madhu_thesis algae photosynthesis

8/12/2019 Madhu_Thesis Algae Photosynthesis

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APPLICATION OF ALGAL CULTURE TECHNOLOGY FOR CARBON DIOXIDE

AND FLUE GAS EMISSION CONTROL

by

Madhu Hanumantha Reddy

A Thesis Presented in Partial Fulfillmentof the Requirements for the Degree

Master of Science

ARIZONA STATE UNIVERSITY

May 2002


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APPLICATION OF ALGAL CULTURE TECHNOLOGY FOR CARBON DIOXIDE

AND FLUE GAS EMISSION CONTROL

by

Madhu Hanumantha Reddy

has been approved

January 2002

APPROVED:

, Chair

Supervisory Committee

ACCEPTED:

Department Chair

Dean, Graduate College


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ABSTRACT

Human activity in the modern world has disturbed the composition of the

atmosphere. This has led to some of the major environmental issues of our time--ozone

depletion, acid rain, and global warming/climate change, which is potentially the most

serious. The use of fossil fuels and practice of deforestation to meet the world's energy

demands has led to increasing concentrations of carbon dioxide and other greenhouse

gases in the atmosphere. Increased CO2and the greenhouse gases in the atmosphere is

such a serious problem for mankind that many research and development approaches are

implemented to reduce CO2emissions. Various processes in reducing CO2emissions

have been used; and photosynthetic technology using microalgae is widely discussed as a

feasible technology. In this study, the feasibility of photosynthetic algal technology using

various culture of microalgae in reducing CO2and flue gases was assessed. The

microalgal species selected for this study exhibited growth under high-CO2

concentration.

Microalgal species were grown in batch column reactors under varying

environmental conditions of (1) light, (2) CO2, and (3) temperature--the essential

nutrients for algal growth. Specific growth rates were compared in evaluating the

efficiency of the algal species, and a maximum growth rate of 0.47 h-1

was obtained for

Scenedesmusspecies. Growth under simulated flue gases with controlled pH yielded

good results, indicating the use of microalgae in reducing the flue gases from power

plants. A flat-plate photobioreactor was used as a pilot test setup with a constant supply

of 5% CO2with varying dilution rates. Carbon fixation of 3.65% was obtained based on

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the input producing 0.17 g C/L-day. CO2uptake of 8.50% was observed during the entire

operation of the flat-plate photobioreactor.

Flat-plate bioreactor efficiency was compared with the tubular bioreactor, which

was used for the same culture at the same conditions. Beaver Creek (BC) microalgal

culture growth was not affected by higher concentrations of CO2, which indicates the

feasibility of this technology. An optimum environmental condition has to be determined

to obtain higher productivity, growth rates, and output.

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To My Parents

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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my research advisor Dr. Paul

Westerhoff for his valuable guidance and encouragement without whom this research

work would have not been possible. Thank you Dr. Qiang Hu for your precious time,

encouragement and guiding me through this project and assisting me in successfully

completing this project. In addition, I would also like to thank Dr. Peter Fox and Dr.

Morteza Abbaszadegan for serving on my committee. I would also like to acknowledge

the financial support provided by the Salt River Project through my advisor.

I would like to thank Mr. Peter Goguen for supporting me in my laboratory work.

Many people have contributed directly and indirectly to this work. I sincerely appreciate

the support they have provided me in pursuing this research. I would especially like to

thank Ms. Lennie Okano for her support and guidance in getting started with the research.

Thank you to all my fellow environmental engineering graduate students for providing

valuable assistance and moral support. Special thanks to Mr. Garry Pearson (Lab stores)

and everyone in Engineering Laboratory Services for providing help in purchasing and

manufacturing. Finally, I would like to extend my gratitude to Ms. Lori Scrutchfield, Ms.

Daisy Eldridge and Ms. Dawn Takeuchi for their patience and help with the daily

administrative details.

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TABLE OF CONTENTS

Page

LIST OF TABLES.............................................................................................................. x

LIST OF FIGURES ........................................................................................................... xi

INTRODUCTION .............................................................................................................. 1

LITERATURE REVIEW ................................................................................................... 4

Atmospheric Air Composition.................................................................................4

Green House Gases and Global Warming ...............................................................5

Coal Fired Power Plant Emissions...........................................................................6

Types of Flue Gases and Effects..............................................................................7

Chemistry of Flue Gases..........................................................................................8

Algal Photosynthesis..............................................................................................11

Tolerance of Algal Species to CO and Flue Gas2 ..................................................12

Photobioreactors ....................................................................................................14

EXPERIMENTAL AND ANALYTICAL METHODS ................................................... 16

Experimental Methods...........................................................................................16

Collection and Culturing Algae ................................................................ 16

Batch Growth Studies ............................................................................... 16

Flat-Plate Photobioreactor ........................................................................ 18

Analytical Methods................................................................................................19

Algal Cells Isolation ................................................................................. 19

Optical Density ......................................................................................... 20

Dry Mass (Suspended Solids)................................................................... 21

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Page

Variation in Carbon Dioxide..................................................................... 22

Variation in Light Intensity....................................................................... 22

Variation in Temperature.......................................................................... 23

pH Measurements ..................................................................................... 24

Algal Growth in Flue Gas ......................................................................... 24

Variation in Flue Gas ................................................................................ 25

CO2Measurement Using Gas Chromatography....................................... 25

RESULTS ......................................................................................................................... 32

Cell Characterization .............................................................................................32

Species Identification................................................................................ 32

Algal Batch Experiments .......................................................................................33

Growth Rates Comparison........................................................................ 33

Effects in CO2Concentration.................................................................... 35

Effects in Light Intensity .......................................................................... 36

Effects in Temperature.............................................................................. 37

Growth in Simulated Flue Gas.................................................................. 38

Continuous Flow Flat-Plate Photobioreactor.........................................................41

Growth Rate for Batch Experiments......................................................... 41

Experimental Results of Photobioreactor Operation ................................ 42

Carbon Analysis Data ............................................................................... 44

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Page

DISCUSSION................................................................................................................... 69

Variations in Growth Rate .....................................................................................69

Growth in Flue Gas................................................................................................70

Comparison of Biomass Production ......................................................................72

Cell Adhesion and Settling in Flat-plate Photobioreactor .....................................73

Comparison of Flat-plate and Tubular Photobioreactor ........................................74

CONCLUSIONS............................................................................................................... 76

REFERENCES ................................................................................................................. 78

APPENDIX

A. RESULTS OF FLAT-PLATE PHOTOBIOREACTOR RUN IN

GREENHOUSE (BY MARIO E. SOTO) .............................................................83

B. CALCULATION OF CARBON BIOMASS IN CO2GAS..............................92

C. CARBON FIXATION CALCULATIONS FOR CONTINUOUS MODE..94

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LIST OF TABLES

Table Page

3.1. Cyanobacterial BG-11 Growth Medium Contents .................................................... 27

3.2. Experimental Conditions Used for Batch Growth Studies ........................................ 28

3.3. Flue Gas Composition of SRP Power Plant............................................................... 29

4.1. Results of Growth Rates for ChlorellaSpecies Using Exponential Fit Method

and Two Point Method Under Varying Environmental Conditions .......................... 47

4.2. Results of Growth Rates for ScenedesmusSpecies Using Exponential FitMethod and Two Point Method Under Varying Environmental Conditions............. 48

4.3. Results of Growth Rates for Mixed Species Using Exponential Fit Methodand Two Point Method Under Varying Environmental Conditions .......................... 49

4.4. pH Range for the Algal Culture at Varying CO2Concentrations.............................. 50

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LIST OF FIGURES

Figure Page

3.1. Plexi glass tank with the glass column used for the experiment................................ 30

3.2. Overview of batch photobioreactor (not to scale)...................................................... 30

3.3. Schematic setup of flat-plate photobioreactor ........................................................... 31

4.1. Comparison of Chlorella, Scenedesmusand Mixed algal cultures (column

diameter: 30 mm; 160 mol/m2-s; 32 oC) @ (A) 5% CO2 / 95% Air and (B) 20%CO2/80% Air. ............................................................................................................. 51

4.2. Calculation of growth rates using exponential fit method for

Scenedesmusspecies @ 20% CO2 / 80% Air (32

o

C; 160 mol/m

2

-s;Column Diameter: 30 mm; Triplicate Data) .............................................................. 52

4.3. Calculation of growth rates using two point method for Scenedesmus

species @ 20% CO2 / 80% Air (32oC; 160 mol/m2-s; Column Diameter:

30 mm; Triplicate Data) ............................................................................................. 53

4.4. Effect of CO2concentration on the growth of (A) Scenedesmusand

(B) Chlorellaspecies (32oC; 160 mol/m2-s; Column Diameter: 30 mm)............... 54

