APPLICATION OF ALGAL CULTURE TECHNOLOGY FOR CARBON DIOXIDE AND FLUE GAS EMISSION CONTROL by Madhu Hanumantha Reddy

A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science

ARIZONA STATE UNIVERSITY May 2002

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

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 CO2 and the greenhouse gases in the atmosphere is such a serious problem for mankind that many research and development approaches are implemented to reduce CO2 emissions. Various processes in reducing CO2 emissions 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 CO2 and 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 Scenedesmus species. 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% CO2 with 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. CO2 uptake 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

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 CO2 and Flue Gas ..................................................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 vii

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 CO2 Measurement Using Gas Chromatography ....................................... 25 RESULTS ......................................................................................................................... 32 Cell Characterization .............................................................................................32 Species Identification................................................................................ 32 Algal Batch Experiments .......................................................................................33 Growth Rates Comparison........................................................................ 33 Effects in CO2 Concentration.................................................................... 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 CO2 GAS..............................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 Chlorella Species Using Exponential Fit Method and Two Point Method Under Varying Environmental Conditions .......................... 47 4.2. Results of Growth Rates for Scenedesmus Species Using Exponential Fit Method and Two Point Method Under Varying Environmental Conditions ............. 48 4.3. Results of Growth Rates for Mixed Species Using Exponential Fit Method and Two Point Method Under Varying Environmental Conditions .......................... 49 4.4. pH Range for the Algal Culture at Varying CO2 Concentrations.............................. 50

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, Scenedesmus and 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 Scenedesmus species @ 20% CO2 / 80% Air (32 oC; 160 mol/m2-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 (32 oC; 160 mol/m2-s; Column Diameter: 30 mm; Triplicate Data) ............................................................................................. 53 4.4. Effect of CO2 concentration on the growth of (A) Scenedesmus and (B) Chlorella species (32 oC; 160 mol/m2-s; Column Diameter: 30 mm)............... 54 4.5. Effect of light intensity on Scenedesmus species growth in (A) 30 mm diameter columns and (B) 50 mm diameter columns (32 oC; 20% CO2) .................. 55 4.6. Effect of temperature on Scenedesmus growth in 50 mm diameter columns (27 oC 42 oC; 20% CO2; 160 mol/m2-s)................................................................ 56 4.7. Effects of (A) Na2SO3 (2 Molar) and (B) NaNO2 (2 Molar) concentrations on Scenedesmus growth (No Aeration; 500 ml Flasks) .................................................. 57 4.8. Algae growth in presence of NOX without pH adjustment (325ppm NO2 / 327ppm NO / bal Nitrogen / 20% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s)....................................................... 58 4.9. Algae growth in the presence of SO2 without pH adjustment (313ppm SO2 / Ultra Zero Air / 20% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s) .............................................................. 59

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Figure

Page

4.10. Effect of SO2 with 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% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s). ................................. 60 4.11. Effect of SO2 and NOX with pH adjustment with 10N NaOH and additional nutrient medium (Potassium Phosphate) on (A) algae growth (B) solution pH (725ppm SO2 / 25ppm NOx / bal Nitrogen / 20% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s) .............................................................. 61 4.12. Comparison of growth curves for Beaver Creek species @ 160 and 245 mol/m2-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 times during 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 entire operation..................................................................................................................... 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 CO2 and 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 CO2 capture 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 (CO2 and 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 CO2 per land area than higher plants (Brown 1996). Capture and utilization of the carbon dioxide and other flue gases by

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 CO2 reduction has been practiced and the CO2 fixation 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 CO2 and 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 CO2 reduction, carbon fixation and biomass production. Comparison of helical tubular photobioreactor (Okano 1999) and flat-plate photobioreactor has been reported. 2

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% CO2, which is 300 - 600 times the normal level of CO2 in the air (Karube et al. 1992).

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 heatabsorbent. 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

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 CO2 levels 6

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. Methane is 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 inground 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 (SO2 and SO3) and nitrogen oxides, NOX (NO and NO2) (Rao and Rao 1996). The concentration of SOX in 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 7

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 NOX is 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 SO2 and NOX interact in the atmosphere. The formation of sulfuric and nitric/nitrous acid is a complex process involving several chemical reactions. SOX: 8

Sulfur dioxide (SO2) is the pre-dominant form of sulfur oxides, and is a colorless and nonflammable gas. The solubility of SO2 in 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 SO2 varies from 3.28 at 0oC to 1.24 at 25oC and 0.56 at 50oC (Wedzicha 1984). SO2 concentrations are most commonly expressed in g/m3 or as a volume-tovolume ratio such as parts per million (ppm). SO2 released 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 SO2 and 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 SO2 by 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 + O2 2SO3 (2) SO3 + H2O H2SO4 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, 9

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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(2) HSO3- (aq) H+ + SO32pK1 = 1.76 pK1 = 7.21

In the aqueous phase, sulfur dioxide exists as three species: [S (IV)] = [SO2. H2O)] + [HSO3- ] + [SO32- ] 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 NO2 is 1.0x10-2 mol/atm. The typical atmospheric partial pressure of NO is 2x10-10 atm and NO2 is 2x10-9 atm. NO reacts rapidly with O2 to 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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reaction with hydroxyl radicals, which are highly reactive and abundant in the atmosphere. HO + NO2 (+M) HONO2 (+M) Aqueous Phase: Nitrogen oxide reacts with oxygen to form nitrogen dioxide, which is a highly reactive, red brown gas, which then reacts with the hydroxyl radical (OH) to form nitric acid (HNO3). The reactions are limited by their dependence upon the partial pressures of NOX present in the atmosphere, and the low solubility of NOX (Warneck 2000). The three equilibria in the aqueous oxidation of NOX are, (1) 2NO2 (g) + H2O (l) 2H+ + NO3- + NO2(2) NO (g) + NO2 (g) + H2O (l) 2H+ + 2NO2(3) 3NO2 (g) + H2O (l) 2HNO3- + NO (g) Algal Photosynthesis Photosynthesis comes from the Greek roots "photo" meaning light and "synthesis" meaning to make something. An example of naturally occurring biological oxidationreduction reactions is the process of photosynthesis. It is a very complex process carried out by green plants and algae. These organisms are able to harness the energy contained in sunlight, and via a series of oxidation-reduction reactions, produce oxygen and carbohydrates, as well as other compounds, which may be utilized for energy as well as the synthesis of other compounds (Karube et al. 1992). CO2 + H2O + light energy (CH2O)n + O2

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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 energy 6O2 + 24 H+ + 24eThe 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+ + H20 NADPH + 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 CO2 and Flue Gas Microalgae have been proved to be most productive carbon dioxide users, which can be grown under varying environmental conditions. To mitigate the CO2 emissions from power plants various cultures of microalgae have been used. Hamasaki et al. (1994) reported testing Nannochloropsis salina, strain NANNP-2, Phaeodactylum tricornutum, strain PHAEO-2 and Tetraselmis sp, strain T-S3 in 10% CO2 and N2 gas at 25oC under 12

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outdoor and indoor conditions in flue gas from power plant. Murakami and Ikenouchi (1997) isolated two green algal strains, Chlorella sp. UK001 and Chlorella littorale and have reported a CO2 fixation rate of 1gCO2/l/day. Chlorella sp. UK001, a unicellular, green freshwater microalga, has been reported to have grown at levels up to 40% CO2, at an optimum temperature of 30oC and 5.5 to 6.0 (Hirata et al. 1996). Okano (1999) isolated the Beaver Creek microalgal culture from Beaver Creek and tested the culture under varying temperature, light and CO2 conditions. The Beaver Creek culture were exposed to temperatures ranging from 25 oC to 37 oC, light intensities ranging from 45 to 141 mol/m2-s, and CO2 concentrations ranging from house air to 20% CO2. Yanagi et al. (1995) investigated the influence of 50 ppm of NOX and SOX and 10% CO2 on the growth of Chorella sp. HA-1, at 380 mol/m2-s at a temperature of 26
o

C; HA-1 did not tolerate 50 ppm SO2 and was not affected by the addition of NO or

NO2. Brown and Zeiler (1994) indicated that high levels of CO2 are easily tolerated by microalgae and initial results indicated that moderate levels of up to 150 ppm SOX and NOX were also well tolerated. Chlorella sp. exhibited growth in simulated flue gas at 20 ppm SOX and 60 ppm SOX and in coal fired flue gas at <10 ppm SOX and 150 ppm NOX (Maeda et al. 1995). Sakai et al. (1995) isolated unicellular Chlorella (Chlorophyta) strains from hot springs of Japan, which exhibited highest specific growth rate at temperature of 42oC with a 40% CO2 concentration.

