1. Developing New Working Protocols and Bioreactor Designs to Enhance Biomass Growth and Energy Yield in Schizochytrium limacinum and C.reinhardtii Jeffrey Yau and Christina George Manhasset Science Research

2. Background The use of bioreactors to combat the growing problem of greenhouse gases has been extensively studied in recent decades. The use of bioreactors to combat the growing problem of greenhouse gases has been extensively studied in recent decades. (Chisti, 2007) http://www.ieagreen.org.uk/newsletter/dec80/images/biofixation.JPG

3. The U.S. has reported a 3.3% increase of carbon dioxide emissions in the past year Mostly due to car emissions and industrial factories. (Hopwood, 2007) http://photos.mongabay.com/08/0423methaneglobal.jpg The red line shows the trend together with seasonal variations. The black line indicates the trend that emerges when the seasonal cycle has been removed. Graph 1

4. Raceway Pond Design A raceway pond is made of a closed loop recirculation channel (Chisti, 2007) http://www.agric.wa.gov.au/content/SUST/BIOFUEL/110407_Biodieselfrom microalgae.pdf Original Design Modified Design Taken: January 17, 2009

5. Tubular Photo Bioreactor Design Consists of straight transparent tubes either made out of glass or plastic (also known as solar collectors) Consists of straight transparent tubes either made out of glass or plastic (also known as solar collectors) (Chisti, 2007) http://www.agric.wa.gov.au/content/SUST/BIOFUEL/110407_Biod ieselfrommicroalgae.pdf Original Design Taken: January 17, 2009 Modified Design

6. Control: Airlift Design A self-contained bioreactor A self-contained bioreactor Utilizes a baffle to re-circulate the bacteria in suspension Utilizes a baffle to re-circulate the bacteria in suspension Taken on: December 21, 2008

7. Organism: S.limacinum Contains pigments for photosynthesis Contains pigments for photosynthesis Known to contain EPA, DHA, and omega-3 fatty acids Known to contain EPA, DHA, and omega-3 fatty acids Reliable source of oil production for biodiesels Reliable source of oil production for biodiesels (Kamlangdee, 2003) http://roweb.cityu.edu.hk/researchreport/2002-2003/Project/020.jpg

8. Organism: C.reinhardtii Contains an enzyme called hydrogenase that allows creation of hydrogen Contains an enzyme called hydrogenase that allows creation of hydrogen (Tiede, 2008) Ability to produce hydrogen under anoxic conditions Ability to produce hydrogen under anoxic conditions (Fouchard, 2005) http://en.wikipedia.org/wiki/Chlamydomonas_reinhardtii Magnified 3000X

9. Tubular Photo Bioreactor design for algal cultures Molina Grima; et al (2001): Experiment on tubular photobioreactors using P. tricornutum Molina Grima; et al (2001): Experiment on tubular photobioreactors using P. tricornutum Tested tubular diameter on the amount of sunlight that penetrated through the culture broth Tested tubular diameter on the amount of sunlight that penetrated through the culture broth

10. Polyunsaturated fatty acids production by Schizochytrium sp. Kamlangdee (2003): Experiment on polyunsaturated fatty acids production by Schizochytrium sp. Kamlangdee (2003): Experiment on polyunsaturated fatty acids production by Schizochytrium sp. Found single isolate reliable in production of DHA Found single isolate reliable in production of DHA

11. Hydrogen as Clean Fuel Via Continuous Fermentation by Anaerobic Photosynthetic Bacteria, Rhodospirillum rubrum Observed the effect of light intensity, agitation, and liquid dilution rate on hydrogen production Observed the effect of light intensity, agitation, and liquid dilution rate on hydrogen production Use of biocatalyst can be considered alternative to Fischer Tropsch synthetic reactions Use of biocatalyst can be considered alternative to Fischer Tropsch synthetic reactions (Najafpour, 2003) http://www.vurup.sk/pc/vol45_2003/issue3-4/pdf/14.pdf

12. Purpose Therefore the purpose of this experiment was to create a bioreactor design that would enhance growth rate and energy yield in Schizochytrium limacinum and C.reinhardtii Therefore the purpose of this experiment was to create a bioreactor design that would enhance growth rate and energy yield in Schizochytrium limacinum and C.reinhardtii

13. Hypothesis Null Hypothesis: No significant difference will be found in the growth of C.reinhardtii and S.limacinum in either bioreactor. Null Hypothesis: No significant difference will be found in the growth of C.reinhardtii and S.limacinum in either bioreactor. Alternate Hypothesis: The growth of C.reinhardtii and S.limacinum will be greatest in the Tubular Photobioreactor when exposed to carbon dioxide, with respect to pH levels. Alternate Hypothesis: The growth of C.reinhardtii and S.limacinum will be greatest in the Tubular Photobioreactor when exposed to carbon dioxide, with respect to pH levels.