4.5. Effect of light intensity on Scenedesmusspecies growth in (A) 30 mm

diameter columns and (B) 50 mm diameter columns (32oC; 20% CO2) .................. 55

4.6. Effect of temperature on Scenedesmusgrowth in 50 mm diameter columns

(27oC 42

oC; 20% CO2; 160 mol/m

2-s)................................................................ 56

4.7. Effects of (A) Na2SO3(2 Molar) and (B) NaNO2(2 Molar) concentrations onScenedesmusgrowth (No Aeration; 500 ml Flasks) .................................................. 57

4.8. Algae growth in presence of NOXwithout pH adjustment

(325ppm NO2 / 327ppm NO / bal Nitrogen / 20% CO2and Compressed

Air; Column Diameter: 30 mm; 160 mol/m2-s)....................................................... 58

4.9. Algae growth in the presence of SO2without pH adjustment(313ppm SO2 / Ultra Zero Air / 20% CO2and Compressed Air;

Column Diameter: 30 mm; 160 mol/m2-s) .............................................................. 59

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Figure Page

4.10. Effect of SO2with pH adjustment with 10N NaOH and additional

Nutrient medium (Potassium Phosphate) on (A) algae growth (B) solution

pH (QGas= 1.94 L/min; 313ppm SO2 / Ultra Zero Air / 20% CO2and

Compressed Air; Column Diameter: 30 mm; 160 mol/m2

-s). ................................. 60

4.11. Effect of SO2and NOXwith pH adjustment with 10N NaOH and additionalnutrient medium (Potassium Phosphate) on (A) algae growth (B) solution pH

(725ppm SO2/ 25ppm NOx / bal Nitrogen / 20% CO2and Compressed Air;

Column Diameter: 30 mm; 160 mol/m2-s) .............................................................. 61

4.12. Comparison of growth curves for Beaver Creek species @ 160 and245 mol/m

2-s (32

oC; 5% CO2; Column Diameter: 30 mm).................................... 62

4.13. Variation in BG-11 nutrient medium flow rate during the reactor run.................... 63

4.14. Optical density variation for Beaver Creek culture at different retention timesduring flat-plate photobioreactor run ......................................................................... 64

4.15. Beaver Creek dry mass cell concentration versus cell optical density .................... 65

4.16. Mass weight curve for Beaver Creek culture........................................................... 65

4.17. Carbon Dioxide concentration measurements at the inlet and outlet of the

photobioreactor........................................................................................................... 66

4.18. Biomass produced per day at different flow rates during the entire operation........ 67

4.19. Biomass produced per day at different retention times during the entireoperation..................................................................................................................... 67

4.20. Cumulative biomass produced during the entire operation of the reactor ............... 68

4.21. Cumulative carbon produced versus the applied carbon during the entire

operation of the reactor .............................................................................................. 68

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Introduction

The most commonly considered indicator of climate change and global warming

is the surface air temperature. Atmospheric CO2and other greenhouse gases, primarily as

a result of the combustion of fossil fuels, are viewed as the components of the climate

system that interact in complex ways over a wide range of timescales. Atmospheric CO2

had been balanced through various cyclic phenomena of the decreasing effect by

photosynthetic fixation by plants and dissolving in the seawater and of the increasing

effect due to releasing from decaying plants and the seawater (Hamasaki et al. 1994). Due

to the anthropogenic emissions, which are primarily from combustion of fossil fuels

greenhouse gases in the atmosphere have been steadily increasing thereby causing great

anxiety in global warming (Matsumoto et al. 1995). The growing evidence that links the

greenhouse gas carbon dioxide (CO2) and global climate change highlights the need to

develop cost effective carbon sequestration schemes. The main challenge of CO2capture

and storage is the high cost of technologies using current state-of-the-art.

Various technologies have been used to mitigate the fossil fuel-fired power plant

stack emissions including the (1) physical-chemical processes, such as wet or dry

absorption and membrane separation techniques and (2) biological methods, in particular

using microalgal photosynthesis (Hamasaki et al. 1994). Chlorophyll in photosynthetic

algae captures light energy, which is used to convert simple molecules (CO2and H2O)

into carbohydrates (sugars and starches) with the release of O2. Microalgae are of

particular interest because of their rapid growth rates, tolerance to varying environmental

conditions and can also fix greater amounts of CO2per land area than higher plants

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microalgae has emerged as a promising technology to help reduce emissions from fossil

fuel-fired powered plants. The carbon fixed by microalgae is incorporated into

carbohydrates, lipids and proteins, so energy, chemicals or foods can be produced from

algal biomass. The energy rich biomass is widely used as a source fuel (liquid and

gaseous), health foods, animal feed and also in producing vitamins and pigments (Negoro

et al. 1991). Processes in conversion of algal biomass to such useful products would

indirectly decrease dependence on fossil fuels.

Indoor and outdoor cultivation of algae using photobioreactors and open ponds

for CO2reduction has been practiced and the CO2fixation using the photobioreactors has

proven to be efficient with higher productivity (Ogbonna and Tanaka 1997). Biomass

productivity of a photobioreactor depends on close alignment of the culture environment

to the needs of the algal culture. Various designs and applications of enclosed

photobioreactors have been utilized. The flat-plate and the helical tubular (Biocoil)

photobioreactors have been widely used due to their higher productivity rates (Tredici

and Zettelli 1998). The design of the photobioreactor depends on various factors such as

the light path, temperature, microbial contaminants such as bacteria, fungi, and other

algae, and cost. In this paper the feasibility of microalgal growth and carbon fixation

under different concentrations of CO2and flue gases and at different environmental

conditions has been described. A lab scale flat-plate photobioreactor, which is

particularly useful for mass cultivation of microalgae, was used to test the feasibility of

gaseous CO2reduction, carbon fixation and biomass production. Comparison of helical

tubular photobioreactor (Okano 1999) and flat-plate photobioreactor has been reported.

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Literature Review

Atmospheric Air composition

The atmosphere, which makes up the largest fraction of the biosphere, is a

dynamic system that continuously adsorbs a wide range of solids, liquids and gases from

both natural and man-made sources (Rao and Rao 1996). The composition of air is

variable with respect to several of its components (e.g. CH4, CO2, H2O) so pure air has

no precise meaning; it is commonly considered to be air that is free of dust, aerosols and

reactive gaseous contaminants of anthropogenic origin. The composition of the major

components in dry air is relatively constant (percent by volume given): nitrogen 78.084;

oxygen 20.946; argon 0.934; carbon dioxide 0.033; neon 0.0018; helium 0.000524;

methane 0.00016; krypton 0.000114; hydrogen 0.00005; nitrous oxide (N2O) 0.00003;

xenon 0.0000087. The concentrations of carbon dioxide, methane, nitrous oxide, the

chlorofluorocarbons and some other species of anthropogenic origin are increasing

measurably with time (Warneck 2000). Relatively clean air, which is free of most

reactive anthropogenic pollution (NO, NO2, SO2, non-methane hydrocarbons, etc.), often

used as a reference sample in the calibration and operation of instruments, is designated

as zero air.

Air pollution is basically the presence of foreign substances in air. Air pollution

comes from many different sources. "Stationary sources" such as factories, power plants,

and smelters; "mobile sources" including cars, buses, planes, trucks, and trains; and

"natural sources" such as wildfires, windblown dust, and volcanic eruptions. The major

source of flue gases is the coal fired power plants that produce approximately 10-20%

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Green House Gases and Global Warming

Energy from the sun drives the Earths weather and climate, and heats the Earths

surface; in turn, the Earth radiates energy back into space.Many chemical compounds

found in the Earths atmosphere act as greenhouse gases. These gases allow sunlight,

which is radiated in the visible and ultraviolet spectra, to enter the atmosphere

unimpeded. When it strikes the Earths surface, some of the sunlight is reflected as

infrared radiation (heat). Greenhouse gases tend to absorb this infrared radiation as it is

reflected back towards space, trapping the heat in the atmosphere.

Global warming refers to an average increase in the Earth's temperature, which in

turn causes changes in climate. Many gases exhibit such greenhouse properties,

including those that occur naturally in the atmosphere, such as water vapor, carbon

dioxide, methane, and nitrous oxide, sulfur oxides, and those that are very powerful and

are man-made, such as chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs),

perfluorocarbons (PFCs), and sulfur hexafluoride (SF6) (Benemann 1992). Atmospheric

concentrations of several important greenhouse gases (carbon dioxide, methane, nitrous

oxide, and most man-made gases) have increased by about 25 percent since large-scale

industrialization began some 150 years ago (Warneck 2000). Each greenhouse gas differs

in its ability to absorb heat in the atmosphere. HFCs and PFCs are the most heat-

absorbent. Methane traps over 21 times more heat per molecule than carbon dioxide, and

nitrous oxide absorbs 270 times more heat per molecule than carbon dioxide (Allen and

Rosselot 1997). Often, estimates of greenhouse gas emissions are presented in units of

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millions of metric tons of carbon equivalents (MMTCE), which weights each gas by its

GWP value, or Global Warming Potential.