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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 CO2 from 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 CO2 transfer from the gas stream to the liquid culture medium due to the extensive CO2 absorbing pathway (Watanabe et al. 1995). Okano (1999) reported a CO2 uptake 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 14

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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 Nannochloropsis sp. (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 CO2 fixation (16.7 g l-1 24 h-1) for Chlorella littorale was 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 CO2 emissions. 15

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 25 oC. CO2 is 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 200250ml 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

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 Products Model 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, CO2 and house (Compressed) air were blended to the desired concentrations for the batch experiments using a ColeParmer (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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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-Strut vertical supporting structures, 1.22 m tall. A Cole-Parmer Gas Proportioner flow meter was used to mix the house air and compressed CO2 source using a CONCOA (Virginia Beach, Virginia) Regulator. The CO2 / air mixture was bubbled at

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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 ColeParmer carboy containers were utilized to hold the BG-11 medium for continuous supply and collection. The photobioreactor also consisted of a Cole-Parmer two-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 vulgaris and Scenedesmus caribeanus, and a mixed culture of Chlorella and Scenedesmus were used in this project for the batch 19

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 26 oC for a week or 15 days until the culture turns deep, dark green color. The culture is then centrifuged at a temperature of 25 oC 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 CO2 enriched air through the column, at increasing step-wise increments of CO2 concentrations. After the cells had adjusted to the different CO2 concentrations, 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 CO2 until visible colonies are formed and then transferred. Optical Density A Shimadzu UV-1601 UV-Visible Spectrophotometer was used to analyze growth rates of algal species by measuring optical density. As the photosynthetic

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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-Scientific Fisherbrand* 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 glassmicro fiber filters, which were previously ashed in an oven at 550 oC 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 THELCO Laboratory oven at 105-110 oC for approximately 24 hours. The filter was taken and cooled to room

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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 CO2 was used during the experiments. In order to provide continuous supply of CO2 for the species, pure CO2 was supplied through Praxair compressed tanks fitted with a CONCOA high purity regulator. The supply of CO2 was 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 Parmer Gas 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 CO2 concentration, the gas flow of house air and the compressed CO2 were adjusted, and setting the float reading on the flow meter. Dividing the CO2 flow 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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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-Parmer mercury 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 Products Model 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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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 Na2SO3 and NaNO2 were 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 SO2 / 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 CO2 using a Cole Parmer Gas 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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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 SO2 and Ultra zero air (Cylinder 1), 325ppm NO2 / 327 ppm NO / bal Nitrogen (Cylinder 2). Each cylinder was used separately and blended with compressed CO2 and compressed air to a desired concentration using the Cole Parmer Gas 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. CO2 Measurement 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 CO2 concentration 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 60 oC and at a flow rate of 25-30 mL/min. The detector and the injector temperature were set at 120 oC and the A injection port was used to inject the CO2 gas.

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A calibration curve using a stock 30% CO2 by moles (balance nitrogen, N2) gas tank from Air Liquide (Laporte, Texas) was developed. Six different injections were made with the stock 30% CO2 gas with CO2 concentrations 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 CO2 entering 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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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 K2HPO4 All 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 NaNO3 7.5 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 Minerals

Trace Minerals:
1 liter 2.86 gms 1.81 gms 0.22 gms 0.39 gms 0.079 gms 0.049 gms Other Components: H3BO3 MnCl2. 4H2 ZnSO4.7H2O NA2MoO4.2H2O CuSO4.5H2O Co(NO3)2.6H2O

Ferric ammonium citrate, 6 mg/ml (100 x) 600 mg per 100 ml H2O Na2CO3 (100 x) 2 gms Na2CO3 per 100 ml H2O K2HPO4 (100 x) 3.05 gms K2HPO4 per 100 ml H2O or 4.0 gms for K2HPO4.3H2O.

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Table 3.2 Experimental Conditions Used for Batch Growth Studies Environmental Conditions Adopted: Algal Cultures used: Chlorella vulgaris, Scenedesmus caribeanus Mixed culture (mix of Chlorella vulgaris and Scenedesmus caribeanus) and Beaver Creek culture. Temperature Range: 27 oC 42 oC Light Intensity : 65 mol/m2-s 275 mol/m2-s Carbon 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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Stopper with 2 holes

56 cm

60 cm

18 cm 116 cm Figure 3.1. Plexi glass tank with the glass column used for the experiment.

50mm

Mixed Gas Stream Air CO2 Flue Gas Flow Meters 750-ml Glass Column

Light Panel

High Purity Gas Control Valves Figure 3.2. Overview of batch photobioreactor (not to scale).

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39.58 cm

10.16 cm

Air

CO2 Flow Meters

69.85 cm Flat plate Photobioreactor

Two headed diastolic pump

Waste product Algae

High Purity Gas Control Valves

Fresh BG-11 Medium

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 and Chlorella vulgaris, which are high-CO2 cultures and belong to a class of Chlorellaceae. Scenedesmus culture 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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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 and Chlorella vulgaris cultures. 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 caribeanus in 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. Scenedesmus species was used for most of the growth studies. Due to the high sensitivity of Chlorella to contamination, it was used for comparison studies with the Scenedesmus culture. Both species were exposed at various conditions of light, temperature and CO2 concentrations, 33

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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 CO2 concentrations. 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% CO2 and at 20% CO2 concentrations are shown in FIG. 4.1. At 5% CO2 Chlorella had a slightly higher growth compared to Scenedesmus and 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 Twopoint 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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rate was calculated by selecting two points in the log phase, which showed faster growth. The specific growth rate , was calculated using the formula:

(h 1 ) =

ln X 2 ln X 1 (t 2 t1 )

Where X1 and X2 are the optical densities at times t1 and t2 respectively. 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 CO2 Concentration U.S. power sector emissions are roughly one-third of the U.S. total, and contribute almost 8 percent of global CO2 emissions (U. S. Department of Energy, 1991). TABLE 3.3 shows CO2 emissions 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 32 oC and 160 35

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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 Chlorella species. In house air, the algal species had low growth rate, while at 5%, 20%, 30% and 40% CO2 the 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% CO2 was maintained. FIG. 4.5 shows growth curves for two sets of experiments carried out with the Scenedesmus species 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 Scenedesmus species 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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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 Scenedesmus species in 50 mm diameter columns, with a temperature range of 27 oC to 42 oC. 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 32 oC, a temperature of 32 oC was used for most of the experiments.

