The Anaerobic Digestion of the Microalgae Species Scenedesmus Obliquus for the Production and Subsequent Concentration o...

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Title:
The Anaerobic Digestion of the Microalgae Species Scenedesmus Obliquus for the Production and Subsequent Concentration of Methane from the Biogas
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1 online resource (93 p.)
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english
Creator:
Morton, Emily L
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Agricultural and Biological Engineering
Committee Chair:
Pullammanappallil, P C
Committee Members:
Phlips, Edward J
Welt, Bruce Ari
Diltz, Robert A

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Subjects / Keywords:
anaerobic -- biogas -- digestion -- methane -- microalgae -- scenedesmus
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
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Agricultural and Biological Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
The rising cost of oil and increased awareness of pollution caused by the use of fossil fuels has created a need to research alternative energy sources. This has become a very significant concern for United State’s military operations abroad. Envoys delivering fuel to deployed bases, have consistently been a vulnerable target for attack. Decreasing the frequency of transporting fuel will offer a reduced the risk of danger to service members. Unique solutions for alternative fuel sources need to be explored with the deployed military’s needs in mind: transportation,ease of use, setup and efficiency. These challenges have lead to the exploration of anaerobic digestion as a solution. In this process, bacteria metabolize organic materials to produces methane gas, which can be used as a replacement for fossil fuels in generators. Anaerobic digestion is a viable option for alternative energy production in remote locations because of its ease of use and versatility of required feedstocks. The bacterial cultures used in these experiments demonstrated the ability to produce a methane gas stream as concentrated at 66% from a combination of algae and molasses as a food source.  This project used the microalgae species Scenedesmus obliquus because it was not a human food source, was quick growing, and has a high nutrient content, and white paper is readily available  These results indicate that an integrated system of algae growth and anaerobic digestion can be a successful option for producing energy in remote locations.
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In the series University of Florida Digital Collections.
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Includes vita.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
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by Emily L Morton.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Pullammanappallil, P C.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 THE ANAEROBIC DIGESTION OF THE MICROALGAE SPECIES SCENEDESMUS OBLIQUUS FOR THE PRODUCTION AND SUBSEQUENT CONCENTRATION OF METHANE FROM THE BIOGAS By EMILY L. MORTON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSI TY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Emily L. Morton

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3 For my Grandmother, Hilda Brown

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4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Pratap Pullammanappallil, for his academic guidance through the process of pursuing a m I would also like to thank Drs. Ed Phlips, Bruce Welt, and Robert Diltz for serving on my committee and Dr. Keith Kozlowski for providing valuable insight and recommendations that were vital in my academic success. My research team, Eila Burr, Kayla Garner, Russ Hallett, Karen Farrington I say thank you for all of the extraordinary help you have been collecting data, setti ng up eq uipment and experiments Without you, this would not have been possible. My parents, Karen and Ray Morton, thank you for all of the encouragement, support, and humor that have kept me on track. My friends, Evan Ged and Melissa Myers, you are inspirations and I hope I have made you proud.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Justification and Rationale ................................ ................................ ...................... 14 Current Sources of Biomass ................................ ................................ ................... 14 Agricultural Residues and Waste ................................ ................................ ..... 15 Non Food Energy Crops ................................ ................................ ................... 15 Aquatic Species ................................ ................................ ................................ 16 Current Forms and Processes for the Production of Biofuels ................................ 16 Et hanol ................................ ................................ ................................ ............. 16 Biodiesel ................................ ................................ ................................ ........... 17 Hydrogen ................................ ................................ ................................ .......... 18 Combustion ................................ ................................ ................................ ...... 19 Gasification (Synthesis Gas) ................................ ................................ ............ 19 Pyrolysis ................................ ................................ ................................ ........... 20 Methane ................................ ................................ ................................ ........... 20 Carbon Recovery ................................ ................................ ............................. 21 Summary ................................ ................................ ................................ ................ 22 2 ALGAE GROWTH AND HARVESTING ................................ ................................ .. 27 Objective ................................ ................................ ................................ ................. 28 Materials And Methods ................................ ................................ ........................... 28 Algae Species ................................ ................................ ................................ .. 28 Media Recipes ................................ ................................ ................................ .. 29 Algae Growth Reactors ................................ ................................ .................... 29 Algae Biomass Concentration Determination ................................ ................... 30 Algae Energy Content ................................ ................................ ...................... 30 Harvesting ................................ ................................ ................................ ............... 31 Base Flocculating ................................ ................................ ............................. 31 Filtration ................................ ................................ ................................ ............ 31 Electro Flocculating ................................ ................................ .......................... 32 Centrifuging ................................ ................................ ................................ ...... 32 Materials And Methods ................................ ................................ ........................... 33 Base Flocculating ................................ ................................ ............................. 33

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6 Centrifuge ................................ ................................ ................................ ......... 33 Results and Discussion ................................ ................................ ........................... 34 Algae Growth ................................ ................................ ................................ .... 34 Algae Energy Content ................................ ................................ ...................... 34 Harvesting Efficiencies ................................ ................................ ..................... 34 Base flocculating efficiency ................................ ................................ ........ 34 Centrifuge separation efficiency ................................ ................................ 34 Cost ................................ ................................ ................................ .................. 35 Base flocculating cost ................................ ................................ ................ 35 Centrifuge separation cost ................................ ................................ ......... 35 Summary ................................ ................................ ................................ ................ 36 3 ANAEROBIC DIGESTION ................................ ................................ ...................... 46 Aquatic feedstocks ................................ ................................ ................................ .. 46 Objective ................................ ................................ ................................ ................. 48 Materials and Methods ................................ ................................ ............................ 48 Reactors ................................ ................................ ................................ ........... 48 Bench scale reactors ................................ ................................ ................. 48 Pilot scale reactor ................................ ................................ ...................... 49 Microbial Inoculum ................................ ................................ ........................... 50 Feeding ................................ ................................ ................................ ............ 50 Failure ................................ ................................ ................................ ........ 50 Standard feeding ................................ ................................ ........................ 51 Sampling ................................ ................................ ................................ .......... 51 Flow rate ................................ ................................ ................................ .... 51 Gas concentrations ................................ ................................ .................... 52 Liquid characteristics ................................ ................................ ................. 52 Results and Discussion ................................ ................................ ........................... 53 Gas Production ................................ ................................ ................................ 53 Gas Concentration ................................ ................................ ........................... 53 Liquid Characteristics ................................ ................................ ....................... 53 Ysi probe ................................ ................................ ................................ .... 53 HPLC ................................ ................................ ................................ ......... 54 Summary ................................ ................................ ................................ ................ 54 4 CARBON RECOVERY ................................ ................................ ........................... 77 Algae ................................ ................................ ................................ ....................... 77 Objective ................................ ................................ ................................ ................. 78 Materials and Methods ................................ ................................ ............................ 78 Measurements ................................ ................................ ................................ ........ 79 Optical density ................................ ................................ ................................ .. 79 Gas composition ................................ ................................ ............................... 79 Results and Discussion ................................ ................................ ........................... 80 Summary ................................ ................................ ................................ ................ 80

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7 LIST OF REFERENCES ................................ ................................ ............................... 86 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 93

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8 LIST OF TABLES Table page 1 1 Ethanol production yields of various feedstocks ................................ ................. 25 1 2 Comparison of bi omass sources for the production of biodiesel ........................ 26 2 1 Chemical composition of P IV metal solution for the Waris Media. ..................... 38 3 1 Es timated methane potential ................................ ................................ .............. 55 3 2 Specific methane yield for organic compounds in Chlorella vulgaris .................. 56 3 3 Gross composition of microalgae species and theoretical methane potential during anaerobic digestion of the total biomass ................................ .................. 57 3 4 Gross composition of microalgae species (Becker, 2004) and methane potential yield a naerobic digestion of the total biomass ................................ ...... 58

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9 LIST OF FIGURES Figure page 1 1 Biomass Resources of the United States. National Renewable Energy Labo ratory. ................................ ................................ ................................ ......... 24 2 1 Aquatic species of algae at various experimental culturing stages. ................... 39 2 2 Base flocculating process flow diagr am ................................ .............................. 40 2 3 Base flocculating technique ................................ ................................ ............... 41 2 4 Growth studies of S. obliquus over 1 month ................................ ....................... 42 2 5 Centrifuge process flow diagram ................................ ................................ ........ 43 2 6 Evodos water color comparison. (Photo courtesy of Emily Morton). ................... 44 2 7 Algae cellular concentration and standard deviation ................................ .......... 45 3 1 Pathway for the conversion of organic biomass to biogas through anaerobic digestion processes. ................................ ................................ ........................... 61 3 2 Process Flow diagram. ................................ ................................ ....................... 62 3 3 Bench scale anaerobic digestion flasks successfully producing biogas. (Photo courtesy of Emily Morton) ................................ ................................ ....... 63 3 4 Pilot scale reactor (Photo courtesy of Emily Morton) ................................ .......... 64 3 5 Manometer (photo courtesy of Emily Morton). ................................ .................... 65 3 6 Feeding schedule of molasses and algae paste ................................ ................. 66 3 7 Gas concentrations and cumulative methane production over time. .................. 67 3 8 Dissolved oxygen concentration and pH of digester liquid over time. ................. 68 3 9 Carbon Dioxide gas concentration and pH of digester liquid. ............................. 69 3 1 0 Liquid samples over time, HPLC retention time of 8.5 minutes; fructose concentration. ................................ ................................ ................................ ..... 70 3 11 Liquid samples over time, HPLC retention time of 10.0 minutes; glucose concentration. ................................ ................................ ................................ ..... 71 3 12 Liquid samples over time, HPLC retention time of 13.2 minutes; lactic acid concentration. ................................ ................................ ................................ ..... 72