In the United States, nearly 85 percent of anthropogenic greenhouse gas emissions

result from the burning of fossil fuels. The United States is a major source of

anthropogenic greenhouse gas emissions (U.S. Department of Energy 1991). Other

sources include the oxidation of soil organic carbon (SOC), and carbon release due to

deforestation (Benemann 1992).

Fossil fuels consist primarily of hydrocarbons, which are made up of hydrogen

and carbon. When burned, the carbon combines with oxygen to yield carbon dioxide. The

amount of carbon dioxide produced depends on the carbon content of the fuel. For each

unit of energy produced, natural gas emits about half, and petroleum fuels about three

quarters, of the carbon dioxide produced by coal (Smoot 1997).

Coal Fired Power Plant Emissions

Coal is the altered remains of prehistoric vegetation that originally accumulated as

plant material in swamps and peat bogs and is the worlds most abundant, safe and secure

fossil fuel, it is also clean and cost-effective. Coal has many important uses, but most

significantly in electricity generation, steel and cement manufacture, and industrial

process heating. Coal has been used as an energy source for hundreds of years. Due to the

growth in global population and improvement in living standards demand for energy has

been increased over the past years. Coal is the single largest fuel source for the generation

of electricity worldwide, currently about 38% of the world's electricity is generated from

coal (Karube et al. 1992). Due to the fossil fuel combustion the atmospheric CO2levels

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are increasing by 3.2 billion (giga) tons of carbon annually (GtC/y), that is about 0.4%

per year (Benemann 1992).

Combustion of fossil fuels produces significant amounts of air pollutants. Due to

the usage of coal, the major types of pollutants emitted into the atmosphere are fly ash,

coal dust, soot, and oxides of sulfur and nitrogen. There is also a possibility of emission

of carbon monoxide and unburnt carbon, if automatic combustion control systems are not

used. Methaneis also emitted during the production and transport of coal. The

greenhouse gases emitted are mainly dominated at the point of consumption or

combustion of coal. Normally, over 90% of greenhouse gas emissions from coal are from

the end-use of the coal.

Due to environmental regulations and for optimum efficiency coal property

specifications are becoming more stringent. Moisture, ash, fixed carbon, volatile matter,

calorific value and sulfur content of coal are key properties that can affect quality. The in-

ground variations of these properties should be assessed to maximize quality assurance

during production.

Types of Flue Gases and Effects

Gaseous emissions discharged through a flue or stacks are called flue gases. As

coal is one of the most impure of fuels, coal combustion produces carbon dioxide and

other greenhouse gases that are suspected to cause climatic warming, and it is a source of

sulfur oxides, SOX(SO2and SO3) and nitrogen oxides, NOX(NO and NO2) (Rao and Rao

1996). The concentration of SOXin the atmosphere depends upon the sulfur content of

the fuel used. The sulfur content of the fuel used varies from less than 1% by weight for

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good quality anthracite to over 4% by weight for bituminous coal (Smoot 1997). About

80% of sulfur in coal is found in flue gases in the form of sulfur dioxide (SO2) and sulfur

trioxide (SO3) and the concentration may vary from 0.05 0.25% and occasionally as

high as 0.4% (Rao and Rao 1996).

Seven oxides of nitrogen can be grouped as N2O, NO, NO2, NO3, N2O3, N2O4, out

of which nitric oxide (NO) and nitrogen dioxide arise from the anthropogenic activities

which are classified as major air pollutants (Rao and Rao 1996). High temperatures, high

pressures and high oxygen concentrations promote the production of NOX. About 90% of

the NOXis in the form of NO. About 30 35% of nitrogen in coal gets converted into

NO, remaining nitrogen in the coal gets converted into molecular nitrogen. NO2 is mostly

formed by oxidation of the NO, which is discharged in combustion products (Rai et al.

2000).

Oxidation of sulfur and nitrogen oxides will cause acid rain. These gases also play

an important role in the environment through forest damage, effects on vegetation, smog

formation, material damage, direct and indirect damage to human health, depletion of the

stratospheric ozone layer and the greenhouse effect (Bank 1998).

Chemistry of Flue Gases

The acidity of acid precipitation is dependent not only on emission levels, but also

on the chemical mixtures with which SO2and NOXinteract in the atmosphere. The

formation of sulfuric and nitric/nitrous acid is a complex process involving several

chemical reactions.

SOX:

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Sulfur dioxide (SO2) is the pre-dominant form of sulfur oxides, and is a colorless

and nonflammable gas. The solubility of SO2in water is 17.7% (w/w) at 0oC and 8.5%

(w/w) at 25oC and the solubility is affected by the presence of neutral salts. Henrys law

coefficient for SO2varies from 3.28 at 0oC to 1.24 at 25

oC and 0.56 at 50

oC (Wedzicha

1984). SO2concentrations are most commonly expressed in g/m3or as a volume-to-

volume ratio such as parts per million (ppm).

SO2released into the air is normally oxidized by a complex series of chemical

reactions, and it is important to consider both solution and gas phase chemistries in the

conversion process. Most of sulfur is converted to SO2and only 1% - 2% leaves the stack

as SO3.

Gas Phase:

There are several potential reactions that can contribute to the oxidation of sulfur

dioxide in the atmosphere, each with varying success. One possibility is photooxidation

of SO2by ultraviolet light. Light in this region of the electromagnetic spectrum has the

potential to excite the molecule and lead to the subsequent oxidation by O2. This reaction

was found to be an unimportant contributor to the formation of sulfuric acid. A second

possibility is the reaction of sulfur dioxide with atmospheric oxygen by the following

reactions (Wedzicha 1984):

(1) 2SO2+ O22SO3

(2) SO3+ H2OH2SO4

Reaction (2) occurs quickly, therefore the formation of SO3(hydrophilic acid) in

the moist atmosphere is assumed to lead to the formation of sulfuric acid. However,

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reaction (1) is very slow in the absence of a catalyst, and is influenced by a number of

chemical and environmental factors.

Aqueous Phase:

The dissociation of sulfur dioxide occurs by a two-fold process:

(1a) SO2(g) + H2O (l)SO2. H2O (aq)

(1b) SO2. H2O (aq)H++ HSO3

- pK1= 1.76

(2) HSO3-(aq)H

++ SO3

2- pK1= 7.21

In the aqueous phase, sulfur dioxide exists as three species:

[S (IV)] = [SO2. H2O)] + [HSO3-] + [SO3

2-]

The establishment of the above equilibrium is dependent upon pH, droplet size,

etc. As the dissociation reaction proceeds forward, the droplet concentration of SO2

decreases, and the Henry's Law equilibrium will shift accordingly to partition more from

the atmosphere into the water droplet (Babich and Stotzky 1980).

NOX:

NO (Nitric Oxide) and NO2(Nitrogen Dioxide) are slightly soluble in water. The

Henry's law constant for NO is 1.9x10-3

mol/atm and for NO2is 1.0x10-2

mol/atm. The

typical atmospheric partial pressure of NO is 2x10-10

atm and NO2is 2x10-9

atm. NO

reacts rapidly with O2to form NO2.

Gas Phase:

Nitrogen dioxide is produced through the reaction of nitrogen oxide with oxygen

in the air. The principal contributor to the formation of nitric acid in the atmosphere is the

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This equation is the net result of two processes. One process involves the splitting

of water, which is an oxidative process that requires light, and is often referred to as the

"light reaction". This reaction may be written as:

12 H2O + light or radiant energy6O2+ 24 H+ + 24e

-

The oxidation of water is accompanied by a reduction reaction resulting in the

formation of a compound, called Nicotinamide Adenine Dinucleotide Phosphate

(NADPH). This reaction is illustrated below

NADP++ H20NADPH + H

++ O

The NADPH formation reaction is linked or coupled to yet another reaction

resulting in the formation of a highly energetic compound, called Adenosine

Triphosphate (ATP). As this reaction involves the addition of a phosphate group to a

compound called, Adenosine Diphosphate (ADP) during the light reaction, it is called

photophosphorylation. The light energy, which is captured, is stored in the form of

chemical bonds of compounds such as NADPH and ATP. The energy contained in both

NADPH and ATP is then used to reduce carbon dioxide to glucose, a type of sugar

(C6H12O6).