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Decreasing temperature did not affect the cell reproduction and all the tests exhibited similar kind of growth. The temperature varies with the environmental conditions and also depends on the kind of light source being used, the more the light intensity the more will be the temperature due to the intensity. Growth in Simulated Flue Gas Addition of NaNO2 and Na2SO3. Out of the entire electric industry, coal-fired power plants contribute 96% of sulfur dioxide emissions, 93% of nitrogen oxide emissions, 88% of carbon dioxide emissions, and 99% of mercury emissions. Scenedesmus species was tested under varying conditions of temperature, CO2 concentration and light intensity, which indicated that the species had the capacity to reproduce under varying conditions. Fresh algal cells isolated from the agar plates were suspended in 500 ml flasks with nutrients. Varying concentrations of 2M Sodium Nitrite (NaNO2) and 2M Sodium Sulfite (Na2SO3) were added to the flasks and the microalgal cells were placed on the shaker under a constant light source of 160 mol/m2-s without aeration. Concentrations of 1 mmol/L, 10 mmol/L, 100 mmol/L, 500 mmol/L, and 1000 mmol/L of SO32- were added to the flasks and the growth was observed every twenty-four hours and was compared with the blank nutrient medium microalgal growth. FIG. 4.7A represents growth for the Scenedesmus species at varying concentrations, at concentrations above 100 mmol/L SO32- a decrease in growth was observed. Similar results were obtained with NaNO2, above 100 mmol/L NO2- the species showed decrease in growth.

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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% CO2 in 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 NOX is 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 SO2 and Ultra zero air was obtained to determine the effect of SO2 on algal flora. Algal cells were aerated with 313 ppm SO2, ultra zero air, house air and 20% CO2 in 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. 39

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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 SO2 within 24 hours. pH remained constant, and the results indicate that SO2 might have an adverse influence on the algal cells, thereby increasing toxicity. Effect of NOX and SO2 with 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 40

41

growth, but after Scenedesmus species 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% CO2 concentration (Okano 1999) in evaluating the performance and carbon dioxide uptake. BC cells were isolated from Beaver Creek and were aerated with varying concentrations of CO2 to test their ability to grow under high CO2 levels. BC cells were similar to Scenedesmus species, 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% CO2 was 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.

41

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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 CO2 fixation (Hu et al. 1998c). Based on the batch tests results on BC algae a constant light intensity of 245 mol/m2-s, 5% CO2 and a temperature of 32 oC 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 42

43

exact mass of algae produced by the process. Suspended solids tests were performed with Beaver Creek culture at 5% CO2 with 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. CO2 uptake. Flat-plate photobioreactor was operated in continuous mode with continuous supply of 5% CO2 into the reactor. Carbon dioxide measurements were done at the inlet and the outlet twice a day to evaluate the CO2 uptake by the algae. FIG. 4.17 shows the average CO2 concentrations 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 CO2 flow 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.

43

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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 flatplate 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 44

45

FIG. 4.21, it can be interpreted that the carbon production increased after 5 days, when the retention time was changed to 1 day. Flat-Plate photobioreactor run in greenhouse (by Mario E. Soto). Batch experiments were run for 7 days, using the flat-plate photobioreactor (Greenhouse) under natural light conditions with constant supply of 5% CO2 and 95% inhouse compressed air. An average growth of 0.5 cm-1 was achieved during 12 hours of sunlight. The growth curve for the batch run is shown in Appendix A (Fig 3). Under batch growth conditions, at an average optical density of 0.5 cm-1, the biomass productivity was observed to be 0.008 gC/L-d. Continuous flow photobioreactor was operated under natural light conditions for a duration of 10 weeks. A retention time of 2 days was initially set to control initial growth of algae keeping the target algal concentration of 1.5 - 2.0 cm-1. Retention time was set to 4 days due to the higher dilution rate, which caused all the algal species removal due to continuous flow. The biomass productivity for the continuous flow flat-plate photobioreactor for 10 weeks at an average optical density of 1.5 cm-1 was 0.043 gC/L-d. A hydraulic retention time of 4 days was maintained during the entire operation of the continuous flow photobioreactor. Appendix A (Fig 4) indicates the fluctuation in the optical density concentration during the entire operation of the photobioreactor. It can also be seen from Appendix A (Fig 5) variation in the sunlight (solar radiation measured in MJ/m2) may have caused the decrease in algal concentration. From Appendix A (Fig 6) we can interpret that algal growth increased during day light, but it started decreasing after sunset which indicates that sunlight is an important factor in algal photosynthesis. 45

46

The factors to be considered which might have significantly affected the algal growth are the clogging of waste lines, solution used to prepare the BG-11 nutrient medium, and the placement of the photobioreactor to the sunlight.

46

TABLE 4.1. Results of growth rates for Chlorella species using exponential fit method and two point method under varying environmental conditions

48

TABLE 4.2. Results of growth rates for Scenedesmus species using exponential fit method and two point method under varying environmental conditions

48

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

49

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Table 4.4 pH Range for the Algal Cultures at Varying CO2 Concentrations
CO2 Conc. Air Species Chlorella Scenedesmus Mixed Chlorella Scenedesmus Mixed Chlorella Scenedesmus Chlorella Scenedesmus Chlorella Scenedesmus Initial pH Final pH 7.65 7.83 8.25 5.93 6.1 6.21 5.6 5.83 5.32 5.89 5.35 5.89 9.26 10.45 11.06 6.91 7.18 7.13 6.38 6.51 6.23 6.32 6.33 6.41

20 30 40

50

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(A)
9 8 7 OD730nm (cm ) 6 5 4 3 2 1 0 0 20 40 60 Time (hrs) 80 100 120 Chlorella Scenedesmus Mixed
-1

(B)
9 8 7 6 5 4 3 2 1 0 0 20 40 60 80 100 Time (hrs)

OD730nm (cm )

-1

Chlorella Scenedesmus Mixed 120 140

Figure 4.1. Comparison of Chlorella, Scenedesmus and Mixed algal cultures (Column Diameter: 30 mm; 160 mol/m2-s; 32 oC) @ (A) 5% CO2/95% Air (QGas= 0.95 L/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% CO2 were done in single columns 51

52

(A)
8 OD730nm (cm-1 ) 6 4 2 0 0 20 40 60 Time (hrs) 80 100 120 y = 1.0368e0.0411x R2 = 0.9103 Column 1 Series2 Expon. (Series2)

(B)
8 OD730nm (cm )
-1

y = 1.0437e0.0423x R2 = 0.9612 Column 2 Series2 Expon. (Series2)

6 4 2 0 0 20 40 60 Time (hrs) 80 100 120

(C)
9 8 7 6 5 4 3 2 1 0 0 20 40 60 Time (hrs) 80 100 120 y = 0.9708e0.0437x R2 = 0.9444 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; 32 oC; 160 mol/m2-s; Column Diameter: 30 mm; Triplicate data). 52

OD730nm (cm-1 )

53

(A)
8 OD730nm (cm-1 ) 6 4 2 0 0 20 40 60 Time (hrs) 80 100 120 Column 1 Two-Point Data Two-Point Fit

(B)
8 OD730nm (cm-1 ) 6 4 2 0 0 20 40 60 Time (hrs) 80 100 120 Column 2 Two-Point Data Two-Point Fit

(C)
9 8 7 6 5 4 3 2 1 0 0 20 40 60 Time (hrs) 80 100 120

OD730nm (cm-1 )

Column 3 Two-Point Data Two-Point Fit

Figure 4.3. Calculation of growth rates using two point method for Scenedesmus species @ 20% CO2 / 80% Air (QGas= 1.90 L/min; 32 oC; 160 mol/m2-s; Column Diameter: 30 mm; Triplicate Data).

53

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(A)

9 8 7

OD730nm (cm )

6 5 4 3 2 1 0 0 20 40 60 80 Time (hrs) 100 120 140

-1

Air 5% CO2 20% CO2 30% CO2 40% CO2

(B)

9 8 7 OD730nm (cm ) 6 5 4 3 2 1 0 0 20 40 60 80 Time (hrs) 100 120 140

Air 5% CO2 20% CO2 30% CO2 40% CO2
-1

Figure 4.4. Effect of CO2 concentration on the growth of (A) Scenedesmus and (B) Chlorella species (32 oC; 160 mol/m2-s; Column Diameter: 30 mm).