14. Methodology Problem: What is the most efficient design for a photo bioreactor to enhance the energy yield and growth rate of Schizochytrium limacinum and C.reinhardtii? Control Group: -Growth medium under normal conditions. -Growth rate in Airlift Bioreactor Independent Variable 1: Growth of Schizochytrium limacinum Dependent Variables: -Oil Extracted from Hexane -Hydrogen collected from C.reinhardtii -Three different sized tubes (0.01m, 0.012m, 0.019m) An ANOVA test will be used to statistically analyze the data (p<.05). The Scheffe post hoc test will be used. Schizochytrium limacinum will be obtained from atcc.org. The Glucose Yeast Extract Medium will contain 1g of Yeast Extract, 1g of Peptone, 5.0g of Glucose, and 1L of 15% Natural Seawater in a 1000mL Volumetric flask. The yeast extract, glucose, and the Natural Seawater will be obtained from Carolina Biological. Peptone will be obtained from Flinn Scientific. Growth Projection: 4 weeks Independent Variable 2: Growth of C.reinhardtii Photo Bioreactor Measurement of Biomass yield (once daily from start of experimentation) using Aquafluor Fluorometer C.reinhardtii will be obtained from Carolina, the Biological Vendor. The C.reinahrdtii will be cultured in a fresh water tank, with a 12 hour supply of light, and oxygen. The algae will be kept at 70 degrees Fahrenheit and cultured with a 10% Algal growth medium. Raceway pond Measurement of Biomass yield using Spectrophotometer (Wavelength at 610nm) Measurement of the effect of carbon dioxide on pH levels (Using Co2 Sensor on GLX and pH paper)

15. Discussion A Tubular Photobioreactor is a suitable environment for growth of algae A Tubular Photobioreactor is a suitable environment for growth of algae Performance of the Tubular Photobioreactor surpassed the control bioreactor Performance of the Tubular Photobioreactor surpassed the control bioreactor Daily exposure to carbon dioxide did not greatly effect pH levels in bioreactors Daily exposure to carbon dioxide did not greatly effect pH levels in bioreactors Growth in Tubular Bioreactor was greater than growth in the Raceway Pond possibly due to more efficient pump Growth in Tubular Bioreactor was greater than growth in the Raceway Pond possibly due to more efficient pump

16. Conclusion Data supports the Alternate Hypothesis Data supports the Alternate Hypothesis The Tubular Photo Bioreactor demonstrated a greater amount of growth as compared to the Raceway Pond The Tubular Photo Bioreactor demonstrated a greater amount of growth as compared to the Raceway Pond There was no significance regarding the Carbon Dioxide levels when pumped through the Tubular, Raceway Pond and Airlift Bioreactors There was no significance regarding the Carbon Dioxide levels when pumped through the Tubular, Raceway Pond and Airlift Bioreactors

17. Limitations Errors while using the Spectrophotometer occurred, causing incongruous data: Possibly caused by cuvette or contaimination Errors while using the Spectrophotometer occurred, causing incongruous data: Possibly caused by cuvette or contaimination Possible errors in GLX Xplorer readings for carbon dioxide Possible errors in GLX Xplorer readings for carbon dioxide Possible bacterial contamination in bioreactors Possible bacterial contamination in bioreactors

18. Future Studies Revision of Tubular Photobioreactor and Raceway Pond designs Testing various tube diameters Using grown C.reinhardtii and S.limacinum from bioreactors to extract hydrogen and oils, respectively, to test energy content