Tolerance of Algal Species to CO2and Flue Gas

Microalgae have been proved to be most productive carbon dioxide users, which

can be grown under varying environmental conditions. To mitigate the CO2emissions

from power plants various cultures of microalgae have been used. Hamasaki et al. (1994)

reported testingNannochloropsis salina, strain NANNP-2, Phaeodactylum tricornutum,

strain PHAEO-2 and Tetraselmissp, strain T-S3 in 10% CO2and N2gas at 25oC under

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Photobioreactors

The low productivity and photosynthetic efficiency of open ponds or outdoor

reactors due to the long light path, which causes self-shadowing thereby utilizing low

CO2from gas streams, and other environmental factors such as temperature, microbial

contaminants has led to the design and development of enclosed indoor photobioreactors

(Watanabe et al. 1995). Exploitation of photosynthetic cells for the production of useful

metabolites requires efficient photobioreactors. The critical design requirement in the

photobioreactor design is the illumination surface area per unit volume; an efficient

photobioreactor has a high surface area to volume ratio (S/V ratio) (Ogbonna and Tanaka

1997). Flat-plate and tubular photobioreactors are the most widely used closed systems of

photobioreactors due to their high S/V ratio (Ogbonna et al. 1995). Selection of the

photobioreactor depends on the ability to maximize the productivity and photosynthetic

efficiency, which has been proved by using the flat-plate and tubular reactors (Tredici

and Zittelli 1998). Each type of photobioreactor has advantages and disadvantages in

terms of potential efficiency of sunlight utilization, effective mass transfer of oxygen and

carbon dioxide, ease of cleaning, and scalability.

The helical tubular photobioreactor has (1) a larger S/V ratio, increasing the

incident light energy input per unit volume, and reducing self-shadowing; (2) easy

control of temperature and microbial contaminants; and (3) better CO2transfer from the

gas stream to the liquid culture medium due to the extensive CO2absorbing pathway

(Watanabe et al. 1995). Okano (1999) reported a CO2uptake of 52.35% by a batch mode

run tubular photobioreactor and a maximum of 3.51% and 0.71 g C/d (0.15 g C/L-d) of

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carbon production in a continuously run tubular photobioreactor. Self or mutual shading

by the algal cells causes the light to be dissipated, have a decreased light-saturated

photosynthetic capacity (light and dark zones) and are expensive to operate (Tredici and

Zittelli 1998 and Grima et al. 1998).

Flat-plate photobioreactors are widely employed due to the narrow light path,

which facilitates maintenance of cell densities higher by more than an order of magnitude

as compared to other photobioreactors (Hu et al. 1996a). Flat-plate reactors are more

promising and are widely used due to (1) adjustable orientations aimed at maximal

exposure to solar energy, (2) reduction in oxygen build up, (3) no dark volumes

compared in large degassers and other photobioreactors and (4) high photosynthetic

efficiency (Gitelson et al. 1996 and Hu et al. 1996c).

An appropriate reactor design is required to obtain maximal cell mass. Flat-plate

photobioreactors were reported to produce high cell population density of Spirulina

platensis with their entire surface area well illuminated(Hu et al. 1996c) and

Nannochloropsissp.(Zou and Richmond 1999). Also, flat-plate photobioreactors reactors

are essentially bubble columns, stirred very effectively by streaming of compressed air,

the rate of flow of which may be accurately controlled to set the optimal rate of mixing

(Hu and Richmond 1996b). A maximum rate of CO2fixation (16.7 g l-1

24 h-1

) for

Chlorella littoralewas attained by using flat-plate photobioreactor in a semi-continuous

mode. Based on the high photosynthetic efficiency, high productivity rates, narrow light

path and mixing conditions, flat-plate photobioreactor is the preferred photosynthetic

algal culture technology in reducing CO2emissions.

15


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Experimental and Analytical Methods

Experimental Methods

Collection and Culturing Algae

Samples of algae were collected from different locations on the bank of Beaver

Creek, which receives water from Montezuma well (upper Sonoran desert of Arizona),

and some high Dissolved Inorganic Carbon (DIC) groundwater inputs (Okano 1999). The

steady water flow into Montezuma Well comes from a warm underground spring, with a

constant temperature of 25oC.

CO2is dissolved into the water as it passes through the

underground, and limestone deposits before entering the well (Okano 1999).

Samples were filtered through Micron Separates, Inc.(Westbro, Massachusetts)

47-mm, 0.22 m nylon filters. The retained media from the filter medium were

suspended in 100ml of BG-11 Cyanobacterial growth medium (TABLE 3.1) in 200-

250ml Erlenmeyer flasks. The flasks were placed on a Lab-Line (Melrose Park,

Illinois) force bench top orbital open-air shaker at 100 rpm under six fluorescent lamps

panel.

To assure homogeneity, unialgal cultures are started from clones. Isolation of

algae should proceed with the idea that a truly unialgal culture should be established as a

clone culture. The technique for isolation of these microscopic species was to carry

individual cells through a series of sterile washes.

Batch Growth Studies

It has been seen that microalgae can grow in controlled conditions in the

laboratory. Temperature, pH, and other important variables can be well controlled

compared to the large-scale systems done in open ponds. Availability and intensity of


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17

light are the major factors controlling productivity of photosynthetic cultures (Lee and

Low 1992, Hu et al. 1998a). Growth studies at different conditions were conducted on

different species (Chlorella, Scenedesmus and Mixed culture) of algae and we found that

the species representative of Montezuma Well was the best for our experiments. The

batch growth experiments were conducted in glass tubes contained in a thermal reactor

consisting of a rectangular, 94-liter, 1.27-cm-thick plexi-glass tank. Glass columns of

diameter 30 mm (400 ml volume) and 50 mm (750 ml volume) were used as fixed

volume photobioreactors to hold the microalgal cultures (FIG. 3.1). The photobioreactor

columns were sterilized in the autoclave for 30 minutes and BG-11 Cyanobacterial

medium was used as a growth medium. A VWR Scientific ProductsModel 71

Immersion Circulator with Analog Controller was used to control the water bath

temperature to the desired level in the reactor for the temperature experiments. In order to

control the pressure coming out of the cylinders, CONCOA (Virginia Beach, Virginia)

High Purity Gas Valves are used. Simulated flue gas, CO2and house (Compressed) air

were blended to the desired concentrations for the batch experiments using a Cole-

Parmer(Vernon Hills, Illinois) Gas Proportioner flow meter with a steel float. The

blended gas was bubbled into the columns using glass diffuser (Capillary) tubes through

Vinyl tubing (FIG. 3.2). A pH meter and a thermometer were used to measure pH and the

temperature in the columns. Six cool 45 watts fluorescent lamps fixed to the panel placed

adjacent to the plexi-glass tank provided light for the experiments. Another light panel

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18

with six cool fluorescent lamps was utilized for some growth experiments, which

required more light intensity.

Table 3.2 describes the experimental procedure adapted for the microalgal species

under study. The environmental conditions were varied and the type of algal cultures

were ran in triplicate comparing the growth of each species.

Batch experiments to test the feasibility in using the algal cultures were conducted

using 500 ml flasks, which were placed on a Lab-Line (Melrose Park, Illinois) force

bench top orbital open-air shaker at 100 rpm under six fluorescent lamps panel.

Flat-Plate Photobioreactor

A schematic diagram of the flat-plate photobioreactor system is shown in FIG.

3.3. The lab-scale flat-plate photobioreactor was made from 1.27 cm thick plexi-glass

sheets with a culture volume holding capacity of 10 L. The tank consisted of rectangular

chamber 69.85 cm long, 3.556 cm wide and 40.132 cm in height. A perforated 0.48 cm

(inner diameter) fluoropolymer tubing with 0.05 mm equally spaced holes with a length

of 68.58 cm was used to bubble the gas mixture in the reactor, which was attached to 40

mm steel tubing with a length of 45.72 cm. Steel weights were used to hold the tubing. A

gasket groove was provided in order to provide maximum air tightness with a dimension

of 0.635 cm wide and 0.158 cm deep. The flat-plate photobioreactor was fixed to two

Uni-Strutvertical supporting structures, 1.22 m tall. A Cole-ParmerGas Proportioner

flow meter was used to mix the house air and compressed CO2source using a

CONCOA(Virginia Beach, Virginia) Regulator. The CO2 / air mixture was bubbled at

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19

a rate of 9.6 L/min with a constant supply of 5% CO2. The gas flow rate was maintained

at this level in order to keep the culture suspended in the medium. Two 20 L Cole-

Parmercarboy containers were utilized to hold the BG-11 medium for continuous

supply and collection. The photobioreactor also consisted of a Cole-Parmertwo-headed

diastolic pump, Model No. 7553-70 with 0.318 cm vinyl tubing supplying fresh BG-11

medium into the photobioreactor and removing the product algal mass out into the

container at an equal flow rate. Continuous supply of fresh BG-11 medium was

maintained into the photobioreactor with a change in the medium flow rate depending on

the algal growth in the reactor. Two light panels with 3, 40 watts GE fluorescent lamps

each were placed closely on each side of the photobioreactor to provide maximum light

intensity.