54

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(A)
8 7 OD730nm (cm )
-1

6 5 4 3 2 1 0 0 20 40 60 80 Time (hrs) 100 120 140 160 65 micro mol/m2-s 120 micro mol/m2-s 160 micro mol/m2-s

(B)
8 7 OD730nm (cm )
-1

6 5 4 3 2 1 0 0 20 40 60 80 100 120 Time (hrs) 65 micro mol/m2-s 120 micro mol/m2-s 160 micro mol/m2-s 200 micro mol/m2-s 245 micro mol/m2-s 275 micro 140 160 mol/m2-s 180

Figure 4.5. Effect of light intensity on Scenedesmus species growth in (A) 30 mm diameter columns (QGas= 1.90 L/min) and (B) 50 mm diameter columns (QGas= 2.60 L/min; 32 oC; 20% CO2).

55

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8 7 6 OD730nm (cm )
-1

5 4 3 2 1 0 0 20 40 60 80

at 27C at 28C at 30C at 32C at 39C at 42C 100 120 140 160 180 200 220 Time (hrs)

Figure 4.6. Effect of temperature on Scenedesmus growth in 50 mm diameter columns (27 oC 42 oC; 20% CO2; 160 mol/m2-s; QGas= 2.60 L/min).

56

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(A)

3 2 1 0 0 20 40 60 80 100 120 140 160 Time (hrs) Blank 1 mmol Sulfite 10 mmol Sulfite 100 mmol Sulfite 500 mmol Sulfite 1000 mmol Sulfite OD730nm (cm ) OD730nm (cm )
-1 -1

(B)

3 2 1 0 0 20 40 60 80 100 120 140 160 Time (hrs)

Blank 10 mmol Nitrite 100 mmol Nitrite 500 mmol Nitrite 1000 mmol Nitrite

Figure 4.7. Effects of (A) Na2SO3 (2 Molar) and (B) NaNO2 (2 Molar) concentrations on Scenedesmus growth (No Aeration; 500 ml Flasks).

57

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5 OD730nm (cm ) 4 3 2 1 0 0 20 40 60 80 100 Time (hrs) Chlorella Scenedesmus Mixed

-1

Figure 4.8. Algae growth in presence of NOX without pH adjustment (QGas= 1.95 L/min;325ppm NO2 / 327ppm NO / bal Nitrogen / 20% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s).

58

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0.7 0.6 OD730nm (cm ) 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 Time (hrs) 8 10 Chlorella Scenedesmus Mixed 12
-1

Figure 4.9. Algae growth in the presence of SO2 without pH adjustment (QGas= 1.94 L/min;313ppm SO2 / Ultra Zero Air / 20% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s; Final pH = 2.6).

59

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(A)
2 OD730nm (cm )
-1

1.5 1 0.5 0 0 10 20 30 40

Chlorella Scenedesmus Mixed

50

Time (hrs)

(B)

12 10 8 pH 6 4 2 0 0 10 20 30 40 50 Time (hrs) Chlorella Scenedesmus Mixed

Figure 4.10. Effect of SO2 with 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% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s). Note. pH reduced initially and was increased with more addition of 10N NaOH to minimize the effect of SO2 on the initial growth of algae. 60

61

(A)
4 OD730nm (cm ) 3 2 1 0 0 20 40 Time (hrs) 60 80 Chlorella Scenedesmus Mixed
-1

(B)

14 12 10 pH 8 6 4 2 0 0 20 40 Time (hrs) 60 80 Chlorella Scenedesmus Mixed

Figure 4.11. Effect of SO2 and NOX with pH adjustment with 10N NaOH and additional nutrient medium (Potassium Phosphate) on (A) algae growth (B) solution pH (QGas= 2.00 L/min; 725ppm SO2 / 325ppm NOx / bal Nitrogen / 20% CO2 and Compressed Air; Column Diameter: 30 mm; 160 mol/m2-s). Note. As pH decreased with the aeration of flue gases, Initial pH was increased with more addition of 10N NaOH and BG-11 nutrient medium to minimize the effect of flue gas on the growth of algae.

61

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8 7 OD730nm (cm ) 6 5 4 3 2 1 0 0 20 40 Time (hrs) 60 80 160 micro mol/m2-s 245 micro mol/m2-s
-1

Figure 4.12. Comparison of growth curves for Beaver Creek species @ 160 and 245 mol/m2-s (32 oC; 5% CO2; Column Diameter: 30 mm; QGas= 0.952 L/min).

62

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25 20 Flow Rate (L/d) 15 10 Flow (L/d) 5 0 0 5 10 Operation Time (days) 15 20

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

63

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6 5 OD730nm (cm )
-1

2 Days

1.5 Days

1 Day

0.5 Day

0.75 Day

1 Day

4 3 2 1 0 0 5 10 Operation Time (days) 15

Optical Density 20 variation

Figure 4.14. Optical density variation for Beaver Creek culture at different retention times during flat-plate photobioreactor run.

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500 400 Dry Wt. (mg/L) 300 200 100 0 0 0.2 0.4 0.6 0.8
-1

y = 249.67x - 13.427 2 R = 0.9644

Dry Wt. Mg/L Linear (Dry Wt. Mg/L) 1 1.2 1.4

OD730nm (cm )

Figure 4.15. Beaver Creek dry mass cell concentration versus cell optical density.
6 Mass Algae Produced (g/d) 5 4 3 2 1 0 0 0.5 1 1.5 2 2.5 HRT (days) Mass Weight

Figure 4.16. Mass weight curve for Beaver Creek cells. 65

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6 5 4 % CO2 3 2 1 0 0 5 10 Operation Time (days) 15 20 Initial Conc Final Conc

Figure 4.17. Carbon dioxide concentration measurements at the inlet and outlet of the photobioreactor.

66

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3.5 3 Biomass (gms C/d) 2.5 2 1.5 1 0.5 0 0 5 10 15 20 25 Flow (L/d)

Biomass gms C/d

Figure 4.18. Biomass produced per pay at different flow rates during the entire operation.

3.5 Biomass (gms C/d) 3 2.5 2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 HRT (days) Biomass gms C/d

Figure 4.19. Biomass produced per day at different retention times during the entire operation.

67

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500 Cum. Bio. Prod. (gms) 400 300 200 100 0 0 5 10 Operation Time (days) 15 20

Cumulative Biomass Prod

Figure 4.20. Cumulative biomass produced during the entire operation of the reactor.
7000 Cum. Carbon Sup. (gms) 6000 5000 4000 3000 2000 1000 0 0 5 10 Operation Time (days) 15 20 Applied Carbon Cumulative Carbon

Figure 4.21. Cumulative carbon produced versus the applied carbon during the entire operation of the reactor.

