Parametric Study of Light Intensity on the Growth Rate Of

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PARAMETRIC STUDY OF LIGHT INTENSITY ON THE GROWTH RATE OF

“CHROOGLOEOCYSTIS SIDEROPHILA” IN A PHOTO-BIOREACTOR

A thesis presented to

the faculty of

the Russ College of Engineering and Technology of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Venkata R. Gidugu

November 2007 2

This thesis titled

PARAMETRIC STUDY OF LIGHT INTENSITY ON THE GROWTH RATE OF

“CHROOGLOEOCYSTIS SIDEROPHILA” IN A PHOTO-BIOREACTOR

VENKATA R. GIDUGU

has been approved for

the Department of Chemical and Biomolecular Engineering

and the Russ College of Engineering and Technology by

David J. Bayless

Professor of Mechanical Engineering

Dennis Irwin

Dean, Russ College of Engineering and Technology 3

Abstract

GIDUGU, VENKATA R., M.S., November 2007, Chemical Engineering

PARAMETRIC STUDY OF LIGHT INTENSITY ON THE GROWTH RATE OF

“CHROOGLOEOCYSTIS SIDEROPHILA” IN A PHOTO-BIOREACTOR (168 pp.)

Director of Thesis: David J. Bayless

In this work, “Chroogloeocystis siderophila” (CS) culture was used to

photosynthetically sequester carbon dioxide produced from natural gas combustion

products in a film-based photo-bioreactor. The photo-bioreactor enhances the natural

process of photosynthesis and also offers the possibility of both an environmentally and

economically sustainable process of sequestering CO2 from flue gas and turning it into

biomass that could be used as an energy source.

The goal of the current research was to quantify CS culture productivity as a

function of specific photon flux intensity from 60 µmol m-2s-1 ≤ average photon flux ≤ 85

µmol m-2s-1 (high) to average photon flux ≤ 30 µmol m-2s-1 (low). The results indicated

that the productivity of CS culture in a photo-bioreactor at high light intensity was greater

than at low light intensity. Possible recommendations for increasing the productivity of

CS culture were made by analyzing each test results, as well as given for further testing – especially with shorter harvesting period, repeating light level productivity testing.

Approved:

David J. Bayless

Professor of Mechanical Engineering 4

Acknowledgments

I would like to express my sincere thanks to all of them who have contributed in any way to the completion of my thesis work.

Firstly, I would like to thank my advisor Dr. David J Bayless for his support, enthusiasm and guidance for this work. I would also like to thank Dr. Gregory Kremer,

Dr. Ben J Stuart, Dr. Morgan Vis and Dr. Stoy for their guidance and valuable time in finishing the work.

I am grateful to Micah McCreeny, Shyler Switzer and each and everyone working in the OCRC group for their wonderful support and help to the project. Specially, I would like to thank my friends Chamila Jayathilake, Sreerupa Basu, and Santosh Vijapur for their help.

I wish to thank my sister, brother and brother-in-law for their caring and supportive environment at all times.

Last, and most importantly, I wish to thank my parents Satyanarayana D Gidugu and Manikyamba Gidugu. I cannot end without thanking my fiancé Madan K Teegala for being there with me at all crucial times, encouraging and helping me. It is to them that I dedicate this Thesis. 5

Table of Contents

Page

Abstract...... 3 Acknowledgments...... 4 List of Tables ...... 7 List of Figures...... 9 Nomenclature...... 14 1. Introduction...... 16 1.1. Carbon Dioxide Sequestration Technologies from Flue Gases ...... 18 1.2. Photo-Bioreactor for Carbon Dioxide Sequestration...... 19 1.3. Uniqueness of the Research...... 21 1.4. Biomass as Biofuel ...... 23 1.5. Applications of Microalgae...... 23 1.6. Research Objective ...... 25 2. Literature Review...... 27 2.1. Microalgae Productivity at Different Light Conditions...... 27 2.2. Effect of Light on “Chroogloeocystis siderophila”...... 32 3. Experimental Setup...... 35 3.1. Burner Setup...... 36 3.2. Growth System...... 38 3.2.1. Initial Mass Loading ...... 39 3.2.2. Loading CS culture into Solution Tank ...... 40 3.3. Growth Surfaces...... 43 3.4. Lighting System...... 44 3.5. Harvesting System...... 47 3.6. Nova Gas Analyzer...... 50 3.7. Data Acquisition System (DA System) ...... 51 4. Test Plan & Operational Procedures...... 52 6

4.1. Average Temperature...... 54 4.2. Average Air Velocity...... 55 4.3. Gas Concentrations...... 56 4.4. Solution Flow Rate ...... 56 4.5. Average pH of the Solution ...... 57 5. Results & Discussion ...... 60 5.1. Deviations in Actual Test Conditions from the Test Plan ...... 60 5.2. Uncertainties in Productivity ...... 61 5.3. Test #1...... 62 5.4. Test #2...... 69 5.5. Test #3...... 74 5.6. Test #4...... 79 5.7. Test #5...... 84 6. Conclusions & Recommendations...... 90 6.1. Recommendations for Future Work...... 92 References...... 97 Appendix A...... 100 Test 1...... 100 Test 2...... 123 Test 3...... 132 Test 4...... 141 Test 5...... 150 Appendix B ...... 159

List of Tables

Table 2.1: Overview of the literature review...... 31 Table 4.1: Average Air Velocity Measurements Before and After Tests...... 56 Table 4.2: Proposed Test Matrix...... 58 Table 5.1: Test Results from the Five Tests ...... 60 Table 5.2: Light Intensity Measurements before the First Test...... 63 Table 5.3: CS culture Productivity from the First Test...... 67 Table 5.4: Light Intensity Measurements after the First Test...... 68 Table 5.5: Outer Light Panel Intensities for the First Test ...... 69 Table 5.6: Light Intensity Measurements before the Second Test...... 70 Table 5.7: The CS culture Productivity from the Second Test ...... 72 Table 5.8: Light Intensity Measurements after the Second Test ...... 73 Table 5.9: Outer Light Panel Intensities for the Second Test...... 74 Table 5.10: Light Intensity Measurements before the Third Test ...... 75 Table 5.11: The CS culture Productivity from the Third Test ...... 77 Table 5.12: Light Intensity Measurements after the Third Test ...... 78 Table 5.13: Outer Light Panel Intensities for the Third Test...... 79 Table 5.14: Light Intensity Measurements before the Fourth Test...... 80 Table 5.15: The CS culture Productivity from the Fourth Test ...... 82 Table 5.16: Light Intensity Measurements after the Fourth Test...... 83 Table 5.17: Outer Light Panel Intensities for the Fourth Test ...... 84 Table 5.18: Light Intensity Measurements before the Fifth Test...... 85 Table 5.19: The CS culture Productivity from the Fifth Test ...... 87 Table 5.20: Light Intensity Measurements after the Fifth Test ...... 88 Table 5.21: Outer Light Panel Intensities for the Fifth Test...... 88 Table A.1: Outer Membrane Coverage’s, a Day after Loading CS Culture...... 100 Table A.2: Outer Membrane Coverage’s, a Day after Loading CS Culture...... 123 Table A.3: Outer Membrane Coverage’s, a Day after Loading CS Culture...... 132 8

Table A.4: Outer Membrane Coverage’s, a Day after Loading CS Culture...... 141 Table A.5: Outer Membrane Coverage’s, a Day after Loading CS Culture...... 150 Table B.1: Light Measurements before the First Test ...... 159 Table B.2: Light Measurements after the First Test ...... 160 Table B.3: Light Measurements before the Second Test...... 161 Table B.4: Light Measurements after the Second Test...... 162 Table B.5: Light Measurements before the Third Test...... 163 Table B.6: Light Measurements after the Third Test...... 164 Table B.7: Light Measurements before the Fourth Test...... 165 Table B.8: Light Measurements after the Fourth Test...... 166 Table B.9: Light Measurements before the Fifth Test...... 167 Table B.10: Light Measurements after the Fifth Test...... 168 9

List of Figures

Figure 1.1: Recent Global Climate Change ...... 17 Figure 1.2: Calvin-Benson Cycle Showing Photosynthesis Process ...... 20 Figure 1.3: Photosynthetic Conversion of Carbon Dioxide ...... 21 Figure 1.4: Demand for Liquid Energy from Renewable Sources in the Future ...... 24 Figure 2.1: Air-Lift Bioreactor Configuration ...... 29 Figure 2.2: The Optical Characteristics of CS Culture ...... 33 Figure 3.1: Picture of the Experimental Setup...... 36 Figure 3.2: Burner Setup...... 37 Figure 3.3: Rectangular and Cylindrical Culture Tanks ...... 39 Figure 3.4: Screener to Screen CS Culture ...... 40 Figure 3.5: Pump and Immersion Heater Switches ...... 41 Figure 3.6: Back View of Photo-bioreactor...... 43 Figure 3.7: Header Pipe Arrangement & Growth Surfaces ...... 44 Figure 3.8: Control Panels and Light Panels ...... 45 Figure 3.9: Light Panel Covered with Mylar Sheet ...... 46 Figure 3.10: Grid and Li-Cor Sensor ...... 46 Figure 3.11: Outer Light Panel Measurements...... 47 Figure 3.12: Harvest Pump ...... 48 Figure 3.13: Nova Gas Analyzer ...... 50 Figure 3.14: DA System ...... 51 Figure 4.1: Representation of Plastic Grid for Light Profile ...... 54 Figure 4.2: Representation of Air Flow Profile ...... 55 Figure 4.3: Expected Results ...... 59 Figure 5.1: Cumulative Mass of CS Culture Harvested during the First Test ...... 68 Figure 5.2: Cumulative Mass of CS Culture Harvested during the Second Test...... 73 Figure 5.3: Cumulative Mass of CS Culture Harvested during the Third Test...... 78 Figure 5.4: Cumulative Mass of CS Culture Harvested during the Fourth Test...... 82 Figure 5.5: Cumulative Mass of CS Culture Harvested during the Fifth Test...... 87 10

Figure 5.6: Productivity Results from the Five Tests ...... 89 Figure 6.1: CS Culture produced as Function of Days for High Light Tests...... 94 Figure 6.2: Membrane after Fourth Harvest from the Fifth Test...... 95 Figure 6.3: Membrane before and one day after Second Harvest for the Fifth Test ...... 95 Figure A.1: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day ...... 100 Figure A.2: Filters from Initial Sample Loading gave 3.15 g of CS Culture...... 100 Figure A.3: One of the Outer Surfaces of a Membrane before and after First Harvest.. 101 Figure A.4: Filters from First Harvest gave 2.46 g of CS Culture...... 101 Figure A.5: Data Collected from DA System from Initial Loading to First Harvest ..... 102 Figure A.6: One of the Outer Surfaces of a Membrane before and after Second Harvest ...... 103 Figure A.7: Filters from Second Harvest gave 14.39 g of CS Culture ...... 103 Figure A.8: Data Collected from DA System from First to Second Harvest ...... 104 Figure A.9: One of the Outer Surfaces of a Membrane before and after Third Harvest 105 Figure A.10: Filters from Third Harvest gave 9.63 g of CS Culture ...... 105 Figure A.11: Data Collected from DA System from Second to Third Harvest...... 106 Figure A.12: One of the Outer Surfaces of a Membrane before and after Fourth Harvest ...... 107 Figure A.13: Filters from Fourth Harvest gave 7.98 g of CS Culture ...... 107 Figure A.14: Data Collected from DA System from Third to Fourth Harvest...... 108 Figure A.15: One of the Outer Surfaces of a Membrane before and after Fifth Harvest 109 Figure A.16: Filters from Fifth Harvest gave 10.93 g of CS Culture ...... 109 Figure A.17: Data Collected from DA System from Fourth to Fifth Harvest ...... 110 Figure A.18: One of the Outer Surfaces of a Membrane before and after Sixth Harvest111 Figure A.19: Filters from Sixth Harvest gave 17.17 g of CS Culture...... 111 Figure A.20: Data Collected from DA System from Fifth to Sixth Harvest ...... 112 Figure A.21: One of the Outer Surfaces of a Membrane before and after Seventh Harvest ...... 113 Figure A.22: Filters from Seventh Harvest gave 11.43 g of CS Culture ...... 113 Figure A.23: Data Collected from DA System from Sixth to Seventh Harvest ...... 114 11

Figure A.24: One of the Outer Surfaces of a Membrane before and after Eighth Harvest ...... 115 Figure A.25: Filters from Eighth Harvest gave 8.5 g of CS Culture ...... 115 Figure A.26: Data Collected from DA System from Seventh to Eighth Harvest...... 116 Figure A.27: One of the Outer Surfaces of a Membrane before and after Ninth Harvest ...... 117 Figure A.28: Filters from Ninth Harvest gave 8.42 g of CS Culture...... 117 Figure A.29: Data Collected from DA System from Eighth to Ninth Harvest...... 118 Figure A.30: One of the Outer Surfaces of a Membrane before and after Tenth Harvest ...... 119 Figure A.31: Filters from Tenth Harvest gave 6.94 g of CS Culture...... 119 Figure A.32: Data Collected from DA System from Ninth to Tenth Harvest ...... 120 Figure A.33: One of the Outer Surfaces of a Membrane before and after Eleventh Harvest ...... 121 Figure A.34: Filters from Eleventh Harvest gave 8.43 g of CS Culture...... 121 Figure A.35: Data Collected from DA System from Tenth to Eleventh Harvest...... 122 Figure A.36: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day .... 123 Figure A.37: Filters from Initial Sample Loading gave 3.13 g of CS Culture...... 123 Figure A.38: One of the Outer Surfaces of a Membrane before and after First Harvest 124 Figure A.39: Filters from First Harvest gave 6.51 g of CS Culture...... 124 Figure A.40: Data Collected from DA System from Initial Loading to First Harvest ... 125 Figure A.41: One of the Outer Surfaces of a Membrane before and after Second Harvest ...... 126 Figure A.42: Filters from Second Harvest gave 6.75 g of CS Culture ...... 126 Figure A.43: Data Collected from DA System from First to Second Harvest ...... 127 Figure A.44: One of the Outer Surfaces of a Membrane before and after Third Harvest ...... 128 Figure A.45: Filters from Third Harvest gave 11.7 g of CS Culture ...... 128 Figure A.46: Data Collected from DA System from Second to Third Harvest...... 129 12

Figure A.47: One of the Outer Surfaces of a Membrane before and after Fourth Harvest ...... 130 Figure A.48: Filters from Fourth Harvest gave 14.28 g of CS Culture ...... 130 Figure A.49: Data Collected from DA System from Third to Fourth Harvest...... 131 Figure A.50: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day .... 132 Figure A.51: Filters from Initial Sample Loading gave 2.66 g of CS Culture...... 132 Figure A.52: One of the Outer Surfaces of a Membrane before and after First Harvest 133 Figure A.53: Filters from First Harvest gave 6.93 g of CS Culture...... 133 Figure A.54: Data Collected from DA System from Initial Loading to First Harvest ... 134 Figure A.55: One of the Outer Surfaces of a Membrane before and after Second Harvest ...... 135 Figure A.56: Filters from Second Harvest gave 6.81 g of CS Culture ...... 135 Figure A.57: Data Collected from DA System from First to Second Harvest ...... 136 Figure A.58: One of the Outer Surfaces of a Membrane before and after Third Harvest ...... 137 Figure A.59: Filters from Third Harvest gave 5.17 g of CS Culture ...... 137 Figure A.60: Data Collected from DA System from Second to Third Harvest...... 138 Figure A.61: One of the Outer Surfaces of a Membrane before and after Fourth Harvest ...... 139 Figure A.62: Filters from Fourth Harvest gave 7.1 g of CS Culture ...... 139 Figure A.63: Data Collected from DA System from Third to Fourth Harvest...... 140 Figure A.64: Outer Surface CS culture Coverage for Membranes 1& 3 after a Day ..... 141 Figure A.65: Filters from Initial Sample Loading gave 1.94 g of CS Culture...... 141 Figure A.66: One of the Outer Surfaces of a Membrane before and after First Harvest 142 Figure A.67: Filters from First Harvest gave 8.1 g of CS Culture...... 142 Figure A.68: Data Collected from DA System from Initial Loading to First Harvest ... 143 Figure A.69: One of the Outer Surfaces of a Membrane before and after Second Harvest ...... 144 Figure A.70: Filters from Second Harvest gave 7.24 g of CS Culture ...... 144 Figure A.71: Data Collected from DA System from First to Second Harvest ...... 145 13

Figure A.72: One of the Outer Surfaces of a Membrane before and after Third Harvest ...... 146 Figure A.73: Filters from Third Harvest gave 8.72 g of CS Culture ...... 146 Figure A.74: Data Collected from DA System from Second to Third Harvest...... 147 Figure A.75: One of the Outer Surfaces of a Membrane before and after Fourth Harvest ...... 148 Figure A.76: Filters from Fourth Harvest gave 8.92 g of CS Culture ...... 148 Figure A.77: Data Collected from DA System from Third to Fourth Harvest...... 149 Figure A.78: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day .... 150 Figure A.79: Filters from Initial Sample Loading gave 1.72 g of CS Culture...... 150 Figure A.80: One of the Outer Surfaces of a Membrane before and after First Harvest 151 Figure A.81: Filters from First Harvest gave 8.88 g of CS Culture...... 151 Figure A.82: Data Collected from DA System from Initial Loading to First Harvest ... 152 Figure A.83: One of the Outer Surfaces of a Membrane before and after Second Harvest ...... 153 Figure A.84: Filters from Second Harvest gave 11.67 g of CS Culture ...... 153 Figure A.85: Data Collected from DA System from First to Second Harvest ...... 154 Figure A.86: One of the Outer Surfaces of a Membrane before and after Third Harvest ...... 155 Figure A.87: Filters from Third Harvest gave 14.3 g of CS Culture ...... 155 Figure A.88: Data Collected from DA System from Second to Third Harvest...... 156 Figure A.89: One of the Outer Surfaces of a Membrane before and after Fourth Harvest ...... 157 Figure A.90: Filters from Fourth Harvest gave 17.69 g of CS Culture ...... 157 Figure A.91: Data Collected from DA System from Third to Fourth Harvest...... 158

Nomenclature

ATP Adenosine Triphosphate

Am Area of the Membrane

°C Degree Centigrade

C-C Carbon-Carbon

CRF-II Carbon Remediation Facility-II

CS Chroogloeocystis siderophila

DA Data Acquisition

EIA Energy Information Administration

E Einstein ft-c foot-candle

GtC Giga tons of Carbon g Grams

≥ Greater Than or Equal to

GPM Gallons per Minute

” Inches

ID Inner Diameter

IPCC Intergovernmental Panel on Climate Change

K Kelvin klx Kilo lux

L Liters 15

LPM Liters per Minute

Mm Final Amount of Algae Produced

Mi Initial Amount of Algae Loaded

< Less Than

≤ Less Than or Equal to

m Meters

µ Micro

MEA Monoethanolamine

NA Not Applicable

nm Nanometers

NaOH Sodium Hydroxide

NADPH Nicotinamide Adenine Dinucleotide Phosphate

N Number of Days of Operation

ppm Parts Per Million

PAR Photo Active Radiation

% Percentage

ρ Productivity

RO Reverse Osmosis

sp Species

USA United States of America

V Volts

W Watt 16

1. Introduction

A subject of substantial concern in the contemporary world is the release of greenhouse gases, most notably carbon dioxide, into the atmosphere. Greenhouse gas effect is believed to be the main reason for global warming. In fact, the

Intergovernmental Panel on Climate Change (IPCC) projects that if the greenhouse gas emissions continue in the present trend, the earth’s mean temperature will increase by

0.2°C every ten years (IPCC 2007). Considering that there has only been a 5°C increase in temperature from the most recent ice age, 0.2°C increase in temperature for ten years is considered large (Fischer and Narain 2003).