The flat-plate photobioreactor was run in a continuous mode day and night in

continuous light, with a constant supply of fresh BG-11 medium. Fresh initial supply of

dense algal culture was used for the continuous mode and the same culture was

maintained throughout the operation. An inlet was provided in order to collect the

samples for pH and temperature measurements. A tubing intercept was provided between

the flow meter and the inlet for inlet gas measurements, whereas the outlet gas was

measured directly near the outlet provided for collecting the sample.

Analytical Methods

Algal Cells Isolation

Two cultures of algae, Chlorella vulgarisand Scenedesmus caribeanus,and a

mixed culture of Chlorellaand Scenedesmuswere used in this project for the batch

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20

growth studies, whereas Beaver Creek (BC) culture was used for the photobioreactor

operation. After collection of the algal species, they were filtered using a Whatman

GF/C 45-mm glass microfiber filter. After filtration, the filter with the attached

microorganisms was transferred into a 100-150 ml Erlenmeyer flask containing 50 ml of

fresh BG-11 medium and then covered with a sponge flask covers in order to avoid

contamination from other microorganisms. The Erlenmeyer flask was then placed on an

orbital shaker, under a light intensity of approximately 140 mol/m2-s, at a room

temperature of 26oC for a week or 15 days until the culture turns deep, dark green color.

The culture is then centrifuged at a temperature of 25oC and at a rotational speed of 5000

rpm for 6 minutes. After the centrifugation the supernatant is discarded, and the culture is

rinsed and resuspended in a fresh BG-11 medium. The fresh culture was then placed in a

50 mm glass column photobioreactor of 750 ml each with BG-11 medium. The glass

column was provided with a 0.1 mm glass capillary tube in order to bubble CO2enriched

air through the column, at increasing step-wise increments of CO2concentrations. After

the cells had adjusted to the different CO2concentrations, they were used for the batch

growth experiments and were further screened onto an agar plate. The agar plates were

kept in an incubator supplied with CO2until visible colonies are formed and then

transferred.

Optical Density

A ShimadzuUV-1601 UV-Visible Spectrophotometer was used to analyze

growth rates of algal species by measuring optical density. As the photosynthetic

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21

pigments absorb the light energy from 400 to 700 nm, a wavelength of 730 nm, which is

outside the pigment absorbance range, was used for all the optical density measurements.

Fisher-ScientificFisherbrand*Standard 10-mm pathlength quartz cuvettes with a

sample holding capacity of 3 ml were used to hold the samples in the spectrophotometer.

For all the batch growth experiments, samples were collected at a 2 - 3 hr interval for the

optical density measurements. After a stationary phase had been reached optical density

measurements were made for every 6 hrs. Optical density measurements for the flat-plate

photobioreactor were made three to four times a day, and also made during change in

dilution rate. For batch growth experiments with Sodium Sulphite (Na2SO3) and Sodium

Nitrite (NaNO2) optical density was measured once a day. Growth rates were compared

and analyzed at different environmental conditions for each species of algae and a

relationship of optical density and time was plotted.

Dry Mass (Suspended Solids)

The dry mass of all the algal species was determined on triplicate culture samples.

Suspended solids tests were conducted on microalgal culture, which was diluted to

approximately 8 different optical density values. Equal volumes of the microalgal

samples were filtered through a 47-mm Whatman(Maidstone, England) GF/C glass-

micro fiber filters, which were previously ashed in an oven at 550o

C for one hour and

weighed for their initial weight. A suction pump was used to filter the samples and after

filtration the filters were dried in a Market Forge Sterilmatic THELCOLaboratory

oven at 105-110oC for approximately 24 hours. The filter was taken and cooled to room

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22

temperature in a desiccator and was weighed to get the final weight (Hu et al. 1998, Lee

and Low 1992, Pirt et al. 1983). The difference in the weights of the filter, after filtration

and before filtration, divided by the sample volume filtered gave the dry mass. A plot of

the optical density at 730nm and dry mass in mg/L was developed.

Variation in Carbon Dioxide

Varying concentrations of CO2was used during the experiments. In order to

provide continuous supply of CO2 for the species, pure CO2was supplied through

Praxaircompressed tanks fitted with a CONCOAhigh purity regulator. The supply of

CO2was regulated to the desired reading using the regulator. Compressed house air

through a centrally housed system was used to obtain desired gas mixture ratio. The two

sources of gas were connected to a Cole ParmerGas Proportioner flow meter.

Depending on the gas, the float and the tubing, the flow rates were determined using the

calibration chart calibrated at 50 PSIG, which was provided with the flow meter.

According to the desired CO2concentration, the gas flow of house air and the

compressed CO2were adjusted, and setting the float reading on the flow meter. Dividing

the CO2flow by the total flow and multiplying by 100 obtained the desired CO2

concentration in percentage.

Variation in Light Intensity

In order to evaluate the amount of light intensity striking the microalgal cells, the

light intensity measurements were done. Light is made up of many different types, at

different wavelengths. The reading therefore is a combined effect of all the wavelengths.

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23

Light intensity was measured using a Quantum Meter, Model LI-189 LI-COR(Lincoln,

Nebraska), which had a quantum sensor (Silicon Photodiode) as a detector. As quantum

sensors measure photosynthetically active radiation (PAR) in the 400 700 nm

waveband, the light meter was used for light intensity measurements in the batch reactor

and the flat-plate photobioreactor. The unit of measurement, depending on the sensor

type for the measurement of light intensity was micromoles per square meter per second

(mol/m2-s). In the batch reactor, light intensity measurements were taken at

approximately equal intervals inside the batch column and then averaged. Similar

readings were taken for the flat-plate photobioreactor. As the reactor had light panels on

its both sides, the sensor was inserted inside the reactor and the readings were noted at

different intervals and were averaged to get the light intensity radiated onto the

microalgae.

Variation in Temperature

In order to monitor the temperature of the culture temperature was measured

using a Cole-Parmermercury thermometer. Temperature measurements for the batch

photobioreactor and the flat-plate photobioreactor were taken directly by inserting the

thermometer inside the culture. The temperature of the plexi-glass water bath was

controlled using the VWR Scientific ProductsModel 71 Immersion Circulator with

manual controller. Temperature in the flue gas experiments was maintained by adding a

buffer and taking direct readings from the culture.

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24

pH Measurements

pH was measured for all the batch and flat-plate photobioreactor experiments

using a Corning pH meter 340. Monitoring of pH was necessary in order to keep the

culture in good condition. pH was measured 6 times a day for the flue gas measurements,

as the pH was decreased with continuous supply of flue gas.

Algal Growth in Flue Gas

Algal growth in the flasks with BG-11 medium along with the addition of Sodium

Nitrite (NaNO2, 2M) and Sodium Sulphite (Na2SO3, 2M) was conducted to analyze the

growth and changes in their cell characteristics. Several flasks with varying

concentrations of Na2SO3and NaNO2were used and the optical density was measured. A

graph of optical density against time is plotted.

Batch growth studies were conducted on different species of microalgae in order

to evaluate their growth rates and changes in their characteristics. Flue gases from Air

Liquide, similar in composition to the SRP coal fired power plants (TABLE 3.3), were

used for all the experiments. The mixed gas lab composition was 725 ppm SO 2 / 320-400

ppm NOX / 320-400 ppm NO / balance Nitrogen (Cylinder 1) and 17 18% O2 / balance

Nitrogen (Cylinder 2). The gases from these 2 cylinders were mixed with compressed

CO2using a Cole ParmerGas Proportioner flow meter. Optical density measurements

were taken at equal time intervals to evaluate the growth rates. Temperature and pH was

maintained by adding 10N Sodium Hydroxide (NaOH).

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25

Variation in Flue Gas

Various mixtures of gases were used in order to find the growth inhibitor of the

microalgae. In order to evaluate the exact composition at which the culture algae can

grow, separate composition of flue gases were used. The compositions were, 313 ppm

SO2and Ultra zero air (Cylinder 1), 325ppm NO2 / 327 ppm NO / bal Nitrogen (Cylinder

2). Each cylinder was used separately and blended with compressed CO2and compressed

air to a desired concentration using the Cole ParmerGas Proportioner flow meter,

which was calibrated at 40 PSIG using a dry meter for calibration of flue gases. 10N

NaOH was used to maintain an optimal pH range for growth.

CO2Measurement Using Gas Chromatography

Carbon Dioxide concentrations were measured by taking 0.5-mL of gas at the

inlet and the outlet of the Flat-plate photobioreactor using a Hamilton(Reno, Nevada)

1-mL gas tight injection syringe. The GOW-MAC(San Jose, California) series 580 Gas

Chromatography (GC) machine was used to measure the gaseous CO2concentration from

the flat-plate photobioreactor. The basic components of a GC were a gas cylinder with

reducing valve, a constant-pressure regulator, a port for the injection of the sample, a

chromatographic column, a detector, an exit line, and a recorder. The gas cylinder

contained helium (carrier gas), which was continuously swept through the

chromatographic column at a temperature of 60oC and at a flow rate of 25-30 mL/min.

