Developing New Developing New Working Protocols Working Protocols

and Bioreactor and Bioreactor Designs to Enhance Designs to Enhance Biomass Growth and Biomass Growth and

Energy Yield in Energy Yield in Schizochytrium Schizochytrium limacinumlimacinum and and C.reinhardtiiC.reinhardtiiJeffrey Yau and Christina Jeffrey Yau and Christina

GeorgeGeorge

Manhasset Science ResearchManhasset Science Research

BackgroundBackground The use of The use of

bioreactors to bioreactors to combat the growing combat the growing problem of problem of greenhouse gases greenhouse gases has been extensively has been extensively studied in recent studied in recent decades.decades.

(Chisti, 2007)(Chisti, 2007)http://www.ieagreen.org.uk/newsletter/dec80/images/biofixation.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)

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

Raceway Pond DesignRaceway 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_Biodieselfrommicroalgae.pdf

Original Design Modified Design

Taken: January 17, 2009

Tubular Photo Tubular Photo Bioreactor DesignBioreactor Design

Consists of straight Consists of straight transparent tubes either made transparent tubes either made out of glass or plastic (also out of glass or plastic (also known as solar collectors)known as solar collectors)

(Chisti, 2007)(Chisti, 2007)

http://www.agric.wa.gov.au/content/SUST/BIOFUEL/110407_Biodieselfrommicroalgae.pdf

Original Design

Taken: January 17, 2009

Modified Design

Control: Airlift DesignControl: Airlift Design

A self-A self-contained contained bioreactorbioreactor

Utilizes a Utilizes a baffle to re-baffle to re-circulate the circulate the bacteria in bacteria in suspensionsuspension

Taken on: December 21, 2008

Organism: S.limacinumOrganism: S.limacinum Contains pigments Contains pigments

for photosynthesisfor photosynthesis Known to contain Known to contain

EPA, DHA, and EPA, DHA, and omega-3 fatty omega-3 fatty acidsacids

Reliable source of Reliable source of oil production for oil production for biodieselsbiodiesels

(Kamlangdee, 2003)(Kamlangdee, 2003) http://roweb.cityu.edu.hk/researchreport/2002-2003/Project/020.jpg

Organism: C.reinhardtiiOrganism: C.reinhardtii

Contains an Contains an enzyme called enzyme called hydrogenase that hydrogenase that allows creation of allows creation of hydrogen hydrogen

(Tiede, 2008)(Tiede, 2008) Ability to Ability to

produce produce hydrogen under hydrogen under anoxic conditionsanoxic conditions

(Fouchard, 2005)(Fouchard, 2005)http://en.wikipedia.org/wiki/Chlamydomonas_reinhardtii

Magnified 3000X

Tubular Photo Bioreactor Tubular Photo Bioreactor design for algal culturesdesign for algal cultures

Molina Grima; et al (2001): Experiment on Molina Grima; et al (2001): Experiment on tubular photobioreactors using tubular photobioreactors using P. P. tricornutumtricornutum

Tested tubular diameter on the amount of Tested tubular diameter on the amount of sunlight that penetrated through the sunlight that penetrated through the culture brothculture broth

Polyunsaturated fatty acids Polyunsaturated fatty acids production by production by

Schizochytrium sp.Schizochytrium sp. Kamlangdee Kamlangdee

(2003): Experiment (2003): Experiment on polyunsaturated on polyunsaturated fatty acids fatty acids production by production by Schizochytrium sp.Schizochytrium sp.

Found single isolate Found single isolate reliable in reliable in production of DHAproduction of DHA

Hydrogen as Clean Fuel Via Hydrogen as Clean Fuel Via Continuous Fermentation by Continuous Fermentation by

Anaerobic Photosynthetic Anaerobic Photosynthetic Bacteria, Rhodospirillum Bacteria, Rhodospirillum

rubrumrubrum Observed the Observed the effect of light effect of light intensity, intensity, agitation, and agitation, and liquid dilution liquid dilution rate on hydrogen rate on hydrogen productionproduction

Use of biocatalyst Use of biocatalyst can be considered can be considered alternative to alternative to Fischer Tropsch Fischer Tropsch synthetic synthetic reactionsreactions

(Najafpour, 2003)(Najafpour, 2003)http://www.vurup.sk/pc/vol45_2003/issue3-4/pdf/14.pdf

PurposePurpose

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

HypothesisHypothesis

Null Hypothesis:Null Hypothesis: No significant difference No significant difference will be found in the growth of will be found in the growth of C.reinhardtii and S.limacinum in either C.reinhardtii and S.limacinum in either bioreactor.bioreactor.