68

Discussion Variations in Growth Rate Growth rates for the algal species are listed in tables 4.1 through 4.3 for Chlorella, Scenedesmus and mixed species. Chlorella species showed a maximum growth rate of 0.053 h-1 (50 mm diameter columns, 34 oC, 200 mol/m2-s) and 0.039 h-1 (30 mm diameter columns, 32 oC, 160 mol/m2-s) at a concentration of 20% CO2 using the exponential method. Scenedesmus species showed a maximum growth rate of 0.047 h-1 (50 mm diameter columns, 34 oC, 200 mol/m2-s) and 0.043 h-1 (30 mm diameter columns, 32 oC, 160 mol/m2-s) at a concentration of 20% CO2. A maximum growth rate of 0.043 h-1for mixed species was obtained with a CO2 concentration of 5%, at 160 mol/m2-s, and at 32 oC. Due to the growth pattern of the microalgae, the method adopted to determine the growth rates varies and a best-fit method is used to fit the growth curve of the microalgae in different environmental conditions. An optimum condition of 20% CO2, 160 200 mol/m2-s, and a temperature of 32 oC was considered as the best atmosphere for the microalgal species which exhibited higher growth rates. The values reported were comparatively less than the ones reported using different species of microalgae. Kurano et al. (1995) obtained specific growth rates of 0.078, 0.078 and 0.079 h-1 for Chlorella littorale in culture vessels of diameter 18 mm (10 ml volume), 140 mm (10 ml volume) and 290 mm (10 ml volume) at a temperature of 25 oC, pCO2 at 0.20 under 1 atm, at 250 mol/m2-s, mixed with agitation and aeration. Hu et al. (1998c) observed a specific growth rate of 0.08 hr-1 using 2000 mol/m2-s for

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Chlorella littorale. Under optimized conditions (25 - 30 oC) Murakami and Ikenouchi (1997) observed a growth rate increase of 0.23 h-1 from 0.19 h-1 for Synechocystis aquatilis SI-2 in 5L reactors. Chlorella cultures (H-84 and A-2) obtained from hot springs of Japan has showed a highest specific growth of 0.24 h-1 at 40 oC, 50 E/ m2-s (E, photosynthetic active radiation) and 20% CO2 in a thermo-regulated chamber (Sakai et al.1995). As light intensity is the major factor in algal growth two fixed volume columns (30mm and 50mm in diameter) were used to determine the effect of light intensity. Growth rates were observed to increase with the increase in light intensity and depended on the type of reactor used which requires an optimum surface to volume ratio. Growth in Flue Gas Growth studies at 20% CO2, 313 ppm SO2 with and without pH adjustment caused death of microalgae. Increase in growth of microalgae was observed initially with pH adjustment, but decreased after a lag phase of one day. Experiments with 20% CO2, 325 ppm NOX and 725 ppm SO2, 325 ppm NOX, 20% CO2 had no effect on algal growth, but showed a longer lag phase due to the cell acclimatization. The lifetime of SO2 in the atmosphere is much shorter than CO2. Temperature and some concentrations of SO2 of the flue gas can be removed before treating with microalgae. The process of removal of SO2 from the stack gases is called flue gas desulfurization (FGD), and the best and commonly used process is the limestone process. Limestone is cheap, abundant and limestone is proved to remove SO2 efficiently. Maeda et al. (1995) conducted SOX and NOX resistance and growth tests with Chlorella T-1 strain, at varying concentrations of SOX and NOX with and without pH 70

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adjustment (CaCO3), at 15% CO2, 35 oC. Actual flue gas after passing through the electrostatic precipitator and desulfurization facility was used. pH decreased to 3.0 thereby causing the death of microalgae above concentrations of 20 ppm SOX and algal growth increased after adjusting pH with addition of CaCO3. Zeiler et al. (1995) observed growth of Monoraphidium minutum (NREL Strain Monor02) exposed to 150 ppm NO and 200 ppm SO2 at ~200 E/m2-s, temperature 25 oC. Brown (1996) demonstrated the utilization of NO by microalgae as a N-source by Monoraphidium minutum with sufficient long gas-liquid contact time. Yoshihara et al. (1996) concluded that microalgae which are tolerant to high CO2 concentrations could be used for the removal of NO from the flue gas. From the results, it can be concluded that microalgal growth can be observed with the treatment of SO2 and NOX mixture, where NOX acts as a N-source for the algal photosynthesis, which in turn increases biomass concentration. Due to the increase in the biomass concentration the pH of the medium increases and the effect of HSO3-, SO32- and H2SO3 species in the aqueous phase is suppressed which causes the decrease in pH. As SO32- (hydrophilic acid) will be oxidized to sulfuric acid in moist atmosphere which is toxic to algal cells and also reduces the pH. Constant monitoring and reducing the effect of the SO32- and HSO3- ions in the medium may increase the algal growth and and maintain the pH of the medium. The results confirm the feasibility of the use of microalga with the flue gases. Sodium Hydroxide was used to control the pH of the medium, but with the SO2 experiments, no effect was observed and the growth was decreased. Various buffers should be used which controls the pH and also acts as a nutrient medium for the algal growth. 71

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Comparison of Biomass Production The flat-plate photobioreactor was operated in continuous mode with BC microalgal cultures and a biomass productivity of 0.17 gC/L-d was obtained, which is low compared with the previous works, conducted using the flat-plate bioreactor. Samson and Leduy (1985), from Karube et al. (1992), obtained the algal productivity of 1.17 g/Ld with multistage, continuous cultivation of Spirulina maxima using 4 tanks (~62L volume) connected in series. A productivity of 2.2 g/L-d (Hu et al. 1996a) and 1.68 2.4 g/L-d (Richmond and Hu 1997) were obtained for Spirulina platensis in using flat-plate bioreactor made of glass consisting of 2.6 cm wide flat tanks, while Tredici and Zittelli (1998) obtained 1.93 g/L-d from a 5.4 L flat panel photobioreactor using the same species. Biomass productivity of a photobioreactor depends on close alignment of the culture environment to the needs of the selected algal culture. The optical density during the entire operation was controlled below 4.0 cm-1 by increasing the dilution rate to obtain maximum uptake of CO2. The low productivity may be attributed to the dilution rate of the culture medium, which decreased the algal cell retention time inside the flatplate photobioreactor. Increasing the retention time and mixing rate could help in obtaining higher productivity with the continuous mode of operation.

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Cell Adhesion and Settling in Flat-plate Bioreactor Cell adhesion was observed after 3 days of initial start of the reactor operation, light penetration was impeded by self-shading and light absorption. Which may be due to the trace element deficiency, mixing, disturbance in culture pH, or temperature fluctuations (Pirt et al. 1982). Research in the past few years has indicated that flat-plate reactors made of glass represent a very promising and efficient photobioreactor type (Hu and Richmond 1996, Hu et al. 1996b). Yellowish green foam was noticed due to the high cell density in the reactor, which indicates the sign of distress, damaged, leaky and unhealthy cells (Hu et al. 1996). Antifoam agents can be added to control the foam production, but the adverse effects and the type of agent to be used for a specific culture was not mentioned. Foam was controlled by increasing the dilution rate which removed all the worn out and dead cells from the photobioreactor. Settling of algal cells was observed due to the lower mixing rates adopted. It is clearly shown that the mixing rate and the optical density or the output rate is directly proportional. Hu et al. (1996b) observed change in mixing method affected the growth rates and output rates. Mixing helps to keep the cells in suspension, distribute nutrients and the generated heat within the reactor, improves CO2 transfer, degases photosynthetically produced oxygen, improves mass transfer between the cells and the liquid broth, and facilitates the movement of cells in and out of the illuminated part of the photobioreactor (Ogbonna and Tanaka, 1997). Excessive mixing causes cell damage and

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breakdown of cells, an optimal mixing rate and mixing method should be selected to achieve maximum cell density and output rates. Comparison of Flat-plate and Tubular Photobioreactor Okano (1999) conducted similar experiments using the same environmental conditions in a tubular photobioreactor using the BC cells. The photobioreactor was tested in four trial runs (1) Microalgae batch mode with continuous light; (2) BG-11 medium batch mode with continuous light; (3) Microalgae continuous mode with continuous light (22 days); and (4) Microalgae continuous mode with day/night lighting conditions. Mode 1 retained the most dissolved CO2 of 52% of the input. Mode 3 showed a maximum carbon fixation of 20.03 % based on the input due to the fresh supply of nutrient medium. Compared with the flat-plate photobioreactor the CO2 uptake in continuous mode was 8.50%, which was lower than the tubular photobioreactor that showed 17.60% CO2 uptake. But the biomass productivity was reported to be approximately equal, the flat-plate photobioreactor had 0.17 gC/L-d where as the tubular photobioreactor had a biomass productivity of 0.15gC/L-d. Tubular photobioreactor has better CO2 transfer from the gas stream to the liquid culture medium due to extensive CO2 absorbing pathway, has easy control of temperature and contaminants (Watanabe et al. 1995) which may be the reason for the higher carbon fixation and uptake rates. Flat-plate photobioreactor used in this research was tested in continuous mode with day/night lighting conditions (18 days). The carbon uptake was approximately 8.50% and the carbon fixation based on the input was 3.65%. Similar conditions were 74