The detector and the injector temperature were set at 120oC and the A injection port

was used to inject the CO2gas.

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26

A calibration curve using a stock 30% CO2by moles (balance nitrogen, N2) gas

tank from Air Liquide(Laporte, Texas) was developed. Six different injections were

made with the stock 30% CO2gas with CO2concentrations of 0%, 6%, 12%, 18%, 24%

and 30% of CO2. A chromatogram was produced and depending on the type of

compound to be observed, a peak was identified and the area reading was taken. The

Calibration curve was plotted with the obtained area reading against the CO2

concentrations. The actual concentration of CO2entering and leaving out of the flat-plate

photobioreactor was obtained using the area obtained from the chromatogram and

comparing the area with the calibration curve.

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27

Table 3.1

Cyanobacterial BG-11 Growth Medium Contents (Lab Protocol)CYANOBACTERIAL GROWTH MEDIA:

BG - 11 LIQUID MEDIUM

1 liter

10ml 100 x BG-11 without Fe, Phosphate, Carbonate

1ml 1000 x Ferric ammonium citrate

1ml 1000 x Na2CO3 1ml 1000 x K2HPO4All these components are kept in the Kelvinator

BG 11 SOLID AGAR PLATES

For agar plates, add to the above:

10ml 1 M TES/NaOH buffer pH 8.2 (Kelvinator)3gms Na-thiosulfate (Solid)

15gms Difco Bacto-agar

Autoclave for 30 mins.

100 x BG 11 without Fe, phosphate, carbonate:

1 liter

149.6 gms NaNO37.5 gms MgSO4.7H2O

3.6 gms CaCl2.2H2O

0.60 gms Citric acid (or 0.89 gm Na-Citrate, dihydrate)

` 1.12 ml NaEDTA, pH 8.0, 0.25 M

100 ml Trace MineralsTrace Minerals:

1 liter

2.86 gms H3BO3 1.81 gms MnCl2. 4H2 0.22 gms ZnSO4.7H2O

0.39 gms NA2MoO4.2H2O

0.079 gms CuSO4.5H2O

0.049 gms Co(NO3)2.6H2O

Other Components:

Ferric ammonium citrate, 6 mg/ml (100 x)600 mg per 100 ml H2O

Na2CO3(100 x)

2 gms Na2CO3per 100 ml H2O

K2HPO4(100 x)

3.05 gms K2HPO4 per 100 ml H2O or 4.0 gms for K2HPO4.3H2O.

27


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Table 3.2

Experimental Conditions Used for Batch Growth StudiesEnvironmental Conditions Adopted:

Algal Cultures used: Chlorella vulgaris, Scenedesmus caribeanus Mixed culture

(mix of Chlorella vulgarisand Scenedesmus caribeanus)andBeaver Creek culture.

Temperature Range: 27oC 42

oC

Light Intensity : 65 mol/m2-s 275 mol/m2-sCarbon Dioxide : 0% CO2- 40% CO2

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Table 3.3

Flue Gas Composition of SRP Power Plant

The average composition of flue gas emission from Navajo Generating Station (NGS) is:

CO2 - 12 15%

SO2 - 15 20% (dry)

NOX - 225 ppm (dry)

O2 - ~7% (dry)

Moisture (Scrubbers running) - 12 15%

N2 - Remainder (basically, other than trace things)

NGS scrubs 100% of their flue gas while CGS only scrubs a half percent

The average composition of flue gas emission from Coronado Generating Station (CGS) is:CO2 - 15.1%

SO2 - 312 ppm (dry)

NOX - 325 ppm (dry)

O2 - 3.7 3.9% (dry)

Moisture -`10 11%

N2 - Remainder.

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30

Stopper with 2 holes

60 cm

50mm18 cm

56 cm

116 cm

Figure 3.1. Plexi glass tank with the glass column used for the experiment.

Air as

asrol Valves

eters

tream

Light Panel

lass

n

750-ml G

Colum

Mixed Gas S

Flow M

High Purity GCont

Flue GCO2

Figure 3.2.Overview of batch photobioreactor (not to scale).

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31

Air CO2

High Purity Gas

Control Valves

Flow

Meters

69.85 cm

39.5

8cm

10.1

6cm

Fresh BG-11

Medium

Waste product

AlgaeTwo headed

diastolic pump

Flat plate Photobioreactor

Figure 3.3.Schematic setup of flat-plate photobioreactor.

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Results

Cell Characterization

Species Identification

Microalgal mass culture has been in development for four decades. The main

factors in culture management include the light availability and temperature. Species, that

can reproduce and grow under varying environmental condition will help in providing

good results and increase the scope of this concept. Two cultures of green algae were

used which are a very diverse group as far as culture requirements are concerned. The

species were identified as Scenedesmus caribeanus andChlorella vulgaris, which are

high-CO2cultures and belong to a class of Chlorellaceae.

Scenedesmusculture is in a multicellular colony, and the cells are arranged in

rows by often with number of 4 (sometimes 2 or rare up to 8 and16), usually grouped in

one plane with long axes of the cells parallel to one another. Neighboring cells contact

tightly by cell wall. The genus is pelagic and often found in all kinds of freshwater water

bodies. It belongs to the genus of Scenedesmaceae, order of Chlorococcales and class of

Chlorophyta (Hu 2001, Komarek 1983 and Fott and Novakowa 1969).

Chlorella is a green coccoid species of single cell, belonging to Chlorophyta

(class), Chlorococcales (order) and Chlorellaceae (family). Cells are slightly ellipsoidal to

spherical and mother cells are approximately spherical. The cell wall is thin. During

spore formation the cell wall does not undergo gelatinization, but breaks up into 2

fragments. The chloroplast is cup-shaped or rarely girdle-shaped. The pyrenoid is

distinct, covered with saucer-shaped starch grains. Vacuoles can be seen when the cells

are young. Lipid granules are present when the cells are old. Reproduction by 2 or 4 (or


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33

rarely 8 or 16) autospores. Young autospores are always slightly ellipsoidal. Fragment of

the mother-cell wall is broadly elongated or irregularly triangular, yet always concave,

two pieces contact with one of the ends. They persist separately from the released

autospores and lie freely in the culture medium. Dimension: cells 4-6 m, sporangia up to

10 m (Hu 2001, Komarek 1983 and Fott and Novakova 1969).

The third culture used for the batch growth studies was a mixture of Scenedesmus

caribeanus andChlorella vulgariscultures. The cultures were mixed to evaluate the

effects of growth on each species. The cultures grew well under varying conditions and

the shape and the size of the culture was not modified when observed under the

microscope.

Beaver Creek (BC) culture was used for the photobioreactor operation. BC culture

was similar to Scenedesmus caribeanusin shape and size. BC culture is in a multicellular

colony, and most of the cells were arranged in rows with number of 4 or 2 with an

antennae on their ends. The culture was freshly isolated and showed higher growth rates.

Algal Batch Experiments

Growth Rates Comparison

The optical density (at 730nm) was measured at equal time intervals in order to

track the growth of the algal cells. These species were tested under varying CO2

concentrations in finding their ability to reproduce and grow well. Scenedesmusspecies

was used for most of the growth studies. Due to the high sensitivity of Chlorellato

contamination, it was used for comparison studies with the Scenedesmusculture. Both

species were exposed at various conditions of light, temperature and CO2concentrations,

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and their growth rates were calculated. Both species reproduced well at a light intensity

of 160 moles/m2-s, temperature of 32 oC and at 20% CO2. But the growth rates showed

variations with changes in temperature, light intensity and CO2concentrations. The third,

a mixed algal culture, which was basically a mixture of Chlorella and Scenedesmus

cultures, was used. The batch tests were run in triplicate, and all the optical density

measurements were done at a 1:5 dilution. The standard error bars are shown to indicate

the variability between each test conducted for a single culture. Growth curves for the

three cultures at 5% CO2and at 20% CO2concentrations are shown in FIG. 4.1. At 5%

CO2 Chlorella had a slightly higher growth compared to Scenedesmusand Mixed species.

All the cultures represented similar kind of growth at 20% CO2.

Batch experiments were conducted using two columns in triplicate with a column

diameter of 30 mm and 50 mm. From TABLE 4.2 for Scenedesmus species at 20% CO2

the growth rate is high in 30 mm column compared to the 50 mm diameter column. It was

observed that the light penetrated deeper into the 30 mm columns and had more intensity.

But with the 50 mm column, due to the cell concentration and the column diameter the

measured light intensity was less.

The optical densities were then plotted against time and the specific growth rate

(h-1

) was calculated using two methods, the Exponential method (FIG. 4.2) and the Two-

point method (FIG. 4.3). The Specific growth rate was obtained by fitting a best possible

exponential fit to the growth curve (FIG. 4.2). The average of the exponential values

from the triplicate data gave the specific growth rate. In Two-Point method, the growth

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35

rate was calculated by selecting two points in the log phase, which showed faster growth.