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10 3 13 Liquid samples over time, HPLC retention time of 15.4 minutes; glycerol concentration. ................................ ................................ ................................ ..... 73 3 14 Liquid samples over time, HPLC retention time of 19.36 minut es; acetate concentration. ................................ ................................ ................................ ..... 74 3 15 Liquid samples over time, HPLC acetate mg/L concentration. ........................... 75 3 16 Liquid samples over time HPLC retention time of 22.8 minutes; ethanol concentration. ................................ ................................ ................................ ..... 76 4 1 S ide arm flasks with S. obliquus (photo courtesy of Karen Farrington) ............ 81 4 2 GC results from flask 1: Biogas. ................................ ................................ ......... 82 4 2 GC results from flask 2: CO 2 /Air mix. ................................ ................................ 83 4 3 Changing pH values for each flask over time. ................................ .................... 84 4 4 Changing Optical density of the flasks over time. ................................ ............... 85

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11 LIST OF ABBREVIATIONS AD anaerobic digestion AFB Air Force Base o C degrees Celsius C/N ratio of carbon to nitrogen CCS carbon capture and storage d day DO dissolved oxygen EOR enhanced oil recovery FID Flame Ionization Detector GC gas chromatrograpy HPLC high pressure liquid chromatography LB Luria Bertani NREL National Renewable Energy Laboratory pH measurement of acidity of a solution TDS total dissolved solids UV/Vis ultraviolet/visible VS volatile solids

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfi llment of the Requirements for the Degree of Master of Science THE ANAEROBIC DIGESTION OF THE MICROALGAE SPECIES SCENEDESMUS OBLIQUUS FOR THE PRODUCTION AND SUBSEQUENT CONCENTRATION OF METHANE FROM THE BIOGAS By Emily L. Morton August 2013 Chair : Pra tap Pullammanappallil Major: Agricultural and Biological Engineering The rising cost of oil and increased awareness of pollution caused by the use of fossil fuels has created a need to research alternative energy sources. This has become a very significa Envoys delivering fuel to deployed bases, have consistently been a vulnerable target for attack. D ecreasing the frequency of transport ing fuel will offer a reduce d the risk of danger to service memb ers. Unique solutions for alternative fuel source s need to be explored with These challenges have lead to the exploration of anaerobic digestion as a solution. In th is process, bacteria metabolize organic materials to produces methane gas, which can be used as a replacement for fossil fuels in generators Anaerobic digestion is a viable option for alternative energy production in remote locations because of its ease o f use and versatility of required feedstocks. The bacterial cultures used in these experiments demonstrated the ability to produce methane gas from a combination of algae and molasses as a food source. This project used the microalgae species Scenedesmus o bliquus because it was not a

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13 human food source, was quick growing, and has a high nutrient content These results indicate that an integrated system of algae growth and anaerobic digestion can be a successful option for producing energy in remote locatio ns.

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14 CHAPTER 1 INTRODUCTION Justification and Rationale Volatile political relationships in the Middle East have once again brought the importance of alternative energy sources to the forefront of the United States concerns. Petroleum base d fuels are a finite global resource with an ever increasing demand. This leads to a critical need for the United States to become more self sustaining with energy generation and processing. a requirement into action. This will shift the use of coal and petroleum based energy systems into more sustainable and renewable options (Exec. Order No. 13,423, 2007). These energy systems have to have versatile capabilities that can be implemented in remote and severe environments. Another concern is the waste streams from these deployed bases; humans waste, food scraps, and miscellaneous building material. The t ransportation and removal of waste from these bases requires a large percentage of the fuel supply. The option to process these wastes, while reducing energy consumption has led researchers to waste processing systems for possible sources of feedstock for energy generation. Current Sources of Biomass Biomass conversion has a high potential to help reduce the dependence on fossil fuels, in a sustainable way Research is trending in the direction of complex integrated systems, where a high percentage of poten t ial energy could be produced. Alternative to other renewable energy options, biomass is versatile; it can be used in direct

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15 combustion, gasification, or biological conversion. The United States has a wide range of climate zones which offer an opportunity for growing diverse biomass feedstocks. This variety offers the opportunity to use non food or feed crops. Figure 1 1 shows the current biomass resources of the United States reported by the National Renewable Energy Laboratory (NREL). This figure indicate s the biomass potential by counties of the United States including the following feedstocks: agricultural and forestry residues, urban wood waste, domestic wastewater treatment, and animal manure. It also highlights the potential of the different climate z ones to be equally productive. Biologically based energy currently contributes to only a small portion of global energy use. The most notable biomass sources are: animal waste, municipal solid waste, agricultural residues, food processing waste, and non food crops, including trees, perennial grasses, and aquatic plants (Demirbas, Ozturk, & Demirbas, 2006) Agricultural Residues and Waste Agricultural residues and wastes consist of a broad variety of harvested and processed biomass from both food and n on food sources. These wastes include corn stover, wood chips, sugarcane bagasse, and beet tailings. In 2005 the United States produc ed 62.4 mi llion tons of forestry residues a nd 34 m illion tons of urban wood waste (National Renewable Energy Laboratory, 2 005) These waste materials can be used in the production of biofuels instead of just being discarded. Non Food Energy Crops Non food crops include perennial grasses and trees. Perennial grasses like switch grass are fast growing, offering more than one harvest per growing season. These grasses do not need the deep ploug h ing that food crops require which reduces energy inputs. The woody biomasses, like poplar trees, are more slowly growing

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16 resources and can take years or decades to mature before harvestin g Tre e s are more suitable for growing in northern climates because they do not require the solar intensity of some of the grasses. Aquatic Species Aquatic plant species include micro and macro algae from both marine and freshwater environments. These a lgae species have become a nuisance in some areas where nitrogen and phosphorous runoff from farmland can cause algae blooms. This excess biomass causes eutrophication of water systems which can be deadly to other plants and animals (Migliore, et al., 2012) This biomass resource needs more research into harvesting algae in the natural environment. In addition to the option to use nuisance algae, it can be grown and harvested in controlled environments. Algae are a viable bioma ss choice because of the fast growth rates small land area requirements. Microalgae, unlike many other resources, can be grown in compressed spaces. As long as there is sufficient light intensity and cell mixing, the algae will grow. C urrent Forms and Pro cesses for the Production of Biofuels Ethanol Ethanol has become a widely known and highly disputed biofuel. The controversy comes from the production of first generation ethanol, which is primarily derived from corn or other food crops. E thanol has a bad reputation because of the where there is concern over availability of adequate farm land for food supplies with the growing need for land for fuel feedstocks Second generation feedstocks have moved toward lignocellulosic materia ls, including; woody materials, agricultural residues and fast growing grasses. These lignocellulosic materials have

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17 acc essible fermentable sugars but are not considered a food or feed crop (Gonzlez Garca, Teresa Moreira and Feijoo 2012 ). Table 1 1 com pares the e thanol yields of feedstocks. The highest yielding crops are corn and wheat with a yield of 0.370 and 0.355 L kg 1 and the lowest yielding crops are apples and grapes with a yield of 0.64 and 0.63 L kg 1 respectively. Biological ethanol producti on is the chemical process of converting sugars to alcohols using microbes Saccharomyces cer e visiae ethanol tolerant yeast, is the primary microbe used for this process because it prefers fermentation to cellular respiration even in the presence of oxyge n, which allows the process to be completed in aerobic conditions. Equation 1 1 shows the stoichiometry of the glucose conversion to ethanol. C 6 H 12 O 6 2 C 2 H 5 OH + 2 CO 2 (1 1) onversion of locally sourced crops to ethanol (Renewable Fuels Association, 2013) In 2012, an estimated 462 million barrels of exported oil was replaced with this ethanol. Biodiesel Biodiesel is diesel fuel which has been produced using biologically ba sed oils. These oils can include animal fat, nut and tree oils, and waste cooking oils. The triglycerides are converted to methyl esters using a base catalyst and an alcohol, usually sodium hydroxide and methanol respectively as seen in equation 1 2

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18 (1 2) Biodiesel offers some positive qualities in comparison to petroleum based diesel products. It has a higher flashpoint allowing for safer transportation and storage (Brown, 2003) Also, t he combustion of biodiesel is more compl ete, reducing carbon monoxide from the atmosphere. Figure 1 2 is the chemical conversion process of a raw oil to biodiesel. Table 1 2 compares different feedstocks for the production of biodies e l and ation needs. Hydrogen There are g reen algae and cyanobacteria species that have the ability to produce molecular hydrogen in controlled conditions This process is called biophotolysis, where water is oxidized and a transfer of electrons to the [Fe] hydr ogenase leads to the synthesis of H 2 This can occur naturally but does not produce large quantities in the environment because oxygen is an inhibitor. This becomes a difficult barrier to overcome because of the generation o f oxygen during photosynthesis ( Melis, 2002) The conversion of glucose to hydrogen gas and carbon dioxide can be seen in equation 1 3 C 6 H 12 O 6 + 6 H 2 O 12 H 2 + 6 CO 2 (1 3)