19. Select Bibliography “Algae Could One Day Be Major Hydrogen Fuel Source.” Science Daily. April 2, 2008. Belarbi, E-H; Molina, E; Chisti, Y. “A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil.” Enzyme and Microbial Technology. Vol. 26, Pgs. 516-529., 2000. Biello, David. “Combating Climate Change: Scaling Back Greenhouse Gas Emissions While Keeping the Lights On.” Scientific American. May 8, 2007. Biello, David. “Pollution-Free Hydrogen SUV Hits the Driveway.” Scientific American. March 20, 2008. Brown, Kenneth. “Producing Renewable Hydrogen From Biomass.” BioCycle. Pg. 54, Vol. 45., January 1, 2004. Bullis, Kevin. “Algae-Based Fuels Set to Bloom.” Technology Review. February 5, 2007. Chisti, Yusuf. “Biodiesel from Microalgae.” Biotechnology Advances. Vol. 25, Pgs. 294-306. February 13, 2007. Chisti, Y; Moo-Young, M. “Clean-in-place systems for industrial bioreactors: design, validation, and operation.” Journal of Industrial Microbiology. Vol. 13, Pgs. 201-207., 1994. Gavrilescu, M; Chisti, Y. “Biotechnology- a sustainable alternative for chemical industry.” Biotechnology Advances. Vol. 23, Pgs. 471-499., 2005. Ghirardi L, Maria; et al. “Cyclic Photobiological Algal H2-Production.” Proceedings of the 2002 U.S. DOE Hydrogen Program Review. Colorado, 2002. Hatcher G, Patrick. “Algae Can Help Us Move Past Ethanol’s Problems.” Richmond Times-Dispatch. March 23, 2008. Kamlangdee, N; Fan, K.W. “Polyunsaturated fatty acids production by Schizochytrium sp. Isolated from mangrove.” Songklanakarin J.Sci. Technol. 25(5): 643- 650., 2003. Knothe, Gerhard. “Analyzing Biodiesel: Standards and Other Methods.” JAOCS. Vol. 83, Pgs. 823-833., 2006. Magrini-Bair, Kimberly; et al. “Fluidizable Catalysts for Producing Hydrogen by Steam Reforming Biomass Pyrolysis Liquids.” Proceedings in the 2002 U.S. DOE Hydrogen Program Review. Golden, Colorado, 2002. Melis, A; Happe, T. “Trails of Green Alga Hydrogen Research- From Hans Gaffron to New Frontiers.” Photosynthesis Research. Vol. 80, Pgs. 401-409., 2004. Molina Grima, E; Acien Fernandez, F.G.; Garcia Camacho, F; Camacho Rubio, F; Chisti, Y. “Scale-up of Tubular Photobioreactors.” Journal of Applied Phycology. Vol. 12, Pgs. 355-368., 1999. Molina Grima, E; Fernandez, J; Acien Fernandez, F.G; Chisti, Y. “Tubular photobioreactor design for algal cultures.” Journal of Biotechnology. Vol. 92, Pgs. 113-131., 2001. Morris, R.E; Pollack, A.K; Mansel, G.E; Lindhjem, C; Jia, Y; and Wilson, G. “Impact of Biodiesel Fuels on Air Quality and Human Health.” National Renewable Energy Laboratory. NREL/SR-540-33793, May 2003. Pahl, Greg. “Biodiesel: Homegrown Oil.” Mother Earth News. Pg. 65., February 1, 2006. Patel-Predd, Prachi. “Hydrogen from Algae.” Technology Review. September 27, 2007. Preston Hubbard, Holly. “Hopes are high, as are the hurdles, for alternative fuel Ambitious target set for biofuels, which have yet to be economically viable.” International Herald Tribune. March 15, 2008. “Renewed Interest in Turning Algae Into Fuel Generated.” Science Daily. January 19, 2008. Sachs D, Jeffrey. “Act Now, Eat Later.” Time. Pg. 44. May 5, 2008. Schwartz I, Evan. “The Algae Alternative.” The Boston Globe. July 12, 2004. Seibert, Michael; et al. “Molecular Engineering of Algal H2 Production.” Proceedings of the 2002 U.S. DOE Hydrogen Program Review. Colorado, 2002. Spolaore, P; Joannis-Cassan, C; Duran, E; Isambert, A. “Commercial Applications of Microalgae.” J Biosci Bioeng. Vol. 101, Pgs. 87-96., 2006.