Though many gases released from industry and vehicles may have a global warming effect, carbon dioxide is considered to be one of the major gases leading to

global warming, with greater than one-third emitted by power plants (Herzog 2001).

According to the Energy Information Administration (EIA), the approximate percentage

of carbon dioxide in greenhouse gas emissions was 84.1% in the USA in 2005 (EIA

2006).

In 2007, Alley, et al. reported that the recent changes observed in the global

climate are the average temperature, sea level and northern hemisphere snow cover. They

found that “[e]leven of the last twelve years (1995 -2006) rank among the 12 warmest

years in the instrumental record of global surface temperature (since 1850)” (Alley, et al.

2007). Figure 1.1 shows the changes in the average global surface temperature, rise in the

sea level and the northern hemisphere snow coverage for the month of March-April 17 relative to the corresponding averages from 1961-1990 (Alley, et al. 2007). Reducing the amount of carbon dioxide percentage in greenhouse gases may reduce the observed global warming effect.

Figure 1.1: Recent Global Climate Change (Alley, et al. 2007)

Linton and Cannell (2002), discuss in their paper that mitigating the rate of global

warming indirectly means reducing the rate of carbon dioxide emissions into the

atmosphere. The change in temperature caused by increasing carbon dioxide leads to

change in the amount of water vapor in the atmosphere, causing clouds, which greatly

magnify the warming effect of carbon dioxide. This carbon cycle and climate system 18 allows the emitted carbon to remain in the atmosphere, leading to global warming. In their work, they estimated that in another 10 years global warming could be reduced to less than 0.2ºC by mitigating the increase in the rate of fossil fuel (coal, oil and natural gas) emissions to less than 0.03 GtC/yr/yr (Linton and Cannell 2002).

1.1. Carbon Dioxide Sequestration Technologies from Flue Gases

In 2005, Stewart and Hessami studied the methods used for carbon dioxide sequestration and their disadvantages followed by a description of photo-bioreactor. They identified the following as the separation processes used to separate carbon dioxide from

flue gases: MEA (monoethanolamine) solvent scrubbing, molecular sieve separation,

desiccant adsorption and membrane technology. The separated carbon dioxide is then

stored by injecting it into underground sequestration sinks. The following are the

methods used for storage purposes: geologic injection (enhanced oil recovery and coal

seams), oceanic injection and oceanic fertilization (enhanced method of oceanic injection

where the carbon dioxide injected is used for marine fertilization). These sequestration

and storage processes have some disadvantages. For example, geologic and oceanic

injections offer a good solution but it is a temporary one, because after a period of time

the injected carbon dioxide gets released into the atmosphere (Stewart and Hessami

2005). According to them “[v]ery little research has been done in establishing the

detriment oceanic fertilization may have on the fragile oceanic eco-system” (Stewart and

Hessami 2005). 19

1.2. Photo-Bioreactor for Carbon Dioxide Sequestration

A new technology called photosynthetic fixation of carbon dioxide using

microalgae has been developed which acts both as a separating and storage option

(Stewart and Hessami 2005). The key parameters which affect the growth of the

microalgae in the photo-bioreactor are light, temperature and carbon dioxide level. As

light is the studied parameter in the present work, the effect of light on the organism will

be discussed here. The process of photosynthesis involves the conversion of light energy

into chemical energy. Photosynthesis reaction given by Purves et al. in 1994 is shown in

Equation 1.1.

6CO2 +12H 2O → C6 H12O6 + 6O2 + 6H 2O (1.1)

Algae absorb light energy through pigments (primarily chlorophyll, carotenes and

phycobilins). Every photosynthesis process in plants involves a light and dark reaction;

the former is driven by light while the latter does not directly use light energy. The light

and heat energy are stored in molecules of adenosine triphosphate (ATP) and

nicotinamide adenine dinucleotide phosphate (NADPH) reduced from an electron carrier

(Purves, et al. 1994). In their book, Purves et al. (1994) described that “[b]y the middle of twentieth century, it was clear that photosynthesis comprises two pathways: one, driven by light, produces ATP; the other uses ATP to produce sugar” (Purves, et al. 1994).

Reactions involved in both the pathways occur in the chloroplast. Light reactions excite the electrons and produce ATP and NADPH, while the products from light reaction enter 20 the Calvin-Benson cycle (also known as dark reactions) to form C-C bonds to produce sugar as shown in Figure 2.1 (Purves, et al. 1994). The type of carbohydrate product produced depends on the type of strain being used.

Figure 1.2: Calvin-Benson Cycle Showing Photosynthesis Process (Purves, et al. 1994)

Research on carbon dioxide fixation with microalgae has occurred for the past 50

years leading to two ways of commercially producing microalgae, namely open ponds

and closed photo-bioreactors (Burlew 1953) & (Lee 2001). It is difficult to maintain

culture parameters in open ponds because they occupy a great amount of space.

According to Lee (2001), it is easy to maintain culture parameters and attain higher

biomass concentration in a photo-bioreactor and that “[t]he need to achieve higher

productivity and to maintain monoculture of algae led to the development of enclosed

tubular and flat plate photobioreactors” (Lee 2001). The flue gases from the stack of a

power plant are sent into the photo-bioreactor which contains microalgae. The photo-

bioreactor uses the natural photosynthesis process which requires water, carbon dioxide 21 and, most importantly, light to produce not only food but also to release oxygen into the atmosphere. The microalgae, which are supplied with nutrients, absorb carbon dioxide using sunlight transmitted by optic cables, which capture sunlight at the surface (Stewart and Hessami 2005). Microalgae are then converted to biomass suitable for applications, some of which are described later in this chapter. A simple overview of the process is shown in Figure 1.3.

Figure 1.3: Photosynthetic Conversion of Carbon Dioxide (Stewart and Hessami 2005)

1.3. Uniqueness of the Research

Many cultures of microalgae were tested in photo-bioreactors like

“[m]onocultures of Chlorella, Spirulina and other microalgae has been successfully

maintained in enclosed tubular and flat plate photobioreactors” (Lee 2001). The uniqueness of the current work is that it utilizes thermophilic cyanobacteria called 22

Chroogloeocystis siderophila (CS) which grows at 50°C in its natural habitat (Brown, et al. 2005). The flue gases from the power plant are released at a high temperature of about

120ºC (Ono and Cuello 2007). Use of the CS culture can result in reduced cooling costs when a photo-bioreactor is installed in a power plant. The film based reactor configuration is unique in photosynthetic bioreactors. This photo-bioreactor helps in having better illumination, control over the reactor parameters, and a high surface area for microalgae growth. The effect of light, while understood for some strains of microalgae and CS culture, are unknown in this reactor configuration. In this study, tests were conducted at high and low light intensities on CS culture. The reason for examining low light intensity is that when solar collectors are used as a light source for the pilot reactor to produce the required light intensity, the light is transmitted by low loss fiber optic cables from the solar collectors to the panels. The cost of solar collectors is very high and the maximum amount of light collected should be distributed to all the panels.

By choosing a low light intensity, it is cost effective to distribute light to all panels with a fewer number of solar collectors when compared to distributing high light intensities. The other reason for using low intensities is to investigate the efficiency of photosynthesis with low light intensities when compared to high light intensity and also to show that the system can be used in regions where there is low sunlight. The ability to use lower light intensities to grow microalgae could increase the range of uses for this bioreactor and lead more economic production of microalgae.

The CS culture that is grown inside the photo-bioreactor may be used as biomass for the production of biofuels. Other applications of algae include waste water treatment, 23 value added health food supplements, fine chemicals, specialty feed for aquaculture and reagents for research (Reith, et al. 2004). In 2006, Sadettin et al. research showed that thermophilic cyanobacteria (Synechococuss sp. and Phormidium sp.) have the potential to remove dyes from waste water (Sadettin, et al. 2006).

1.4. Biomass as Biofuel

In 2002, Puppan stated that biomass has a great future scope in the field of

biofuels. The demand for liquid energy from renewable resources in the coming years is shown in Figure 1.4. The advantages of biomass include that it is biodegradable,

sustainable and also causes less pollution when compared with fuels being used. Puppan

says that “[t]he greenhouse effect calculated for biodiesel was only 55% that for diesel

fuel” (Puppan 2002).

The most commonly used biofuels are biodiesel and ethanol (Puppan 2002).

According to Puppan “[b]iodiesel is made by using vegetable oils, animal fats, algae or even recycled cooking greases” (Puppan 2002). Also, microalgae with high lipid content produces higher biodiesel than commercially used oilseed crops (rapeseed, soybean oil) utilizing less amount of water (Sheehan, et al. 1998).

1.5. Applications of Microalgae

Carbon dioxide can be used to produce biomass by utilizing microalgae culture.

Microalgae cultures are grown in bioreactors in the presence of light and carbon dioxide,

where the microalgae utilize carbon dioxide and convert it to biomass.

Figure 1.4: Demand for Liquid Energy from Renewable Sources in the Future (As quoted by (Puppan 2002) taken from the source (Connemann 1999))

According to Apt and Behrens in 1999, recent advancements in biotechnology developed a wide range of products in the commercial market, specifically developing in the field of pharmaceutical research. They have listed products, including “algal-derived long-chained polyunsaturated fatty acids, mainly docosahexaenoic acid, for use as supplements in human nutrition and animals” (Apt, et al. 1999), and nutritional products prepared from the cell extracts of Chlorella (high nutrition), Dunaliella (high salinity) and Spirulina (high alkalinity) which have a wide scope of nutritional and monetary advantages (Apt, et al. 1999) & (Lee 2001).

Microalgae have great potential in the pharmaceutical industry as bioactive compounds. Schwartz, et al. in 1990 points out that “A large number of bioactivities have 25 been reported in algae, including anticancer, antimicrobial, anti-HIV, antiviral and various neurological activities” (quoted in (Apt, et al. 1999)). In 1997, Hon-Nami et al. showed that Tetraselmis sp. (Tt-1) has applications in the paper industry. This species showed very good effectiveness when used as a pulp additive for density, gas permeability, smoothness, ink absorptivity except the tensile index (Hon-Nami, et al.

1997).

1.6. Research Objective

The goal of the overall project is to sequester carbon dioxide produced from

natural gas combustion products in a photo-bioreactor via the concept of photosynthetic

conversion with Chroogloeocystis siderophila (CS) culture by varying light as a

parameter. This particular species of thermophilic cyanobacteria can tolerate a

temperature of 50°C and is also resistant to the other gases present, namely carbon

monoxide, SOx, NOx as well as other acidic compounds (Chakravarti, et al. 2001).

The objectives of the research presented in this thesis are as follows:

• To study the productivity of CS culture by conducting tests at light intensity 60

µmol m-2s-1 ≤ average ≤ 85 µmol m-2s-1 and at a minimum light intensity of

average ≤ 30 µmol m-2s-1 inside the photo-bioreactor and perform an initial

evaluation of the system based on the previous tests.

• To infer possible effects of light intensity on CS culture productivity. 26

A brief summary of each chapter is as follows: Chapter 2 presents a review of the relevant literature on the algal productivity with different levels of light intensities in bioreactors and how the parameters for this study’s research objectives were targeted.

Chapter 3 explains the experimental setup used to achieve the research objectives. The test plan and the operational procedures are discussed in Chapter 4 and the test results are discussed in Chapter 5, while the conclusions drawn from the study and recommendations for future work are made accordingly in Chapter 6.

2. Literature Review

As mentioned in Chapter 1, the research goal was to sequester carbon dioxide within a photo-bioreactor using CS culture while observing the effect of light intensity on the CS culture’s growth rate. In this chapter, the effect of light intensity on different species of microalgae and different designs of bioreactors are discussed as well as the effect of characteristics of light on the growth of CS culture.

Microalgae absorb energy from light to carry out the process of photosynthesis.

Chlorophyll pigment present in microalgae stores energy absorbed from light in its cells during photosynthesis. The photons emitted from the wavelength range of 400-700 nm

(visible region of the spectrum) are absorbed by chlorophyll; therefore, the light emitted in that particular wavelength is known as the growth light or photosynthetic active radiation (PAR) (Purves, et al. 1994). One way to measure the photosynthetic photon flux density by the light emitted is to measure the number of photons emitted per unit area per unit time.

2.1. Microalgae Productivity at Different Light Conditions

Cyanobacteria productivity at varying light conditions in different bioreactor designs is discussed here. In the past, researchers have used different light sources and units, and some did not mention the type of light source that they have used. Since each study used different bulbs, it is inappropriate to convert the light units from earlier studies to µmol m-2s-1. In 2003, Wiedner et al. observed the growth rate of Microcystis PCC 7806 28 from a PAR of 10 to 403 µmol m-2s-1, maintaining a light : dark cycle of 12 hours : 12

hours. The strain was cultivated in a flat glass vessel maintaining a temperature of 22°C

and pH in the range from 8.0 - 8.6. Microcystis PCC 7806 showed a limited growth rate

below a PAR of 80 µmol m-2s-1 and a constant growth rate between 80 µmol m-2s-1 to 403

µmol m-2s-1, indicating that the strain’s growth rate was not saturated at 403 µmol m-2s-1

(Wiedner, et al. 2003). In their study about the growth characteristics of Synechococcus

PCC 7942, Kajiwara et al. (1997) conducted four tests with varying light intensities from

3 klx to 10 klx in a batch reactor, maintaining a pH of 8.0. The duration of each

experiment was 150 hours and the strain was supplied with 5% of carbon dioxide. They

observed that there was photo-limitation on the growth rate of the species at 3 klx, and the

growth rate increased by approximately 30% at a PAR of 5.4 klx. The highest growth rate

among the four tests was observed at a PAR of 8 klx; there was also a decrease in growth

rate at a PAR of 10 klx (Kajiwara, et al. 1997). These results showed that the species

attained photo-inhibition between 8 and 10 klx.

In 1997, studies conducted by Hon-Nami et al. showed that the Tetraselmis sp.

(Tt-1) grown in a tubular reactor at 25°C and under a light intensity of 10 klx gave a

productivity of 10 – 15 g/m2-day maintaining a light and dark cycle of 14 hour : 10 hour

(Hon-Nami, et al. 1997). Rhee and Lee (1999), studied the effect of light limitation and

light super saturation on Anabaena Flos-Aquae in a turbidostat. They conducted twelve

tests from 5 Wm-2 to 160 Wm-2 at a pH of 8.0 while maintaining a temperature of 20°C.

The growth rate at 5 Wm-2 was 0.44 day-1, and at a light intensity of 20 Wm-2, growth rate

increased to 0.99 day-1. The growth rate started to decrease from 22 Wm-2 and went down 29 to 0.56 day-1 at 160 Wm-2 (Rhee and Lee 1999). This showed that photo-inhibition for the

species started at 22 Wm-2.

In 2005, the work of Berzin et al. on a novel “triangular” air-lift bioreactor with

two strains of green algae showed that light and dark cycles could have a significant effect on algal productivities. They defined the air-lift bioreactor as a “pneumatic

contacting device in which fluid circulation takes place in a defined cyclic pattern

through channels” (Berzin, et al. 2005). Flue gas was supplied from the bottom of the bioreactor to the riser tube. This provided uniform mixing of the algae solution inside the

bioreactor, thus preventing fouling. The liquid flows from the gas separator and returned

to the down comer. The down comer and the riser were turbulent regions with maximum gas holdup in the riser. The riser was considered as the dark region and down comer as the light region as shown in Figure 2.1.