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19 Combustion Combustion is the process in which biomass is burned between 800 1000 C in the presence of a ir, and the chemical energy stored in the biomass is transferred to heat and mechanical power through various process options; boilers, steam turbines, generators, stoves. This process is most efficient with biomass with <50% moisture content (McKendry, 20 02) Gasification (Synthesis Gas) Gasification is a chemical process where both organic and inorganic carbonaceous materials are converted to hydrogen, carbon dioxide, and carbon monoxide. This chemical reaction is achieved at high temperatures (>700C) wi thout inducing combustion. The mixture of gas produced is called synthesis gas, or syngas. This syngas can be used directly in gas engines or refined for hydrogen and a host of other energetic hydrocarbons (McKendry, 2002). Ideally, the biomass used in the gasification process needs to have a moisture content below 15%, which would add a considerable amount of drying to the process if a biomass source like algae was chosen (Kadam, 2002). The chemical process to convert material to syngas consists of four di fferent stages: d ehydration; water is driven off the material (100 C ) pyrolysis; volatiles are released leaving char, combustion; volatiles react with oxygen to produce carbon dioxide, gasification; the char reacts with carbon and steam to produce the syn gas. There is also a reversible water gas shift reaction which will transfer oxygen these steps can be seen in equations 1 4 through 1 9 :

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20 Carbon oxygen reaction: C + O 2 CO (1 4) Boudouard reaction: C + CO 2 2 CO (1 5) Carbon water reaction: C + H 2 O H 2 + CO (1 6) Hydrogenation reaction: C + 2 H 2 CH 4 (1 7) Gas phase reactions Water gas shift reaction: CO + H 2 O H 2 + CO 2 (1 8) Methanation: CO + 3 H 2 CH 4 + H 2 O (1 9) Pyrolysis Pyroysi s is the thermochemical process where biomass is heated to 500 C in the absence of oxygen and converted to bio oil or bio crude. This bio oil can be used directly in engines but it has a poor thermal stability and can be very corrosive. The use of microalg ae in pyrolsis is based on the chemical properties of the cells. A high lipid cell will offer a higher yield of bio oil. This need for lipid accumulation is a reoccurring theme in algae biofuel research. Miao et al. were able to produce a bio oil compara ble to fossil fuel with a high heating value of 41 MJ kg 1 and a density of 0.92 kg L 1 (2004). Methane Methane gas, most widely known as natu ral gas, is currently used in roughly 50% of American households (U.S. Energy Informa tion Administration, 2012) While it is a popular energy source, it is also a damaging greenhouse gas and is responsible for trapping radiation within the atmosphere. In the combustion of methane gas for energy, CO 2 is produced, but it is 20 times less harmful in the atmosphere than methane over 100 years (Environmental Protection Agency, 2010) If methane is being produced

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21 naturally, it is beneficial to capture it and use it as a fuel source, reducing methane as a greenhouse gas (2010) Biologically produced methane gas is produced through microbial anaerobic digestion. Some examples of naturally occurring anaerobic digestion process include; swamps, landfills, and wastes from large herds of domesticated animals. These are t he started collecting methane gas produced from the decomposing trash and are using it as fuel to run equipment, or even to sell back to municipalities (Themelis & Ulloa, 2007) This process takes trash and produces energy while removing tons of harmful methane gas from the atmosphere. Methane gas can also be a valuable product instead of a polluting waste stream. Engineering controlled anaero bic digestion systems to produce high concentrations of methane gas has become a global research topic. A thermophilic process, running between 4 5 60 C will kill pathogens so the solids can be land applied as fertilizer. Anaerobic digestion does not requir e aeration and constant mixing like aerobic digestion. The microbes used in anaerobic digestion do not create an excess of biomass as a result of their metabolism either, which would cause a large processing waste stream. Carbon Recovery The United States is currently responsible for 22% of all global CO 2 production caused by humans. These emissions include transportation, industrial, and residential producers which account for 5.7 Gtons of gas a year (Kadam, 2002) Coal burning power plants present an int eresting opportunity to capture the otherwise polluting gasses, while utilizing the carbon for more energy. Plants in the U.S. are capturing only

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22 2 for use in food products or oil recovery. These processes do not sequester t he carbon, because it is again released into the atmosphere. Kadam modeled a process where 2500 acres of cultivated algae can process 210 ktons of CO 2 a year. Growing algae with the addition of inorganic carbon from waste CO 2 presents an opportunity to rec ycle carbon. Summary Using anaerobic digestion as an alternative to produce energy is highly advantageous compared with other options. Unlike ethanol production, which requires feedstocks to undergo costly and sometimes toxic pretreatment steps, anaerobic digestion on a large scale would not require these additions. The production of biodiesl would not be practical because of the amounts of raw oil required, and the use of hazardous methanol. If waste grease and vegetable oil was chosen, substantial pretre atment would be required for a functional process, as well as a large waste stream. The thermal conversion technologies are best suited for woody biomass which requires years to grow, leaving a deployed base to rely on locally grown materials. The ability to use high moisture content feedstocks reduces upfront cost of the process. This is a valuable option for deployed bases by reducing equipment and chemicals that would have to be transported. Anaerobic digestion is also a versatile process in which the m icrobes can adapt to a wide variety of feedstocks and loading rates. This is another key point for the deployable bases when considering waste disposal and conversion to fuel. When considering the options for feedstocks for the anaerobic digestion proces s, the main consideration was the amount of land area the biomass would require for growth, the amount of water, the production rate of the biomass, and heavy equipment

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23 needed for planting and harvesting the crops. The microalgae offer the base the opportu nity to utilize biomass at a very fast rate with minimum waste material, equipment.

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24 Figure 1 1 Biomass Resources of the United States. National Renewable Energy Laboratory.

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25 Table 1 1 Ethanol production yields of various feedstocks Biomass feedstocks Ethanol yield L kg 1 dry biomass Corn stover 0.47 Rice straw 0.46 Wheat straw 0.43 Forest residue 0.34 Saw dust 0.42 Apples 0.064 Barley 0.33 Cellulose 0.259 Corn 0.355 0.37 Grapes 0.063 Jerusalem artichoke 0.083 Molasses 0.28 0.288 Oats 0.265 Potatoes 0.096 Rice (rough) 0.332 Rye 0.329 Sorghum (sweet) 0.044 0.86 Sugar beets 0.088 Sugarcane 0.16 0.187 Sweet Potatoes 0.125 0.143 (Chandra, Takeuchi, & Hasegawa, 2012) & (Brown, 2003)

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26 Ta ble 1 2 Comparison of biomass sources for the production of biodiesel Crop Oil yield (L acre 1 ) Land area needed (M acre) a Percent of existing cropping area a Corn 70 623 846 Soybeans 180 240 326 Canola 482 90 122 Jatropha 766 57 77 Coconut 1088 40 5 4 Oil palm 2408 18 24 Microalgae (70% oil by wt.) 55401 0.8 1.1 Microalgae (30% oil by wt.) 23755 1.8 2.5 a For meeting 50% of the transportation needs in the United States (Christi, 2007).

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27 CHAPTER 2 ALGAE GROWTH AND HARVESTING Macro and micro species of algae have been frequently mentioned in biofuel research because of the potential for fast growth and valuable biomass characteristics. Microalgae are photosynthetic and mostly unicellular organisms that convert carbon dioxide t o biomass through metabolic processes. There are about 8,000 species of green algae, living in both marine and freshwater environments (Packer, 2009) For the remainder of this discussion, green microalgae will be considered. Microalgae offer considerable advantages for use in biofuels as a feedstock. This aquatically growing feedstock allows for an easy introduction of nutrients when compared to soil. The transfer of algal biomass to processing facilities can be achieved with a pump and pipes, instead of t ractor trailers that other biomass sources may require. Reproduction is achieved by cell fission instead of sexual cycles of terrestrial plant. Due to its rapid growth rate, harvesting can be adapted to meet production rates on demand, creating a reduced n eed to store large quantities of biomass. Algal growth is facilitated by an average of 1.2x10 17 0.1 0.5% of solar energy is being captured by biological systems from photosynthesis, leaving an abundance o f uncollected solar potential (Packer, 2009) The challenges of using algae as a biomass feedstock have become the focus of recent research efforts. Algae growth requires high amounts of nitrogen and phosphorous, which could strain an aquatic ecosystem if the water is being pulled directly from surface water sources. The nitrogen and phosphorus requirements for microalgae have been estimated to be between 3.2 to 6.5 tons N acre 1 year 1 which is in a range between 55 to 111 times higher than canola. This high nutrient requirement

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28 leads researchers to consider growing algae in wastewater which also provides a treatment option Wastewater treatment is a highly plausible way to grow microalgae because of the high availability of nitrogen and phosphorous in the water. There are currently problems with disposing of the biomass after the treatment process. Algae can become an invasive species if allowed to enter local waterways so the biomass has to be treated before it can be disposed of. The addition of this process to water treatment facilities has prompted more anaerobic digestion research with the main feedstock being algae. Optimizing the digestion process reduces biomass waste while offering an energy source. Objective The objective of this study was to determine the optimal processes for growing and harvesting of the microalgae species Scenedesmus obliquus Various methods for harvesting the algae were tested and subsequently compared for separation efficiency, time, and cost. Materials And Methods Al gae Species The algal species used in this study was supplied by Dr. Ju e rgen Polle from Brooklyn College, State University of New York from a previous collaborative effort with researchers at Tyndall AFB This algae sample was collected from the Delaware River and determined to be Scenedesmus obliquus. This species of algae has a cellular composition of 50 56% proteins, 10 17% lipids, and 0.59 0.69 carbohydrates (Becker, 2004)