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adopted during the entire reactor operation. The low efficiency of the reactor may be due to cell adhesion, contamination, settling and increased dilution rate. With optimal conditions for growth flat-plate photobioreactors can be used to attain maximum productivity and CO2 fixation. Flat-plate photobioreactor has more advantages compared to the tubular photobioreactor due to its front surface exposed directly to the light source which receives the major thrust of intensity, and the side walls are also illuminated by diffuse and reflected light of rather low photon flux densities, which is very effective for photosynthesis (Hu et al. 1998a). The dissolved oxygen path corresponding to the height of the reactor prevents O2 buildup, a serious problem associated with many types of enclosed reactors. Flat panel reactors may be easily cleaned, both from outside and inside, all panels being readily accessible. Also, these 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.

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Conclusion Algal cultures used under varying environmental conditions showed good results and the response to varying concentrations of CO2 has proved that the algal technology can be scaled-up to industrial-size. Chlorella and Scenedesmus culture were tested under varying concentrations and the growth rates were reported. Beaver Creek culture had higher growth rates and was used for the flat-plate photobioreactor operation. The flat-plate photobioreactor was run in continuous mode with varying HRT and the CO2 uptake was 8.50%. The carbon fixation based on the input was 3.65% and the carbon production was 0.17 g C/L-d during the continuous run. The CO2 uptake and the carbon retainment was lower compared to the tubular photobioreactor operation reported by Okano (1999) in continuous mode. The lower performance was due to several factors, which caused the lower productivity, uptakes and lower growth rates. The factors affecting the growth and biomass production should be studies to increase the efficiency. The industrial size photobioreactors can be used for the industrial flue gas emissions. The outdoor operation of the various reactors may increase the gas removal efficiency of the power plants. Inclined flat-plate photobioreactors in series may help in reducing the emissions from the power plants. Due to the higher temperatures of the flue gas which kills algae, physical-chemical treatment like cooling towers, scrubbers, absorption columns, filters should be used for the temperature and other flue gases control. As the algal cultures showed growth at varying CO2 concentrations, CO2 gas can be directly treated. The flue gases also contain SOX, NOX, CO, mercury and other gases, which affect algal growth. Proper treatment of these gases before entering the photobioreactors should be considered. The treatment with NOX had no effect on the

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algal culture. Desulfurization of SOX will decrease the effect of SOX, which decreases the pH of the solution. Several other factors like cell adhesion, rate of mixing, reactor size should be considered before adopting the industrial size reactors. The pilot system to be considered for future work should have higher surface to volume ratio (S/V), higher mixing rates, higher light intensity should be provided. Inclined flat-plate photobioreactor has proven to be efficient and research with these photobioreactors should be considered.

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Murakami, M., and Ikenouchi, M. (1997). The Biological CO2 Fixation and Utilization Project by Rite (2) Screening and Breeding of Microalgae with High Capability in Fixing CO2. Energy Conversion Management, 38, 493-497. Nagase, H., Eguchi, K., Yoshihara, K., Hirata, K. and Miyamoto, K. (1998). Improvement of Microalgal NOX Removal in Bubble Column and Airlift Reactors. Journal of Fermentation and Bioengineering, 86(4), 421-423. Negoro, M., Shioji, N., Miyamoto, K. and Miura, Y. (1991). Growth of Microalgae in High CO2 Gas and Effects of SOX and NOX. Applied Biochemistry and Biotechnology, 28-29, 877-886. Ogbonna, J.C., Yada, H., and Tanaka, H. (1995). Light Supply Coefficient: A new Engineering Parameter for Photobioreactor Design. Journal of Fermentation and Bioengineering, 4, 369-376. Ogbonna, J.C., and Tanaka, H. (1997). Industrial-size Photobioreators. Chemtech, 27, 4349. Ogbonna, J.C., Soejima, T., and Tanaka, H. (1998). An Integrated Solar and Artificial Light System for Internal Illumination if Photobioreactors. Journal of Biotechnology, 70, 289-297. Okano, L. (1999). Photosynthetic Algal Culture Technology for Carbon Dioxide Emission Control and Growing of Renewable Fuels. Masters Thesis, Arizona State University, Department of Civil Engineering, Tempe. Pirt, S.J., Lee, Y., Walach, M.R., Watts-Pirt, M., Balyuzi, H.M.H., and Bazin, J.M. (1983). A Tubular Bioreactor for Photosynthetic Production of Biomass from Carbon Dioxide: Design and Performance. Chemical Technology Biotechnology, 33B, 35-58. Rai, LC., Kumar, H.D., Mohn, F.H., and Soeder, C.J. (2000). Services of Algae to the Environment. Journal of Microbiology and Biotechnology, 10(2), 119-136. Rao, M.N., and Rao, H.V.N. (1996). Air Pollution. Tata McGraw-Hill Publications, New Delhi: 4-14, 221-255. Richmond, A., and Qiang, H. (1997). Principles for Efficient Utilization of Light for Mass Production of Photoautotrophic Microorganisms. Applied Biochemistry and Biotechnology, 63-65, 649658.

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Sakai, N., Sakamoto, Y., Kishimoto, N., Chihara, M., and Karube, I. (1995). Chlorella Strains from Hot Springs Tolerant to High Temperature and High CO2. Energy Conversion Management, 36, 693-696. Smoot, D. (1997). Coal Combustion. Wiley Encyclopedia of Energy and the Environment, Vol. 1, Wiley Interscience, 405-413. Tredici, M., and Zittelli, G. (1998). Efficiency of Sunlight Utilization: Tubular Versus Flat Photobioreactors. Biotechnology and Bioengineering, 57(2), 187-197. U. S. Department of Energy. (1991). Clean Coal Technology Demonstration Program: Program Update 1990 (as of December 31, 1990), report DOE/FE-0219P, Washington, D.C. Warneck, P. (2000). Chemistry of the Natural Atmosphere. Orlando, Fl: Academic Press, 119-126, 541-574, 611-652. Watanabe, Y., de la Noue, J., and Hall, D.O. (1995). Photosynthetic Performance of a Helical Tubular Photobioreactor Incorporating the Cyanobacterium Spirulina platensis. Biotechnology and Bioengineering, 47, 261-269. Wedzicha, B.L. (1984). Chemistry of Sulphur Dioxide in Foods. Elsevier Applied Science Publishers, New York: 10-43, 128-139. Wodzinski, R.S. and Alexander, M. (1978). Effect of Sulfur Dioxide on Algae. Journal of Environmental Quality, 7(3), 358-360. Yanagi, M., Watanabe, Y., and Saiki, H. (1995). CO2 Fixation by Chlorella sp. HA-1 and its Utilization. Energy Conversion Management, 36, 713-716. Yoshihara, K., Nagase, H., Eguchi, K., Hirata, K., and Miyamoto, K. (1996). Biological Elimination of Nitric Oxide and Carbon Dioxide from Flue Gas by Marine Microalga NOA-113 Cultivated in a Long Tubular Photobioreactor. Journal of Fermentation and Bioengineering, 82(4), 351-354. Yun, Y., Park, J., and Yang, I. (1996). Enhancement of CO2 Tolerance of Chlorella Vulgaris by Gradual Increase of CO2 Concentration. Biotechnology Techniques, 10(9), 713-716. Zeiler, K., Heacox, D., Toon, S., Kadam, K., and Brown, L. (1995). The Use of Microalgae for Assimilation and Utilization of Carbon Dioxide from Fossil Fuel-fired Power Plant Flue Gas. Energy Conversion Management, 36, 707-712.

