Biodiesel Production From Algae Through Ultrasonication

University of Southern California, Undergraduate Symposium for Scholarlyand Creative Work, April 9-11, 2012Biodiesel Production from AlgaeUndergraduate Student Researchers: Avril Pitter, Kirsten Rice, and Kristen SharerGraduate Student Researcher: Stephanie OlagueFaculty Advisor: Professor Massoud PirbazariResearch Scientist: Dr. Varadarajan RavindranLaboratory Coordinator: Mr. Erik HernandezSonny Astani Department of Civil and Environmental Engineering

Introduction Algae fuel is a goodalternative to fossil fuels in atime of high oil prices,increasing energy needs, andcompetition between fooddemands and biofuel sourcesHarvest cycle of 1 to 10 daysDoes not require freshwater –can be produced using oceanor wastewaterBiodegradable and relativelyharmlessCan produce 300 times moreoil per acre than conventionalcrops, such as rapeseed,palm, soybean, and jatropha

Background of Algal Strains Chlamydomonas reinhardtii: Most widely used laboratoryspecies. Single celledchlorophyta. Grows in freshwaterand soil, and has a reported lipidcontent of about 21%.Haematococcus droebakensis Single celled chlorophyta. Growsin freshwater, and has a reportedlipid content of nearly 33%.Platymonas Single celled chlorophyta. Growsin freshwater, and has a reportedlipid content of kensisPlatymonas

Trans-esterification of TriglyceridesTrans-esterification is a chemical reaction between triglyceride and alcohol in the presence of acatalyst. It consists of a sequence of three reactions where triglycerides or triacylglycerides [TAGs]are converted at first to diglycerides and then to monoglycerides, eventually followed by theconversion of monoglycerides to glycerol (MacDougal et al., 2011). The esters corresponding to thechains R1, R2 and R3 are formed. Three moles of alcohol are stoichiometrically required for eachmole of triglyceride, but a higher molar ratio of five to six is often employed for maximum esterproduction depending on process conditions (Sharma et al., 2008). Some of the fatty acid methylesters (FAMEs) may be transformed to diesel hydrocarbons without oxygen species.MacDougall, K.M., McNichol, J., McGinn, P.J., O’Leary , S.J. B., and Melanson, J.E. (2011). Triacylglycerol profiling of microalgae strainsfor biofuels feedstock by liquid chromatography–high-resolution mass spectrometry. Analytical and Bioanalytical Chemistry, 401, 2609–2616.Sharma, Y.C., Singh, B., and Upadhyaya, S.N. (2008). Advancements in development and characterization of biodiesel: A review. Fuel, 87, 23552373.

Research Objectives Explore a cleaner alternative to petroleum diesel by extracting oilfrom algae and converting it into biodieselIncrease the efficiency and practicality of algae production byoptimization of important growth variablesCompare the growth rate and lipid content of different algalspecies under the same growth conditionsDetermine the chemical composition of the algae biodieselthrough gas chromatography

Experimental Techniques Bioreactor Studies: Three high-yielding algal species were tested for producingbiodiesel by photosynthesis --- Chlamydomonas reinhardtii, Haematococcus droebakensis,and Platymonas. The growth rates were determined by gravimetric methods under varyingconditions.Lipids Extraction Studies: The energy-rich lipids were extracted from the algae byultrasonication for the disruption of algal cells by cavitation at high frequencies(greater than 20,000Hz) using 1,2-dichloroemethane and methanol (2:1 vol.)according to modified Folch’s technique (Folch et al., 1957).Esterification: Chemical conversion by trans-esterification of algal oil from lipids tofatty acid methyl esters (FAMEs) and to biodiesel hydrocarbons.*Folch, J., Lees, M., and Stanley, G.J. S. (1957). A simple method for isolation of total lipids from animal tissues.Journal of Biological Chemistry, 226 (1), 497-509.Photosynthesis bioreactorsAlgal biomassUltrasonication for lipidsdisruption andextraction by phaseseparation

Experimental Techniques (continued) Analysis of important biodiesel products by gas chromatography (GC)and gas chromatography-mass spectrometry (GC-MS)Gas chromatography employing a Varian 3900 instrument equipped witha fame ionization detector, and a DB-5 capillary column (30 m x 0.32 mmx 0.25 µm), with temperature programming.Gas chromatography-mass spectrometry using a Bruker 400 GC incombination with a Bruker Daltonics MS-300 detector, equipped with aDB-5 capillary column for separation and identification of products.Gas chromatographyGas chromatography-mass spectrometry