Liquid Flow

Riser Tube Gas Separator

Down Comer Gas Flow

Figure 2.1: Air-Lift Bioreactor Configuration (Berzin, et al. 2005)

It was observed that having a larger ratio of cross sectional areas of riser to downcomer produced smaller algal growth rate. They tested different ratios at 250 µE m-2

s-1 for a reactor height of 1 m and gas velocity of 0.334 cm/s. The two strains (Dunaliella parva and Dunaliella tertiolecta) were supplied with 8% carbon dioxide in the inclined air-lift bioreactor (Berzin, et al. 2005).

In 1958, Krauss and Sorokin studied the growth rate of five different species of green algae at different light conditions. The five strains, Chlorella pyrenoidosa,

Chlorella vulgaris, Scenedesmus obliquus, Chlamydomonas reinhardti (each grown at a temperature of 25°C) and Chlorella pyrenoidosa (a high temperature strain grown at

39°C), were grown in the test tubes at a pH of 6.8 and were supplied with 4% carbon

dioxide in air mixture. The growth rate of the strains was observed by varying light

intensity from 0 to 10,000 ft-c. The light inhibition was observed from 250 ft-c for

Chlorella vulgaris and 500 ft-c for the rest of the species growing at 25°C. Chlorella

pyrenoidosa, at 39°C, did not show growth inhibition until 3,000 ft-c (Krauss & Sorokin

1958).

In 2007, Ono and Cuello studied the effect of light intensity on CS culture in four

Pyrex flasks. The Pyrex flasks were supplied with 5% carbon dioxide while BG-11

(growth medium) was maintained at an initial pH of 7.5. Tests were conducted at four

different light intensities, starting from an average of 36.9 µmol m-2s-1 to 246.1 µmol m-

2s-1 and maintaining a light and dark cycle of 16 hours : 8 hours. Experiments lasting 18

days and 28 days were conducted at 246.1 µmol m-2s-1, while experiments lasting 8 days were conducted for 246.1 µmol m-2s-1, 200 µmol m-2s-1 and 100 µmol m-2s-1. It was 31 observed that the optimum light intensity for CS culture was 200 µmol m-2s-1, and that the

species survived at an average light intensity of 36.9 µmol m-2s-1 and 246.1 µmol m-2s-1

(Ono and Cuello 2007). An overview of the effect of varying light intensity on microalgae productivity is shown in Table 2.1.

Table 2.1: Overview of the literature review

Reactor Light Author Strain Light Intensity Growth Rate Type Conditions Limited < 80 Wiedner µmol m-2s-1 Flat Glass Microcystis 10 µmol m-2s-1 12 h: 12h et al. Constant: Vessel PCC 7806 403 µmol m-2s-1 L/D Cycle (2003) 80 - 403 µmol m-2s-1 Kajiwara Experiment Photo- Batch Synechococcus 3 klx, 5.4 klx, 8 et al. duration: limitation:3 klx Reactor PCC 7942 klx & 10klx (1997) 150 hrs Highest:8 klx

Hon-Nami Tubular Tetraselmis 14h: 10h et al. 10 klx 10–15 g/m2-day Reactor sp. L/D Cycle (1997)

Rhee Photo- 5 Wm-2 – 160 12 light and Lee Turbidostat Flos-Aquae inhibition:22 Wm-2 tests (1999) Wm-2

Ono and Pyrex 16: 8 Cuello CS culture 246.1 µmol m-2s-1 Survived Flasks L/D Cycle (2007)

Ono and Pyrex 200 µmol m-2s-1 16: 8 Maximum: 200 Cuello CS culture Flasks 100 µmol m-2s-1 L/D Cycle µmol m-2s-1 (2007)

Ono and Pyrex 16: 8 Cuello CS culture 36.9 µmol m-2s-1 Survived Flasks L/D Cycle (2007)

From this literature review, it can be deducted that the growth rate of microalgae varies with varying light intensity. Each species has a light limited and light inhibited growth and the limits vary with the type of microalgae strain. Each species has a particular light intensity at which its growth rate is highest. The work of Berzin et al.

(2005) showed that productivity of species depends on the light and dark cycles maintained in the bioreactor (Berzin, et al. 2005).

In 1998, Sheehan et al. discussed microalgae growth in open ponds. They worked on sequestering carbon dioxide (> 90 %) in 1000 m2 open ponds with targeted growth rate of microalgae about 50 g/m2-day. They achieved their target on some occasions and the overall growth rate attained was very low, approximately 10 g/m2-day. This was due to a

decrease in growth rate on cold climate days (Sheehan, et al. 1998).

2.2. Effect of Light on “Chroogloeocystis siderophila”

Three characteristics of light that are critical for the growth of microalgae in the photo-bioreactor are spectrum, intensity and duration (Whiting 2006). In 2007, Ono and

Cuello used a Beckman DU640 spectrophotometer in their studies to measure the optical characteristics of CS culture. The species exhibited a peak at a wavelength of 658 nm, indicating absorption of maximum energy at that particular wavelength (Ono and Cuello

2007). The species falls in the red region of the visible spectrum as shown in Figure 2.2. 33

Figure 2.2: The Optical Characteristics of CS Culture (Ono and Cuello 2007)

CS culture shows good growth characteristics with sunlight, which has the full

spectrum of light. According to an article by Birn (2003), during typical daylight, sun has

a color temperature ranging between 5500-6500 K and the sun has a color temperature

ranging between 5000-5400 K at direct noon (Birn 2003) & (Color Temperature 2006).

According to Kay (2007), 5000 K is set as a reference color temperature for sunlight

(Kay 2007). In the present work, to replicate the sunlight, photo-bioreactor was illuminated with fluorescent bulbs which have a color temperature of 5400K.

It is required to know the light intensity at which CS culture growth is optimum in order to conduct the tests. According to Bayless et al. (2006), the CS culture growth does

not saturate at 50 µmol m-2s-1 (Bayless, et al. 2006). Therefore, a high light intensity of

60 ≤ average ≤ 85 µmol m-2s-1 including external illumination was targeted in the photo-

bioreactor for three out of five tests conducted. 34

To find the growth characteristics of CS culture at low light intensities, Ono and

Cuello (2007) conducted tests at an average light intensity of 36.9 µmol m-2s-1 on CS culture and observed that the species survived at low light intensity. Brown et al. (2005) conducted experiments at a light intensity of 30 µmol m-2s-1, showing that the species grows at low light intensities (Brown, et al. 2005). Therefore, two additional sets of tests

were conducted at a low light intensity averaging ≤ 30 µmol m-2s-1.

The more light the species receives, the more its photosynthetic rate increases.

However, there is maximum intensity at which the species becomes photo-inhibitive. The tests conducted by Ono and Cuello (2007) showed that the species exhibited good growth rate at a high illumination of 246.1 µmol m-2s-1 (Ono and Cuello 2007).

3. Experimental Setup

To study the effect of light intensity on the growth of CS culture in a photo- bioreactor, the major setup includes a photo-bioreactor with a variable light source. The photo-bioreactor consists of a Bunsen burner, growth system, growth surfaces, lighting system, harvesting system, Nova gas analyzer and data acquisition (DA) system.

Combusted gases from the Bunsen burner pass through the recirculation duct and on to the growth surfaces in the photo-bioreactor. Growth surfaces, covered with CS culture flowing on both sides from the growth system, were illuminated with light panels on each side. The gas concentrations in the photo-bioreactor were analyzed using a Nova gas analyzer and were recorded in DA system. Growth surfaces are harvested depending on the amount of CS culture populated over them. The test rig was developed by previous researchers; this researcher’s contribution to the test rig development was in the improvement of design of the headers.

The photo-bioreactor shown in Figure 3.1 is constructed of ¼’’ plexiglass supported on a wooden frame which is closed with a bolted plexiglass lid with a rubber gasket around it to avoid any leakage of gas. The glass lid has 12 holes from which air flow measurements were taken with a pitot tube and harvesting operations were performed; these holes were closed with bolts when the photo-bioreactor was being operated. Figure 3.1 shows the significant parts of the equipment, labeled, which are explained later in this chapter. 36 Solenoid Valve Air & Gas Rotameters Recirculation Duct Main Control Panel Light Panels Timer Switches

D.A System

Nova Gas Analyzer

Fin Strip Heaters Burner Air Circulation Blower Figure 3.1: Picture of the Experimental Setup

3.1. Burner Setup

A Bunsen burner, which is operated manually, was used for combustion of natural gas to produce carbon dioxide required for growth of the CS culture. A picture showing the burner nozzle, thermocouple, pilot valve, gas line and air line is shown in Figure 3.2. 37

Burner Nozzle

Thermocouple

Air Line

Pilot valve

Gas Line

Figure 3.2: Burner Setup

The air and natural gas required for combustion pass through the air and gas lines.

The air and gas rotameters which are situated above the main control panel were adjusted to 68 (19.4 LPM) and 0.8 LPM, where the range of the air rotameter varies from 0 - 150

(0 - 42.75 LPM) and the gas rotameter varies from 0.4 - 5 LPM. The thermocouple present inside the plastic panel of the burner duct was heated with a propane torch by purging the natural gas line; this was accomplished by turning the knob to the pilot position and clicking it 10 - 15 times. When the knob on the ignition module is turned to the “on” position produces a flame from the burner nozzle which is red in color is produced. If the carbon monoxide level inside the photo bioreactor is more than 40 ppm, the burner is shut off and fin strip heaters are switched on to provide the required heat to the photo-bioreactor. 38

To start the burner, the air rotameter was set at 20 (5.7 LPM) and the gas rotameter was set at 2 LPM. The fan speed was set to low position on the knob. After starting the burner, the rotameter settings were adjusted to the desired values to produce the maximum amount of carbon dioxide while maintaining carbon monoxide level below

40 ppm. The flue gases in the photo-bioreactor provide enough heat to maintain the temperature from 46 - 52°C.

Three snap disks are located on the gas line. The snap disk located on the top of the burner duct functions promptly by shutting off the flame when the duct is overheated or when the thermocouple becomes cold (i.e. when the flame of the burner is shut off) by breaking the circuit of the solenoid valve; the solenoid valve immediately closes without allowing the gas to flow through it. When the solenoid valve receives heat, the valve is opened for the gas to pass through it, but when the cork does not receive any heat, the solenoid valve stops the supply of gas.

3.2. Growth System

The CS culture grown in the culture tanks shown in Figure 3.3 were isolated from

the Yellowstone National Park, Montana (Brown, et al. 2005). They were maintained at a

pH of ~ 7.5 - 8.2, temperature of 50°C and were illuminated with a light bulb. Before starting each test, the tanks were scraped and approximately 50 L of CS culture solution

was taken from any of the three cylindrical tanks which were populated with a good

amount of CS culture. Approximately 37.5 L was loaded into solution tank by screening

the solution and the other 12.5 L was loaded in the harvest tank for initial sample loading.

Figure 3.3: Rectangular and Cylindrical Culture Tanks

3.2.1. Initial Mass Loading

Initial mass quantification was performed after completely loading 37.5 L of

culture into the solution tank. A 12.5 L sample of the CS culture solution was loaded into

the harvest tank and run through the filters to determine the mass of culture per liter of solution. The solution was collected onto coarse filters (100 µm), fine filters (0.5 µm) and

Whatman filter paper (6 µm) by running the harvest pump until a clear solution was

observed in the harvest tank. The filters and the Whatman filter paper were removed from

the filter casings and were dried in the oven for three to five days; the weight of the

sample CS culture was determined from the difference between the weight of filters and

Whatman filter paper before and after drying.

The uncertainty calculated from the initial loading error analysis for 100 ml

samples was 2.75%. However, it is assumed that the error would reduce for larger

samples of population, even though the technique of loading would not be the same. 40

3.2.2. Loading CS culture into Solution Tank

The solution tank can handle up to 16 gallons of CS culture solution.

Approximately 37.5 L of CS culture solution was screened through a screener, shown in

Figure 3.4, to break microalgae into very small particles to avoid blocking flow in the header. The growth solution loaded into the solution tank was added with 50% Reverse

Osmosis (RO) water and 50% growth medium (BG-11+HEPES) to increase the solution above the safe level in the solution tank.

Figure 3.4: Screener to Screen CS Culture

The solution was supplied to the growth surfaces via plumbing within the system.

The solution was pumped to the three growth surfaces with the help of three hoses attached to each of the stainless steel headers. The solution from the solution tank was pumped into three hoses with the help of diaphragm pumps controlled by a transformer operating at 120 V. Only two pumps were operated, while the third pump acted as a backup pump. The desired flow rate was achieved by setting the transformer between 40-

45 percent of the maximum output voltage. The pumps delivered solution to the growth surfaces at 0.8-1 GPM through each hose. A flow rate of 1-1.2 GPM was maintained when loading the CS culture to get acceptable coverage on the membranes. The 41 immersion heater is installed in the solution tank to maintain the solution temperature from 47-50°C. A knob is provided to control the temperature of the immersion heater and hence the solution temperature.

The air and gas rotameters described earlier in Section 3.1 are located on the main control panel as shown in Figure 3.5. The combusted gases were circulated in the photo- bioreactor with the help of a Fantech Model FX8FL air circulation blower. The fan is controlled using a knob which is located on the main control panel. The gases pass through the insulated recirculation ducts which do not dissipate heat. The air velocity measurements inside the photo-bioreactor were taken with a Pitot tube at 12 points, at two levels of depth, one at 8” and another at 14” in the photo-bioreactor through the holes on the top of the glass lid.

Gas Rotameter

Air Rotameter

Fan Knob

Solution Pump Immersion Switch Heater Switch

Figure 3.5: Pump and Immersion Heater Switches 42

After the CS culture solution was pumped to the growth surfaces, the organism populates on the membranes over a period of time. The nutrient solution dripping from the membrane surfaces gets collected continuously into the solution tank from the three drain valves which are connected to both solution tank and harvest tank. Immediately after loading, pH was measured for the CS culture loaded into the solution tank. If the pH value was below 6.5, then appropriate amount of NaOH solution was added to the CS culture solution to maintain pH of the CS culture solution above 6.5. The pH measurements were taken twice a day.

Twelve inches above the immersion heater is considered as the “safe level” at which the level of solution was always maintained. The temperature inside the solution tank was measured with a thermocouple. The solution tank has an outlet to pump the solution onto the growth surfaces and a bypass at the bottom to drain the solution. The water condensed in the system is drained from the photo-bioreactor twice each day. The approximate amount of condensed water collected on any given day was 1.5 L. The amount of water drained in the morning was 10% more than in the night. The reason could be due to decrease in temperature in the photo-bioreactor at night. Figure 3.6 shows the back view of the photo-bioreactor and it also shows the spray nozzle valve and nozzle used for harvesting operation which is described in detail in Section 3.5. 43

Drain Valve of Solution Spray Nozzle Tank Valve

Solution Nozzle Pump Hoses

Solution Tank Solution Tank

Harvest Tank

Figure 3.6: Back View of Photo-bioreactor

3.3. Growth Surfaces

The carbon dioxide gas produced inside the photo-bioreactor was absorbed by CS

culture grown on growth surfaces, made of a specific fabric called Omnisil 1000

membrane as shown in Figure 3.7. The membranes were inserted into 24’’ long and 2’’

ID stainless steel headers. The header arrangement consists of a stainless steel insert, 3

rods and a plastic shim. The shim is resting on the insert with a steel plate on it. The

header is closed on both the ends with stainless steel plates with a rubber gasket in

between the plate and header ends to improve sealing. The plates were tightened to the

header with nuts and bolts. To have enough tension for the solution to flow on the

membranes, springs were used to hold the membranes to the frame. The headers, along

with the membranes, were placed inside the photo-bioreactor between the light panels 44

Steel Header Tape Shim Omnisil 1000 Membrane Insert

Figure 3.7: Header Pipe Arrangement & Growth Surfaces

and were aligned using guides which are fixed to the wall of the photo-bioreactor and the

light panels.

3.4. Lighting System

The photo-bioreactor is illuminated with four light panels: two panels are placed

inside the reactor and the other two panels are placed outside the reactor, each one facing

a different side of the membrane. The outer two panels are blocked by the wall of the

photo-bioreactor, giving less light intensity when compared to the inner panels. Each panel consists of 6 fluorescent lights of 45 watts each and is completely sealed to avoid any electric shock due to the passage of water into the panels. The light panels were switched on by using the timer switches located on Control Panel 2 as shown in Figure

3.8. Timer switches were adjusted for 12 hour light and 12 hour dark cycle. The switches

are automatically switched on at 8:00 AM in the morning and automatically switched off

at 8:00 PM in the evening.

The outside light panels are connected to the Light Control Panel Boxes 1 & 2

which are below the transition area (where the gases enter) and the inner lighting panels

are connected to the Light Control Panel Box 3 which is above another transition area 45

(where the gases leave). From there the wires are connected to the ballast which is below the photo-bioreactor and are plugged in electrically through the main control panel. The bulb’s light intensity decays up to 30% after a warm up of 2 hours and remain at steady state afterwards. The light measurements were taken after the 2 hour warm up period by placing the light measuring plastic grid in the membrane slots as shown in Figure 3.8.

