The growing demand for the use of fossil fuels in various industries have been reported as the major factor causing high CO2 levels in the atmosphere resulting in global warming1. The world is presently confronted with the crisis of fossil fuels depletion and environmental degradation and it is estimated that in the next 50 years, fossil fuels must have been exhausted2. Therefore, biofuels becomes an alternative fuel option for industries and transportation.
Microalgae are photosynthetic micro-organisms that require light energy and carbon dioxide for the production of high value compounds ranging from carotenoids to polyunsaturated fatty acids3,4. Microalgae may be destined to different applications, such as; biofuel production and wastewater purifications5. Biofuel production can take place either under autotrophic or mixotrophic conditions. Microalgae are known as “efficient solar energy converters” that can produce a great variety of metabolites6. Also, the high productivity of microalgae justified by its shorter generation time and higher oil content compared to crops is a clear sign of how significantly microalgae can contribute to the large scale production of biofuel7.
Microalgae have been studied as alternative fuel sources that is renewable, economical and environmentally friendly8. Biodiesel production from microalgae is an emerging technology considered by many as a very promising source of energy, mainly because of its competition for land in the generation of biofuels, as some species contain up to 60% of overall mass by fatty acids or lipids. This oil from algae can be extracted, processed and converted into biodiesel used as transportation fuel using current available technology. Biodiesel from algae lipids is highly degradable and non-toxic9. Microalgae like higher plants produced storage lipids in the form of triacylglycerols (TAGs), although triacylglycerols could be utilized in producing a wide range of chemicals. Biodiesel can be synthesized from triacylglycerols through a simple transesterification reaction in the presence of methanol. Biodiesel can be used in unmodified diesel engines and it has series of advantages over the conventional diesel fuel, because it is biodegradable, renewable and produces less sulphur compounds and particulate emissions when burned10. This study was carried out to produce a biodegradable oil that will serve as an alternative source to replace the fossil fuels which are known for their negative impact on the environment. This study was aimed at producing an affordable biodiesel from Chlorella vulgaris as well as analyzing the qualities of the biodiesel produced. Fuel produced from this alga will serve as alternative fuel that can replace the fossil fuels due to its less negative impact on the atmosphere.
MATERIALS AND METHODS
Sampling site: The study was carried out from June-November, 2015. Sampling of water was carried out in Kano Metropolis from Sharada industrial run-off waste waters. This pond is located at 8.521°N and 11.981°E. Water in this pond was traced to have resulted from the Sharada Industrial Estate, where industrial wastes were deposited into the water. The pond measures about 220 m long and 50 m wide.
Sample collection: Water samples were collected using phytoplankton sample bottles as described by Indabawa11.
Isolation and identification of microalga: The study was carried out in the Laboratory of the Department of Botany, Bayero University, Kano, Nigeria. The identification of the microalga was done as described by Anaga and Abu12 using standard psychological keys, charts and illustrations by Hilary and Erica13 and Marvin14. Chlorella vulgaris was isolated from the water samples, this was achieved by repeated sub-culturing of the microalga on the culture media as described by Agwa et al.15 and Makoto16. In this method, single-cell isolation by micropipette was employed, where glass capillary micropipettes was used for the isolation. A light microscope equipped with a binocular lenses and a graduated stage was used for the identification and isolation procedures.
Preparation of the culture media: The culture media was used for culturing process of the microalga BG-11 media. The media was prepared in the Plant Biology Department laboratory of Bayero University Kano.
Purification of microalga: The two paths of purification methods adopted in this research were physical separation of the Chlorella vulgaris microalga from contaminants and the use of antibiotics to kill contaminants such as; bacteria and viruses.
Antibiotic treatment: The antibiotic solution was prepared as described by Guillard17. In this method, 100 mg of penicillin, 25 mg of dihydrostreptomycin sulfate and 25 mg of chloramphenicol was dissolved in 10 mL of distilled water and sterilized by filtration.
Culturing and sub-culture of the microalga: Chlorella vulgaris was cultured, revived and maintained in BG-11 media using a 1000 mL capacity flask and a designated 10 L capacity photo-bioreactor as described by Ripka et al.18 and Anderson19 employed by Indabawa11. The sub-culture of the microalgal sample was achieved by serial dilution, where the initial 1 mL of the sample was enriched with 9 mL of BG-11 media and was allowed to stand for 14 days. The cell was in the log phase of its growth during these days. The cells were exposed to a light period of about 8 h as described and employed by Indabawa11.