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29 Media Recipes Algae media recipes were developed by original suppliers of the algae and manipulated to provide heavier concentrations of certain nutrients that could provide additional biomass growth potential. This media was used in the lab scale production of algae. The base media that w as used is comm only known as Waris Medium. The components o f these media can be seen in Table 2 1. Large scale growth was achieved with two, 10,000 gallon outdoor pools. These pools were the continuation of the lab scale growth reactors and included tap water to propag ate biomass growth. 100 g of Miracle Gro plant fertilizer was added weekly to the pools, unless the cell concentration was noticeably reduced; then more fertilizer was added. Algae Growth Reactors Algae were grown in various reactors both in controlle d laboratory settings and uncontrolled outdoor greenhouse settings Indoor reactors were all supplemented with artificial grow lights (Growlight compact fluorescent 125 W, 6400 K full spectrum; T5 High Output Fluorescent grow light strips, 6500 K full spect rum 24 W, 2000 lumens) in order to provide various lighting schemes that mimicked the traditional solar radiation or provided additional larger doses of solar radiation dependent on need via 12 hour timers and were provided with a 1 .5 % CO 2 :9 8.5 % air mixtu re to provide inorganic carbon. The flasks of algae from the lab were then poured into 50 gallon tubs in an outdoor greenhouse. These tubs had the same gas concentrations bubbling through but only received natural solar radiation. The 10,000 gallon reacto rs were setup outside in direct sunlight. Supplementary 1.5% CO 2 :98.5% air mixture was bubbled through the pools to provide inorganic

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30 carbon. These pools had two underwater pumps running continuously to circulate the cells, as well as one pump that would a erate the water in the center of the pool. Photos of the algae growth stages used in this demonstration can be seen in Figure 3 1. Algae Biomass Concentration Determination Concentrations of algae were determined using a Varian Cary 50 UV/Vis spectrophoto meter set at a wavelength of 640 nm. Standard mass calibration curves were generated for each species using four known quantities of algae in order to produce a linear (or quadratic) calibration curve. Algae Energy Content An IKA C5000 Bomb Calorimeter was used to determine the total energy content of the algae. A 2 gram sample of dried algae was used, with a benzoic acid table for calibration. This method does not take into consideration at what ranges there are combustion reactions but instead gives a total energy contained in the sample.

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31 H arvesting Algae, being an aquatic feedstock, require considerable dewatering before conversion to a biofuel, especially the thermal conversion processes The challenges with harvesting a significant amount of al gae lie in the low cell density, small cell size, and electronegative surface charges (Brennan & Owende, 2010) These issues are limiting the success of algal biofuels. Currently, algae is being harvested using the following technologies; chemical floccul ation, filtration, centrifugation, and electro flocculation. These methods for cell removal each have disadvantages that may limit the subsequent conversion steps. Base Flocculating Chemical flocculating by altering the pH of the algal slurry is a highly recognized process because of the >80% removal efficiency (Brennan & Owende, 2010) The addition of a base, KOH or NaOH, to a pH of above 10 will cause the cells to settle, allowing the water to be siphoned off. This increased pH of the paste is ideal whe n the biofuel conversion process is not a biological one such as gasification or direct combustion. In biological processes where ethanol or methane is produced, the pH may need to be neutralized before it can be used as a feedstock to ensure the health of the process microbes. Altering the pH also leaves a hazardous waste stream of the remaining water that would have to be treated before disposal. Filtration Filtration can be used for large species of algae, but Scenedesmus obliquus has a cell size of <3 0 m, which can only be collected using microfiltration processes. This is only recommended for low volumes of algal slurry, and would be more cost efficient

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32 than centrifugation for small production processes (American Water Works Association, 2011) Elec tro Flocculating Electricity can be used to flocculate algae out of suspension us ing a small current to disrupt the naturally occurring negative charges of algae cell surfaces (Banerjee, et al., 2013) The current is produced u sing an electrode submerged in the algae slurry. This method is an effective form of separatio n, but may rupture algae cells, which would result in a loss of valuable intracellular nutrients. Centrifuging Centrifugation is the chosen method of harvesting for high value algal products and extended life of the cells This process involves expensive equipment and possible maintenance, as well as high energy costs. This process promises to yield the highest recovery efficiency of >95%, and can offer an increas e in paste concentration of 15% total suspended solids (Brennan & Owende, 2010) This process would allow for the recycle of separated water back into production pools, reducing the waste and requirement for additional water and nutrients. Each of these h arvesting processes can be improved with the addition of filters or drying steps. Sun drying is an inexpensive and effective method but requires long drying times, and large drying surfaces. If the algal paste is not consumed quickly after harvesting, it c an degrade and spoil.

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33 Materials And Methods Base Flocculating The low concentration algae water was pumped from the large growth pools into a 50 gallon conical tank, with a valve on the bottom. A 10N KOH solution was made using 85 90% KOH pellets. This solution was used to adjust the pH to 11 this generally required ~100 mL per 30 gallons. The tank was stirred and allowed to sit overnight. The algae cells settled to the bottom of the tank, allowing for a large percentage of water to be siphoned off the top for treatment and disposal. The remaining algae layer was poured into plastic holding pans that were lined with The Absorber cloths. These hydrophilic cloths are made out of synthetic material that prevents the algae cells from passing through once t reated with the KOH solution. The plastic gardening pans would allow excess water to fall out of the bottom, while leaving a thick algae paste in The Absorber cloths. The algae paste was left to filter for at least 4 hours. The paste was then placed into storage containers for later use. Figure 2 2 is a process flow diagram of the growth through harvesting process with the KOH base, and Figure 2 3 shows the equipment in use. Centrifuge The large pools were brushed and stirred to insure the highest concen tration of algae. The pool water was pumped through underground pipes to a 200 L holding tank located next to the Evodos centrifuge. The Evodos operating instructions were followed and pool water was pumped into the centrifuge at a rate of 50 L per hour. T his rate was based on the clarity of the outlet water, and was adjusted with a small valve. The Evodos was run for an average of 8 hours a day at 3000 G and could centrifuge 400 L of pool water a day. The outlet water was pumped into another algae pool for recycling.

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34 The centrifuge drum was emptied each afternoon where the algae paste was removed and stored in a fridge for use. Figure 2 4 shows the process flow diagram from growth to harvesting with the centrifuge. Results and Discussion Algae G rowth The d oubling time of the Scenedesmus obliquus was of 0.85 d 1 where at the end of seven days the liquid was split in half and replaced with new media. This would half the cellu lar concentration of the flasks, so the algae cells can propagate again to the initi al concentration. Figure 2 5 is the cellular concentration of the algae during the growth and splitting of the flasks over one month. Algae E nergy C ontent The Scenedesmus obliquus was determined to have an average calorie content of 17724.77 J g 1 This energy content is 80% of the reported average value for S. obliquus (Weyer, Bush, Darzins, & Wilson, 2010) This reduction in energy could be related to the amount and type of fertilizer used and differences in light intensity. Harvesting Efficiencies Base flocculating efficiency Base flocculating is a proven and reliable process for the harvesting of microalgae. This process proved to have 90% cell recovery efficiency. The addition of the hydrophilic cloths allowed for an 8 % cell concentration in paste with a standard deviation of 5.13% Centrifuge separation efficiency The Evodos type 10 centrifuge had a cell recovery efficiency of 99%. This machine has a main drum for the collection of cells during the run and four trays th at

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35 collect any remaining cells in the fan blades after the removal of the drum. The drum proved to have the highest paste concentration with an average of 33 % cells with a standard deviation of 0.83% The cells captured by the trays did not receive the ful l force of the centrifuge so the paste had a cell density of only 3 %. Figure 2 6 is a comparison of the dark green inlet water to the light green outlet water. The remaining green color was determined to be dye from the addition of the Miracle Gro fertili zer to the algae pools. Figure 2 7 is a graph of the separation efficiencies of the two processes. The Evodos had a higher cellular concentration in the paste as well as a lower standard deviation than the base flocculation treatment. Cost Base flocculatin g cost To calculate the cost of the base treatment, the price of the KOH pellets from Fischer Scientific was used. The cost of the disposal of the treated waste water was not included. This addition will increase the cost of the process depending on the lo cation of the lab and any government regulations, the volume of the water to be disposed of, and the exact pH of the wastewater. The cost of algae harvesting algae with KOH is $123 kg 1 VS algae including five man hours. Centrifuge separation cost To calc ulate the cost of the centrifuge process, electricity consumption, run time, peak electricity hours, and the average weight of the separated algae was taken into consideration. The cost of the peak power charge was taken from Gulf Power, which is the elect ricity company Tyndall AFB uses. The energy consumption of the Evodos was taken from the operational manual, and accounted for the average volume of water the

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36 machine was treating. The cost of running the Evodos is $110.86 kg 1 VS including the 2 man hours and the 8 hours of electricity use. Considering a 20 year uniform payment loan with a 7% interest, the annual cost of the Evodos can be calculated using the following equation: (2 1) Where: P : price i : interest n : loan length in years (National Council of Examiners for Engineering and Surverying 2008) The cost of the Evodos is $57,000 and with this loan would r equire an annual payment of $4,269. Summary The cellular concentration of the algae was determined using the UV/Vis and a quadratic calibration curve. This calibration curve does not account for the addition of the Miracle Gro plant fertilizer which ha s a blue dye included in the mix. The blue dye caused an over estimation of the cellular concentration For the harvesting of the microalgae species S obliquus the Evodos type 10 had a higher separation efficiency as compared to the base flocculation pro cess. While the Evodos is a more expensive process when the fully burdened cost of the machinery is

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37 taken into account, it offers the opportunity to have a large scale continuous system. The base flocculation process is much more time and manpower intensiv e. The extra processing time of filtering with the hydrophilic cloths can present an opportunity for the algae to spoil, and the possibility of competing bacteria being introduced into the digestion system.

