Jeffrey Yau and Christina George Manhasset Science Research 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

Background 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

Mostly due to car emissions and industrial factories. (Hopwood, http://photos.mongabay.com/08/0423methaneglobal.jpg 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) 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

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

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

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

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

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

Tubular Photo Bioreactor design for algal cultures 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

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

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 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

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

Hypothesis 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.

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)

Growth of C.reinhardtii in Airlift Compared to Tubular Photobioreactor and Raceway Pond (25% Medium) Time (Day) Transmittance 70 75 80 85 90 95 100 1/8/09 1/9/09 1/10/09 1/11/09 1/12/09 1/13/09 1/14/09 1/15/09 1/16/09 1/17/09 1/18/09 1/19/09 1/20/09 1/21/09 1/22/09 1/23/09 Airlift Tubular Raceway N=14 Graph 1: Comparison of the growth of C.reinhardtii, between the Airlift, Tubular and raceway pond Photo Bioreactors for Trial 1. There was a significant increase in growth in the Tubular Photo Bioreactor as compared to the Airlift and Raceway Pond. (p=.019)

Growth of C.reinhardtii in Tubular Photobioreactor vs. Raceway Pond Time Transmittance 60 65 70 75 80 85 90 95 100 1/8/09 1/9/09 1/10/09 1/11/09 1/12/09 1/13/09 1/14/09 1/15/09 1/16/09 1/17/09 1/18/09 1/19/09 1/20/09 1/21/09 1/22/09 1/23/09 Tubular Raceway N=14 Graph 2: Comparison of the growth of C.reinhardtii between the Raceway Pond and Tubular Photo Bioreactor. There was a significant increase in growth in the Tubular Photobiroeactor as compared to the Raceway. (p=.019)

Growth of C.reinhardtii after CO2 Exposure Between Tubular Photobioreactor and Raceway Pond Time (Day) Transmittance 70 75 80 85 90 95 100 1/15/09 1/16/09 1/17/09 1/18/09 1/19/09 1/20/09 1/21/09 1/22/09 1/23/09 Tubular Raceway N=7 Graph 3: Comparison of the growth of C.reinhardtii between the Tubular and the Raceway Pond after CO2 exposure. No significant difference was found.

Photobioreactor and Raceway Pond Growth of C.reinhardtii after CO2 Exposure in Airlift Compared to Tubular Photobioreactor and Raceway Pond Time (Day) Transmittance 60 65 70 75 80 85 90 95 100 1/15/09 1/16/09 1/17/09 1/18/09 1/19/09 1/20/09 1/21/09 1/22/09 1/23/09 Airlift Tubular Raceway N=7 Graph 4: Comparison of the growth of C.reinhardtii between the Airlift, Raceway, and Tubular Photo Bioreactor after carbon dioxide exposure for Trial 1. There was no significant difference in growth in all bioreactors.

4.9 18 2.7 5 10 15 20 25 30 Airlift Tubular Raceway Percent Change in Absorbance of C.reinhardtii in Bioreactors for Trial 1 Bioreactors Percent (%) Graph 5: Shows the percent change in absorbance of C.reinhardtii in all three bioreactors for Trial 1. The Tubular Photobioreactor (red) grew C.reinhardtii with the most percent change in absorbance out of all three bioreactors while the Raceway Pond grew the organism with the least percent change in absorbance.

Percent Change in Absorbance of C Percent Change in Absorbance of C.reinhardtii in Bioreactors for Trial 2 1.5 3.9 9.7 2 4 6 8 10 12 Airlift Tubular Raceway Bioreactors Percent (%) Graph 6: Shows the percent change in absorbance of C.reinhardtii in all three bioreactors for Trial 2. The Raceway Pond (light blue) grew C.reinhardtii with the most percent change in absorbance out of all three bioreactors while the Airlift grew the organism with the least percent change in absorbance.

Growth of C.reinhardtii in Bioreactors (10% Medium) Time (Day) Transmittance 80 82 84 86 88 90 92 94 96 98 100 1/26/09 1/27/09 1/28/09 1/29/09 1/30/09 1/31/09 2/1/09 2/2/09 2/3/09 2/4/09 2/5/09 Airlift Tubular Raceway N=9 Graph 7: Comparison of the growth of C.reinhardtii, between the Airlift, Tubular and Raceway Pond photo Bioreactors for Trial 2. There is no significant difference between the bioreactors. However, comparing the transmittance numbers (1/29-2/5) to the carbon dioxide levels before exposure, there is a significance. (p=.015)

Growth of C.reinhardtii After CO2 Exposure in Bioreactors (Trial 2) Time (Day) Transmittance 80 82 84 86 88 90 92 94 96 98 100 1/29/09 1/30/09 1/31/09 2/1/09 2/2/09 2/3/09 2/4/09 2/5/09 Airlift Tubular Raceway N=6 Graph 8: Comparison of the growth of C.reinhardtii between the Airlift, Raceway, and Tubular Photo Bioreactor after carbon dioxide exposure for Trial 2. No significance was found when compared to carbon dioxide levels measured.

Discussion A Tubular Photobioreactor is a suitable environment for growth of algae Performance of the Tubular Photobioreactor surpassed the control bioreactor 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

Conclusion Data supports the Alternate Hypothesis 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

Limitations 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 bacterial contamination in bioreactors

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

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