Inner Light Panels Control Membrane Panel 2 Slots

Outer Light Panels

Figure 3.8: Control Panels and Light Panels

For all low light tests, light intensity was lowered by switching on only the

required number of lights. The intensity of the panels was further reduced by covering the

panels with Mylar sheets as shown in Figure 3.9. The light intensity of the panels was

varied by varying the size of the Mylar sheet. External illumination also adds for the light

intensity in the photo-bioreactor, increasing it by 4±2 µmol m-2s-1. For the last test, all the

lights of the outer panels were switched on and were covered with Mylar sheets to get

measurements in the same range as the inner light panels. 46

Fluorescent Bulbs

Mylar sheet

Figure 3.9: Light Panel Covered with Mylar Sheet

A light intensity from 60 ≤ average ≤ 85 µmol m-2s-1 for the first two tests was achieved with four of six lights in use in each panel, as well as external illumination. For the last test, all six lights of the outer panels were in use and were covered with Mylar sheets to achieve the desired light intensity, since the outer panel levels were not within the targeted range due to obstruction by the wall of the photo-bioreactor. The intensity of light was measured using a Li-Cor sensor model Q 27057 placed in the hole of a plastic grid and connected to a light meter LI-250A as shown in Figure 3.10. The plastic grid was placed in the membrane slots for taking the light measurements before and after the test. The Li-Cor sensor was placed perpendicular to the rays of the light to measure the light intensity.

Li-Cor Light Sensor in Meter Grid Li–Cor Sensor

Figure 3.10: Grid and Li-Cor Sensor

For all the five tests, the light measurements were taken once every four days for outer panels to make sure that the measurements were in the same range as the beginning of the test. The arrangement of the grid hanging to the outer light panels by hooks is shown in Figure 3.11.

Hooks clamping the grid to the outer light panel

Figure 3.11: Outer Light Panel Measurements

3.5. Harvesting System

The harvest tank has a capacity of 16 gallons and is connected to the harvest

pump. The harvest pump is switched on by plugging a cord into the electrical connection

of a 208 VAC wall socket. The pump has a priming bolt on the front and a filter bypass

line which is connected to the harvest tank as shown in Figure 3.12. The flow is

distributed between the filter bypass line and the filter line in order to keep the desired

flow rate and pressure. The outlet line from the filters is securely placed into the harvest

tank through the lid so that the back pressure does not cause water spillage.

Before each harvest, the immersion heater was switched off and the solution from the solution tank was transferred into the harvest tank by closing the drain valve of the solution tank and opening the drain valve of harvest tank. The solution tank was filled with 50% RO water and 50% (BG-11+HEPES) up to the safe level and again the 48

Harvest Pump Switch Pump Motor

Pump Rotameter

Filter Bypass Line

Pump Primer

Figure 3.12: Harvest Pump

immersion heater was switched on. This process serves the purpose of giving nutrients to

the CS culture. This procedure was followed for all the harvests except for the first

harvest of the first test and first harvest of the second test.

Three different harvesting methods were used for the five tests. The first one was

standard harvesting method, where the solution from the harvest tank was sent through

three headers at a high flow rate to shear off the CS culture saturated on the growth

surfaces. The solution flow into the filters was regulated by the pump rotameter which is

ranging from 0 - 40 GPM and there is a pressure gauge connected to the pipe line which ranges from 0 - 100 psi. A flow rate as high as 10 – 30 GPM was used to shear the CS

culture from the membranes. This method has a disadvantage of not properly washing the

CS culture populated on the membranes. Therefore, CS culture which was still sticking to

the membranes was hand scraped with a rod inserted into the photo-bioreactor by

opening the bolts of the lid; this method is called hand scraping method. This method 49 took much time (approximately two hours) and manual energy while also damaging the membranes. Also, taking the bolts off for long time increased carbon monoxide levels.

The first two test’s harvesting operations were mostly done with the hand scraping method.

The last harvesting method, which is called the nozzle spray method, was used from the third test onwards. A nozzle spray of Swagelok model SS-43S4 connected to the harvest lines as a bypass was inserted into the photo-bioreactor through the bolts on top of the lid and was used to shear the CS culture by closing the harvest line half way through to attain the desired flow rate. The sheared CS culture solution from the bottom of the photo-bioreactor was collected into the harvest tank by closing the drain valve of the solution tank. This operation took approximately half an hour, but has the drawback of CS culture sticking to the wall of the photo-bioreactor and to the light panels. This was overcome by cleaning the panels and the wall of the photo-bioreactor with the nozzle immediately after harvesting the membranes. This method gave better results without damaging the membrane cloth. Also, while performing the harvesting operation, the sprayer washed off most of the CS culture sticking to the bottom of the reactor. Although this method does not solve the problem of decreasing the carbon monoxide levels when the bolts of the lid were removed, this problem also occurred with the hand scraping method.

The solution collected in the harvest tank was circulated into the coarse and fine filters. The CS culture solution is run back and forth from the filter casings to the harvest tank. This process was continued until all the CS culture was collected and the rest of the 50 solution was vacuum pumped through Whatman Filter Paper. After finishing the harvesting operation, the solution tank was filled with a fresh batch of 50% RO water and

50% (BG-11+HEPES). Immediately after filling the tank, pH measurements were taken.

If the pH value was below 6.5, an appropriate amount of NaOH solution was added to the

CS culture solution to maintain pH of the CS culture solution above 6.5.

3.6. Nova Gas Analyzer

The Nova Model 375WP portable analyzer, shown in Figure 3.13, samples a gas

stream from transition area and measures the amount of carbon dioxide, carbon monoxide, and oxygen in the stream. The Nova gas analyzer was calibrated before

starting each test. Samples were drawn from the photo-bioreactor into the analyzer

through a series of filters by a built-in vacuum pump. The vacuum pump condenses the

gases and removes the moisture with the help of a peristaltic pump and the gases are sent

to the analyzer. The analyzer uses an infrared detector for carbon dioxide and

electrochemical sensors for analyzing oxygen and carbon monoxide concentrations. The

values were recorded automatically into the data acquisition system every two minutes.

The readings were also taken in a notebook manually every two hours.

Carbon monoxide Oxygen

Carbon dioxide

Figure 3.13: Nova Gas Analyzer 51

3.7. Data Acquisition System (DA System)

The DA system shown in Figure 3.14, is connected to the Nova Gas Analyzer,

which shows the level of carbon monoxide in the photo-bioreactor ranging from

0 – 400 ppm, as well as percentages of carbon dioxide and oxygen, and temperature of

the photo-bioreactor in degrees centigrade. The maximum permissible level of carbon monoxide in the reactor was set to 40 ppm in the carbon monoxide alarm setting. When

the carbon monoxide level reaches the maximum setting, the DA system stops the burner

and shuts the solenoid valve acting as a safety device. The light levels inside the photo-

bioreactor can also be measured using a digital multimeter connected to the DA system.

The data were collected in the DA system and the values were recorded in a computer to

record the gas levels and temperature inside the photo-bioreactor.

Thermocouple Inputs

Carbon monoxide Power Switch Alarm

Computer Burner Reset connected to Button the DA System

Nova Gas Analyzer

Figure 3.14: DA System 52

4. Test Plan & Operational Procedures

The objectives of this research presented in this thesis as discussed in Chapter 1 are:

• To study the productivity of CS culture by conducting tests at light intensity 60 ≤

average ≤ 85 µmol m-2s-1 and at a minimum light intensity of average ≤ 30 µmol

m-2s-1 inside the photo-bioreactor and perform an initial evaluation of the

repeatability of the system based on previous tests.

• To infer possible effects of light intensity on CS culture productivity.

The key parameter of the test plan was light intensity; this parameter was varied

across the 5 tests that were performed as indicated below:

i) Two tests to estimate the growth of CS culture at 60 ≤ average ≤ 85 µmol m-2s-1. ii) Two tests to estimate the growth of CS culture at average ≤ 30 µmol m-2s-1.

iii) A test to estimate the growth of CS culture at 60 ≤ average ≤ 85 µmol m-2s-1.

The two high light tests at 60 ≤ average ≤ 85µmol m-2s-1 were conducted first, followed by the others listed in order. On the whole, three tests were conducted at high light intensity to show that the results of the system at those conditions were valid and reliable. Two tests were conducted at a low light intensity of average ≤ 30 µmol m-2s-1 to

show that the tests were repeatable at the same conditions. At least 80% of the light

measurements were expected to fall between 60 to 85 µmol m-2s-1 for high light intensity

tests and between 27 to 33 µmol m-2s-1 for low light intensity tests. Light and dark cycles

are required for the CS culture to follow the Calvin-Benson cycle. Therefore, light and

dark cycles were maintained in the photo-bioreactor taking into consideration a typical 53 day of summer in the state of Ohio. To simulate day and night cycle, a light and dark cycle of 12 hours : 12 hours was maintained in the photo-bioreactor.

After stopping the tests and prior to initial mass loading, light intensity measurements of the fluorescent bulbs in the photo-bioreactor were conducted in the following manner: a plastic grid shown in Figure 4.1 (matrix of 8 x 3 equally spaced holes, which were similar in diameter to the Li-Cor quantum sensor model Q 27057) was placed in place of one of the membranes with the support of guides. The light intensities were measured by placing the Li-Cor sensor perpendicular to the surface of light panel and connecting it to the light meter LI-250 A. The measurements were taken only once because the standard deviation of a light measurement taken three times at the same point was less than 5% of the overall standard deviation.

The light intensity measurements were taken once every four days for the outer panels. Since the photo-bioreactor was closed during operation, the inner light panel measurements were not taken. Each light intensity measurement was repeated every four days at the same time of a day as the first measurement for third, fourth and fifth test and with 1-2 hours difference of the initial measurement time for first and second test. Light measurements were taken as quickly as possible to introduce no significant effect on the growth of CS culture. To measure the outer panel light intensities, the panels were inclined and the distance between the plastic grid and the light panel was maintained as in the photo-bioreactor.

Figure 4.1: Representation of Plastic Grid for Light Profile

The light intensity tests were conducted keeping the photo-bioreactor in a steady

state. The key parameters characterizing the steady state of the photo-bioreactor are

discussed below.

4.1. Average Temperature

The average temperature of the air in the photo-bioreactor was set to 46 - 52 ºC.

However, due to the dark cycle, temperature at night sometimes decreased to 44 ºC for

couple of hours; the effective decrease in temperature at night time seemed to have negligible effect on the growth of CS culture. The temperature measurements were recorded in DA system during the test.

The temperature in the solution tank was targeted at 47-50 ºC. The temperature inside the solution tank was measured with a thermocouple every hour. More than 90% of the temperature measurements were maintained within the targeted range throughout the five tests. Therefore, the effect of solution tank temperature on the growth rate of CS culture was considered negligible. 55

4.2. Average Air Velocity

The average air velocity of the flue gases emitted from a power plant stack is

around 1.2 ms-1; this condition was simulated in the photo-bioreactor with the fan setting

at 8. The targeted average gas velocity measurement was 1.16 ± 0.2 ms-1. The velocity

measurements were taken at 12 points as shown in Figure 4.2. At each of the 12 points,

velocity measurements were taken at two levels of depth, one at 8 inches and another at

14 inches. Due to the turbulence in the photo-bioreactor, there was significant variation in

velocity from one point to another. The minimum at any point did not go below 0.4 ms-1, except at point 3 and a depth level of 14’’ there was no air flow, thus, limiting the velocity measurements to only 11 out of the 12 points at 14’’ depth. Since adequate CS culture was observed even in absence of air flow, it is acceptable to consider the average velocity only from 11 points. Therefore, the effect of average air velocity on the growth rate of CS culture was considered negligible. The measurements were taken with a pitot tube once before starting the test and again after finishing the test, as shown in Table 4.1.

Figure 4.2: Representation of Air Flow Profile 56

Table 4.1: Average Air Velocity Measurements Before and After Tests

Test # Air Velocity Before Air Velocity After Average Velocity Test (ms-1) Test (ms-1) (ms-1) 1 0.86±0.09 0.72±0.06 0.79±0.05 2 0.96±0.07 0.9±0.06 0.93±0.02 3 0.93±0.06 0.92±0.07 0.92±0.005 4 0.95±0.07 1.0±0.07 0.98±0.02 5 1.03±0.16 1.05±0.11 1.04±0.007

4.3. Gas Concentrations

The concentration of the gases in the photo-bioreactor was carefully monitored.

The carbon monoxide concentration in the photo-bioreactor during operation never

exceeded 40 ppm (EPA standards). To be on the safe side, the concentration of carbon

monoxide was always maintained as low as possible.

Flue gases released from the stack of a typical coal-fired power plant is 14% by

volume and that released from medium to large power plants contains 3-15% carbon

dioxide by volume (Chakravarti, et al. 2001). Thus, maintaining a concentration of at

least 9 ± 0.5% in the photo-bioreactor was desirable. On an average, the achievable

carbon dioxide concentration in the photo-bioreactor with the current burner system

ranges from a minimum of 8.3% to a maximum of 11.5% by maintaining CO below 40

ppm.

4.4. Solution Flow Rate

A solution flow rate of 0.8 - 1.0 GPM per membrane was maintained with the manually operated solution flow meters. To observe the growth rate of CS culture at a 57 lower solution flow rate, the fifth, sixth and seventh harvest of the first test was maintained at solution flow rate of 0.6 GPM. For the third, fourth and fifth test, a solution flow rate of 1.0 – 1.2 GPM was maintained for the first hour of the test to get good coverage. The CS culture grown on the membrane surface was sheared using the three methods of harvesting as described in Section 3.5.

4.5. Average pH of the Solution

Acidic environment is not adequate for the growth of CS culture and so a pH above 6.3 was maintained throughout the tests. Out of the five tests conducted, two high light intensity and a low light intensity tests were maintained at an average pH from 6.3 to 6.9. An average pH of 7.3 to 7.6 was maintained for another low light intensity and a high light intensity test. Kajiwara et al. (1997) observed that Synechococcus PCC7942 showed little growth when maintained at a pH of 5.4 and showed early growth when maintained at a pH of 7.4-8.0 (Kajiwara, et al. 1997). This shows that variation in pH also varies the growth rate of microalgae. It was observed that with increase in pH, there was increase in CS culture growth.

Approximately 12.5 L of CS culture for each membrane was loaded in the solution tank. As the productivity of CS culture also depends on the area covered on each membrane, measures were taken to improve the membrane coverage. The percentage of

CS culture covering the membrane after loading was measured with a measuring tape.

The days of operation of each test varied depending on the growth of CS culture and light intensity in the photo-bioreactor. For each test, CS culture growth was quantified using visual inspection and also by pictures taken once a day after loading, once before and 58 after performing harvest and once after the CS growth was collected on the filters. The matrix that was proposed for the five tests is shown in Table 4.2.

Table 4.2: Proposed Test Matrix

Test # Light Intensity Mass of CS culture Productivity (µmol m-2s-1) initially loaded (g) (g/m2-day) 1 60 ≤ Average ≤ 85 2 60 ≤ Average ≤ 85 3 Average ≤ 30 4 Average ≤ 30 5 60 ≤ Average ≤ 85

Harvesting operation was performed to shear CS culture populated on both sides

of the membrane. Dr. Morgan Vis from Plant Biology Department of Ohio University

and professors from the CRF- II group helped in quantifying loading the CS culture and

its growth before performing harvesting operation. To determine the productivity of CS

culture better, all the tests were subjected to at least two intermediate harvests. The CS

culture harvested was collected in coarse and fine filters and dried in the oven for 3-5 days.

The CS culture productivity was determined by adding the mass estimated from the initial, intermediate and final harvestings denoted as Mm and subtracting the initial

amount of CS culture loaded (Mi), divided by the area of the membrane (Am), which is

0.929 m2, and the number of days of operation (N). The mass of CS culture from the final

harvest was the sum of the culture collected in filters and also CS culture grown on the

membranes. The productivity (ρ) of CS culture from each test was calculated as a ratio, as

shown below.

M − M ρ = m i Am ∗ N 59

The acceptable range to quantify the products from each test at the same light intensity conditions was that the variation in productivity from a test to test at the same light intensity condition should be less when compared to the variation in the productivity between the averages of the tests at low and high light intensity. Expected results from the tests at low light intensity and the high light intensity tests are shown in Figure 4.3.

3.5

3 -day) 2 2.5

1.5

0.5 Productivity (g/m 0 0 20406080 Light Intensity (µmol m-2s-1)

Figure 4.3: Expected Results

5. Results & Discussion

Five tests were conducted to examine the effects of light intensity on the productivity of CS culture growth in the photo-bioreactor. Three tests were conducted at

60 ≤ average ≤ 85 µmol m-2s-1 and two tests were conducted at an average ≤ 30 µmol m-

2s-1. The results of the five tests are discussed in this chapter and summarized in Table

5.1. Details of operational conditions and pictures of testing can be found in Appendix A.

While the range of tests were not exhaustive to define the effect of light level in a

statistically significant way, the data is useful to indicate the effects and lead to

recommendations for future testing.

5.1. Deviations in Actual Test Conditions from the Test Plan

Light measurements of the outer panels for the first two high light intensity tests

did not meet the targeted conditions. The reason for getting lower values than the targeted

range is due to blockage by the wall of the photo-bioreactor as described in Section 3.4.