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38 Table 2 1 Chemical composition of P IV metal solution for the Waris Media. Component Amount (mg L 1 deionized water) Final Concentration (mM) Na 2 2 O 750 2 FeCl 3 2 O 97 0.36 MnCl 2 2 O 41 0.21 ZnCl 2 5 0.037 CoCl 2 2 O 0.2 0.0084 Na 2 MoO 4 2 O 0.4 0.017

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39 Figure 2 1 Aquatic species of algae at various experimental culturing stages. A) Benchtop flasks (50 2000 mL) B) Open pond reactor (~50 gallons) C) Open pond reactor (~10,000 gallons) (Photos courtesy of Robert Diltz and Emily Morton). C B A

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40 F igure 2 2 Base flocculating process flow diagram

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41 Figure 2 3 Base flocculating technique A) 30 gallon hopper after the addition of KOH, B) Settled algae before filtration, C) Water filtering out through hydrophilic cloths, D) Collected paste (Photos courtesy of Eila Burr). C A D A B

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42 Figure 2 4 Growth studies of S. obliquus over 1 month -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0 100 200 300 400 500 600 700 Time (hours) Ln(Concentration/Concentration(0))

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43 Figure 2 5 Centrifuge process flow diagram

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44 Figure 2 6 Evodos water color comparison. (Photo courtesy of Emily Morton). Inlet water Outlet water

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45 Figure 2 7. Algae cellular concentration and standard deviation 0 5 10 15 20 25 30 35 40 Base Treatment Centrifuge Treatment % Algae Cells in Harvested Paste

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46 CHAPTER 3 ANAEROBIC DIGESTION Anaerobic digestion (AD) is the conversion of organic materials to methane gas his naturally occurring process has been adapted and is being engineered to offer the highest methane gas production potential. The advantages of using AD as an energy conversion process stem from the simplicity of the system low costs, and low residual w aste Figure 3 1 shows the chemical conversion steps in the AD process. The United States is currently emitting 2.18 million tons of methane gas from animal manure and 0.51 million tons of methane from wastewater treatment processes (National Renewable En ergy Laboratory, 2005). The global warming potential of methane is 23 times more harmful than carbon dioxide over the course of 100 years (IPCC 2001). This methane potential can be removed from the environment by digesting the biomass in controlled systems T able 3 1 shows the methane potential for different agricultural residues where the biomass would generally be left to rot, producing methane gas. Aquatic feedstocks The most cost effective use of algal biomass produced from wastewater treatment is the c onversion to methane biogas from AD. Benemann and Oswald have reported an approximate electricity generation from algal biogas of 1kWh electricity per kg algal VS (1996). AD is most appropriate for high moisture content feedstocks, between 80 90%, making i t a viable option for algal biomass conversion. AD of algae can be problematic because of the high proportion of protein s in the cells, causing a low Carbon/ N itrogn (C/N) ratio. These high protein levels also may cause an increased

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4 7 production of ammonium, which is inhibitory to AD bacteria. Marine species of algae contain intercellular salts which also have inhibitory affects. If a saltwater species were used as a feedstock, the digestion bacteria would have to be slowly adapted or the bacterial culutre wou ld have to be culitvated from the sediment of brackish swamps or bogs (Brennan & Owende, 2010) Specific methane yields for carbohydrates, lipids, and protein s are calculated from the composition of Chlorella vulgaris and are shown in table 3 2. Table 3 3 shows the gross composition of select microalgae species that are currently being studied for AD, as well as the theoretical methane potential. This table shows average methane potential of 54 L methane per gram VS. Table 3 4 shows the methane yield of di fferent species of microalgae based on the process parameters. This table shows a significantly reduced yield when compared with the theoretical potential.

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48 Objective The United States Air Force is currently looking for alternative energy resources that cou ld be produced onsite of deployable base camps. The key component in choosing a biofuel process is the ease of use, the ability to ship and construct conversions technologies in remote locations, and the amount of heavy machinery that would be required for planting and harvesting. A demonstration to digest microalgae was chosen because of the high production rates of the biomass and the logical assumption that the low maintenance AD process would meet mission requirements. Materials and Methods Figure 3 2 is the process flow diagram beginning with the growth of the algae to the biogas concentration analysis. Reactors In this demonstration a pilot scale reactor was used and several bench scale reactors were used for various experiments and to insure the av ailability of bacterial culture in the event of contamination. Bench scale reactors The bench scale reactors consisted of 1 L and 500 mL flasks with rubber stoppers and can be seen in figure 3 3. A liquid sampling port and a gas flow port were drilling in to the rubber stoppers. Each of these reactors was placed in a water bath at 50 C to maintain a thermophilic temperature. The rubber stoppers were tightly fitting and sealed with Parafilm to insure anaerobic conditions. After the addition of the inoculum liquid, the flasks were sparged with nitrogen gas for 1 minute. The gas flow ports were connected to water displacement vessels and the gas production was noted daily. Liquid samples were also taken two to three times a week for High performance

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49 liquid ch romatography ( HPLC) analysis. The pH was also monitored after liquid samples were taken. Each day the flasks were shaken by hand to release gas bubbles and mix the settled feedstock. The bubbles indicate the successful production of biogas. Pilot scale r eactor A 600 L cylindrical reactor was used in this anaerobic digestion process. This large reactor was located at Tyndall AFB in Panama City, Florida. It was placed outside on a concrete slab under an awning to limit exposure to bad weather. The reactor w as fitted with 3 band heaters that maintained a thermophilic temperature between 53 58 C manually, with a thermocouple probe inserted into the liquid. The bands were wrapped under insulation and metal siding. Figure 3 4 shows the exterior walls of the reac tor as well as the insulation. This photo was taken during the addition of the heating bands. A circulation pump was used to mix the liquid contents, introduce feedstock into the digester, and to retrieve liquid samples. This pump also allowed for the rel ease of any bubbles that may have been trapped in the liquid. The lid of the digester was sealed with a gasket and tightened down with screws to insure an airtight seal. A gas outlet port was positioned on the lid of the reactor with tubing connected to a gas manometer. Figure 3 5 shows the manometer. This outlet prevented an accumulation of pressure in the digester and allowed for gas production monitoring. The gas meter measured gas flow through the manometer by the use of a liquid float switch connected to a solenoid. The solenoid would open to release the gas after ~0.67 L of gas was collected and each opening of the solenoid would be counted. The manometer was designed for the use in anaerobic digestion outlet gas flow by Dr. Pratap Pullamanappallil fro m the University of Florida, and has proven to be a reliable measurement tool (Koppar & Pullammanappallil, 2008)

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50 Microbial Inoculum The anaerobic digestion inoculum was received from the University of Florida. This thermophi lic mix of bacteria was previously used in the digestion of sugar beet tailings, and had proven to be a robust consortium, capable of producing high concentrations of methane gas (Koppar & Pullammanappallil, 2008) The bacteria l consortium was received after an extended period of reduced f eeding. The bacterial colony was activated into a growth and gas production by a slow introduction of black strap molasses. Feeding Molasses was chosen because it is an inexpensive feed sourc e at $1 per gallon (US Department of Agriculture, 2013) high in sucrose, and would not add excess liquid volume to the digester (Curtin, 1983) A step feeding scheduled was used to activate the bacteria with molasses; 0.5 g L 1 VS followed by an increase of 0.5 g L 1 VS until the goal of 2 g L 1 VS was achieved. The gas flow rate was monitored to determine when the next step of feeding was required. Over a period of 3 5 days, t he gas production would stop and another dose of mol asses was added. Failure During the activation of the microbes, a catastrophic failure occurred when the heating blankets surrounding the digester were set too high which caused the majority of liquid to boil out of the digester. In order to determine wh ether a viable consortium of bacteria survived through the extreme heating process, a cell count was done with the use of serial dilution on Petri dishes using Luria Bertani (LB) agar kept at 50 C. The dishes were kept in anaerobic conditions using a seale d j ar and oxygen scrubbing packets.

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51 Along with the remaining digester liquid a growth media was created using the components listed in table 3 5 These components were mixed in tap water that had been sparged with nitrogen gas for 1 minute. The composit ion of the trace minerals and vitamins were referenced from the growth media used in the start up of thermophilic digestion discussed in Suwannoppadol et al (2012). Trace vitamins and minerals were added to the growth multi vitamins. This media recipe is listed in table 3 6 and adapted from Suwannoppadol et al (2012). Standard feeding A circulation pump was used to both mix the digester liquid, and to introduce food sources into the digester. A small volume of liqui d was pumped from the digester into a five gallon bucket where the molasses, and later the algae, could be suspended and pumped back into the digester. This suspension of feedstock into the inoculum reduced the contact with oxygen and any clogging in the p ump. Figure 3 6 is the feeding schedule of the molasses and algae paste weights. Once the production ra te of the off gas from the consumption of molasses was steady, algae paste was slowly introduced. Again, a step feeding schedule was used to slowly intro duce the new food source. This was done in 0.25g L 1 VS increments. The gas flow rate was used as an indication for the next feeding. Sampling Flow rate The biogas flow rate was measured by a manometer attached to the outlet port of the digester, this met count of the meter was noted daily.