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Zou, N., and Richmond, A. (1999). Effect of Light-path Length in Outdoor Flat-plate Reactors on Output Rate of Cell Mass and of EPA in Nannochloropsis sp. Journal of Biotechnology, 70, 351-356.

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APPENDIX A RESULTS OF FLAT-PLATE PHOTOBIOREACTOR RUN IN GREENHOUSE (BY MARIO E. SOTO)

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GROWTH OF MONTEZUMA WELL ALGAE ON BG-11 NUTRIENT SOLUTION, 5 % CO2 AND NATURAL LIGHT CONDITIONS Research Report by Mario Esparza-Soto Spring of 2001 ABSTRACT Arizona native algae were successfully grown in a 20-liters flat-plate reactor under natural light and continuous flow conditions for seventy days. After several changes in operation conditions, it was determined that the reactor was operated at an optimum retention time of 4 days and an algae concentration of 1.5 to 2.0 cm-1. Several factors were determined to influence the performance of the reactor: amount of sunlight, direct/indirect sunlight, water quality of nutrient solution. Preliminary Tubular Batch Experiments Several tubular batch columns were run in the laboratory from 01-11-01 to 02-2001. Algae were grown in BG-11 nutrient solution and a mixture of 95 % in-house compressed air and 5 % compressed CO2. The algae culture was illuminated 24-hours with a panel of 4 fluorescent lights. Each tubular batch reactor was started at the stationary phase of algae growth by suspending 20 ml of centrifuged algae (15 min at 1800 rpm) in 800 ml of BG-11. Centrifuged algae were collected from the previous batch reactor as algae reached their stationary phase. Figure 1 shows the growth curve of algae in the tubular batch reactors. The reactors were started at an approximate OD730 of 0.5 cm-1. Lag growth phase lasted approximately 10 to 12 hours. The stationary growth phase OD730 was approximately 4.5

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cm-1 and was reached after 2 days, however most of the batch reactors were stopped after 4 to 5 days. Stationary-phase algae was centrifuged as previously described and used as seed for the new batch reactor. Total Suspended Solids Total suspended (TS) and volatile solids (VS) were measured on stationary-phase algae. Increasing volumes of suspended algae (10 to 30 ml) were vacuum-filtered through weighted glass-fiber filters (GF/C, Whatman, combusted at 550 C for 3 hours). TS and VS were estimated following the Standard Method 2540 B and C for total and volatile suspended solids, respectively. Figure 2 shows the plot of TS vs. OD730. The regression line was TS (mg/l) = 216.45 * OD730 (cm-1) (R2 = 0.92). The VS fraction of TS was 94 +/- 2.2 (n = 9). Batch Experiment Under Natural Light Conditions Batch experiments were performed at the greenhouse located at the roof of the Life Science Building. The reactor used was a flat-plate reactor, which was first run under batch conditions, 5 % CO2 and natural light (normal day and night cycle). A batch cycle of 7 days was run to acclimate the algae to the new light conditions. The flat-plate reactor was started by suspending centrifuged algae from the last tubular batch reactor in 20 liters of BG-11 nutrient solution. The initial OD730 was approximately 0.35 cm-1. Figure 3 shows the growth curve of the algae in the batch flat-plate reactor under natural light conditions. The lag-growth phase of algae lasted for approximately the first 24 hours. Once algae entered the exponential growth phase, it followed the following cycle: daytime growth, overnight non-growth. This sequence was repeated the last seven 85

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days monitored, however, the growth rate of 0.5 cm-1 per 12 hours of sunlight achieved from day two to day five slowed down to 0.3 cm-1 after the sixth day. Slower growth rate was attributed to the algae reaching the stationary phase of growth. Continuous Flow Reactor After running the flat-plate reactors under batch conditions for seven days, the system was shifted to continuous flow. The targeted algae concentration for the continuous-flow condition was between 1.5 and 2 cm-1 because of it was demonstrated that algae were growing at their fastest (exponential) rate at this concentration range under batch conditions (Figure 4). Batch-grown algae were centrifuged (1800 rpm for 20 minutes) and diluted accordingly in 20 liters of new BG-11 nutrient solution to reach an OD730 of 2.15 cm-1. A batch of 20 liters of BG-11 nutrient solution were made and pumped through the reactor. Retention time was adjusted by changing the flowrate to the desired value. Figure 4 shows the variation of algae concentration as OD730 during the ten weeks the continuous flow reactor was operated. Each vertical line represents a week of operation. Retention time was initially set at 2 days, but within the first two days of operation, algae were washed out the system as the OD730 decreased from 2.15 to 1.24 (Figure 4). Retention time was then increased to 4 days after the third day and kept constant until the end of the experiment. After retention time was increased to 4 days the algae recovered by increasing OD730 from 1.45 to 2.0 cm-1 within two days (Figure 4). However, after the middle of the first week, OD730 decreased from 2.0 to approximately 1.1 cm-1 at the end of the second week. This decrement was caused by a front of bad 86

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weather that reached the city, making the days very clouded for a period of seven consecutive days and decreasing from 25 to 75 % the total daily solar radiation available to algae (Figure 5). The total daily solar radiation measured at the Encanto weather station, located on Central Phoenix (southeast of intersection of Thomas Rd. & 19th Ave), are plotted in Figure 5 for the period of the experiment. After the end of the second week, the bad weather was gone and solar radiation was available through most of the day. During the third and fourth weeks, the algae concentration increased from 1.1 to a maximum of 1.9 cm-1 because of several reasons. First, algae were growing very fast because of higher solar radiation than previous two weeks. Second, algae started forming big clumps and clogging waste lines, which were pumping out waste algae directly from the reactor, instead of collecting overflow waste. At the beginning of the fifth the problem of the clogging of waste lines was solved by washing the lines every two or three days to keep them free of algae clumps. During this period of time algae concentration stabilized around 1.5 cm-1. During the sixth and the seventh weeks algae concentration decreased to 1.0 cm-1 because the BG-11 stock solution was prepared with chlorinated tap water instead of deionized water for at least two retention times (8 days). This stress period covered from approximately the middle of the fifth week to the middle of the sixth week six. By the end of this period, the BG-11 stock solution was prepared correctly and the system started recovering by the beginning of the eight week. During the last two weeks of operation the algae concentration observed an increasing trend from 1.1 cm-1 at the end of 87

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the tap-water stress period to approximately 1.4 cm-1 when the system was shut down. If the system was operated longer, the algae concentration may have completely recovered to the optimum concentration of 1.5 - 2 cm-1. Figure 6 shows the observed daily pattern of the algae growth curve during three days. Algae grew only during daylight, as expected, but the algae concentration peaked approximately at 3 p.m. and by sunset the concentration decreased. This may have been caused by the position of the reactor in the greenhouse. The reactor was located close to the west wall of the greenhouse and other greenhouse room was located to this side, which filtered sunlight and did not allowed direct contact with the reactor after 3 p.m. The same happened in the morning during the first three hours after sunrise, the sunlight was obstructed by the east wall (concrete) of the greenhouse and did not fall directly over the reactor. Therefore, approximately 40 % of sunlight potential (12-14 hours of sunlight; 5-6 hours of non-direct sunlight) was lost by structure interference. If this 40 % of sunlight were used more efficiently a higher algae concentration may have obtained at the end of each day. On the other hand, algae concentration decreased overnight because of the continuous flow conditions of the system and the lack of sunlight for growth. For optimal reactor operation, the algae concentration has to be approximately the same each morning to be sure that the algae is not being washed out the system if it continuously decrease each morning. On the other hand, if the algae concentration continuously increase each morning it may be a signal of accumulation within the system.