Table 5.1: Test Results from the Five Tests

Light Light Amount of Total CS # CS culture Test Intensity Intensity after CS culture culture Days Growth # before Test Test Initially Produced Operated Rate (µmol m-2s-1) (µmol m-2s-1) Loaded (g) (g) (g/m2-day) 1 62.8 ± 11.7 69±8.6 9.45 114.03 62 1.82 2 69±8.6 68±8.7 9.39 46.54 22 1.82 3 29.6±0.4 29.7±0.5 7.98 29.93 24 0.98 4 29.4±0.6 29.7±0.5 5.81 37.18 24 1.41 5 74.2±2.1 73.9±1.6 5.15 59.52 17 3.44 61

Also, carbon dioxide percentage in the photo-bioreactor changed from the proposed conditions by up to ± 3%. The reason for the change in carbon dioxide percentage was due to change in gas flow rate while performing harvesting operation following the hand scraping method.

5.2. Uncertainties in Productivity

The results of CS culture productivity with high light and low light intensities

showed that the CS culture was light dependent, with the growth rate of CS culture at a

light intensity of 60 ≤ average ≤ 85 µmol m-2s-1 exceeding the growth rate at an average

light intensity ≤ 30 µmol m-2s-1, as expected. The uncertainty in the growth rate of CS culture at both the light intensities is due to the time gap between each harvest, area of the membrane covered with CS culture, mass of CS culture initially loaded and the mass of

CS culture from each harvest.

Except for the first test, harvesting operations for all the remaining four tests were

performed at the same time of the day. The percentage of CS culture coverage on the

membranes varied from test to test when initially loaded. For all the tests, the first harvest

was conducted after the membranes were completely populated with CS culture.

Therefore, the total area of the membrane was considered while calculating the

productivity. As described in Section 3.2.1, there could be uncertainty less than 2.75% in

the amount of CS culture loaded. The dry weight measurements of the filters were taken

immediately after removing the filters from the oven, giving less chance for error. The

results from each test are discussed below. 62

5.3. Test #1

The first test of CS culture productivity was performed with the average PAR between 60 to 85 µmol m-2s-1 at all tested points on the photo-bioreactor light panels. The

data for the test was shared with graduate student Chalermsak Dasaard, who performed a

substantial portion of the test as a part of his MSME project. The inner panel levels were

within the targeted range but the outer panels were not due to obstructions with the wall

of the photo-bioreactor as mentioned in Chapter 3 (Section 3.4). Only 25% of the points

for the membrane 1, side 1 and 46% of the points for membrane 3, side 2 were in the

targeted range for the outer panels. The light intensities for the four inner panels were

within the targeted range. The average light intensity values measured for each panel in

µmol m-2s-1 are shown in Table 5.2. The overall average light intensity of all the panels was 62.8 ± 11.7 µmol m-2s-1, which is within the targeted range. These measurements

show a lot of variation for the same membrane side from one test to another.

The solution flow rate maintained at the beginning of the test was 0.8 GPM.

Pictures of outer membranes before and after harvest, filters and the data collected from

DA system for each harvest of this test are shown in Appendix A. The light

measurements taken before the test are shown in Appendix B as Table B.1.

Table 5.2: Light Intensity Measurements before the First Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 55.7 5.3 2 60.9 1.6 2 1 60.9 1.6 2 82 6.5 3 1 72.3 6.2 2 45.3 21.7 Total 62.8 11.7

Approximately 9.45 g of CS culture were initially loaded into the solution tank.

The CS culture solution coverage for each side of the membrane is shown in Appendix A

as Table A.1. The pictures of initial coverage of the outer membranes one day after

loading are shown in Figure A.1 and the filters weighing 3.15 g of CS culture initially

loaded are shown in Figure A.2.

The first harvest was performed with the standard harvesting method (described in Section 3.5) nine days after initial loading. This method did not visibly shear off an

appreciable amount of CS culture from the membranes. The pictures of one of the outer

membranes before and after the first harvest are shown in Figure A.3, filters before

drying in the oven are shown in Figure A.4 and the data collected from the DA system

are shown in Figure A.5. Approximately 2.46 g of CS culture were obtained from the first

harvest. An average pH of 6.6 was maintained over the period. The water in the solution

tank was not transferred into the harvest tank for this harvest; instead RO water was

added to the harvest tank for performing the harvesting operation. 64

The second harvest was performed after five days from the first harvest by scraping (as described in Section 3.5). As shown in Figure A.6, most of the CS culture from the membranes was removed. Approximately 14.39 g of CS culture were collected

in filters as shown in Figure A.7. The average pH maintained over the period from the

first harvest to the second harvest was 6.83 and the data collected from the DA system

are shown in Figure A.8. In total, 16.85 g of CS culture were produced from 14 days of

operation.

Bolts were removed while performing the second harvest, which changed gas

concentration levels due to change in pressure in the reactor and remained at those levels

until the third harvest. The carbon dioxide level inside the reactor went down to an

average of 8.3% from an average of 9.4% preceding the harvest. The same method of

harvesting was used for all subsequent harvesting operations. One of the outer

membranes before and after the third harvest is shown in Figure A.9.

For the third harvest, 9.63 g of CS culture was collected in the filters, which are

shown in Figure A.10. The average pH of the solution maintained over the period of 7

days was 6.7 and the data collected from DA system are shown in Figure A.11. After the

third harvest, carbon dioxide concentrations were at 8.0-8.5% and then after changing the

settings in the gas rotameter to 0.8, the concentrations of gases returned to the targeted

value of 9 ± 0.5%.

Eight days after the third harvest, the fourth harvest was performed. The pictures

of one of the outer membranes before and after the fourth harvest are shown in Figure

A.12. Approximately 7.98 g of CS culture were produced as shown in Figure A.13. An 65 average pH of 6.6 was maintained over the period. The data obtained from the DA system are shown in Figure A.14.

The fifth, sixth and the seventh harvests were conducted to observe the growth of

CS culture with a solution flow rate maintained at 0.6 GPM instead of 0.8 GPM. The

seventh harvest was for the growth condition initially maintained at 0.6 GPM and then

switched to 0.8 GPM after observing that the growth seemed to be lower at that flow rate.

The fifth harvest was conducted seven days after the fourth harvest; an average pH of the

solution maintained was 6.64 over the period. One of the outer membranes before and

after the fifth harvest is shown in Figure A.15. Approximately 10.93 g of CS culture were

collected in the fifth harvest as shown in Figure A.16 and the data collected in the DA

system are shown in Figure A.17. Approximately 17.17 g was obtained during the sixth

harvest. One of the outer membranes before and after the sixth harvest is shown in Figure

A.18 and the filters are shown in Figure A.19. An average pH of 6.63 was maintained

over the period. Data collected in the DA system over a period of seven days are shown

in Figure A.20. After five days from the sixth harvest, the seventh harvest was performed.

The pictures of the membranes before and after the seventh harvest are shown in Figure

A.21. The filters gave a mass of 11.43 g of CS culture as shown in Figure A.22. An

average pH of 6.5 was maintained over the period. The data collected in the DA system

are shown in Figure A.23.

The last four harvests were performed at a reduced interval to observe the effect

on the CS culture growth rate. Specifically, the eighth harvest was performed after 3 days

from the seventh harvest. The membranes were dried for four hours before performing 66 the harvesting operation to see if drying the membranes before harvesting made the harvesting operation easier. One of the outer membranes before and after the eighth harvest is shown in Figure A.24. Approximately 8.5 g of CS culture were collected in the

filters as shown in Figure A.25. An average pH of 6.34 was maintained over the period.

The data collected from the DA system are shown in Figure A.26.

The ninth harvest was performed after four days from the eighth harvest. One of

the outer membranes before and after the ninth harvest is shown in Figure A.27.

Approximately 8.42 g of CS culture were collected during this harvest, and the filters are

shown in Figure A.28. An average pH of 6.27 was maintained over the period. The data

collected from the DA system are shown in Figure A.29. Fewer amounts of CS culture were collected from the tenth harvest among the four harvests. One of the outer membranes before and after the tenth harvest is shown in Figure A.30. Approximately

6.94 g of CS culture were collected in the filters as shown in Figure A.31. An average pH

of 6.43 was maintained over the period. The data collected from the DA system are

shown in Figure A.32.

The final harvest was performed three days after the tenth harvest. One of the

outer membranes before and after the eleventh harvest is shown in Figure A.33.

Approximately 8.43 g of CS culture were collected in the filters as shown in Figure A.34.

An average pH of 6.48 was maintained over the period. The data collected from the DA

system are shown in Figure A.35.

The final harvest was completed by removing the membranes and measuring the residual mass of CS culture. The mass obtained from the CS culture left on the three 67 membranes is 7.75 g. The growth rate for this harvest may have been influenced by the addition of CS culture sticking to the bottom of the photo-bioreactor. The amount of CS culture collected on the membranes was much higher when compared to other tests. A total of 16.18 g, including membranes weight was obtained from the final harvest. The

CS culture productivity results from each harvest for the first test are shown in Table 5.3.

The productivity from beginning of the test to the second harvest was 1.29 g/m2-

day, which was less than the other harvests. The productivity from the fourth harvest was

only 1.07 g/m2-day. The total test produced 114.03 g of CS culture from 62 days of

operation. The overall growth rate from this test was 1.82 g/m2-day. The days during

growth of CS culture in this test is shown by the plot in Figure 5.1. The plot shows that

smaller harvest intervals had higher mass of CS culture harvested.

Table 5.3: CS culture Productivity from the First Test

Number of Days CS culture Growth Rate Harvest # Average pH Operated Before Harvest Produced (g) (g/m2-day) 1 9 2.46 0.29 6.60 2 5 14.39 3.10 6.83 3 7 9.63 1.48 6.70 4 8 7.98 1.07 6.60 5 7 10.93 1.68 6.64 6 7 17.17 2.64 6.63 7 5 11.43 2.46 6.50 8 3 8.50 3.05 6.34 9 4 8.42 2.27 6.27 10 4 6.94 1.87 6.43 11 3 8.43 3.02 6.48 Overall 62 114.03 1.84 6.58 Values

120

100 Small Intervals 80

60 Large Intervals Culture Harvested (g) 40 CS 20

Mass ofMass 0 0 10203040506070 Date of Harvest (Days from Start)

Figure 5.1: Cumulative Mass of CS Culture Harvested during the First Test

The light measurements taken after the test are shown in Appendix B as Table

B.2. Only 60% of the points for the membrane 1, side 1 and 38% of the points for

membrane 3, side 2 were within the targeted range for the outer panels. The average light intensity values taken after the test are shown in Table 5.4. The overall average light

intensity of all the panels was 69 ± 8.6 µmol m-2s-1, which is within the targeted range.

Table 5.4: Light Intensity Measurements after the First Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 59.5 4.4 2 72.9 5.3 2 1 72.4 5.4 2 76.9 5.7 3 1 75.4 5.4 2 54 10.8 Total 69 8.6

The outer panel light intensities for this test were taken with four out of six lights in use. The measurements were taken for a period of 22 days out of the 62 days of operation. The measurements were taken at different times during the day to observe the change in light intensities. The values were within less than 10% variation of the overall average value as shown in Table 5.5.

Table 5.5: Outer Light Panel Intensities for the First Test

Membrane1,Side 1 Membrane 3, Side 2 Date (µmol m-2s-1) (µmol m-2s-1) 1/22/2007 80.4 81.7 1/26/2007 81.9 82.6 1/30/2007 80 81.1 2/3/2007 78.9 80.3 2/7/2007 78.2 79.6 2/11/2007 78 79.3 2/15/2007 81 80.6 Overall Average 79.8 80.7 5.4. Test #2

The second test of CS culture productivity was performed with PAR between 60

to 85 µmol m-2s-1 at all tested points on the photo-bioreactor light panels. A repeat of the

first test’s conditions was performed immediately after stopping the test, and so the light measurements taken after stopping the first test were considered as the light measurements taken before starting the second test. The average light intensity values measured in µmol m-2s-1 for each panel are shown in Table 5.6.

A solution flow rate of 0.8 GPM was maintained throughout the test. Pictures of

outer membranes before and after harvest, filters and the data collected from DA system

for each harvest of this test are shown in Appendix A. Light measurements taken before 70 the test are shown in Appendix B as Table B.3. All the subsequent harvesting operations were conducted at the same point of a day following loading of CS culture.

Table 5.6: Light Intensity Measurements before the Second Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 59.5 4.4 2 72.9 5.3 2 1 72.4 5.4 2 76.9 5.7 3 1 75.4 5.4 2 54 10.8 Total 69 8.6

Approximately 9.39 g of CS culture were initially loaded into the solution tank.

The CS culture solution coverage for each side of the membrane is shown in Appendix A

as Table A.2. The pictures of initial coverage of the outer membranes one day after

loading are shown in Figure A.36 and the filters weighing 3.13 g of CS culture are shown

in Figure A.37. All the harvests in this test were performed following the scraping method.

The first harvest was performed four days after initial loading, because shearing of CS culture from the membranes was easier. One of the outer membranes before and

after the first harvest is shown in Figure A.38. The pictures of filters before drying in the

oven are shown in Figure A.39 and the data collected from the DA system are shown in

Figure A.40. Approximately 6.51 g of CS culture were obtained from the first harvest and

an average pH of 6.5 was maintained over the period. The water in the solution tank was 71 not transferred into the harvest tank for this harvest; instead RO water was added to the harvest tank for performing the harvesting operation.

The second harvest was performed seven days after the first harvest. The pictures of one of the outer membranes taken before and after the second harvest are shown in

Figure A.41. Approximately 6.75 g of CS culture were produced as shown in Figure

A.42. An average pH of 6.65 was maintained over the period. The data obtained from the

DA system are shown in Figure A.43.

The third harvest was performed six days after the second harvest. One of the

outer membranes before and after the third harvest is shown in Figure A.44.

Approximately 11.7 g of CS culture were collected in the filters as shown in Figure A.45.

An average pH of 6.7 was maintained over the period. The data obtained from the DA

system are shown in Figure A.46.

CS culture showed an early growth rate after the third harvest, therefore the final

harvest was performed five days after the third harvest. One of the outer membranes

before and after the final harvest is shown in Figure A.47. Approximately 14.28 g of CS culture were collected in the filters as shown in Figure A.48. An average pH of 6.7 was maintained over the period. The data collected from the DA system are shown in Figure

A.49.

The final harvest was completed by removing the membranes and measuring the residual mass of CS culture. The mass obtained from the CS culture left on the three

membranes was 7.3 g. The amount of CS culture collected on the membranes was much 72 higher when compared to other tests. A total of 21.58 g, including membrane weights was obtained from the final harvest.

The CS culture productivity results from each harvest for this test are shown in

Table 5.7. The total test produced 46.54 g of CS culture from 22 days of operation. The

overall growth rate from this test was 1.82 g/m2-day. A less productivity of about 1.04

g/m2-day was observed from the second harvest. The days during growth of CS culture in

this test is shown by the plot in Figure 5.2.

Table 5.7: The CS culture Productivity from the Second Test

Number of Days CS culture Growth Rate Harvest # Average pH Operated Before Harvest Produced (g) (g/m2-day) 1 4 6.51 1.75 6.5 2 7 6.75 1.04 6.65 3 6 11.70 2.10 6.7 4 5 14.28 3.07 6.7 Overall 22 46.54 1.92 6.64 Values

The light measurements taken after the test are shown in Appendix B as Table

B.4. As explained earlier, the inner panels levels were within the targeted range but the

outer panels were not due to obstruction with the wall of the photo-bioreactor. Only 50%

of the points for the membrane 1, side 1 and 33% of the points for membrane 3, side 2

were within the targeted range for the outer panels. The average light intensity values for

each panel taken after the test are shown in Table 5.8. The overall average light intensity

of all the panels was 68 ± 8.7 µmol m-2s-1, which is within the targeted range.

Culture Harvested (g) Harvested Culture 15 CS 10

5 Mass of of Mass 0 0 5 10 15 20 25 Date of Harvest (Days from Start)

Figure 5.2: Cumulative Mass of CS Culture Harvested during the Second Test

Table 5.8: Light Intensity Measurements after the Second Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 58.9 4.2 2 73.3 5.4 2 1 73.5 4.9 2 75.4 6.05 3 1 75.7 5.6 2 53.5 10.6 Total 68 8.7

The outer panel light intensities for this test were taken with four of six lights in use. The measurements were taken at different times during the day to observe the change in light intensities. The values were within less than 10% variation of the overall average value as shown in Table 5.9. 74

Table 5.9: Outer Light Panel Intensities for the Second Test

Membrane 1, Side 1 Membrane 3, Side 2 Date (µmol m-2s-1) (µmol m-2s-1) 2/16/2007 79 80.6 2/20/2007 78.9 80.3 2/24/2007 79.4 79.9 2/28/2007 80.4 81.1 3/4/2007 78.8 79.3 3/8/2007 79.9 80.4 3/10/2007 80.1 80.8 Overall Average 79.5 80.3

5.5. Test #3

The third test of CS culture productivity was performed with the average PAR less than or equal to 30 µmol m-2s-1 at all tested points on the photo-bioreactor light

panels. More than 80% of the light measurements were between 27-33 µmol m-2s-1. The average light intensity values for each panel taken after the test are shown in Table 5.10.

The overall average light intensity of all the panels was 29.6 ± 0.4 µmol m-2s-1, which is

within the targeted range.