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52 Gas concentrations The biogas concentrations were measured using a Landtech GEM 5000 gas meter. The meter was connected directly to the outlet tubing and wo uld pump biogas through the meter for 90 seconds. This tool measured CH 4 O 2 CO 2 CO, and H 2 S and was used daily to monitor the progress of the digester. A HP/ Agilent 6890 gas chromatograph (GC) with an FID detector was used to monitor the bench scale digesters. The GC used a Supelco Carboxen 1010Plot # 24246 capillary column (30mx0.32mm) and argon as a carrier gas. A 250 L gas sample was used a Supelco Analytical Scotty Gas biogas mix of 1%: H 2 O 2 CO, CH 4 CO 2 with a 95% balance of N 2 (Cat No. 22561) calibration gas was used. Liquid characteristics A YSI probe was used to measure the liquid characteristics of the dige ster. This meter measures pH, salinity, temperature, dissolved oxygen, and total dissolved solids. Each day, a 500 mL liquid sample was taken from the digester and quickly measured with the probe. The probe was allowed to equilibrate for 5 minutes before r eadings were taken. Liquid samples were also analyzed regularly with high performance liquid chromatography (HPLC). An HP/ Agilent 1100 HPLC was used with a Phenomenex Rezex 8u 8%, 300x7.8 mm column with an Alltech guard column attached. The mobile phase was 0.005 N H 2 SO 4 isocratic me thod with a flow rate of 0.5 mL min 1 and an injection volume of 50 L. This had a run time of 25 minutes. A 2 g L 1 sucrose solution was used as a sugar baseline and comparison.

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53 Results and Discussion Gas Production The gas p roduction from the anaerobic digester was monitored for a total of 6 months, with a focus on March through June of 2013, when the consortium was healthy. The total cumulative amount of biogas that was produced was 9480 L with a cumulative methane value of 3768 L. This volume of methane equates to roughly 38.6 kWh (Eriksson, 2012) Figure 3 7 shows the gas concentrations from the digester as well as the cumulative methane production over the three month project. Gas Concentrati on From Figure 3 7 the gas concentrations at the beginning of the project show oxygen concentration in the digester matching atmospheric concentrations. The gradual reduction of oxygen and the gradual increase of CO 2 and CH 4 display a healthy and producti ve anaerobic system. The highest methane concentration was 66% the correlating CO 2 concentration of 27%. Liquid Characteristics Ysi probe Figure 3 6 shows the liquid characteristics of the digester from the YSI. Total dissolved solids (TDS), dissolved oxy gen (DO), and pH were recorded. The changes in pH are proportional to the CO 2 in the system as seen in figure 3 8 The pH of the system began at 8.8 and with the increased concentration of CO 2 the pH decreased to an a verage value of 7.9. Figure 3 9 shows the affect an increase in DO has on the pH of the system. When the DO of the system increases, the bacteria switch to aerobic digestion, which is the preferred metabolic pathway.

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54 HPLC The HPLC was able to show a transitioning sugar to alcohol pattern. Th e figures 3 10 through 3 1 6 show the concentrations of the liquid components each day the digester was sampled. The figures are representations of HPLC peaks, each graph is showing a single retention time and the compound those peaks represent. These value s for figures 3 10 through 3 14 and 3 16 were left in the units mAU instead of being converted to a concentration curve because the changes in the values were valuable, instead of the exact concentrations. Figure 3 15 is the concentration of acetate chang ing over time in the units mg/L because the calibration curve was reliable This graph shows a correlation between high concentrations of methane gas with a high availability of acetate in the digester liquid around day 100 of the experiment. The acetate c oncentration decreases quickly, which causes the concentration of methane gas in the headspace to also decrease. It can also be noted that on day 97, 600g of algae paste was added to the digester. Over the course of the experiment, the changes in concentra tions show the metabolic processes of the bacteria. Summary The digestion of the microalgae Scenedesmus obliquus has proven to be an option for the production of methane gas for energy use. The algae cells were a good feedstock because of their low pretre atment requirements, and ability to pump into the digester. Anaerobic digestion is a plausible option for energy generation in remote environments.

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55 Table 3 1. Estimated methane potential Biomass Average methane production potential L kg 1 Volatile Solids L kg 1 Total Solids Corn waste 338 290 Wheat straw 290 243 Rice straw 302 232 Sugarcane waste 278 206 (Chandra, Takeuchi, & Hasegawa, 2012)

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56 Table 3 2 Specific methane yield for organic compounds in Chlorella vulga ris Substrate Composition L CH 4 g 1 VS Proteins CH 2.18 O 0.17 N 0.08 0.851 Lipids CH 1.82 O .011 1.014 Carbohydrates (CH 1.67 O 0.83 ) n/6 0.4515 (Angelidaki and Sanders, 2004) (Becker, 2007 )

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57 Table 3 3 Gross composition of microalgae species and th eoretical methane potential during anaerobic digestion of the total biomass Species Proteins (%) Lipids (%) Carbs (%) CH 4 (L CH 4 g 1 VS ) Euglena gracilis 39 61 14 18 0.53 0.8 54.3 84.9 Chlamydomonas reinhardtii 48 17 0.69 44.7 Chlorella pyrenoidosa 57 2 6 0.8 53.1 Chlorella vulgaris 51 58 12 17 0.63 0.76 47.5 54.0 Dunaliella salina 57 32 0.68 53.1 Spirulina maxima 60 71 13 16 0.63 0.74 55.9 66.1 Spirulina platensis 46 63 8 14 0.47 0.69 42.8 58.7 Scenedesmus obliquus 50 56 10 17 0.59 0.69 42.2 46.6 (Becker, 2004)

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58 Ta ble 3 4 Gross composition of microalgae species (Becker, 2004) and methane potential yield anaerobic digestion of the total biomass Reactor Feedstock T (C) HRT (d) Loading Rate (g VS L 1 d 1 ) Methane Yield (L CH 4 g VS 1 ) CH 4 (%vol) References Batch 11L Algae sludge ( Chlorrella Scenedesmus) 35 50 3 30 1.44 2.86 0.17 0.32 62 64 Golueke et al., 1957 Algal biomass 35 28 1 0.42 72 Chen, 1987 Spriulina 35 28 0.91 0.32 0.31 Dunaliella 35 28 0.91 0.44 0.45 CSTR 2 5L Tetraselmis (fresh) 35 14 2 0.31 72 74 Asinari Di San Marzano et al., 1982 Tetraselmis (dry) 35 14 2 0.26 72 74 Tetraselmis (dry) + NaCl 35 g/L 35 14 2 0.25 72 74 Batch 5L Chlorella vulgaris 28 31 64 0.31 0.35 68 75 Sanchez and Travieso, 1993 Semi Cont. daily fed 10L Spirulina maxima 35 33 0.97 0.26 68 72 Samson and LeDuy, 1982 FedBatch 2L Spirulina maxima 15 52 5 40 20 100 0.25 0.34 46 76 Samson and LeDuy, 1986 CSTR 4L Chlorella Scenedesmus 35 10 2 6 0.09 0.136 69 Yen and Brune, 2007

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59 Table 3 5 Chemical composition of P IV metal solution. Component Amount (mg L 1 deionized water) Final Concentration (mM) Na 2 2 O 750 2 FeCl 3 2 O 97 0.36 MnCl 2 2 O 41 0.21 ZnCl 2 5 0.037 CoCl 2 2 O 0.2 0.0084 Na 2 MoO 4 2 O 0.4 0.017

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60 Table 3 7. Trace vitamins and minerals found in multi vitamin. Component Amount (g L 1 water) nitrilotriacetic acid 12.8 FeCl 3 6H 2 O 1.35 MnCl 4 H 2 O 0.1 CoCl 2 6H 2 O 0.024 CaCl 2 2H 2 O 0.1 ZnCl 2 0.1 CuCl 2 2H 2 O 0.025 H 3 BO 3 0.01 Na 2 MoO 4 4H 2 O 0.024 NaCl 1 .0 NiCl 2 6H 2 O 0.12 Na 2 SeO 3 5H 2 O 4.0 mg Na 2 WO 4 2H 2 O 4.0mg (Suwannoppadol et al., 2012)

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61 Figure 3 1 Pathway for the conversion of organic biomass to biogas through anaerobic dig estion processes.

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62 Figure 3 2 Process Flow diagram.

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63 Figure 3 3 Bench scale anaerobic digestion flasks successfully producing biogas. (P hoto courtesy of Emily Morton)

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64 Figure 3 4 Pilot scale reactor (Photo courtesy of Em ily Morton)

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65 Figure 3 5 Manometer (photo courtesy of Emily Morton).

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66 Figure 3 6. Feeding schedule of molasses and algae paste 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 54 62 68 71 72 75 78 83 85 90 96 107 114 121 126 127 129 134 139 141 142 146 148 149 153 154 155 156 160 Feeding Schedule Molasses (g) Algae paste (g) weight (g)

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67 Figure 3 7 Gas concentrations and cumulative methane production over time. 0 500 1000 1500 2000 2500 3000 3500 4000 0 10 20 30 40 50 60 70 60 70 80 90 100 110 120 130 140 150 160 Gas Percentage (%) Time (days) Gas Production CH4 CO2 O2 Cumulative methane Cumulative Methane (L)

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68 Figure 3 8 Dissolved oxygen conc entration and pH of digester liquid over time. 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 0 5 10 15 20 25 40 60 80 100 120 140 160 DO (%) Time(days) YSI Probe DO (%) pH pH

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69 Figure 3 9 Carbon Dioxide gas concentration and pH of digester liquid. 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 0 10 20 30 40 50 60 70 40 60 80 100 120 140 160 CO2 Concentration Time (days) Carbon Dioxide and pH CO2 pH pH

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70 Figure 3 10 Liquid samples over time, HPLC retention time of 8.5 minutes; fructose concentration. 0 10000 20000 30000 40000 40 60 80 100 120 140 mAU time (d) 8.5 min (Fructose) Digestate Sucrose (2 g/L)

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71 Figure 3 11 Liqui d samples over time, HPLC retention time of 10.0 minutes; glucose concentration. 0 100 200 300 400 500 600 700 800 50 70 90 110 130 150 170 mAU time (d) 10.0 minutes (Glucose) Digestate Sucrose (2 g/L)