Scaling up of microalgal culture: The microalgal cells were pre-cultivated in 100 mL liquid medium in a 250 mL Erlenmeyer flask with BG-11 media on a shaking platform at a rotation speed of 100 rpm. The pre-cultivated microalgal cells were used as inoculums for 10 L capacity photo-bioreactor cultivation. All cultivations were illuminated under a light intensity of 35 μ mol1 m2 sec1 at a cycle of 8 h light and dark cycle at 23±2 for 20 days. The culture was sub-cultured again into 1000 mL Erlenmeyer flask, where 100 mL of the culture was placed into 900 mL. At this juncture, the culture apparatus was designed in order to introduce aeration of 8 h as at the same time it was exposed to 8 h photoperiods. The cell density of the microalgal growth rate was taken daily by measuring the optical density of the algal culture at 540 nm using a spectrophotometer (Spectronics D20) as described by Anaga and Abu12.
Measuring of microalgal growth: The growth pattern or growth rate of the microalgal culture was measured by optical density (OD) at 540 nm, during the cultivation period with visible ultraviolet spectrophotometer as employed by Indabawa11.
Harvesting of microalgal biomass: Chlorella vulgaris was harvested by flocculation method as described by Vandamme et al.20.
Lipid extraction from the microalga: Lipid was extracted from the microalgal biomass by solvent extraction method21.
Purification of the microalgal oil: Lipid extracted from the microalga was purified by distillation method as employed by Chen et al.22.
Production of fatty acid methyl ester (FAME): Transesterification of the lipid was carried out according to the method employed by Indhumathi et al.23.
Washing and drying of biodiesel: It was noticed that the biodiesel obtained as a result of the transesterification of the biodiesel process of the microalgal oil contained some small amount of methanol, glycerin, soap, catalyst and other impurities. In order to remove the unwanted materials from the oil, water was warmed to about 45°C and was passed through the esters to allow all the soluble materials to stick to the bottom of the vessel. Water was then removed from the vessel periodically until the pH of the biodiesel became relatively neutral. The biodiesel was afterwards still looking cloudy which was an indication of the presence of water. To get rid of the water, it was then heated to a temperature of 100°C until all moisture was completely removed23.
FT-IR spectrum analysis of the biodiesel: The NEAR FT-IR machine was used for the analysis of the oil extracted from the microalgal Chlorella vulgaris. The stage of the FTIR machine was thoroughly cleaned using a cotton bud that had been dipped in petroleum ether. The machine was then test run to ensure no impurities remained on the stage. The sample was then loaded onto the stage when the machine had finished the test running process and was declared ready for use as displayed on the computer screen. A drop of the sample was loaded on the equipment by the use of a clinical syringe. The peak values of the analysis were marked and the reading was recorded accordingly11.
Physico-chemical characterization of the microalgal oil: Physico-chemical properties of Chlorella vulgaris biodiesel (Iodine value, acid value, saponification value, flash point, fire point, cloud point, pour point, density, refractive index and pH) were determined in order to compare the biodiesel produced with standards.
Determination of iodine value [ASTM D974(01)]: Iodine value [ASTM D974 (01)] was achieved using the method employed by Indabawa11.
Determination of acid value and free fatty acid value (ASTM D974): About 2 g of oil was measured and poured in a beaker. About 50 mL of a neutral solvent prepared by the mixture of ethanol and petroleum ether was taken and poured into the beaker containing the oil sample. The resulting mixture was stirred vigorously for 30 min. About 0.56 g of potassium hydroxide pellet was measured and placed in a conical flask and 0.1 M potassium hydroxide was prepared, 3 drops of phenolphthalein indicator was added to the sample and was titrated against 0.1 M potassium hydroxide until a pink coloration was observed. The acid value (AV) was calculated using the following relations as employed by Indhumathi et al.23:\
|| Volume of standard alkali used
|| Normality of standard alkali used
|| Weight of oil used
Determination of saponification value (ASTM 5558-95): A freshly prepared solution of alcoholic potassium hydroxide was made by dissolving potassium hydroxide pellet in ethanol. About 2 g of oil was measured and poured into a conical flask. About 25 mL of the ethanolic potassium hydroxide was then added to it. Blank solution was used as well. The sample was covered and placed in a steam water bath and was allowed for 30 min, while shaking the sample periodically, 1 mL of phenolphthalein indicator was added to the mixture.
The mixture was titrated against 0.5 M Hydrogen chloride (HCL) and the titer value was noted. The saponification value (SV) was then obtained using the following relations according to Indhumathi et al.23:
|| Volume of HCL used in titration with the blank
|| Volume of HCL used in titration with the oil
|| Weight of oil used
|| 2 g
|| 192.61 mg g1
Determination of flash point and fire point (ASTM D6751): Flash point is the temperature at which a combustible mixture can be formed above the liquid fuel. An ignition source is required to determine a flash point. An ignition was placed on measured microalgal oil in an open flame. A thermometer was placed in order to record the temperature changes. When the source of ignition was removed, the vapor ceased to burn and this temperature was recorded as the flash point. The flash point was measured as the temperature for which the vapor continued to burn for at least 5 sec after the source of ignition was removed at an open flame11. The fire point was assumed to be almost 10°C higher than the flash point. The standard flash point is between 130-205°C.