A solution flow rate of 1.0-1.2 GPM was maintained for the first hour of the test

to get a good coverage. A solution flow rate of 0.8-1.0 GPM was maintained throughout

the test to get better coverage. Pictures of outer membranes before and after harvest,

filters and the data collected from DA system for each harvest of this test are shown in

Appendix A. The light panels were covered with Mylar sheets for this test to attain the

targeted light intensity. The light measurements taken before the test are shown in

Appendix B as Table B.5. 75

Table 5.10: Light Intensity Measurements before the Third Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 29.6 2.6 2 30 1.5 2 1 30.4 1.9 2 29.2 1.9 3 1 29 1.9 2 29.2 3.9 Total 29.6 0.4

Approximately 7.98 g of CS culture were initially loaded into the solution tank.

The CS culture solution coverage for each side of the membrane is shown in Appendix A

as Table A.3. The pictures of initial coverage of the outer membranes one day after

loading are shown in Figure A.50 and the filters weighing 2.66 g of CS culture were

shown in Figure A.51. To maintain same period between each harvest, a gap of six days

was followed.

The first harvest was performed six days after initial loading. All the harvests in

this test were performed following the nozzle spray method. The pictures of one of the outer membranes before and after the first harvest are shown in Figure A.52 and filters

before drying in the oven are shown in Figure A.53. Data collected from the DA system

are shown in Figure A.54. Approximately 6.93 g of CS culture were obtained from the

first harvest. An average pH of 6.75 was maintained over the period.

The second harvest was performed six days after the first harvest. An average pH

of 6.9 was maintained over the period. The pictures of the one of the outer membranes

taken before and after the second harvest are shown in Figure A.55. Approximately 6.75 76 g of CS culture were collected in the filters from the second harvest as shown in Figure

A.56 and the data collected from the DA system are shown in Figure A.57.

The third harvest was performed 6 days after the second harvest. Over the six day

period, it was observed that there was no significant growth of CS culture on one side of

second membrane due to no solution flow on that particular side. The initial flow

coverage on that side of the membrane was 60%. However, the flow characteristics

changed for that particular side after performing the first harvest and the second harvest.

The pictures of one of the outer membranes before and after the third harvest are shown

in Figure A.58. A total of 5.17 g of CS culture were collected in the filters as shown in

Figure A.59. An average pH of 6.92 was maintained over the period. Data collected from

the DA system are shown in Figure A.60. The productivity for the third harvest was 0.91

g/m2-day. Considering that there was no growth on one side of the membrane, the

productivity calculated from five sides of the membranes for the third harvest was 1.11

g/m2-day.

After performing the third harvest, it was observed that all sides of the membranes

showed adequate growth of CS culture. The final harvest was performed six days after

the third harvest. The pictures of one of the outer membranes before and after the fourth

harvest are shown in Figure A.61. A total of 7.1 g of CS culture were collected from the

filters as shown in Figure A.62. An average pH of 6.92 was maintained over the period.

The data collected from the DA system are shown in Figure A.63.

The final harvest was completed by removing the membranes and measuring the residual mass of CS culture. The mass obtained from the CS culture left on the three 77 membranes for this test is 3.09 g, which is less when compared to the previous tests. This could be due to shearing of CS culture from the membranes with high pressure water

during the nozzle spray harvesting method.

A total of 11.03 g, including membrane weights was produced from the final

harvest. The CS culture productivity from each harvest for the third test is shown in Table

5.11. The CS culture settled at the bottom of the tank was separately collected in filter

paper for the final harvest. Approximately 0.84 g of CS culture was collected from the

bottom of the photo-bioreactor. The total test produced 29.93 g of CS culture from 24

days of operation. The overall growth rate from this test was 0.98 g/m2-day. The days

during growth of CS culture in this test is shown by the plot in Figure 5.3.

Table 5.11: The CS culture Productivity from the Third Test

Number of Days CS culture Growth Rate Harvest # Average pH Operated Before Harvest Produced (g) (g/m2-day) 1 6 6.93 1.24 6.75 2 6 6.81 1.22 6.9 3 6 5.17 1.11 6.92 4 6 7.1 1.27 6.92 Overall 24 29.93 1.21 6.87 Values

culture harvested (g) harvested culture 10 CS 5

Mass of Mass 0 0 5 10 15 20 25 30 Date of Harvest (Days from Start)

Figure 5.3: Cumulative Mass of CS Culture Harvested during the Third Test

The light measurements taken after the test are shown in Appendix B as Table

B.6. More than 80% of the light measurements were between 27- 33 µmol m-2s-1. The average light intensity values of each panel taken after the test are shown in Table 5.12.

The overall average light intensity of all the panels was 29.7 ± 0.5 µmol m-2s-1, which is within the targeted range.

Table 5.12: Light Intensity Measurements after the Third Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 29.2 2.6 2 30.2 1.7 2 1 30.4 1.9 2 29.6 2.0 3 1 29.5 1.9 2 29 4.1 Total 29.7 0.5

The outer panel light intensities for this test were taken with two of six lights on and with Mylar sheets on them. Once, the measurements were not taken due to unavailability of a light meter. The measurements were taken at the same time of day following the first measurement to maintain uniformity. The values were within less than

10% variation of the overall average value as shown in Table 5.13.

Table 5.13: Outer Light Panel Intensities for the Third Test

Membrane 1, Side1 Membrane 3, Side 2 Date (µmol m-2s-1) (µmol m-2s-1) 5/2/2007 38.5 41.8 5/6/2007 38.4 41.4 5/10/2007 38.4 41.7 5/14/2007 38.6 41.7 5/18/2007 NA NA 5/22/2007 38.8 42.6 5/26/2007 39.1 42.4 Overall Average 38.6 41.9

5.6. Test #4

The fourth test, a repeat of the third test’s conditions was conducted with the

average PAR less than or equal to 30 µmol m-2s-1. More than 80% of the light

measurements were between 27-33 µmol m-2s-1. The average light intensity values for

each panel taken after the test are shown in Table 5.14. The overall average light intensity

of all the panels was 29.4 ± 0.6 µmol m-2s-1, which is within the targeted range.

A solution flow rate of 1.0-1.2 GPM was maintained for the first hour of the test

to get a better coverage. A solution flow rate of 0.8-1.0 GPM was maintained throughout

the test to get better coverage. Pictures of outer membranes before and after harvest,

filters and the data collected from DA system for each harvest of this test are shown in 80

Appendix A. The light measurements taken before the test are shown in Appendix B as

Table B.7.

Table 5.14: Light Intensity Measurements before the Fourth Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 28.9 2.0 2 30 1.7 2 1 30.3 1.9 2 29.6 1.8 3 1 29 1.4 2 28.7 4.0 Total 29.4 0.6

Approximately 5.81 g of CS culture were initially loaded into the solution tank.

The CS culture solution coverage for each side of the membrane is shown in Appendix A

as Table A.4. The pictures of initial coverage of the outer membranes one day after

loading are shown in Figure A.64 and the filters weighing 1.94 g of CS culture were

shown in Figure A.65. All the harvests in this test were performed using nozzle spray

method. To maintain same period between each harvest, a gap of six days was followed.

The first harvest was performed six days after initial loading. The pictures of one

of the outer membranes taken before and after the first harvest are shown in Figure A.66,

filters before drying in the oven are is shown in Figure A.67. The data collected from the

DA system are shown in Figure A.68. A total of 8.1 g of CS culture were obtained from

the first harvest. An average pH of 7.3 was maintained over the period. 81

The second harvest was performed six days after the first harvest. The average pH maintained over the period was 7.15. Pictures of one of the outer membranes taken before and after the second harvest are shown in Figure A.69. A total of 7.24 g of CS culture were collected in the filters as shown in Figure A.70 and the data collected from the DA system are shown in Figure A.71.

The third harvest was performed six days after the second harvest. The growth of

CS culture looked better when compared to the second harvest. The pictures of one of the

outer membranes before and after the third harvest are shown in Figure A.72. A total of

8.69 g of CS culture were collected in the filters as shown in Figure A.73. An average pH

of 7.59 was maintained over the period. The data collected from the DA system are

shown in Figure A.74.

The membranes looked very much populated four days after the third harvest. To

maintain a gap of six days for all the harvests, the final harvest was performed six days

after the third harvest. The pictures of one of the outer membranes before and after the

fourth harvest are shown in Figure A.75. The CS culture sticking to the bottom of the

photo-bioreactor was not separately measured, since the amount of CS culture collected

from the third test was very less when compared to the weight obtained from the filters.

A total of 8.92 g of CS culture were collected in filters including the CS culture sticking

to the bottom of the photo-bioreactor as shown in Figure A.76. An average pH of 7.5 was

maintained over the period. The data collected from the DA system are shown in Figure

A.77. The productivity of CS culture from each harvest for this test is shown in Table

5.15. 82

The final harvest was completed by removing the membranes and measuring residual mass of CS culture. The mass obtained from the CS culture left on the three

membranes was 4.23 g. A total of 13.15 g, including the membrane weights was obtained

from the final harvest. The total test produced 37.18 g of CS culture from 24 days of

operation. The overall growth rate from this test was 1.41 g/m2-day. The days during

growth of CS culture in this test is shown by the plot in Figure 5.4.

Table 5.15: The CS culture Productivity from the Fourth Test

Number of Days CS culture Growth Rate Harvest # Average pH Operated Before Harvest Produced (g) (g/m2-day) 1 6 8.1 1.45 7.3 2 6 7.24 1.3 7.15 3 6 8.69 1.56 7.59 4 6 8.92 1.60 7.5 Overall 24 37.18 1.47 7.4 Values

15 Culture Harvested(g) 10 CS 5

Mass of 0 0 5 10 15 20 25 30 Date of Harvest (Days from Start)

Figure 5.4: Cumulative Mass of CS Culture Harvested during the Fourth Test 83

The light measurements taken after the test are shown in Appendix B as Table

B.8. More than 80% of the light measurements were between 27-33 µmol m-2s-1. The average light intensity values for each panel taken after the test are shown in Table 5.16.

The overall average light intensity of all the panels was 29.7 ± 0.5 µmol m-2s-1, which is

within the targeted range.

Table 5.16: Light Intensity Measurements after the Fourth Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 29.1 2.6 2 30.2 1.3 2 1 30.3 1.9 2 29.6 1.7 3 1 29.9 1.7 2 29.0 4.4 Total 29.7 0.5

The outer panel light intensity measurements for this test were carried out as

taken for the third test. The measurements were taken at the same time of day following

the second measurement to maintain uniformity. Once, the measurements were not taken

due to unavailability of a light meter. The values are within less than 10% variation of the overall average value as shown in Table 5.17. 84

Table 5.17: Outer Light Panel Intensities for the Fourth Test

Membrane 1, Side 1 Membrane 3, Side 2 Date (µmol m-2s-1) (µmol m-2s-1)

5/30/2007 42.6 45.9 6/3/2007 NA NA 6/7/2007 38.9 42.5 6/11/2007 39.3 42.6 6/15/2007 38.6 41 6/19/2007 38.5 41.7 6/23/2007 38.5 41.9 Overall Average 39.3 42.6

5.7. Test #5

The fifth test, a repeatability test, of CS culture productivity was performed with

the average PAR between 60 to 85 µmol m-2s-1 at all tested points on the photo-bioreactor

light panels. This test was a repeatability test to observe if the first two tests conducted

were repeatable. More than 80% of the light measurements were between 60 to 85 µmol

m-2s-1. The average light intensity values of each panel taken after the test are shown in

Table 5.18. The overall average light intensity of all the panels was 74.2 ± 2.1 µmol m-2s-

1, which is within the targeted range.

A solution flow rate of 1.0-1.2 GPM was maintained for the first hour of the test

to get a better coverage. A solution flow rate of 0.8-1.0 GPM was maintained throughout

the test to get better coverage. The pictures of outer membranes before and after harvest,

filters and the data collected from DA system for each harvest of this test are shown in

Appendix A. The light measurements taken before the test are shown in Appendix B as 85

Table B.9. The outer panels were covered with Mylar sheets with all the six lights in use for this test.

Table 5.18: Light Intensity Measurements before the Fifth Test

Average Standard Membrane # Side Light Intensity Deviation (µmolm-2s-1) 1 1 72.6 4.2 2 72.4 5.6 2 1 74.6 4.8 2 76.3 5.1 3 1 76.9 4.9 2 72.3 13.5 Total 74.2 2.1

Approximately 5.15 g of CS culture were initially loaded into the solution tank.

The CS culture solution coverage for each side of the membrane is shown in Table A.5.

The pictures of initial coverage of the outer membranes one day after loading are shown in Figure A.78 and the filters weighing 1.72 g of CS culture are shown in Figure A.79.

All the harvests in this test were performed following the nozzle spray method.

The first harvest was performed four days after initial loading. The pictures of one

of the outer membranes before and after the first harvest is shown in Figure A.80, and the

filters weighing 8.88 g of CS culture before drying in the oven is shown in Figure A.81

and the data collected from the DA system are shown in Figure A.82. An average pH of

7.55 was maintained over the period.

The second harvest was performed five days after the first harvest. The membranes looked very much populated during this period. The pictures of one of the outer membranes taken before and after the second harvest are shown in Figure A.83. 86

Approximately 11.67 g of CS culture were collected in the filters as shown in Figure

A.84. An average pH of 7.48 was maintained over the period. The data collected from the

DA system are shown in Figure A.85.

The membranes looked very much populated four days after the second harvest.

Therefore, the third and the fourth harvests were performed with four days gap. The

pictures of one of the outer membranes before and after the third harvest are shown in

Figure A.86. The membranes were completely sheared for both the harvests. The filters

weighing 14.3 g of CS culture before drying in the oven is shown in Figure A.87. An

average pH of 7.47 was maintained over the period. The data collected from the DA

system are shown in Figure A.88. While performing harvesting operation, it was

observed that shearing the CS culture from the membranes was difficult during the third

harvest than in the first and the second harvest.

The final harvest was conducted four days after the third harvest. The pictures of

one of the outer membranes before and after the final harvest is shown in Figure A.89 and the filters weighing 17.69 g of CS culture before drying in the oven is shown in

Figure A.90. An average pH of 7.6 was maintained over the period. The data collected from the DA system are shown in Figure A.91. The membranes were completely sheared after the harvesting operation. Also, it is observed from this harvest that shearing the CS culture from some parts of the membranes became much more difficult. The CS culture productivity from each harvest for the fifth test is shown in Table 5.19.

The final harvest was completed by removing the membranes and measuring the residual mass of CS culture. The mass obtained from the CS culture left on the three 87 membranes was 6.98 g. A total of 24.67 g, including the membrane weights, was obtained from the final harvest. The total test produced 59.52 g of CS culture from 17

days of operation. The overall growth rate from this test was 3.44 g/m2-day. The days

during growth of CS culture in this test is shown by the plot in Figure 5.5.

Table 5.19: The CS culture Productivity from the Fifth Test

Number of Days CS culture Growth Rate Harvest # Average pH Operated before Harvest Produced (g) (g/m2-day) 1 4 8.88 2.39 7.55 2 5 11.67 2.51 7.48 3 4 14.3 3.85 7.47 4 4 17.69 4.76 7.6 Overall 17 59.52 3.32 7.52 Values

Culture Harvested (g) 20 CS 10

Mass of Mass 0 0 5 10 15 20 Date of Harvest (Days from Start)

Figure 5.5: Cumulative Mass of CS Culture Harvested during the Fifth Test

The light measurements taken after the test are shown in Appendix B as Table

B.10. More than 80% of the light measurements were between 65 to 80 µmol m-2s-1. The average light intensity values of each panel taken after the test are shown in Table 5.20. 88

The overall average light intensity of all the panels was 73.9 ± 1.6 µmol m-2s-1, which is

within the targeted range.

Table 5.20: Light Intensity Measurements after the Fifth Test

Average Standard Membrane # Side Light Intensity Deviation (µmol m-2s-1) 1 1 72.8 4.3 2 72.4 5.6 2 1 73.9 4.7 2 75.7 6 3 1 76.4 5.2 2 72.7 13.7 Total 73.9 1.6

The outer panel light intensities for this test were taken with all the lights in use.

The measurements were taken at the same time of day following the first measurement to

maintain uniformity. The values are within less than 10% variation of the overall average

value as shown in Table 5.21

Table 5.21: Outer Light Panel Intensities for the Fifth Test

Membrane 1, Side 1 Membrane 3, Side 2 Date (µmol m-2s-1) (µmol m-2s-1) 6/27/2007 94.10 101.70 7/1/2007 94.21 101.96 7/5/2007 93.74 102.64 7/9/2007 94.38 101.83 7/13/2007 94.30 101.67 Overall Average 94.1 101.9

The productivity from the five tests is shown by a plot in the Figure 5.6. It is observed that the productivity for the first two tests was same. The fifth test, which was performed to ensure repeatability of the first two tests, gave an additional 1.62 g/m2-day

(89%) increase in productivity to the average productivity of the first two tests. Though the third and fourth tests were performed at same light conditions, the productivity of the fourth test increased by 0.43 g/m2-day (44%) compared to the third test. The increase in productivity for the fourth and fifth tests could be due to fewer number of days

maintained between the harvests and also due to the maintenance of pH of the CS culture solution between 7.4-7.6. Also, for the fifth test the outer panels were with almost same illumination as the inner panels. The conclusions about the results are made in the

Chapter 6 and recommendations are also made accordingly.