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72 Figure 3 1 2 Liquid samples over time, HPLC retention time of 13.2 minutes; lactic acid concentration. 0 500 1000 1500 2000 2500 50 70 90 110 130 150 mAU time (d) 13.2 minutes (Lactic Acid) Digestate

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73 Figure 3 1 3 Liquid samples over time, HPLC retention t ime of 15.4 minutes; glycerol concentration. 0 1000 2000 3000 4000 5000 6000 7000 50 60 70 80 90 100 110 120 130 140 150 mAU time (d) 15.4 minutes (Glycerol) Digestate Sucrose (2 g/L)

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74 Figure 3 1 4 Liquid samples over time, HPLC retention time of 19.36 minutes; acetate concentration. 0 1000 2000 3000 4000 5000 6000 50 60 70 80 90 100 110 120 130 140 150 mAU time (d) 19.36 minutes (acetate) Digestate

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75 Figure 3 1 5 Liquid samples over time, HPLC acetate mg/L concentration 0 0.2 0.4 0.6 0.8 1 1.2 50 60 70 80 90 100 110 120 130 140 150 Concentration mg/L time (d) acetate

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76 Figure 3 1 6 Liq uid samples over time, HPLC retention time of 22.8 minutes; ethanol concentration. 0 200 400 600 800 1000 1200 50 60 70 80 90 100 110 120 130 140 150 mAU time (d) 22.8 minutes (Ethanol) Digestate Sucrose (2 g/L)

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77 CHAPTER 4 CARBON RECOVERY In 2010, the United States released nearly 6,000 million metric tons of carbon dioxide, which is 84 % of the total gas emitted into the atmosp here through human activities (United States Department of State, 2007) Human produced carbon dioxide is primarily from coal fueled power plants, transportation, and industry processes. Power plants produce 38% of the CO 2 that is being released in the form of flue gas (United States Department of State, 2007) Flue gas is primarily between 3% and 15% CO 2 noting that coal fired power plants produce a higher percentage of CO 2 Coal combustion power p lants account for 0.95 kg CO 2 per 1kWh of electricity produced (DOE, 2000). Currently, the processes for collecting CO 2 are limited by the high cost of capture, the difficulty of storage, and the low efficiency of current technologies (Chi, O'Fallon, & C hen, 2011) A current process of carbon capture and storage (CCS) aims to pump carbon dioxide gas into geologic formations, with the expectation that the gas will remain there indefinitely. It is also used for enhanced oil recovery (EOR), where the U.S. pe troleum industry has injected over 600 million tons of CO 2 into wells, producing 245,000 barrels of oil per day (Meyer, 2010) This process uses the CO 2 for the enhanced recovery of oil, but it is released back into the environ ment after the liquid petrol has been collected. Algae There has be en a high interest in the use of microorganisms as a biofuel resource because of the potentially high productivity and the possibility for CO 2 fixation. Specifically, both marine and fresh water species of microalgae are being utilized to

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78 produce high volumes of biomass. Large scale productions have been considered which would offer up to 51.9 tons acre 1 year 1 with high efficiency raceways (Christi 20 07). Photobioreactor production system s result ed in 60.7 tons acre 1 year 1 and it has been theorized that a maximum value of 106 tons acre 1 year 1 could be obtained (Carlsson et. al., 2007). F lue gases contain NO x and SO x which have proven not to be harmful to the growth of the algae them selves, but they can cause a harmful change in the pH of the aqueous media, resu lting in slow growth or death (Packer, 2009) Packer also states that an increased CO 2 in bubbling gas from 0.038% to 1.0% is responsible for doubling the algae biomass produce d which is why CO 2 is supplemented in most algae production systems These engineered systems could offer a way to capture carbon doixide in the form of biomass which can be utilized for the production of pharamcuticals, food, and fuels. Objective The ob jective of these experiments is to determine the plausibility of using microalgae to recover inorganic carbon from the outlet gas of an anaerobic digester. This will obtain a more concentrated methane gas stream. Scenedesmus obliquus was the species used i n these experiments. Four different gas concentrations were used to determine if the growth rate of the algae were negatively affected and if the concentration of CO 2 decreased. Materials and Methods Four 500mL side arm flasks were used as bioreactors t o grow algae. Each flask contained 180mL of Waris media with 20mL of a healthy Scenedesmus obliquus culture from previous experiments. The media also contained a pH 10 buffer solution. The flasks were placed on a shaker table with artificial grow lights fo r a 12 hour automatic

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79 cycle (Growlight compact fluorescent 125 W, 6400 K full spectrum; T5 High Output Fluorescent grow light strips, 6500 K full spectrum 24 W, 2000 lumens). Flask 1 had a sealed balloon filled with biogas produced from the pilot scale re actor; flask 2 had a 1 .5 % CO 2 :9 8.5 % air mix; flask 3 was just stopped with a sponge to reduce evaporation; and flask 4 was a control flask where the 1 .5 % CO 2 :9 8.5 % air mix was bubbled through continuously. Measurements Optical density The growth rate of the cultures was monitored daily. Concentrations of algae were determined using a Varian Cary 50 UV/Vis spectrophotometer set at a wavelength of 600 nm. A Spec 20 was also used to measure the flasks that were sealed wit h balloons. Standard mass calibration curves were generated for each species using four known quantities of algae in order to produce a linear (or quadratic) calibration curve. Gas composition A HP/ Agilent 6890 gas chromatograph with an FID detector that was equipped with a Supelco Carboxen 1010Plot # 24246 capillary column (30mx0.32mm) and argon as a carrier gas to monitor the bench scale digesters. A 250 L gas sample was used and a Supelco Analytical Scotty Gas mix of 1%: H 2 O 2 CO, CH 4 CO 2 with a 95 % balance of N 2 (Cat No. 22561) calibration gas was used to simulate biogas components. The GEM 5000+ was used at the end of the experiments to determine the final gas concentrations remaining the in biogas balloon and the CO 2 /air mix.

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80 Results and Di scussion The gas samples that were taken over the course of the experiment show a change in the concentration of the methane, carbon dioxide, nitrogen, and oxygen. These changing levels could be attributed to the release of gas during the sampling. In fig ure 4 2 the side arm flasks can be seen. The most problematic issue that was faced in the use of microalgae for carbon sequestration is the pH control in the liquid. In this experiment, the cell concentration of the algae may have been too low to handle t he inorganic carbon load, which caused the pH to drop dramatically, killing any healthy algal cells. The initial addition of the buffer solution helped in the maintenance of a healthy pH range, but constant pH monitoring and correction need to be considere d for any future work. Flask 2, the flask with sealed CO 2 /Air mix, had the worst least amount of cellular growth. The low initial concentration of 13% CO 2 was did not provide enough inorganic carbon to sustain the culture for the duration of the experimen t. Summary This opportunity to use the growth of microalgae as a carbon recycling process appears to be plausible. The main considerations are the use pH of the algae media, and the recapture of the methane stream. Unlike many current flue gas algae syste ms where the gas bubbles through the algae media, and then escapes into the atmosphere, this system would require the methane gas to be recaptured. A large scale system would require research into recapture processes.

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81 Figure 4 1 Side arm flasks with S obliquus (photo courtesy of Karen Farrington)

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82 Figure 4 2 GC results from flask 1: Biogas. 0 100 200 300 400 500 600 700 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 peak area RT (min) S. obliquus/biogas exp (flask #1 -biogas) 2d 3d 7d 9d H 2 O 2 CO N 2 CH 4 CO 2

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83 Figure 4 2 GC results from flask 2: CO 2 /Air mix. 0 50 100 150 200 250 300 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 peak area RT (min) S. obliquus/biogas exp (flask #2 -CO2 mix) 2d 3d 7d 9d H 2 O 2 CO N 2 CH 4 CO 2 H 2 O 2 CO N 2 CH 4 CO 2

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84 Figure 4 3 Changing pH values for each flask over time. 5 5.5 6 6.5 7 7.5 8 8.5 9 0 1 2 3 4 5 6 7 8 9 10 pH time (d) S. obliquus/biogas exp 1 2 3 4

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85 Figure 4 4 Changing Optical densit y of the flasks over time. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 1 2 3 4 5 6 7 8 9 10 OD 600nm time (d) S. obliquus/biogas exp 1 2 3 4

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86 LIST OF REFERENCES Biogas as veh icle fuel: a European overview. 2003, Stockholm, Sweeden. J.K Edzwald, Ed., 2000. Water Quality and Treatment: A Handbook on Drinking Water, New York. C. Asina ri Di San Marzano, A. Legros, H. Naveau, E. Nyns Biomethanation of the marine algae Tetraselmis International Journal of Sustainable Energy (1982), pp. 263 272. Banerjee, C., Ghosh, S., Sen, G., Mishra, S., Shukla, P., & Bandopadhyay, R. (2013). Study o f algal biomass harvesting using cationic guar gum from the natural plant source as flocculant. Carbohydrate Polymers 675 681. C. Banerjee, S. Ghosh, G. Sen, S. Mishra, P. Shukla, R. Bandopadhyay Study of algal biomass harvesting using cationic guar g um from the natural plant so urce as flocculant Carbohydrate Polymers (2013), pp. 675 681 J.C. Benemann, J.C. Weissman, B.L. Koopman, W.J. Oswald Energy prodcution by microbial photosynthesis Nature (1977), pp. 19 23 J. Benemann, W. Oswald Systems a nd economic analysis of microalgae ponds for conversion of CO2 to biomass Pittsburgh: Pittsburgh E nergy Technology Center (1996) L. Brennan, P. Owende Biofuels from microalgae A review of technologies for production, processing, and extractions of bio fuels and co produc ts Renewable and Sustainable Energy Reviews (2010), pp. 557 577 C. R. Brown. 2003. Biorenewable Resources Ames: Blackwell Publishing Company. W.J. Catallo, T.F. Shupe, T.L. Eberhardt Hydrothermal processing of biomass from invasice aquatic plants Biomass & Bioenergy (2008), pp. 140 145 R. Chandra, H. Takeuchi, T. Hasegawa Methane production from lignocellulosic agricultural crop wastes: A review in context to second generation of biofuel production Renewable and Sustainable E nergy Reviews (2012), pp. 1462 1476

