Transcript Schizochytrium limacinum C.reinhardtii Developing New Working

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
http://photos.mongabay.com/08/0423methaneglobal.jpg
Graph 1
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.
•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)
Raceway Pond Design
Original Design
http://www.agric.wa.gov.au/content/SUST/BIOFUEL/110407_Biodieselfrom
microalgae.pdf
Modified Design
Taken: January 17, 2009
A raceway pond is made of a closed
loop recirculation channel
(Chisti, 2007)

Tubular Photo Bioreactor Design
Original Design
Modified Design
http://www.agric.wa.gov.au/content/SUST/BIOFUEL/110407_Biod
ieselfrommicroalgae.pdf
Consists of straight transparent
tubes either made out of glass or
plastic (also known as solar
collectors)
(Chisti, 2007)

Taken: January 17, 2009
Control: Airlift Design

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A self-contained
bioreactor
Utilizes a baffle to
re-circulate the
bacteria in
suspension
Taken on: December 21, 2008
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
Contains an enzyme
called hydrogenase
that allows creation
of hydrogen
(Tiede, 2008)
 Ability to produce
hydrogen under
anoxic conditions
(Fouchard, 2005)

http://en.wikipedia.org/wiki/Chlamydomonas_reinhardtii
Magnified 3000X
Tubular Photo Bioreactor design for
algal cultures

Molina Grima; et al (2001): Experiment on
tubular photobioreactors using P.
tricornutum
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Tested tubular diameter on the amount of
sunlight that penetrated through the culture
broth
Polyunsaturated fatty acids
production by Schizochytrium sp.
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Kamlangdee (2003):
Experiment on
polyunsaturated
fatty acids
production by
Schizochytrium sp.
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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
Photo Bioreactor
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
Raceway pond
Measurement of
Biomass yield (once
daily from start of
experimentation) using
Aquafluor
Fluorometer
Measurement of
Biomass yield using
Spectrophotometer
(Wavelength at
610nm)
Dependent Variables:
-Oil Extracted from
Hexane
-Hydrogen collected from
C.reinhardtii
-Three different sized
tubes (0.01m, 0.012m,
0.019m)
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.
Measurement of the
effect of carbon
dioxide on pH levels
(Using Co2 Sensor on
GLX and pH paper)
An ANOVA test will be used to statistically analyze the data (p<.05). The Scheffe post hoc test will be used.
Growth of C.reinhardtii in Airlift Compared to Tubular Photobioreactor
and Raceway Pond (25% Medium)
N=14
100
Transmittance
95
90
Airlift
Tubular
Raceway
85
80
75
70
Time (Day)
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
N=14
100
95
Transmittance
90
85
Tubular
Raceway
80
75
70
65
60
Time
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
N=7
100
Transmittance
95
90
Tubular
Raceway
85
80
75
70
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
Time (Day)
Graph 3: Comparison of the growth of C.reinhardtii between the Tubular and the Raceway Pond after
CO2 exposure. No significant difference was found.
Growth of C.reinhardtii after CO2 Exposure in Airlift Compared to Tubular
Photobioreactor and Raceway Pond
N=7
100
95
Transmittance
90
85
Airlift
Tubular
Raceway
80
75
70
65
60
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
Time (Day)
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.
Percent Change in Absorbance of C.reinhardtii in Bioreactors for Trial 1
30
Percent (%)
25
20
18
15
10
5
4.9
2.7
0
Airlift
Tubular
Raceway
Bioreactors
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.reinhardtii in Bioreactors for Trial 2
12
9.7
Percent (%)
10
8
6
3.9
4
2
1.5
0
Airlift
Tubular
Raceway
Bioreactors
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)
100
N=9
98
Transmittance
96
94
92
Airlift
90
Tubular
Raceway
88
86
84
82
80
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
Time (Day)
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)
100
N=6
98
96
Transmittance
94
92
Airlift
90
Tubular
Raceway
88
86
84
82
80
1/29/09
1/30/09
1/31/09
2/1/09
2/2/09
2/3/09
2/4/09
2/5/09
Time (Day)
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
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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
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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
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

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
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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
Select Bibliography
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“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): 643650., 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.