Results The graph shows Haematococcus growthin Bold’s Basal Medium for 39 days.Overall growth between additions ofnutrients predominantly showedexponential and declining growthperiods. Note that more Bold's BasalMedium was added on days 18 and 32.Haematococcus droebakensis, was thebest algal species among the speciestested based on the results of the FAME(fatty acid methyl esters) analyses usinggas chromatography and massspectrometry techniques.Adding more nutrients before thestabilization of deathphases attained more algae for futureoil extraction (Day 32 was in growingphase).After ultrasonication, the lipids wereextracted by the Folchs method. The lipidcontent was 8.6% by weight.Haematococcus Growth in Bolds Basal Medium0.25Algae Mass (grams) 0.20.150.10.0500 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42Time (days)Haematococcus droebakensis growth in Bold’s BasalMedium*Day 13 results most likely errorGreen residuecontaining lipids

ResultsHaematococcus0.03ChlamydomonasAlgal Mass (grams)0.025Platymonas0.02 0.0150.010.00500246810121416Time (days)Growth patterns of Haematococcus droebakensis, Chlamydomonasreinhardtii, and Platymonas in Bold’s Basal Medium for 15 days.Overall growthbetween additions ofnutrientspredominantlydepicted exponentialand declining growthperiods.

Intensity and Peak HeightResultsRetention Time (min)Gas chromatography spectrum showing major organic compounds typically foundin algal biodiesel produced by the species Platymonas

Intensity and Peak HeightResultsRetention Time (min)Gas chromatography-mass spectrometry spectrum for identifying the majororganic compounds typically found in algal biodiesel produced by the speciesPlatymonas

ResultsIdentification of major components of biodiesel produced by the speciesPlatymonas from gas chromatography-mass spectrometry studiesRetentiontime (min)9.1910.6411.2211.6012.7214.2614.69CompoundCAS Number H50C26H54C28H58C30H62C31H64Molecularweight (Daltons)282310338366394422436

Summary and Conclusions Chlamydomonas reinhardtii manifested the fastest growth rate amongvarious algal strains.Platymonas yielded the most amount of diesel hydrocarbons per unitof biomass. Hence it requires more investigations to arrive ataccurate yields to chose the best algal strain.The oil or hydrocarbon content of the Platymonas species is about 3545% of the dry weight based on preliminary estimates. The otherspecies tested have lower yields at less than 15-20% of dry weight.The diesel hydrocarbons identified by gas chromatography-massspectrometry were higher molecular weight alkanes (predominantlyunbranched) in the C20 to C31 range. No esters were present insizable amounts as they were probably transformed into purehydrocarbons.More research is required to draw firm conclusions.

Future Research and Recommendations Test algal strains in greater detail to compare growth rate,lipid content, and hydrocarbon yields.Investigate carbon dioxide injection to observe kinetics ofbiomass growth and relation to possible carbonsequestration applicability.Study biomass growth at different temperatures and nutrientconcentrations.Improve ultrasonication process to increase lipid extractionefficiency.Evaluate the use of metallic oxides (barium and zirconiumoxides) and enzymes (lipase) to imrpove esterification andhydrocarbon yields.

AcknowledgementsWe greatly appreciate the guidance and help provided by Professor G.K.Surya Prakash (Director), Dr. Aditya Kulkarni and Dr. Thomas Mathew, LokerHydrocarbon Research Institute, Department of Chemistry, regarding the gaschromatography and mass spectroscopy studies for identification andquantification of biodiesel components.We express our sincere thanks to Professor Oscar Aparicio, ComputationalBiology Laboratories, Department of Biological Sciences, for his help in theultrasonication studies relating to the production of biodiesel.We gratefully acknowledge the partial funding for the project provided by theProvost Undergraduate Research Associates Program and the Viterbi MeritResearch Program of the University of Southern California.

phases attained more algae for future oil extraction (Day 32 was in growing phase). After ultrasonication, the lipids were extracted by the Folchs method. The lipid content was 8.6% by weight. 0 0.05 0.1 0.15 0.2 0.25 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Algae Mass (grams) Time (days) Haematococcus Growth in Bolds Basal .