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87 P. Chen Factors influencing meth ane fermentation of micro algae PhD thesis, University of Calibornia Berkley (1987) Z. Chi, J.V. O'Fallon, S. Chen Bicarbonate produced from carbon capture for algae culutre Trends in Biotechnlogy (2011), pp. 537 541 Y. Christi Biodiesel from microalgae Biotechnology Advances (2007), pp. 294 306 P. Collet, A. Helias, L. Lardon, M. Ras, R. Goy Life cycle assessment of microalgae culture coupled to biogas production Bioresource Tec hnology (2011), pp. 207 214 L.V. Curtin Molasses in Animal Nutrition West Des Moines: National Feed Ingredients Association (1983) F. Delrue, Y. Li Beisson, P. Setier, C. Sahut, A. Roubaud, A. Froment, G. Peltier Comparison of various microalgae liqui d biofuel production pathways based on energetic, economic and environmental criteria Bioresource Technology (2013) A. Demirbas, T. Ozturk, M. Demirbas Recovery of energy and chemical from carbonaceous materials Journal of Energy Sources(2006), pp. 14 73 1482 Department of Energy, 200. Carbon Dioxide Emissions from the Generation of Electric Power in the United States, Washington DC E. Ehimen, J. Holm Nielsen, M. Poulsen, J. Boelsmand Influence of different pre treatment routes on the anaerobic di gestion of a filamentous algae Renewable Energy (2013), pp. 476 480 Environmental Protection Agency Methane and Nitrous Oxide Emissions from Natural Sources ( 2010)

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88 O. Eriksson Environmental technology assessment of natural gas compared to biog as Rijeka: InTech (2012) C. Golueke, W. Oswald, H. Gotaas Anaerobic digestion of algae Applied Microbiology (1957), pp. 47 55 S. Gonzlez Garca, M. Teresa Moreira, G. Feijoo, R.J. Murphy Comparative life cycle assessment of ethanol production from f ast growing wood crops (black locust, eucalyptus and poplar) Biomass and Bioenergy (2012), pp. 378 388 X.M. Guo, E. Trably, E. Latrille, H. Carrere, J.P. Steyer Hydrogen production from agricultural waste by dark fermentation: A review International Journal of Hydrogen Energy (2010), pp. 10660 10673 R. Honda, J. Boonnorat, C. Chiemchaisri, W. Chiemchaisri, K. Yamamoto Carbon dioxide capture and nutrients removal utilizing treated sewage by concentrated microalgae cultivation in a membrane photobior eactor Bioresource Technology (2012), pp. 59 64 H. Hsueh, H.Chu, Y. Su A batch study on the bio fixation of carbon dioxide in the absorbed solutuion from a chemical wet scrubber by hot spring and marine algae Chemosphere (2007), pp. 878 886 H. Hsueh, W. Li, H. Chen, H. Chu Carbon bio fixation by photosynthesis of Thermosynechococcus sp.CL 1 and Nannochloropsis oculta Photochemistry and Photobiology (2009), pp. 33 39 K. Kadam Environmental implications of power generati on via coal microalgae cofi ring Energy (2002), pp. 905 922 G. Kocar Anaerobic Digesters: From Waste to Energy Crops as an Alternative Energy Sourc e Energy sources (2008), pp. 660 669

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89 A. Koppar, P. Pullammanappallil Single stage, batch, leach bed, thermophilic anaerobic dig estion of spend sugar beet pulp Bioresource Technology (2008), pp. 2831 2839 P. McKendry Energy production from biomass (p art 2): conversion technologies Bioresource Technology (2002), pp. 47 54 A. Melis Green alga hydrogen productionL process, chal lenges, and prospects International Journal of Hydrogen Energy (2002), pp. 1217 1228 J.P. Meyer Summary of Carbon Dioxide Enhanced Oil Recovery (CO2EOR) Injected Well Technology Plano American Petroleum Insitute (2010) X. Miao, Q. Wu High yield bio oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides Journal of Biotechnology (2004), pp. 85 93 G. Migliore, C. Alsis, A. Sprocati, E. Massi, R. Ciccoli, M. Lenzi, A. Wang Anaerobic digestion of macroalgal biomass and sediments sourced from the Orbertello lagoon Italy Biomass and Bioenergy (2012), pp. 69 77 A. Molino, F. Nanna, Y. Ding, B. Bikson, G. Braccio Biomethane production by anaer obic digestion of organic waste Fuel (2013), pp. 1003 1009 National Council of Examiners for Engineering and Surveying Fundamentals for Engineer ing Supplied Reference Handbook (2008) National Renewable Energy Laboratory A Geographic Perspective on the Current Biomass Resource Availability in the United States Department o f Energy (2005) A.D N'Yeurt, D.P. Chynoweth, M.E. Capron, J.R. Stewart, M.A. Hansan Negative carbon via Ocean Afforestation Process Safety and Enviornmental Protection (2012), pp. 467 474

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90 J.C. Ogbonna, H. Tanaka Light requiremnt and photosynthetic ce ll culitvation Delevopment of porcesses for efficient light utilization in photobioreactors Journal of Applied Phycology (2000), pp. 207 218 W. Oswald, C. Golueke Biological transformtion of solar energy Advances in Applied Microbiology (1960), pp. 2 23 242 M. Packer Algal capture of carbon dioxide; biomass generation as a tool for greenhouse gas mitigation with reference to New Zealand energy strategy and policy Energy Policy (2009), pp. 3428 3437 J. Park, R. Craggs, A. Shilton Wastewater treat ment high rate alg al ponds for biofuel production Bioresource Technology (2011), pp. 35 42 S. Park, Y. Li Evaluation of methane production and macronutrient dedgadation in the anaerobic co digestion of algae biomass residue and lipid waste Bioresource Technology (2012), pp. 42 48 Renewable Fuels Association. (2013). Pocket Guide to Ethanol 2013 Washington, DC. R. Samson, A. LeDuy Biogas production from anaerobic digestion of Spirulina maxima algal biomass Biotechnology and Bioenergy (1982), pp. 1 919 1924 R. Samson, A. LeDuy Detailed study of anaerobic digestion of Spirulina maxima algae biomass Biotechnology and Bioenergy (1986), pp. 1014 1023 E. Sanchez, L. Travieso Anaerobic digestion of Chlorella vulgaris for energy production Resour Cons erv Recycle (1993), pp. 127 132 B. Sialve, N. Bernet, O. Bernard Anaerobic digestion of microalgae as a nexessary step to make microalgal biodiesel sustainable Biotechnology Advances (2009), pp. 409 416

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91 B.T. Smith, R.H. Davis Sedimentation of algae flocculated using naturally avaliable magnesium based flocculants Algal Research (2012), pp. 32 39 S. Suwannoppadol, G. Ho, R. Cord Ruwisch Distribution of methanogenic potential in fractions of turf grass used as inoculum for the start up of thermop hilic anaerobic digestion Bioresource Technology (2012), pp. 124 130 N.J. Themelis, P.A. Ulloa Methane generation in landfills Renewable Energy (2007), pp. 1243 1257 U.S. Energy Information Administration Annual Energy Review Washington D.C.: Ener gy Information Administration (2012) United States Department of State Sugar Byproducts, Imports, and Prices US Department of Agriculture (2013) United States Department of Agriculture Energy Balance for the Corn Ethanol Industry USDA (2010) C. V asseur, G. Bougaran, M. Garnier, J. Hamelin, C. Leboulanger, M. Le Chevanton, E. Fouilland Carbon conversion efficiency and population dynamics of a marine algae bacteria consortium growing on simplified synthetic digestate: First step in a bioprocess c oupling algal pro duction and anaerobic digestion Bioresource Technology (2012), pp. 79 87 A. Vergara Fernandez, G. Vargas, N. Alarcon, A. Velasco Evaluation of marine algae as a source of biogas in a two stage anaerobic reactor system Biomass and Bioe nergy (2008), pp. 338 344 K.M. Weyer, D.R. Bush, A. Darzins, B.D. Wilson Theoretical Maximum Algal Oil Production Bioengineering Resources (2010), pp. 204 213 R.H. Wiffels, M.J. Barbosa An Outlook on Microalgal Biofuels Science (2010), pp. 796 799

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92 A.C. Wikie Anaerobic Digestion: Biology and Benefits Dairy Manure Management: Treatment, Handling, and Community Relations (2005), pp. 63 72 H. Yen, D. Brune Anaerob i c co digestion of algal sludge and waste paper to produce methane Bioresource Technology (2007), pp. 130 134

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93 BIOGRAPHICAL SKETCH Emily Morton received her Bachelor of Science in Biological Engineering in 2010 from North Carolina State University. In the fall of 2011 she returned to academia to pursue her Master of Sc ience in Biological Engineering under Dr. Pullammanappallil while on contract for the United States Air Force with Applied Research Associates in Panama City, FL. She graduated in the summer of 2013. After completion of her graduate studies, she aspires t o continue her research in the field of alternative energy with an emphasis on biomass to renewable fuels