration from 4,400 ppm to 4,758 ppm, the biomass productivity increased from 161 to 188%(g/L/d) while further increase in CO₂ concentration (to 7,929 ppm) showed a decrease of 163% for the green microalgae Chlorella.  Similarly, the Haematococcus and Scenedesmus species showed increased growth rates as the concentration of CO₂ was increased from 4,400 ppm to 4,758 ppm,but a further increase to 7,929 ppm resulted in lower growth rates. These findings as well as the findings of this study show that an optimal carbon dioxide concentration exists.

Microalgae Oil Content Using CO₂as a Carbon Source

The oil yield results are depicted in Table 6.  Analysis of the variance (ANOVA) was performed on the oil yield data as shown in Table 9.  The effects of microalgae type on oil yield were significant at the 0.003 level.  However, the effect of CO₂ concentration and the interactions between microalgae type and CO₂ concentration were not significant.  Tukey’s grouping was used to test the differences among the levels of each parameter as shown in Table 10.  The two microalgae Chlorella saccharophilaand Tetraselmissuecica were significantly different from one another at the 0.05 level.  The highest mean oil yield (4.18%)was obtained from the freshwater microalgae species.  The CO₂ concentrations were not significantly different from one another at the 0.05 level.  The highest mean oil yield (3.16%) was obtained with the 3% CO₂ concentration.

Effect of Microalgae Type

The effect of the microalgae type on the oil content is illustrated in Figure 7.  Chlorella saccharophilaachieved the highest oil yields at all CO₂ concentration.  It produced average oil yields of 4.71, 3.90 and 3.59% while Tetraselmissuecica produced average oil yields of 2.09, 1.01 and 2.43% at the 3%, 6% and 9% CO₂ concentrations, respectively.  These results are similar to those of Pittman et al. [47] which indicated that the marine microalgae produce much lower oil yields compered to freshwater microalgae.  The oil yields obtained from Chlorella saccharophilawere 4 times higher than those obtained from Tetraselmissuecica, despite the higher biomass yields obtained from the Tetraselmissuecicaspecies. 

Sharma et al. [48] stated that the occurrence and extent to which lipids are produced by microalgae is species/strain specific.  Pittman et al. [47] stated that different species use their energy for different metabolic pathways.  In this study a trade-off between cell generation and lipid accumulation was seen among the species.  The marine species used most of its energy for cell generation as opposed to lipid while the freshwater spices used most of its energy for oil accumulation as opposed to cell generation.

Demirbas [7], Moheimani, [33], Sobczuk et al., [49], Sukenik et al., [50], Wagenan et al., [51] and Pagnanelli et al., [52] reported a lipid content in the range of 36-47% and 15-23% for Chlorella saccharophilaand Tetraselmissuecicaspecies, respectively.  The differences in the lipid content are attributed to the different nutrient systems used and the culture age before harvest. 

Effect of Carbon Dioxide Concentration

The effect of carbon dioxide concentration on the oil yield is illustrated by Figure 8.  As the carbon dioxide concentration was increased from 3% to 9%, the oil yield decreased from 4.7% to 3.6%for the freshwater microalgae (Chlorella saccharophila).  On the other hand as the carbon dioxide concentration was increased from 3% to 6% the oil yield decreased from 2.1% to 1.0%for the marine microalgae (Tetraselmissuecica).  However, a further increase in CO₂ concentration to 9% increased the oil yield to 2.43%. 

It should be noted that the trends in Figure 9 are the opposite of the trends of cell yield shown in Figure 5.  The higher oil yield obtained for the freshwater microalgae at the 3% CO₂ concentration can be attributed to the trade-off of lower cell generation, and the lower oil yields obtained when the CO₂ concentration was increased to 9% is a result of increased cell division.  Similarly, the variation in lipid content for the marine (Tetraselmissuecica) microalgae species can also be attributed to the variation in biomass yield caused by variation in CO₂ concentration and the tolerance of the species to the acidity caused by higher CO₂ concentrations.  The results showed that from an economic stand point, the 3% CO₂ concentration is the most optimal condition for lipid accumulation in both species.  

Similar results were reported in the literature.  Widjaja et al. [36] noted that the microalgae species Chlorella vulgaris in lipid yields of 20%, 28% and 25% at the CO₂ concentration of 0, 0.33 and 0.83%, respectively.  Huang and Su [53] noted that for the microalgae species Chlorella vulgaris grown using 0%, 15% and 50% CO₂ concentrations resulted in lipid yields of 34%, 35% and 36%, respectively.  The findings are similar to those obtained in this study since they indicate that varying the CO₂ concentration does not significantly influence the lipid content.  The variation in oil yield can be attributed to the varying cultivation periods, variation in nutrient systems and the effectiveness of the oil extraction methods used.

Effect of Carbon Source

Biomass

Oil yield

The oil yields obtained with NaHCO₃ and 3% CO₂ as carbon sources are shown in Figure 9.  The oil yield achieved by Chlorella saccharophila 3% CO₂ as a carbon source (4.71%) concentration was significantly lower than that obtained using NaHCO₃ (12.91%) as the carbon source.  This suggests that sodium bicarbonate is the preferred carbon source for Chlorella saccharophila that prompts lipid accumulation in the cells.  Whereas, the marine Tetraselmissuecica species is better at utilizing the CO₂ as a carbon source for both the generation of cells and the accumulation of lipids.  Tetraselmissuecica resulted in higher cell and oil yields using the CO₂ carbon source as opposed to NaHCO₃.  However, the values are not significantly different from one another.  The media continuing CO₂ can result in pH fluctuations and cause conditions that are not suitable for proper cell function (lipid accumulation).  The presence of NaHCO₃ in the media helps regulate the pH so that the environment is not too acidic.   

Mukund et al. [57] tested Chlorella, Chlorococcum sp. and Desmococcus sp. and noted that the photosynthetic activity is strongly influenced by bicarbonate and that it results in increased lipid accumulation.  Nayak et al. [58] reported a lipid yield of 16% in Scenedesmussp IMMTCC-6 when it was supplied with CO₂ only, while addition of NaHCO₃ to the medium resulted in an increase in lipid yield to 22%.  Dhakal et al. [59] noted that the freshwater microalgae species Chlorella vulgaris produces a lipid yield of 22% using NaHCO₃ as the carbon source.  The oil yields obtained in this study were lower than those reported in the literature.  This may be attributed to the different cultivation parameters, cultivation growth period and the fact that different species respond differently to the nutrients supplied. 

CONCLUSION

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