Figure 5.6: Productivity Results from the Five Tests

6. Conclusions & Recommendations

The results of CS culture growth rate at high and low light intensities indicated

that light intensity may have an effect on CS culture productivity. Although the exact amount of experimental error is difficult to quantify, the data indicates that there is a difference in the average CS culture productivity at a light intensity of 60 ≤ average ≤ 85

µmol m-2s-1 and average ≤ 30 µmol m-2s-1. However, there are possible confounding

effects that could make such a conclusion invalid. For example, the increase in

productivity from the fourth and the fifth test could be due to maintenance of pH from

7.4-7.6 and the frequency of harvesting also seemed to affect the productivity. In

addition, the high productivity of the fifth test may have been affected by the higher

illumination of the outer panels compared to the first and second test.

The decrease in productivity from the average of the first two tests to the third test

was 0.84 g/m2-day (46%). This indicates that there was a decrease in productivity from

high light to low light intensity. With a change in pH of the solution, 0.43 g/m2-day

(44%) increase in productivity was observed from the third to the fourth test. If we could

attribute a 0.71 g/m2-day (44%) increase in productivity in the fifth test to a change in

pH, the remaining 0.91 g/m2-day increase in productivity in the fifth test compared to the

average of first two tests might be attributable to the increase in light intensity of the

outer panels and to the fewer number of days maintained between the harvests.

From the first test it was observed that the light measurements taken before the

test had significant variation across the light measurement points. The reason for this 91 variation could be due to the usage of an un-calibrated multimeter instead of a LI 250A light meter. The growth rate from the beginning of the test to the second harvest was only

1.29 g/m2-day. Not changing the solution in the solution tank during harvesting operation

could be one of the reasons for lower growth rate. Also, the productivity from the fourth

harvest was only 1.04 g/m2-day. The reason for getting such low productivity could be

due to eight day period maintained between the third and the fourth harvest.

When the solution was not changed in the solution tank after the first harvest, the

productivity from the second harvest was only 1.04 g/m2-day during the second test. This

once again proved that if water in the solution tank is not changed for each harvest,

growth rate decreases. From the third test, it was observed that productivity of the third

harvest was only 0.91 g/m2-day. This decrease in growth rate was due to no solution flow

on one side of the membrane. When calculated for only five sides of the membrane, the

productivity was 1.11 g/m2-day, which was closer to the other harvest productivities.

As the number of harvests increased, the amount of CS culture produced

increased. This can be observed from the productivity results of the final harvest of the

second, third, fourth and fifth test. The productivity from the final harvest of the second

test was 3.07 g/m2-day, which was 1.25 g/m2-day (69%) higher than the overall average

productivity of the second test. The productivity from the final harvest of the third test

was 1.27 g/m2-day, which was 0.29 g/m2-day (30%) higher than the overall average

productivity of the third test. The productivity from the final harvest of the fourth test was

1.6 g/m2-day, which was approximately 0.19 g/m2-day (14%) higher than the overall

average productivity of the fourth test. An increase in productivity was observed from the 92 second harvest to the final harvest of fifth test because of the fewer number of days maintained between the harvests. The highest increase in productivity of 1.34 g/m2-day

(53%) was observed from the second to the third harvest in the fifth test.

The amount of CS culture loaded for each test decreased from the first test to last

test, starting from 9.45 g to 5.15 g. This was due to frequent usage of CS culture from the

culture tanks. It was observed that as the test period increased, the flow on the

membranes increased. This could be due to the change in the characteristics of shim

material due to the heat in the photo-bioreactor and also due to the harvesting operation.

Sometimes after performing the harvesting operation, it was observed that there was no

solution flow on the membrane, even though there was initially a good flow.

As described in Chapter 2, the targeted growth rate of microalgae in 1000 m2 open

ponds was about 50 g/m2-day. The overall growth rate attained was approximately 10

g/m2-day (Sheehan, et al. 1998). From the fifth test, it was observed that there was still

6.56 g/m2-day productivity, which needs to be achieved to simply reach attained growth

rate, let alone the targeted conditions.

6.1. Recommendations for Future Work

By observing the productivity results from low light and high light tests, it is recommended to repeat at least one low light at average ≤ 30 µmol m-2s-1 and a high light

of 60 ≤ average ≤ 85 µmol m-2s-1, at a pH level (7.4-7.6) to observe the validity of

productivity results of the CS culture in a photo-bioreactor. It is suggested to keep the harvesting period less than six days (perhaps two days) for the low light tests, to see the effect of rapid harvesting on the productivity of CS culture. 93

Before starting a test it is recommended to work on the headers to get good coverage on both sides of the membrane. The end plates should be tight enough not to allow water to flow from the end. The flow on the membranes should be such that, if there is no coverage on one side of the membrane in some parts, there should be flow on the other side of the membrane in the same region. If there is no good coverage, the insert should be tilted and tested in different inclinations to attain good flow.

It was observed from the last two tests that there was good CS culture growth

when maintaining a pH of 7.4-7.6. Therefore, it is recommended to maintain the pH of

the solution above 7.4 and conduct tests to observe the CS culture growth rate between a

pH of 7.6-8.2 (since the culture tanks are maintained at that pH value). Through

maintaining a higher pH it is expected to increase growth rate. It is recommended to change the solution in the solution tank after each harvest to maintain adequate nutrient supply for the CS culture solution in the solution tank. This will diminish the possibility

of attaining a lower growth rate.

From the first, second and fifth tests it was observed that there was better growth

of CS culture from fewer days of operation between harvests. Figure 6.1 shows the mass of CS culture produced as a function of days between harvests for high light tests (1, 2 &

5). The standard deviations are taken for the harvests performed (with the same number

of days between the harvests) more than three times. A higher standard deviation for the

four and seven days between the harvests was observed. The reason for this could be the

different harvesting operations performed, not changing the nutrient solution, and higher 94 pH and increase in light levels for the outer panels. All the low light tests harvests were performed for a six day gap and hence are not plotted.

14 12.94 12 11.7 11.12 10.45 10 Culture (g) Culture 8.47 8 CS 7.98

6 Mass of 4 2.46 2

0 012345678910

Number of Days between Harvests

Figure 6.1: CS Culture produced as Function of Days for High Light Tests Also, from the fourth and fifth tests, it was observed that shearing the CS culture from the membranes became tougher as the number of days of operation increased.

Membranes from the fifth test were still covered with CS culture in some parts after the

fourth harvest, as indicated in red marks in Figure 6.2. In future tests it is recommended

to maintain a shorter duration between initial loading and first harvest (i.e., perhaps 3

days instead of 4-9 days) and to find the harvest period between intermediate harvests

(may be a one day gap for a high light test and two days gap for a low light test). Figure

6.3 shows the membrane before and one day after the second harvest for the fifth test.

Figure 6.2: Membrane after Fourth Harvest from the Fifth Test

Figure 6.3: Membrane before and one day after Second Harvest for the Fifth Test The productivity of CS culture calculated at 60 µmol m-2s-1 by doubling the productivity from the third test is 1.96 g/m2-day, which is greater than the productivity from the average of first two tests. From a comparison of the fourth and the fifth test which were maintained at a pH between 7.4-7.6, the productivity of CS culture calculated at 60 µmol m-2s-1 by doubling the productivity from the fourth test is 2.82 g/m2-day, which is not greater than the productivity from the fifth test. The reason for this could be due to higher light intensity for outer panels, fewer days between harvests and increase in pH for the fifth test. Therefore, it is recommended to find the photosynthetic efficiency at

30 and 70 µmol m-2s-1.

The maximum light intensity in the photo-bioreactor with six lights on all panels including external illumination was between 110 to 125 µmol m-2s-1. Since it is observed from the fifth test that there was an additional increase in CS culture productivity with 96 higher light intensity for outer panels, this indicated that there was an increase in CS culture growth with increase in light intensity. Therefore, it is recommended to conduct light intensity tests between 110 to 125 µmol m-2s-1. Also, according to Ono and Cuello

(2007) the species shows a good growth rate up to approximately 250 µmol m-2s-1. The growth rate data between light conditions of 110 to 125 µmol m-2s-1 could be helpful in

finding the growth characteristics of CS culture in the present photo-bioreactor at three different light intensities. 97

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Purves, William K, Gordon H Orians, and H Craig Heller. Life: The Science of Biology. 4th edition. Massachusetts: Sinauer Associates, 1994.

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100

Appendix A

Test 1 Table A.1: Outer Membrane Coverage’s, a Day after Loading CS Culture

Side 1 Side 2 Membrane 1 70 30 Membrane 2 95 30 Membrane 3 65 40

Figure A.1: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day

Figure A.2: Filters from Initial Sample Loading gave 3.15 g of CS Culture 101

Harvest Number: First Average pH of the solution: 6.6

Days of Operation: 9 Solution tank temperature: 48.7 °C

Harvesting Method: Standard Harvest Solution flow rate: 0.8 GPM

Figure A.3: One of the Outer Surfaces of a Membrane before and after First Harvest

Figure A.4: Filters from First Harvest gave 2.46 g of CS Culture 102

Figure A.5: Data Collected from DA System from Initial Loading to First Harvest

103

Harvest Number: Second Average pH of the solution: 6.83

Days of Operation: 5 Solution tank temperature: 48.6 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.6: One of the Outer Surfaces of a Membrane before and after Second Harvest

Figure A.7: Filters from Second Harvest gave 14.39 g of CS Culture

104

Figure A.8: Data Collected from DA System from First to Second Harvest

105

Harvest Number: Third Average pH of the solution: 6.7

Days of Operation: 7 Solution tank temperature: 49.1 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.9: One of the Outer Surfaces of a Membrane before and after Third Harvest

Figure A.10: Filters from Third Harvest gave 9.63 g of CS Culture

106

Figure A.11: Data Collected from DA System from Second to Third Harvest 107

Harvest Number: Fourth Average pH of the solution: 6.6

Days of Operation: 8 Solution tank temperature: 47.6 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.12: One of the Outer Surfaces of a Membrane before and after Fourth Harvest

Figure A.13: Filters from Fourth Harvest gave 7.98 g of CS Culture

108

Figure A.14: Data Collected from DA System from Third to Fourth Harvest

109

Harvest Number: Fifth Average pH of the solution: 6.64

Days of Operation: 7 Solution tank temperature: 47.1 °C

Harvesting Method: Hand scraping Solution flow rate: 0.6 GPM

Figure A.15: One of the Outer Surfaces of a Membrane before and after Fifth Harvest

Figure A.16: Filters from Fifth Harvest gave 10.93 g of CS Culture

110

Figure A.17: Data Collected from DA System from Fourth to Fifth Harvest 111

Harvest Number: Sixth Average pH of the solution: 6.63

Days of Operation: 7 Solution tank temperature: 47.5 °C

Harvesting Method: Hand scraping Solution flow rate: 0.6 GPM

Figure A.18: One of the Outer Surfaces of a Membrane before and after Sixth Harvest

Figure A.19: Filters from Sixth Harvest gave 17.17 g of CS Culture

112

Figure A.20: Data Collected from DA System from Fifth to Sixth Harvest

113

Harvest Number: Seventh Average pH of the solution: 6.5

Days of Operation: 5 Solution tank temperature: 47.6 °C

Harvesting Method: Hand scraping Solution flow rate: 0.6 – 0.8 GPM

Figure A.21: One of the Outer Surfaces of a Membrane before and after Seventh Harvest

Figure A.22: Filters from Seventh Harvest gave 11.43 g of CS Culture

114

Figure A.23: Data Collected from DA System from Sixth to Seventh Harvest

115

Harvest Number: Eighth Average pH of the solution: 6.34

Days of Operation: 3 Solution tank temperature: 47.0 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

(After drying for 4 hours)

Figure A.24: One of the Outer Surfaces of a Membrane before and after Eighth Harvest

Figure A.25: Filters from Eighth Harvest gave 8.5 g of CS Culture

116

Figure A.26: Data Collected from DA System from Seventh to Eighth Harvest

117

Harvest Number: Ninth Average pH of the solution: 6.27

Days of Operation: 4 Solution tank temperature: 47.7 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.27: One of the Outer Surfaces of a Membrane before and after Ninth Harvest

Figure A.28: Filters from Ninth Harvest gave 8.42 g of CS Culture

118

Figure A.29: Data Collected from DA System from Eighth to Ninth Harvest

119

Harvest Number: Tenth Average pH of the solution: 6.43

Days of Operation: 4 Solution tank temperature: 48.4 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.30: One of the Outer Surfaces of a Membrane before and after Tenth Harvest

Figure A.31: Filters from Tenth Harvest gave 6.94 g of CS Culture

120

Figure A.32: Data Collected from DA System from Ninth to Tenth Harvest

121

Harvest Number: Eleventh Average pH of the solution: 6.48

Days of Operation: 3 Solution tank temperature: 48.3 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.33: One of the Outer Surfaces of a Membrane before and after Eleventh

Harvest

Figure A.34: Filters from Eleventh Harvest gave 8.43 g of CS Culture

122

Figure A.35: Data Collected from DA System from Tenth to Eleventh Harvest

123

Test 2 Table A.2: Outer Membrane Coverage’s, a Day after Loading CS Culture

Side 1 Side 2 Membrane 1 20 60 Membrane 2 100 40 Membrane 3 90 50

Figure A.36: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day

Figure A.37: Filters from Initial Sample Loading gave 3.13 g of CS Culture 124

Harvest Number: First Average pH of the solution: 6.5

Days of Operation: 4 Solution tank temperature: 48.4 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.38: One of the Outer Surfaces of a Membrane before and after First Harvest

Figure A.39: Filters from First Harvest gave 6.51 g of CS Culture

125

Figure A.40: Data Collected from DA System from Initial Loading to First Harvest 126

Harvest Number: Second Average pH of the solution: 6.65

Days of Operation: 7 Solution tank temperature: 47.9 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.41: One of the Outer Surfaces of a Membrane before and after Second Harvest

Figure A.42: Filters from Second Harvest gave 6.75 g of CS Culture

127

Figure A.43: Data Collected from DA System from First to Second Harvest 128

Harvest Number: Third Average pH of the solution: 6.7

Days of Operation: 6 Solution tank temperature: 48.0 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.44: One of the Outer Surfaces of a Membrane before and after Third Harvest

Figure A.45: Filters from Third Harvest gave 11.7 g of CS Culture 129

Figure A.46: Data Collected from DA System from Second to Third Harvest 130

Harvest Number: Fourth Average pH of the solution: 6.7

Days of Operation: 5 Solution tank temperature: 47.6 °C

Harvesting Method: Hand scraping Solution flow rate: 0.8 GPM

Figure A.47: One of the Outer Surfaces of a Membrane before and after Fourth Harvest

Figure A.48: Filters from Fourth Harvest gave 14.28 g of CS Culture 131

Figure A.49: Data Collected from DA System from Third to Fourth Harvest

132

Test 3 Table A.3: Outer Membrane Coverage’s, a Day after Loading CS Culture

Side 1 Side 2 Membrane 1 65 55 Membrane 2 60 60 Membrane 3 99 99

Figure A.50: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day

Figure A.51: Filters from Initial Sample Loading gave 2.66 g of CS Culture 133

Harvest Number: First Average pH of the solution: 6.75

Days of Operation: 6 Solution tank temperature: 48.9 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.52: One of the Outer Surfaces of a Membrane before and after First Harvest

Figure A.53: Filters from First Harvest gave 6.93 g of CS Culture

134

Figure A.54: Data Collected from DA System from Initial Loading to First Harvest 135

Harvest Number: Second Average pH of the solution: 6.9

Days of Operation: 6 Solution tank temperature: 48.1 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.55: One of the Outer Surfaces of a Membrane before and after Second Harvest

Figure A.56: Filters from Second Harvest gave 6.81 g of CS Culture 136

Figure A.57: Data Collected from DA System from First to Second Harvest 137

Harvest Number: Third Average pH of the solution: 6.92

Days of Operation: 6 Solution tank temperature: 48.3 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.58: One of the Outer Surfaces of a Membrane before and after Third Harvest

Figure A.59: Filters from Third Harvest gave 5.17 g of CS Culture 138

Figure A.60: Data Collected from DA System from Second to Third Harvest 139

Harvest Number: Fourth Average pH of the solution: 6.92

Days of Operation: 6 Solution tank temperature: 49.6 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.61: One of the Outer Surfaces of a Membrane before and after Fourth Harvest

Figure A.62: Filters from Fourth Harvest gave 7.1 g of CS Culture 140

Figure A.63: Data Collected from DA System from Third to Fourth Harvest

141

Test 4 Table A.4: Outer Membrane Coverage’s, a Day after Loading CS Culture

Side 1 Side 2 Membrane 1 75 35 Membrane 2 60 90 Membrane 3 65 95

Figure A.64: Outer Surface CS culture Coverage for Membranes 1& 3 after a Day

Figure A.65: Filters from Initial Sample Loading gave 1.94 g of CS Culture 142

Harvest Number: First Average pH of the solution: 7.3

Days of Operation: 6 Solution tank temperature: 48.8 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.66: One of the Outer Surfaces of a Membrane before and after First Harvest

Figure A.67: Filters from First Harvest gave 8.1 g of CS Culture 143

Figure A.68: Data Collected from DA System from Initial Loading to First Harvest 144

Harvest Number: Second Average pH of the solution: 7.15

Days of Operation: 6 Solution tank temperature: 48.6 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.69: One of the Outer Surfaces of a Membrane before and after Second Harvest

Figure A.70: Filters from Second Harvest gave 7.24 g of CS Culture 145

Figure A.71: Data Collected from DA System from First to Second Harvest 146

Harvest Number: Third Average pH of the solution: 7.59

Days of Operation: 6 Solution tank temperature: 49.2 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.72: One of the Outer Surfaces of a Membrane before and after Third Harvest