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6 5 OD730nm (cm )
-1

4 3 2 1 0 0 12 24 36 Time (hrs) 48 60 72 Reactor 1 Reactor 2

Figure 1. Algae Growth Curve: Tubular Batch Reactor, 5 % CO2, and 24-hr Fluorescent Light Illumination.

500 400 TS (mg/L) 300 200 100 0 0 0.5 1 1.5

-1

y = 216.45x 2 R = 0.9241

2.5

OD730nm (cm )

Figure 2. Total Solids of Algae Versus Optical Density at 730nm.

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3.5 3 OD730nm (cm )

-1

2.5 2 1.5 1 0.5 0 2/20/01 2/21/01 2/22/01 2/23/01 2/24/01 2/25/01 2/26/01 2/27/01 2/28/01 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 AM AM AM AM AM AM AM AM AM

Figure 3. Algae Growth Curve: Flat-Plate Reactor, Batch Conditions, Natural Light Conditions, BG-11 Nutrient Solution, and 5 % CO2.
2.2 2.0 OD730nm (cm ) 1.8 1.6 1.4 1.2 1.0
2/28/01 12:00 AM 3/7/01 12:00 AM 3/14/01 3/21/01 3/28/01 12:00 12:00 12:00 AM AM AM 4/4/01 12:00 AM 4/11/01 4/18/01 4/25/01 12:00 12:00 12:00 AM AM AM 5/2/01 12:00 AM 5/9/01 12:00 AM
-1

Figure 4. Algae Growth Curve: Flat-Plate Reactor Under Continuous Flow, Natural Light Conditions, BG-11 Nutrient Solution and 5 % CO2.

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30

Solar Radiation (MJ/m ) OD730nm (cm )

-1

20

10

0 2/28/01 3/7/01 3/14/01 3/21/01 3/28/01 4/4/01 4/11/01 4/18/01 4/25/01 5/2/01 5/9/01 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 AM AM AM AM AM AM AM AM AM AM AM

Figure 5. Total Daily Solar Radiation Measured at the Encanto Weather Station Located in Central Phoenix for the Duration of the Continuous Flow Experiment

1.50 1.40 1.30 1.20 1.10 1.00

4/30/01 12:00 AM 5/1/01 12:00 AM 5/2/01 12:00 AM 5/3/01 12:00 AM

Figure 6. Daily Trend of the Algae Growth Curve. Same Conditions as B.

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APPENDIX B CALCULATION OF CARBON BIOMASS IN CO2 GAS

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Ideal gas law: Ideal gas constant:

pV = nRT R = 0.08205783 L-atm/mole-K

Measured values in Continuous mode: pCO2 = 0.051 atm (5.20 % of Injection air) T = 305K (32 oC) CO2 gas flow rate = 0.132 L/min Solving equation:

molCO2 0.0510atm n p = = = 0.00203 V RT (0.08205783L atm / mol K )(305K ) L Mass concentration of C per liter
0.00203 molCO2 12 gC gC 1molC )*( ) = 0.0243 *( L molC L 1molCO2

Mass of C entering the system 0.0243 gC L 1440 min gC * (1.32 )*( ) = 46.20 min L day day

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APPENDIX C CARBON FIXATION CALCULATIONS FOR CONTINUOUS MODE

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Measured values

Initial OD in the system

= 0.2430 cm-1 = 47.24 mg biomass/L

Final OD out of the system = 1.3945 cm-1 = 334.73 mg biomass/L Percent Carbon in biomass = 53.55 % Carbon Input into the system = 46.20 gC/day Carbon Retained in the system = 34.24 gC/day Amount of biomass production per day in the system
(249.67 * OD 13.427)
i =1 n

mgbiomass L * (Q )* L day

1 ( OD)
i =1 n

mgbiomass day

Amount of carbon production per day in the system Average( mgbiomass 0.5355mgC gC gC *( )*( )) = 1.69 1000mgC day mgbiomass day

Percentage of carbon fixed based on input 1.69 gCproduced day * 100% = 3.65% gCinput 46.2 day

Percentage of carbon fixed based on retainment 1.69 gCproduced day *100% = 4.93% gCretained 34.24 day

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Time 0 3 11 16 23 27 33 37 39 43 47 49 51.5 60 60 63 68 75 85 90 90 96 107 111 116 121 121 129 135 140 145 155 161 165 168.5 170.5 179 181 183 187.5 193

Time in da OD Actual OD 0 0.0486 0.243 0.125 0.0692 0.346 0.458333 0.1162 0.581 0.666667 0.155 0.775 0.958333 0.215 1.075 1.125 0.2553 1.2765 1.375 0.5335 2.6675 1.541667 0.6677 3.3385 1.625 0.7241 3.6205 1.791667 0.9094 4.547 1.958333 0.9867 4.9335 2.041667 0.6599 3.2995 2.145833 0.6594 3.297 2.5 0.6772 3.386 2.5 0.5745 2.8725 2.625 0.575 2.875 2.833333 0.6357 3.1785 3.125 0.7098 3.549 3.541667 0.7885 3.9425 3.75 0.8116 4.058 3.75 0.3943 1.9715 4 0.4309 2.1545 4.458333 0.6667 3.3335 4.625 0.7209 3.6045 4.833333 0.7239 3.6195 5.041667 0.73 3.65 5.041667 0.6577 3.2885 5.375 0.7332 3.666 5.625 0.7845 3.9225 5.833333 0.812 4.06 6.041667 0.6322 3.161 6.458333 0.7717 3.8585 6.708333 0.8114 4.057 6.875 0.681 3.405 7.020833 0.7265 3.6325 7.104167 0.7564 3.782 7.458333 0.8011 4.0055 7.541667 0.5093 2.5465 7.625 0.6055 3.0275 7.8125 0.6425 3.2125 8.041667 0.556 2.78

Biomass flow L/DamgC/L gC/day 47.24281 5.5 104.1092 0.5726 72.95882 5.5 112.9175 0.621046 131.6313 5.5 121.3228 0.667275 180.0673 5.5 124.4207 0.684314 254.9683 5.5 127.0098 0.698554 305.2768 5.5 128.0656 0.704361 652.5677 5.5 131.0028 0.720515 820.0963 5.5 131.5446 0.723495 890.5032 5.5 131.7123 0.724418 1121.822 5.5 132.117 0.726643 1218.32 5.5 132.2409 0.727325 810.3592 7.33 131.5191 0.964035 809.735 7.33 131.5175 0.964023 831.9556 7.33 131.5748 0.964443 703.7501 7.33 131.1952 0.961661 704.3743 7.33 131.1974 0.961677 780.1491 7.33 131.4362 0.963427 872.6518 7.33 131.6723 0.965158 970.897 7.33 131.8745 0.96664 999.7339 7.33 131.9264 0.967021 478.7974 11 130.0512 1.430564 524.487 11 130.361 1.433971 818.8479 11 131.5413 1.446955 886.5085 11 131.7035 1.448739 890.2536 11 131.7118 1.44883 897.8685 11 131.7284 1.449012 807.6128 11 131.5118 1.44663 901.8632 11 131.737 1.449107 965.9036 11 131.8652 1.450518 1000.233 11 131.9273 1.4512 775.7799 11 131.4236 1.44566 949.9247 11 131.8348 1.450183 999.4842 11 131.926 1.451186 836.6994 11 131.5866 1.447453 893.4993 11 131.7189 1.448908 930.8249 11 131.7971 1.449768 986.6262 11 131.9032 1.450935 622.3577 22 130.8747 2.879244 742.4489 22 131.3233 2.889113 788.6379 22 131.4601 2.892122 680.6556 22 131.1119 2.884462

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