This document summarizes a capstone project presentation on maximizing the lipid yield of Scenedesmus dimorphus for biodiesel production. The project aims to determine if CO2 improves algae growth and lipid content, and to optimize lipid content by varying urea concentration and nutrient deprivation time. Experiments are proposed to cultivate algae with and without CO2, under different urea levels, and to extract lipids, produce biodiesel via transesterification, and analyze results. The literature review covers algae biodiesel production and factors that influence lipid yield like temperature, nutrients and nitrogen limitation.

1. Polythecnic University of Puerto Rico Chemical Engineering Department Course – CHE- 5916 Capstone Project Presentation Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel Group: Sara Currás Medina Gustavo Mendez Santos Carlos A. Ramos Encarnación Germano Salazar Benites Advisor: Dr. Alessandro Anzalone Date: July 24, 2009

2. Acknowledgment Prof. Sylvia M. VélezVillamil Biology Department University of Puerto Rico at Humacao Prof. Edgardo González, Ph.D. Bureau of Forest Services, Director Department of Natural and Environmental Resources Alessandro Anzalone, Ph.D. – Advisor Chemical Engineering Department, Director

3. Agenda

4. Problem Statement

5. Introduction

6. Introduction: Algae Fuel Introduction Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

7. ResearchDescription

8. ResearchObjectives Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

9. Research Contributions Determine if the use of CO2 during algae cultivation is beneficial to its growth and lipid content. Maximize the lipid content varying the urea concentration and nutrient depravation time. Detailed documentation of the process.

10. Literature Review

12. Grows practically anywhere, ensuring no competition with food crops.

13. Excellent bioremediation agents - they have the potential to absorb massive amounts of CO2 and can play an important role in sewage and wastewater treatment.

15. Microscopic (µm)

16. Saline or fresh water

17. Grow extremely quickly

18. Large amounts of lipids within their cell structure

19. Multicellular

20. Macroscopic (up to 60 m)

21. Saline or fresh water

22. Grow extremely quickly

24. Microalgae Strain Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

25. Scenedesmusdimorphus

26. Production of Lipids from Microalgae Light (Photons) CO2 O2 Microalgae (Photosynthetic CO2 fixation) Nutrients (N, P, Si) Biomass (carbon) Lipid storage Carbohydrate storage

27. Cultivation

28. Cell growth

29. Temperature

31. CO2 Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

33. Contributes to the biomass production.

34. Influence on algae lipid yield

35. Response to nitrogen limitations, mechanism of survival, increase lipid content.

37. Each strain prefers different source.

38. Kansas State University

39. Best source for Scenedesmusdimorphusgrowth.

41. Flocculation

42. Flocculation

43. Extraction

44. Lipid Extraction Concentrated Algae Cell Disruption Mechanical Chemical Press Solvents Filtering Distillation Algae Oil Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

45. Lipid Extraction - Solvents

46. Transesterification

47. Transesterification Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

48. Transesterification Reaction

50. Proposed Experiment Co2 vs. non Co2 Algae Culture Daily Cell Count N Measure After 17 days Biomass Wt. Lipid Wt. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

51. Cell Count

52. Nitrogen Measure

53. Biomass and Lipid Weight Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

54. Proposed Experiment Optimal Lipid Yield Algae Culture 1.2, 1.8, 2.4 g/L Urea Daily Cell Count N Measure After N Consumption Biomass Wt. Lipid Wt. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

55. Cell Count

56. Nitrogen Measure

57. Biomass and Lipid Weight Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

58. Proposed Experiment Biodiesel Production Algae Culture Daily Cell Count After a fixed period Process the oil Obtain Biodiesel Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

59. Biomass and Lipid Weight Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

61. Stock Culture Prepare the Stock Culture (SC) in a beaker of 1000mL. 1. algae 1000mL SC

62. Stock Culture Leave the algae to grow and reproduce for about 7 days before using it. 12:12 2. Agitation SC 90°F Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

63. Growth Media Prepare the Growth Media(GM) in an Erlenmeyer of 1000mL. 1. GM

67. Measuring Nitrogen Concentration Spectrophotometer 250mL Culture Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

75. Measure the weight of the sample.

76. Compare the weight of the dry lipids vs. the dry biomass. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

78. Biodiesel Measure the growing rate every day. 1. Measure the nitrogen consumption every day. 2. 90°F When the culture is prepared to be processes, flocculate the sample 3. 12:12 With a 1.5µm filter , filtrate the sample using a vacuum. 4. For every gram of biomass add 18 ml of a mixture of Hexane/Isopropyl (3:2) 5. After agitation, filtrate the sample again. 6. Separate by distillation the solvent from the oil. 7. CO2 or Air

79. Transesterification 1. Add the extracted oil to a 1 liter flask. 2. In another flask mix KOH with ethanol 3. Heat the ethanol to dissolve KOH if needed. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

80. Transesterification 4. Mix KOH and ethanol blend into algae oil and agitate vigorously. 5. After 120 minutes of reaction time, allow time for separation. The mixture will separate into two layers biodiesel on top, glycerin on bottom. Biodiesel Glycerin 6. Separate the biodiesel on another flask. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

81. Transesterification 8. Allow 24-48 hours for water to settle, biodiesel will float to the top and become clearer. 7. Place the biodiesel in a glass column and spray water into the top.. Biodiesel Water 9. Separate the biodiesel from water. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

82. References “Oil Crisis”. Health and Energy. 13 July 2009 <http://healthandenergy.com/oil_crisis.htm>. Sheehan, John, Terri Dunahay, John Benemann, and Paul Roessler. A Look Back at the U.S. Department of Energy’s Aquatic Species Program—Biodiesel from Algae. Colorado: NationalRenewableEnergyLaboratory, 1998. “Scenedesmus Dimorphus-Algae Culture”. Algae Depot. 2009. 12 June 2009 <http://www.algaedepot.com/servlet/the-1/Scenedesmus-dimorphus--dsh--Algae/Detail>. “Kunikane. S, M. Kakeko, and R. Maehara. Growth and Nutrient Uptake of Green Alga, Scenedesmus Dimorphous, Under a Wide Range of Nitrogen/Phosphorous Ratio-I. Setsunan, Japan: University of Setsunan, 1984. Shen, Ying, Zhijian Pei, Wenqiao Yuan, and Enrong Mao. Effect of Nitrogen and Extraction Method on Algae Lipid Yield. Kansas State: University of Kansas State, 2009. Tzann, Stelios T. “Non Mechanical Methods”. Tutorial on Cell Disruption. 3 June 1996. 7 July 2009 <http://128.113.2.9/dept/chem-eng/Biotech Environ/DOWNSTREAM/disrupt.htm>. Maximization of ScenedesmusDimorphusLipid Yield for the Production of Biodiesel

83. Thanks for listening!