Figure A.73: Filters from Third Harvest gave 8.72 g of CS Culture

147

Figure A.74: Data Collected from DA System from Second to Third Harvest 148

Harvest Number: Fourth Average pH of the solution: 7.5

Days of Operation: 6 Solution tank temperature: 49.2 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 GPM

Figure A.75: One of the Outer Surfaces of a Membrane before and after Fourth Harvest

Figure A.76: Filters from Fourth Harvest gave 8.92 g of CS Culture 149

Figure A.77: Data Collected from DA System from Third to Fourth Harvest

150

Test 5 Table A.5: Outer Membrane Coverage’s, a Day after Loading CS Culture

Side 1 Side 2 Membrane 1 95 60 Membrane 2 50 50 Membrane 3 90 35

Figure A.78: Outer Surface CS Culture Coverage for Membranes 1& 3 after a Day

Figure A.79: Filters from Initial Sample Loading gave 1.72 g of CS Culture 151

Harvest Number: First Average pH of the solution: 7.55

Days of Operation: 4 Solution tank temperature: 48.9 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 – 1.0 GPM

Figure A.80: One of the Outer Surfaces of a Membrane before and after First Harvest

Figure A.81: Filters from First Harvest gave 8.88 g of CS Culture 152

Figure A.82: Data Collected from DA System from Initial Loading to First Harvest 153

Harvest Number: Second Average pH of the solution: 7.48

Days of Operation: 5 Solution tank temperature: 48.4 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 – 1.0 GPM

Figure A.83: One of the Outer Surfaces of a Membrane before and after Second Harvest

Figure A.84: Filters from Second Harvest gave 11.67 g of CS Culture 154

Figure A.85: Data Collected from DA System from First to Second Harvest 155

Harvest Number: Third Average pH of the solution: 7.47

Days of Operation: 4 Solution tank temperature: 48.5 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 – 1.0 GPM

Figure A.86: One of the Outer Surfaces of a Membrane before and after Third Harvest

Figure A.87: Filters from Third Harvest gave 14.3 g of CS Culture 156

Figure A.88: Data Collected from DA System from Second to Third Harvest 157 Harvest Number: Fourth Average pH of the solution: 7.6

Days of Operation: 4 Solution tank temperature: 48.2 °C

Harvesting Method: Nozzle spray Solution flow rate: 0.8 – 1.0 GPM

Figure A.89: One of the Outer Surfaces of a Membrane before and after Fourth Harvest

Figure A.90: Filters from Fourth Harvest gave 17.69 g of CS Culture 158

Figure A.91: Data Collected from DA System from Third to Fourth Harvest

159 Appendix B

Table B.1: Light Measurements before the First Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 Row 1 59.29 64.05 61.91 63.81 62.62 60.95 58.1 65.95 Row 2 53.57 58.33 54.05 54.76 50.72 49.52 45.95 54.29 Row 3 50.48 52.38 50.95 50.48 50.95 50.95 55.95 56.91 Side 2 Row 1 60.48 60.95 61.19 61.91 60.95 62.14 59.52 60.95 Row 2 60.48 60.72 59.05 58.33 56.19 58.57 60 61.67 Row 3 63.1 61.91 60.95 61.91 61.91 63.1 62.86 61.91 Membrane 2 Side 1 Row 1 60.48 60.95 61.19 61.91 60.95 62.14 59.52 60.95 Row 2 60.48 60.72 59.09 58.33 56.19 58.57 60 61.67 Row 3 63.1 61.91 60.95 61.91 61.91 63.1 62.86 61.91 Side 2 Row 1 71.19 69.29 79.29 90.24 90.24 89.05 85.72 86.91 Row 2 73.1 85.72 66.67 83.1 84.29 84.76 82.86 83.1 Row 3 81.43 72.38 83.34 85.72 84.53 86.19 84.76 84.29 Membrane 3 Side 1 Row 1 87.62 87.86 75 76.19 73.33 75.24 69.53 70.48 Row 2 73.1 67.14 68.81 69.05 64.52 70 63.57 70.48 Row 3 69.29 67.62 67.86 69.05 67.38 75.24 74.52 82.38 Side 2 Row 1 70 60 63.81 65 66.19 59.53 63.81 66.19 Row 2 71.43 76.19 78.57 38.1 41.19 42.38 21.91 27.38 Row 3 20.24 10.95 18.81 24.52 22.62 24.05 28.57 24.76

160 Table B.2: Light Measurements after the First Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 Row 1 51.4 57.8 60.2 61.9 62.6 61 58.7 50.8 Row 2 54.8 61.3 63.4 66.1 66 64.7 64.3 56.2 Row 3 52.3 58.8 60.7 61.4 61.6 60.6 59 52.7 Side 2 Row 1 64.3 73.6 76.9 78.9 79 77.7 74.1 65.5 Row 2 67.3 74.9 77.7 79.1 79.1 77.7 74.2 65.1 Row 3 66.2 70.7 74.9 75.6 74.9 73.4 68.5 61.6 Membrane 2 Side1 Row1 61.3 68.6 69.7 72.8 72.8 70.9 67.6 59 Row 2 70.2 76.2 78.3 79.8 78.7 76.8 74.9 67.7 Row 3 69.4 75 77.5 78 77.3 75.3 74.2 66.2 Side 2 Row 1 61.8 70.9 75.1 77.2 77.8 77.3 74.6 67.8 Row 2 67.8 75.8 78.9 80.5 81.6 79.7 77.8 72.2 Row 3 73.5 79.5 84.3 85.5 84.3 85.2 79.9 77.3 Membrane 3 Side 1 Row 1 65.7 75.4 78.5 80.6 79.8 78.6 75.3 66.4 Row 2 69.4 78 82.5 82.8 82.3 80.6 78.4 70.9 Row 3 66.6 74.4 76 78 76.5 76.5 71.8 64.8 Side 2 Row 1 30.5 45.5 55.5 60.3 62.8 63 62.6 58.8 Row 2 31.8 47.6 57.5 62.4 64.8 65.4 64.5 63.3 Row 3 29.3 41.7 50.6 55.1 56.7 56.7 55.8 55.2

161 Table B.3: Light Measurements before the Second Test

162 Table B.4: Light Measurements after the Second Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 Row 1 51.3 57.8 59.7 61.4 62.2 60.6 57.9 49.8 Row 2 54.6 61.1 63.1 65.1 64.5 64.1 61.7 55.4 Row 3 52.3 58.3 60.4 60.9 60.9 59.5 57.4 52.9 Side 2 Row 1 64.5 74.3 77.3 79.1 79.3 77.7 74 64.9 Row 2 67.2 75.1 78 79.7 79.6 78.5 74.8 65.2 Row 3 66.8 71.4 74.6 76.3 75.6 73.4 68.9 62.7 Membrane 2 Side 1 Row1 63.3 71.2 75 75.7 76 74.8 70.4 60.5 Row 2 70.2 76.5 78.3 79.7 78.9 76.9 75 67.7 Row 3 69.4 75 77.5 78 77.3 75.3 74.2 66.2 Side 2 Row 1 60.6 68.7 71.9 75.7 76.7 75.4 74.7 66.1 Row 2 66.9 74.5 76.4 79.7 79.2 79.3 76.4 69.6 Row 3 70.1 76.3 82.3 83.8 84.7 84.2 80.5 75.5 Membrane 3 Side 1 Row 1 65.2 75.8 78.2 81.2 79.9 79 76.1 66 Row 2 69.5 78.4 82.7 83.4 83.7 81.2 78.1 70.4 Row 3 66.6 74.6 76 78.4 77.4 76.7 72.3 65.6 Side 2 Row 1 30.8 44.9 54.3 59.9 61.9 62.4 61.7 58.9 Row 2 31.3 46.8 56.3 61.8 63.8 64.5 63.4 62.5 Row 3 28.9 41.7 50.1 55 56.8 56.8 55.9 55.6

163 Table B.5: Light Measurements before the Third Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 Row 1 28.42 31.66 32.06 32.38 32.2 32.6 31.32 29.7 Row 2 28.75 30.32 31.64 31.14 31.63 31.8 30.74 29.22 Row 3 24.8 27.5 28 29.22 27.88 27.8 25.79 22.81 Side 2 Row 1 28.9 28.15 29.3 29.88 29.8 29.22 28 28.53 Row 2 31.3 29.06 29.99 30.32 30.16 29.6 27.84 28.2 Row 3 34 30.64 31.6 32.13 31.8 31.7 30.07 31.54 Membrane 2 Side 1 Row 1 28.2 28 28.8 28.65 28.43 28.4 26.8 26.6 Row 2 30.9 29.96 31.25 29.79 31.2 31.3 31.1 31.9 Row 3 30.6 30.96 32.66 33.5 33.02 32 31.9 32.85 Side 2 Row 1 27.3 28.6 27.8 28.55 28.74 27.3 28.3 29.3 Row 2 28.4 27.96 28.86 30 28.38 30.2 29.6 29.4 Row 3 25.7 28 32.5 32.8 33.1 32.3 31 27.3 Membrane 3 Side 1 Row 1 28.3 26.6 28.2 27.8 28 29.9 28.7 29.9 Row 2 27.6 27.6 27.6 27.3 28.2 28 28.9 29.5 Row 3 29.2 33.3 34 32.3 28.1 29.9 28.9 27 Side 2 Row 1 30.89 31.37 32.05 31.64 30.8 31.4 32.6 25.7 Row 2 31.57 30.9 31.16 32.28 32.3 32.78 32.25 23.9 Row 3 27 27.6 28.24 28.8 27.3 26.9 24.7 15.6

164 Table B.6: Light Measurements after the Third Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 Row 1 27.93 30.2 31 30.86 31.2 31 30.22 28.4 Row 2 27.4 29.5 32.5 32.73 31.64 32.3 29.3 29.95 Row 3 24.9 28.9 29.4 29.4 29.5 26.7 25.3 22.1 Side 2 Row 1 31.3 31.31 29.6 29.5 29.53 29.3 27.8 29.3 Row 2 30.93 30.9 30.77 30.7 30.52 30.6 27.6 26.2 Row 3 35 30.02 31.96 32.3 31.33 30.3 28.9 29.3 Membrane 2 Side 1 Row 1 30.48 29.61 28.3 28.02 28.48 28.92 26.8 26.76 Row 2 30.97 30.52 31.36 29.72 31.46 31.8 31.91 29.18 Row 3 30.38 30.7 32.54 33.28 33.63 32.7 31.62 32.17 Side 2 Row 1 28.34 28.94 28.92 29.54 29.58 27.29 28.93 29.37 Row 2 26.85 29.2 28.49 30.47 29.17 30.63 30.96 29.55 Row 3 24.9 28.2 32.7 32.65 33.3 32.65 31.16 27.9 Membrane 3 Side 1 Row 1 26.2 26.3 30.1 30.69 29.87 31.39 30.19 30.62 Row 2 27.3 27.6 27.5 27.98 28.97 29.19 29.84 30.11 Row 3 29 33.2 33.6 32.66 29.14 29.13 29.3 27.84 Side 2 Row 1 23.89 32.56 30.62 31.32 30.88 31.27 32.17 31.54 Row 2 23.3 32.62 32.43 32.47 32.72 31.14 30.27 30.64 Row 3 14.89 24.35 26.28 28.05 28.56 28.18 29.02 28.25

165 Table B.7: Light Measurements before the Fourth Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 28.14 30.36 31.23 31.41 31.09 31.1 29.89 28.26 27.2 29.2 29.93 30.88 30.73 30.15 29.8 29.78 25.6 27.93 28.5 27.56 28.3 28.17 25.5 23.17 Side 2 30.06 31.07 29.54 29.74 29.9 30.77 29.95 29.38 29.42 30.72 30.7 30.22 31.26 29.28 28.8 28.09 35.73 30.75 31.12 31.62 30.02 28.02 28.27 26.43 Membrane 2 Side 1 28.53 28.13 29.18 29.39 26.33 27.9 27.35 28.38 31.3 29.82 30.48 30.02 30.43 30.9 30.62 31.35 30.12 30.87 30.62 33.61 34.83 32.43 31.38 32.4 Side 2 27.85 29.87 29.36 30.18 30.99 29.75 30.37 28.04 29.83 29.31 30.97 30.63 29.82 30.61 28 29.11 24.52 29.62 29.63 32.92 32.79 29.82 28.98 26.74 Membrane 3 Side 1 28.2 26.83 30.19 29.9 28.3 29.85 28.18 27.88 27.71 28.43 28.9 28.6 29.8 28.12 28.42 28.86 27.6 28.5 30.54 32.2 32.54 30.56 30.01 27.75 Side 2 25.56 32.09 31.05 29.56 30.21 31.18 31.5 31.81 23.36 30.93 30.41 31.63 30.9 30.62 30.38 30.07 14.75 21.17 26.3 28.17 29.96 30.4 28.93 28.11

166 Table B.8: Light Measurements after the Fourth Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 29.56 31.43 31.89 32.06 32 31.91 28.99 28.6 28 30.22 31 31.47 31.83 30.1 28.9 28.79 24.4 27.9 27.99 28.7 28.8 27.3 25.3 22.14 Side 2 29.73 30.17 29.45 30.04 30.43 29.67 29.76 29.4 29.33 29.64 29.69 29.89 30.03 29.56 27.64 29.98 34.46 31.67 31.13 31.86 32 30.23 29.32 28.99 Membrane 2 Side 1 27.86 29 28.43 28.45 28.9 27.7 27.4 29.1 32.4 30.02 30.65 30.9 30.76 30.34 29.45 30.6 29.76 29.87 30.66 33.67 34.3 33.8 32.78 31.8 Side 2 28.2 27.89 28.7 28.75 29 29.73 28.76 29.6 30.49 28.9 29.86 28.11 30.16 30.04 29.9 30.3 25.3 28.67 30.23 32.8 33.1 32.74 29.76 28.5 Membrane 3 Side 1 29.6 28.5 29.5 30.04 29.58 28.2 28.4 30.9 27.9 27.99 28.7 29.21 30.34 27.3 29.11 31.1 29.8 32.98 33.2 33.16 32.25 30.5 28.79 30.77 Side 2 24.6 32.12 31.6 30.05 31.3 30.8 30.98 32.76 22.89 32.3 32.9 32.3 32.67 31.8 31 31.4 13.98 22.3 27 27.89 28.9 29.2 28.12 27.45

167 Table B.9: Light Measurements before the Fifth Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 65.06 70.17 72.6 73.5 73.7 72.9 72.8 71.3 62.9 74.42 76.55 77.3 77.4 77.3 77.85 74.74 64.1 68.58 70.3 70.3 70.3 70.2 69.53 64.87 Side 2 62.9 70.11 73.8 75.2 75.8 74.65 70.4 63.1 68.3 73.8 77.3 80.2 80.83 80.3 75.6 68 62.6 69.5 73.5 76 76.5 74.2 71.2 62.6 Membrane 2 Side 1 65 72.8 74.6 75.5 76.7 74.3 71.9 63.5 71 77.88 78.7 80.3 80.6 78.8 78 71.2 69.7 74.09 77.5 79.4 78.9 78.5 75.2 66.8 Side 2 64.07 71.63 74.37 74.8 75.57 74.58 72 65.15 71 77.41 80.62 81.31 81.47 79.11 77.3 72.75 80.12 80.01 80.24 82.36 81.8 80.5 80.6 74.2 Membrane 3 Side 1 66.19 76.4 79.9 80.8 81.7 79.4 78.5 67.8 71.24 74.5 80.5 83.41 82.7 81.6 78.1 74.8 68.34 74.1 79.35 80.6 79.95 79.3 76 71.7 Side 2 45.71 68.4 80.46 80.43 81 80.1 80.18 77.55 44.86 68.35 80.69 84.6 84.62 84.9 84.98 83.57 37.17 54.41 68.2 71.49 74.3 73.68 73 72.71

168 Table B.10: Light Measurements after the Fifth Test

Membrane 1 1 2 3 4 5 6 7 8 Side 1 66.26 71.37 73.8 74.7 74.9 74.1 74 72.5 64.1 75.62 77.75 78.5 78.6 78.5 79.05 75.94 65.3 69.78 71.5 71.5 71.5 71.4 70.73 66.07 Side 2 65 69.21 74.9 75.3 74.9 73.75 69.5 62.2 67.4 74.9 78.4 79.3 79.93 79.4 74.7 67.1 63.7 69.6 74.6 77.1 78.6 73.3 72.3 61.7 Membrane 2 Side 1 64.7 72.5 75.3 76.2 75.4 74 71.6 63.2 70.7 75.7 78.4 79 80.3 78.5 76.7 70.9 67.4 73.79 77.2 78.1 77.6 77.2 74.9 66.5 Side 2 62.57 70.13 72.87 74.3 74.07 73.08 70.5 63.65 69.5 75.91 79.12 79.81 79.97 78.61 75.8 71.25 78.62 82.51 82.74 83.86 83.8 83 82.1 75.7 Membrane 3 Side 1 65.29 75.5 78.6 80.9 79.8 78.5 77.6 66.9 70.34 78.6 81.6 82.5 82.8 81.7 80.2 71.9 67.44 73.2 78.45 79.7 78.9 78.4 75.1 70.8 Side 2 46.01 68.7 80.76 80.73 81.3 80.4 80.48 77.85 45.16 68.65 80.99 84.9 85.92 86.2 85.28 83.87 37.47 54.71 68.5 71.79 74.6 73.98 73.3 73.01