Determination of cloud point (ASTM D2500): A sample of the biodiesel was placed in a test tube and then placed in a cooling bath after it had been heated to about 40°C. When the biodiesel started to form cloud below the test tube, its temperature was quickly measured and taken as the cloud point. This procedure was repeated 3 times and the mean value was recorded23.
Determination of pour point (ASTM D97): A sample of the biodiesel was placed in the freezer for 24 h. The biodiesel was removed after the period and placed in a beaker containing warm water of about 10°C to melt. The temperature at the bottom of the test tube at which the biodiesel starts to pour is recorded as the pour point of the biodiesel11.
Determination of specific gravity/density (ASTM D1298) by hydrometer method: This procedure was used by Indhumathi et al.23 to evaluate the specific gravity of the biodiesel.
Determination of refractive index: This was done using an Abti refractometer as employed by Indabawa11.
Determination of pH: A portable pH meter was used to determine the acidity or basicity of the oil.
Statistical analysis: Percentage abundance was calculated for the alga species, mean was calculated for the weight of algal biomass and volume of biodiesel obtained. Plot of the optical density was done using Microsoft Excel version 2013.
Species of alga isolated from sharada industrial wastewater: Microalgae sampled from the water include members from three classes of microalgae; Class Chlorophyceae: Spirogyra sp., Scenedesmus sp., Chlorella sp., Chlamydomonas sp., Zygnema and Volvox sp. Class Diatoms: Nitzschia sp. and Class Cyanophyceae: Anabaena sp. and Oscillatoria sp. (Table 1). The frequency of the algae species varies with Oscillatoria sp. and Spirogyra sp. having the highest occurrence of 20%.
Alga biomass production: There was no significant difference (p>0.05) in the weight of the algal biomass produced from C. vulgaris for a period of 21 days of culturing, the highest biomass obtained for the period was 50 g while the least was 49 g.
Chlorella vulgaris growth curve in BG-11 media over 21 day period
Algae species present in the water in order of abundance (%)
Weight of algal biomass and the volume of biodiesel obtained from C. vulgaris in 21 days
Physico-chemical characterization of Chlorella vulgaris biodiesel, test limit and ASTM standards
Also, no significant difference (p>0.05) was observed in the volume of biodiesel produced from C. vulgaris for the period of 21 days which ranges from 5.011-5.211 mL from approximately 50 g of C. vulgaris biomass (Table 2).
Optical density: The optical density of the microalgal culture was estimated using the spectrophotometer (Spectronics D20) for 25 days and the measurement was initiated from the 5th day after inoculation. The growth curve of the optical density (absorbance) was plotted against the number of days as indicators of the growth (Fig. 1). Rapid growth rate was observed from day 8 through day 9 from 0.25-0.6 wavelengths, respectively with the highest growth rate on day 20 and 21 (1.32 cm).
FT-IR characterization of biodiesel: The NEAR FT-IR characterization of the biodiesel indicated highest peak region of 1745 wavelength (cm1) at 50.922 absorbance followed by 2924 cm1 at 55.190 absorbance which was as a result of C-H stretch. Carbonyl stretch C=O of esters appears however; as aliphatic from 1750-1735 cm1. The peak band of 3010 cm1 was olefinic =C-H stretch. The bands between 600-1200 cm1 indicated the presence of cis-alkene. The peak 2855 was formed as a result of C-H stretch. C–H bend or scissoring from 1470-1450 cm1 indicated the presence of saturated hydrocarbons (alkanes). The peak 1380 indicated C-H rock methyl. The band 724 indicated the presence of long chain methyl bonds while the peak 1100 indicated the presence of unsaturated C–O bond (Fig. 2).
FT-IR spectrum analysis of the Chlorella vulgaris biodiesel
Physico-chemical qualities of biodiesel: The 11 physico-chemical qualities of biodiesel produced from C. vulgaris all fell within the ASTM acceptable limit with saponification value of 192 mg KOH g1, an acid value of 0.64 mg KOH mg1 and iodine value of 85 mg g1 mI2. Also, the biodiesel had a refractive index of 10, fire point of 105°C and pour point of 4°C. A pH of 7.2 was obtained while ASTM value is 7.0 and density at 40°C was obtained to be 848 g mL1. Cloud point of +2°C was observed while specific gravity at 40°C was measured to be 0.855g cm3 with a flash point of 115°C (Table 3).
About 9 species of algae were found in water samples collected from Sharada Industrial wastewater, Kano. These species are Spirogyra sp., Scenedesmus sp., Chlorella sp., Chlamydomonas sp., Zygnema sp., Volvox sp. Nitzschia sp. Anabaena sp. and Oscillatoria sp.