Alternate Hypothesis:Alternate Hypothesis: The growth of The growth of C.reinhardtii and S.limacinum will be C.reinhardtii and S.limacinum will be greatest in the Tubular Photobioreactor greatest in the Tubular Photobioreactor when exposed to carbon dioxide, with when exposed to carbon dioxide, with respect to pH levels.respect to pH levels.

MethodologyMethodologyProblem: 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)

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 Airlift Compared to Tubular PhotobioreactorGrowth of C.reinhardtii in Airlift Compared to Tubular Photobioreactor and Raceway Pond (25% Medium)and Raceway Pond (25% Medium)

Time (Day)

Tra

nsm

itta

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/09

Airlift

Tubular

Raceway

N=1N=144

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 in Tubular Photobioreactor vs. Raceway PondGrowth of C.reinhardtii in Tubular Photobioreactor vs. Raceway Pond

Time

Tra

nsm

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nce

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Tubular

Raceway

N=1N=144

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 COGrowth of C.reinhardtii after CO22 Exposure Between Tubular Exposure Between TubularPhotobioreactor and Raceway PondPhotobioreactor and Raceway Pond

Time (Day)

Tra

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itta

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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=N=77

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.

Growth of C.reinhardtii after COGrowth of C.reinhardtii after CO22 Exposure in Airlift Compared to Tubular Exposure in Airlift Compared to Tubular Photobioreactor and Raceway PondPhotobioreactor and Raceway Pond

Time (Day)

Tra

nsm

itta

nce

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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=7N=7

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.

4.9

18

2.7

0

5

10

15

20

25

30

Airlift Tubular Raceway

Percent Change in Absorbance of C.reinhardtii in Bioreactors for Trial 1

Bioreactors

Per

cen

t (%

)

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.

Percent Change in Absorbance of C.reinhardtii in Bioreactors for Trial 2

1.5

3.9

9.7

0

2

4

6

8

10

12

Airlift Tubular Raceway

Bioreactors

Per

cen

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)

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 in Bioreactors (10% Medium)

Time (Day)

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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=N=99

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.

Growth of C.reinhardtii After CO2 Exposure in Bioreactors (Trial 2)

Time (Day)

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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=N=66

DiscussionDiscussion A Tubular Photobioreactor is a suitable A Tubular Photobioreactor is a suitable

environment for growth of algaeenvironment for growth of algae Performance of the Tubular Performance of the Tubular

Photobioreactor surpassed the control Photobioreactor surpassed the control bioreactorbioreactor

Daily exposure to carbon dioxide did not Daily exposure to carbon dioxide did not greatly effect pH levels in bioreactorsgreatly effect pH levels in bioreactors

Growth in Tubular Bioreactor was greater Growth in Tubular Bioreactor was greater than growth in the Raceway Pond than growth in the Raceway Pond possibly due to more efficient pumppossibly due to more efficient pump

ConclusionConclusion Data supports the Alternate HypothesisData supports the Alternate Hypothesis The Tubular Photo Bioreactor The Tubular Photo Bioreactor

demonstrated a greater amount of demonstrated a greater amount of growth as compared to the Raceway growth as compared to the Raceway PondPond

There was no significance regarding There was no significance regarding the Carbon Dioxide levels when the Carbon Dioxide levels when pumped through the Tubular, Raceway pumped through the Tubular, Raceway Pond and Airlift BioreactorsPond and Airlift Bioreactors

LimitationsLimitations

Errors while using the Spectrophotometer Errors while using the Spectrophotometer occurred, causing incongruous data: Possibly occurred, causing incongruous data: Possibly caused by cuvette or contaiminationcaused by cuvette or contaimination

Possible errors in GLX Xplorer readings for Possible errors in GLX Xplorer readings for carbon dioxidecarbon dioxide

Possible bacterial contamination in bioreactorsPossible bacterial contamination in bioreactors

Future StudiesFuture 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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