The specific growth rate , was calculated using the formula:

)(

lnln)(

12

121

tt

XXh

=

Where X1and X2are the optical densities at times t1and t2respectively. FIG. 4.3 shows

triplicate growth curve for Scenedesmus species using two-point method. The growth

rates for each species under different conditions using two-point and exponential method

is shown in TABLES 4.1, 4.2 and 4.3. Exponential fit for all the curves yielded good

results compared to the two-point method. Most of the curves showed exponential growth

with a short lag phase and increased log phase, but some did not show this trend due to

lack of cell growth. This was due to the cell adjustment or due to the presence of dead

cells or unhealthier algal cells.

Effects of CO2Concentration

U.S. power sector emissions are roughly one-third of the U.S. total, and contribute

almost 8 percent of global CO2emissions (U. S. Department of Energy, 1991). TABLE

3.3 shows CO2emissions for Navajo and Coronado Power Generating Stations, which

range from 10 16%. Algal cells after isolation were tested with varying CO2

concentrations. All the cultures reproduced well at these concentrations, which were later

tested at concentrations from trace amounts in house air to 40%. Batch experiments were

run for Scenedesmus and Chlorella species in triplicate with house air, 5% (QGas= 0.952

L/min), 20% (QGas= 1.90 L/min), 30% (QGas= 2.2 L/min)and at 40% (QGas= 2.4 L/min)

CO2. Temperature and light intensity was constantly maintained at 32oC and 160

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36

mol/m2-s. Initial and final pH is recorded in TABLE 4.4. For accuracy optical density

measurements to track growth were diluted at 1:5.

FIG. 4.4 shows the effects of carbon dioxide on the growth for Scenedesmus and

Chlorellaspecies. In house air, the algal species had low growth rate, while at 5%, 20%,

30% and 40% CO2the algal cells showed similar growth. As carbon dioxide is one of the

main sources in algal metabolic process, the variation in carbon dioxide concentration

showed no effect on the growth. As shown in FIG. 4.4, clearly indicates the decrease or

increase in dissolved carbon dioxide in the nutrient medium does not affect the growth of

the algal species.

Effects of Light Intensity

Two plywood structure supporting three sets of two cool 34 watts GE Fluorescent

lamps were used for the light intensity measurements. The light source was placed

adjacent to the plexi-glass tank, and a temperature of 32 oC and a constant supply of 20%

CO2was maintained. FIG. 4.5 shows growth curves for two sets of experiments carried

out with the Scenedesmusspecies in two different columns of varying diameter, a 30 mm

and a 50 mm diameter column. Two bulbs (65 mol/m2-s), four bulbs (120 mol/m2-s),

six bulbs (160 mol/m2-s), eight bulbs (200 mol/m2-s), ten bulbs (240 mol/m2-s) or

twelve bulbs (275 mol/m2-s) were used to obtain variation in light intensity for the

microalgal cultures. FIG. 4.5A represents the growth curve for the Scenedesmusspecies

in 30 mm diameter columns at 65, 120 and 160 mol/m2-s. At 160 mol/m2-s the cell

reproduced well and growth compared at 65 and 120 mol/m2-s. FIG. 4.5B represents the

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37

growth curve in 50 mm diameter columns, similar kind of growth was observed. Growth

curves for light intensities above 160 mol/m2-s did not have much effect on cell growth.

Specific growth rates (TABLE 4.2) for tests done in 30 mm column were higher

when compared to the 50 mm columns. Higher growth rate of algal cells may be due to

the light penetration inside the columns even at higher cell densities. It was also possible

that dense cell concentrations blocked light penetration causing decrease in the growth.

Microalgal cells need sufficient light for their photosynthesis, which is one of the main

element for their growth. Above 160 mol/m2-s, the growth remained the same and the

reproduction rate was not affected.

Effects of Temperature

Temperature is one of the main factors in influencing algal growth, some

microalgal cells will grow at a certain temperature range, most of the algal cells have

preferred temperature ranges (Ogbonna and Tanaka 1997). Batch tests were carried out at

a constant light intensity of 160 mol/m2-s with a constant supply of 20% CO2, but the

temperature of water bath was varied. Growth curves are shown in FIG. 4.6 for

Scenedesmusspecies in 50 mm diameter columns, with a temperature range of 27oC to

42oC. At 42

oC, the microalgal cells did not show any signs of growth and had a long lag

phase. Due to the high temperature the cells could not reproduce and turned to brown

color, which gradually changed to off-white color, and the cells started settling even after

continuous bubbling. As the microalgal cells were acclimatized at 32oC, a temperature of

32oC was used for most of the experiments.

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Effect of NOx.

The major component of oxides of nitrogen is Nitric Oxide (NO), which has low

solubility in water and is difficult to remove by conventional methods. The algal species

was tested in house air with 325 ppm Nitrogen Dioxide (NO2), 325 ppm NO, balance

nitrogen and 20% CO2in 30 mm diameter columns at 160 mol/m2-s. Batch experiments

were then carried out with the Scenedesmus, Chlorella, and Mixed species that were

adjusted to the gas mixture in single columns. The algal cells reproduced well (FIG. 4.8).

The pH and the growth rates are tabulated in TABLES 4.1, 4.2, and 4.3.

The cells showed a longer lag phase in getting acclimated to the gas

concentration. The pH and the growth rates represent that NOXis not the only factor in

decreasing the algal growth in simulated flue gases. As nitric oxide is poorly soluble in

water, it is essential to use a reactor that provides sufficient gas-liquid contact time.

Effect of SO2.

Oxides of Sulfur are formed when fuel containing sulfur (mainly coal and oil) is

burned, and during metal smelting and other industrial processes. The largest fraction of

sulfur oxides is sulfur dioxide (SO2), which is common and pervasive air pollutant. A

cylinder containing 313 ppm SO2and Ultra zero air was obtained to determine the effect

of SO2on algal flora. Algal cells were aerated with 313 ppm SO2, ultra zero air, house air

and 20% CO2in 30 mm diameter columns at 160 mol/m2-s. FIG. 4.9 indicates the effect

on Scenedesmus, Chlorella and Mixed algal photosynthesis, by treatment with SO2. pH

decreased from 6.2 to 2.6, and the algal cells turned into off-white color and had a

pungent odor.

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Sulfur dioxide is highly soluble in water, which reduces the pH of the solution

thereby decreasing the cell growth. FIG. 4.10 represents growth curves for tests by pH

adjustment with 10 N NaOH and additional nutrient medium (Potassium Phosphate) at

constant light supply of 160 mol/m2-s. Similar gas mixture of 313 ppm SO2, Ultra zero

air, house air and 20% CO2, and the initial pH was maintained at 6 (FIG. 4.10B). Algal

cells showed some signs of growth at initial stages but the growth was inhibited by 313

ppm SO2within 24 hours. pH remained constant, and the results indicate that SO 2might

have an adverse influence on the algal cells, thereby increasing toxicity.

Effect of NOXand SO2with pH adjustment.

Flue gas, which is mainly a mixture of oxides of Nitrogen, Sulfur and Carbon

dioxide was aerated in a column filled with the nutrient medium and microalgae to

evaluate the effect on growth. A cylinder with 725 ppm SO2, 320 - 400 ppm NOx, 320 -

400 ppm NO, and balance Nitrogen was blended with house air and 17 - 18% O2, 20%

CO2. 10N NaOH and additional BG-11 nutrient medium (Potassium Phosphate) was

added to adjust the pH of the nutrient medium. Batch growth experiments with a constant

light intensity of 160 mol/m2-s were run using Scenedesmus, Chlorella and mixed

species in 30 mm fixed volume photobioreactors.

Initial pH of 12 was maintained at the start to avoid sudden pH drop after aerating

which may affect the initial algal growth of the cells. pH dropped from 12 to

approximately 6 within a few hours after the test started and remained constant through

out the test period (FIG. 4.11B). Longer lag phase was observed (FIG. 4.11A) due to the

cell acclimation to the blended gas mixture. All the species represented similar kind of

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growth, but after Scenedesmusspecies attained its stationary phase pH started to

decrease.

Continuous Flow Flat-Plate Photobioreactor

Growth Rate for Batch Experiments

The Beaver Creek culture, (BC culture) was used for flat-plate photobioreactor

tests. Similar culture of algae was used to test the performance of tubular photobioreactor

at 5% CO2concentration (Okano 1999) in evaluating the performance and carbon dioxide

uptake. BC cells were isolated from Beaver Creek and were aerated with varying

concentrations of CO2to test their ability to grow under high CO2levels. BC cells were

similar to Scenedesmusspecies, which were oblong or fusiform in shape. Batch tests

were run at a light intensity of 160 mol/m2-s and 245 mol/m2-s to evaluate the cell

growth under varying light intensities for the flat-plate photobioreactor (FIG. 4.12). A

constant temperature of 32 oC, 5% CO2was maintained inside the 30 mm columns. At

245 mol/m2-s the BC cells reproduced well and showed higher growth rate compared to

160 mol/m2-s and are provided in TABLE 4.3.