A checklist of the optical density of the C. vulgaris growth obtained which showed that the lowest absorbance observed at day 4 and 5, respectively was as result of the initiation stage for which the algal cells starts to adapt the media environment in which they were introduced. Optimum culture temperature used in this study yielded maximum biomass and this was in agreement with findings of Converti et al. 24, who stated that Chlorella vulgaris showed a promising growth in the designed photo-bioreactor from 5-23 days with tangible biomass obtained from the batch cultures, this biomass was higher than the 41.64 g observed by Al-lwayzy et al.9, who produced biodiesel in laboratory scale from C. vulgaris in freshwater body for diesel engine. This indicated that C. vulgaris from non-sterile ponds contain high amount of lipids. The high amount of lipids suggested that C. vulgaris has good potential to be used as an economically viable sources of renewable oil and biodiesel production. This was in conformity with the findings of Al-lwayzy et al.9 and Senthil et al.25, while Converti et al.24 declared that high lipid productivity of C. vulgaris depends on the culture conditions such as; optimum temperature of 25-30°C and increased CO2 concentration.
The presence of biodiesel was detecting using the Fourier transform infrared spectroscopy (FT-IR). The NEAR FT-IR characterization of the biodiesel indicated peak region at 1745 cm1, this was due to the presence of carbonyl ester. This indicated the presence of aliphatic aldehydes which however was in accordance with the findings of Cortes26. Ester was however present in the biodiesel, the ester present is palmitoleic acid (C17H32O2). Another peak value of 2924 cm1, which was as a result of C-H stretch at 2924, alkyl was therefore suspected to be present. The values ranging from 2000-2500 cm1 indicated the presence of unsymmetrical internal alkynes (4-octyne). The peak band of 3010 cm1 was due to olefinic = C – H stretch. This indicated the presence of an aromatic compound or alkene while the bands between 600 and 1200 cm1 indicated the presence of cis-alkene. The peak 2855 was formed as a result of C-H stretch. This was suspected to be a carbonyl compound (ketone, H–C=O stretch 2830-2695 cm1) as a result of unsaturated C-H bond. This was also confirmed by Barbara27. The 1162 band was due to C-O stretch and this indicated the presence of unsaturated fatty ester oil27.
The saponification value was in agreement with standard value for biodiesel set by the American System for Testing Materials (ASTM D). The acid value of the biodiesel obtained was within the ASTM range, but higher than an acid value of 0.4 g obtained by Vijayaraghavan and Hemanathan28 from freshwater algae and 0.37 mg KOH g1 from Chlorella protothecoides in the study by Xu et al.29. Iodine value also fell within the ASTM range while the fire point obtained was lower than the ASTM D7651 value of 140-215°C. The pour point in this study was in agreement with the ASTM D97, but higher than the pour point observed by Xu et al.29 in biodiesel produced from Chlorella protothecoides. The pH observed in this study was slightly higher than the ASTM standard of 7.0 for biodiesel. Density and specific gravity were in conformity with ASTM D6751 standard. The flash point obtained in this study was similar to the previous findings observed by Xu et al.29 with a flash point of 115°C for Chlorella protothecoides, but higher than flash point of 98°C obtained by Vijayaraghavan and Hemanathan28. The flash point in this study was lower than the ASTM standard of 130°C. The result indicated that majority of the physico-chemical properties of biodiesel produced from Chlorella vulgaris were in line with the ASTM standard except for the flash point that was 10% lower and the pH which was slightly higher. This indicated that the transesterification process was approximately complete and the quality of the C. vulgaris biodiesel obtained from this work was up to standard. Studies on the cost implication of producing biodiesel from Chlorella vulgaris will help provide information for policy makers in the oil industry as this study did not capture that aspect.
CONCLUSION AND RECOMMENDATION
Chlorella vulgaris was isolated from industrial run-off waste waters. C. vulgaris produced biodiesel with high yield per algae biomass as about 50 g of C. vulgaris biomass generated 5.1 g of lipid which was converted to biodiesel from simple transesterification reaction. FT-IR spectrum revealed that C. vulgaris lipids were converted to fatty acid methyl ester (FAME) at 1745, while the standard value for ester bond formation was obtained at 1744. BG 11 media is recommended for biomass production of Chlorella vulgaris. FTIR is recommended for easy and accurate characterization of biodiesel. Chlorella vulgaris is recommended for use as it is economical and viable sources of renewable oil and biodiesel production.
This study discovered that Chlorella vulgaris biomass can be used to produce an economical and biodegradable quality biodiesel. BG 11 media is the best media for the production of Chlorella vulgaris biomass. This study will help researchers in the transport industry uncover the critical areas for mass production of biodiesel from Chlorella vulgaris.
The authors appreciate the laboratory staff, Department of Botany, Bayero University, Kano.