Initial and final pH of the medium at 160 mol/m2-s was 5.65 and 7.05, and at

245 mol/m2-s was 6.05 and 7.31. A light intensity of 245 mol/m2-s was selected for the

flat-plate photobioreactor tests based on the results from the batch tests.

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Experimental Results of Photobioreactor Operation

Mode of operation and optical density.

Flat-plate reactor has been tested to achieve ultrahigh-cell-density culture, with

significantly higher rates of CO2fixation (Hu et al. 1998c). Based on the batch tests

results on BC algae a constant light intensity of 245 mol/m2-s, 5% CO2and a

temperature of 32oC was selected for the reactor operation. Flat-plate photobioreactor

performance was tested in a continuous mode with constant supply of BG-11 medium in

continuous light. Growth curve results (FIG. 4.12) from batch tests showed a higher

optical cell density in the geometric growth phase (0.6 cm-1

1.5 cm-1

) and higher initial

reproduction rate. Flat-plate reactor was started with a continuous supply of BG-11

culture medium with an initial retention time of 2 days for initial growth of algal cells.

Algal cells reproduced well and had an optical density of approximately 1.0 cm-1

within

two days. In order to prevent settling and to keep the cells in geometric growth phase, the

hydraulic residence time (HRT) was varied during the entire operation of the reactor

(FIG. 4.13). pH and temperature was monitored and the optical density variation at

different retention times during the entire operation is shown in FIG. 4.14. The main goal

was to keep the algal cells in the geometric growth phase to increase carbon dioxide

uptake. As can be noted from FIG. 4.14, optical density was decreased with the change in

the retention time and also due to the contamination by dead cells.

Dry mass and optical density.

Optical density measurement is an indication of cell growth; to obtain the exact

cell mass at certain optical density dry mass test was performed. Dry mass tests gives the

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exact mass of algae produced by the process. Suspended solids tests were performed with

Beaver Creek culture at 5% CO2with a light intensity of 245 mol/m2-s which yielded a

linear relationship between the optical density and cell density (FIG. 4.15). The result

obtained was used to calculate the dry mass, biomass and carbon production with the

information that the Beaver Creek algae contain 53.55 % carbon (Okano 1999).

Based on the results obtained from the dry mass measurements, the mass of algae

produced in grams per day was calculated by using the linear relationship equation. FIG.

4.16 shows maximum quantity of algae produced during the different retention times. A

retention time of 0.5 day produced higher mass, this relationship gives the optimal

retention time to be used for a photobioreactor.

CO2uptake.

Flat-plate photobioreactor was operated in continuous mode with continuous

supply of 5% CO2into the reactor. Carbon dioxide measurements were done at the inlet

and the outlet twice a day to evaluate the CO2uptake by the algae. FIG. 4.17 shows the

average CO2concentrations at the inlet and outlet of the flat-plate photobioreactor. The

algal cells and water matrix consumed only 8.50% of the input on average.

To keep the algal cells suspended in the reactor, a higher CO2flow rate was fed

into the system, which caused the system to be less efficient in retaining the dissolved

CO2.Another limitation to be considered was the reactors height, which was the main

factor in reducing the contact time.

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Carbon Analysis Data

Dry mass relationship obtained was used to calculate the biomass production in

the system. The biomass production was calculated using the optical density

measurements and the flow rates applied during the operation. The carbon fixation based

on the input was approximately 3.65% for the flat-plate photobioreactor, whereas Okano

(1999) reported 3.56% for the tubular photobioreactor. The carbon retainment in the flat-

plate photobioreactor was 4.93% lower than 20%, which was obtained for the tubular the

tubular photobioreactor (Okano 1999). The carbon fixation based on the input was

reported to analyze the uptake of the algal cells for their photosynthesis to the applied

carbon. Biomass productivity of 0.17g C/L-d was obtained for the flat-plate

photobioreactor, whereas Okano (1999) reported 0.15 g C/L-d for the Tubular

photobioreactor run in continuous mode. Biomass productivity was lower compared with

the other studies reported using tubular and flat-plate photobioreactors. Performance and

carbon fixation in the flat-plate photobioreactor was decreased due to settling, cell

adhesion which inhibited light intensity, dilution rate. Calculations of carbon biomass

production are shown in APPENDIX B and APPENDIX C.

FIG. 4.18 and 4.19 show the variation in biomass produced at different flow rates

and retention time in the photobioreactor. Based on the results it was showed that at a

retention time of 0.5 day the biomass produced in grams per day was higher. The

cumulative biomass production in grams was obtained with incrementing the biomass

produced for the entire operation (FIG. 4.20). FIG. 4.21 shows the difference between the

carbon applied and the cumulative carbon produced during the photobioreactor run. From

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TABLE 4.1. Results of growth rates for Chlorella species using exponential fit

method and two point method under varying environmental conditions


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TABLE 4.2. Results of growth rates for Scenedesmus species using exponential fit

method and two point method under varying environmental conditions

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TABLE 4.3. Results of growth rates for Mixed species using exponential fit methodand two point method under varying environmental conditions

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Table 4.4

pH Range for the Algal Cultures at Varying CO2ConcentrationsCO2Conc. Species Initial pH Final pH

Air Chlorella 7.65 9.26

Scenedesmus 7.83 10.45

Mixed 8.25 11.06

5 Chlorella 5.93 6.91


Mixed 6.21 7.13







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(A)

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100 120Time (hrs)

OD730nm

(cm

-1)

Chlorella

Scenedesmus

Mixed

(B)

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100 120 140

Time (hrs)

OD730nm

(cm

-1)

Chlorella

Scenedesmus

Mixed

Figure 4.1.Comparison of Chlorella, Scenedesmusand Mixed algal cultures (Column

Diameter: 30 mm; 160 mol/m2-s; 32 oC) @ (A) 5% CO2/95% Air (QGas= 0.95L/min) and (B) 20% CO2/ 80% Air (QGas= 1.90 L/min).

Note.Figure 4.1B. is plotted without error bars as the experiments at 20% CO2were done

in single columns

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(A)

y = 1.0368e0.0411x

R2= 0.9103

0

2

4

6

8

0 20 40 60 80 100 120

Time (hrs)

OD730nm

(cm-1)

Column 1

Series2

Expon. (Series2)

(B)

(C)

y = 1.0437e0.0423x

R2= 0.9612

0

2

4

6

8

0 20 40 60 80 100 120

Time (hrs)

OD730nm

(cm-1)

Column 2

Series2

Expon. (Series2)

y = 0.9708e0.0437x

R2= 0.9444

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100 120

Time (hrs)

OD730nm

(cm-1)

Column 3

Series2

Expon. (Series2)

Figure 4.2.Calculation of growth rates using exponential fit method for Scenedesmus

species @ 20% CO2 / 80% Air (QGas= 1.90 L/min; 32oC; 160 mol/m2-s; Column

Diameter: 30 mm; Triplicate data).

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(A)

0

2

4

6

8

0 20 40 60 80 100 120

Time (hrs)

OD730nm

(cm-1)

Column 1

Two-Point Data

Two-Point Fit

(B)

0

2

4

6

8

0 20 40 60 80 100 120

Time (hrs)

OD730nm

(cm-1)

Column 2

Two-Point Data

Two-Point Fit

(C)

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100 120

Time (hrs)

OD730nm

(cm-1)

Column 3

Two-Point Data

Two-Point Fit

Figure 4.3.Calculation of growth rates using two point method for Scenedesmusspecies

@ 20% CO2 / 80% Air (QGas= 1.90 L/min; 32oC; 160 mol/m2-s; Column Diameter:

30 mm; Triplicate Data).

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(A)

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100 120 140Time (hrs)

OD730nm

(cm

-1)

Air

5% CO2

20% CO2

30% CO2

40% CO2

(B)

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100 120 140Time (hrs)

OD730nm

(cm

-1)

Air

5% CO2

20% CO2

30% CO2

40% CO2

Figure 4.4.Effect of CO2concentration on the growth of (A) Scenedesmusand (B)

Chlorellaspecies (32oC; 160 mol/m2-s; Column Diameter: 30 mm).

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(A)

0

1

2

3

4

5

6

7

8

0 20 40 60 80 100 120 140 160

Time (hrs)

OD730nm

(cm

-1)

65 micro mol/m2-s

120 micro mol/m2-s

160 micro mol/m2-s

(B)

0

1

2

3

4

5

6

7

8

0 20 40 6

madhu_thesis algae photosynthesis

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