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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 UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2013

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© 2013 Emily L. Morton

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For my Grandmother, Hilda Brown

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ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Pratap Pullammanappallil, for his academic

guidance through the process of pursuing a master’s. 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, setting up equipment 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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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 Ethanol ............................................................................................................. 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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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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LIST OF REFERENCES ............................................................................................... 86

BIOGRAPHICAL SKETCH ............................................................................................ 93

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LIST OF TABLES

Table page 1-1 Ethanol production yields of various feedstocks ................................................. 25

1-2 Comparison of biomass sources for the production of biodiesel ........................ 26

2-1 Chemical composition of P-IV metal solution for the Waris Media. ..................... 38

3-1 Estimated 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 anaerobic digestion of the total biomass ...................................... 58

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LIST OF FIGURES

Figure page 1-1 Biomass Resources of the United States. National Renewable Energy

Laboratory. ......................................................................................................... 24

2-1 Aquatic species of algae at various experimental culturing stages.. ................... 39

2-2 Base flocculating process flow diagram .............................................................. 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

3-10 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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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 minutes; 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 Side 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: CO2/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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LIST OF ABBREVIATIONS

AD anaerobic digestion AFB Air Force Base

oC 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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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment 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: Pratap 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 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 methane gas from a combination of algae and molasses as a food source.

This project used the microalgae species Scenedesmus obliquus because it was not a

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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 locations.

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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

Government’s concerns. Petroleum based 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.

With President Bush’s signing of Executive Order 13,423 in 2007, a requirement

of energy reduction and ‘Green’ initiatives for energy production has pressed the military

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

transportation 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 potential energy could be produced. Alternative to

other renewable energy options, biomass is versatile; it can be used in direct

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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 indicates 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 zones 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 non-food sources. These wastes include corn

stover, wood chips, sugarcane bagasse, and beet tailings. In 2005 the United States

produced 62.4 million tons of forestry residues and 34 million tons of urban wood waste

(National Renewable Energy Laboratory, 2005). 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 ploughing that food crops require which reduces

energy inputs. The woody biomasses, like poplar trees, are more slowly growing

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resources and can take years or decades to mature before harvesting. Trees 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 algae 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 biomass 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.

Current Forms and Processes 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. Ethanol has a bad reputation because of the

‘food for fuel ’argument, 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 materials, including; woody materials,

agricultural residues, and fast-growing grasses. These lignocellulosic materials have

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accessible fermentable sugars but are not considered a food or feed crop (González-

García, Teresa Moreira and Feijoo, 2012). Table 1-1 compares the ethanol 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 production is the chemical process of converting sugars to

alcohols using microbes. Saccharomyces cerevisiae, ethanol tolerant yeast, is the

primary microbe used for this process because it prefers fermentation to cellular

respiration even in the presence of oxygen, which allows the process to be completed in

aerobic conditions. Equation 1-1 shows the stoichiometry of the glucose conversion to

ethanol.

C6H12O6 2 C2H5OH + 2 CO2 (1-1)

Currently, 10% of the nation’s gasoline supply comes from the conversion 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 based 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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(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, the combustion of biodiesel is more complete, 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 biodiesel and

the land area needed to meet 50% of the U.S.’s transportation needs.

Hydrogen

There are green 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]-hydrogenase leads to the

synthesis of H2. 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 of oxygen during photosynthesis (Melis, 2002).

The conversion of glucose to hydrogen gas and carbon dioxide can be seen in equation

1-3

C6H12O6 + 6 H2O 12 H2 + 6 CO2 (1-3)

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Combustion

Combustion is the process in which biomass is burned between 800-1000 °C in

the presence of air, 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, 2002).

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 (>700°C) without

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 different

stages: dehydration; 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 syngas.

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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Carbon-oxygen reaction: C +

O2 CO (1-4)

Boudouard reaction: C + CO2 2 CO (1-5) Carbon-water reaction: C + H2O H2 + CO (1-6) Hydrogenation reaction: C + 2 H2 CH4 (1-7) Gas phase reactions Water-gas shift reaction: CO + H2O H2 + CO2 (1-8) Methanation: CO + 3 H2 CH4 + H2O (1-9)

Pyrolysis

Pyroysis 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 microalgae 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 comparable

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 natural gas, is currently used in roughly

50% of American households (U.S. Energy Information 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,

CO2 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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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 the

leading contributors to methane’s damaging effects on the environment. Landfills have

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 anaerobic digestion systems to produce high concentrations of

methane gas has become a global research topic. A thermophilic process, running

between 45-60°C will kill pathogens so the solids can be land applied as fertilizer.

Anaerobic digestion does not require 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 CO2 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 interesting 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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2% of the country’s produced CO2, for use in food products or oil recovery. These

processes do not sequester the carbon, because it is again released into the

atmosphere. Kadam modeled a process where 2500 acres of cultivated algae can

process 210 ktons of CO2 a year. Growing algae with the addition of inorganic carbon

from waste CO2 presents an opportunity to recycle 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

pretreatment 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 microbes 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 process,

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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needed for planting and harvesting the crops. The microalgae offer the base the

opportunity to utilize biomass at a very fast rate with minimum waste material,

equipment.

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

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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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Table 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 areaa

Corn 70 623 846 Soybeans 180 240 326

Canola 482 90 122

Jatropha 766 57 77

Coconut 1088 40 54

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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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 to 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 tractor 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 need to store large quantities of biomass. Algal growth

is facilitated by an average of 1.2x1017 watts of sunlight hitting the Earth’s surface. Only

0.1-0.5% of solar energy is being captured by biological systems from photosynthesis,

leaving an abundance of 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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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

Algae Species

The algal species used in this study was supplied by Dr. Juergen 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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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 was used is commonly known as Waris Medium. The

components of 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

propagate 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 controlled 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 spectrum 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% CO2:98.5% air mixture 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 reactors were setup outside in direct sunlight. Supplementary

1.5% CO2:98.5% air mixture was bubbled through the pools to provide inorganic

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carbon. These pools had two underwater pumps running continuously to circulate the

cells, as well as one pump that would aerate 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

spectrophotometer 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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Harvesting

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 algae 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

flocculation, 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 when 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 <30 μm, which can only be collected using microfiltration processes. This

is only recommended for low volumes of algal slurry, and would be more cost efficient

32

than centrifugation for small production processes (American Water Works Association,

2011).

Electro-Flocculating

Electricity can be used to flocculate algae out of suspension using a small current

to disrupt the naturally occurring negative charges of algae cell surfaces (Banerjee, et

al., 2013). The current is produced using an electrode submerged in the algae slurry.

This method is an effective form of separation, 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 increase 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 harvesting 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 can degrade and spoil.

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 treated 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 concentration 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. This 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.

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 Growth

The doubling 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 cellular concentration of the flasks, so the algae cells can propagate again to

the initial concentration. Figure 2-5 is the cellular concentration of the algae during the

growth and splitting of the flasks over one month.

Algae Energy Content

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 that

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 full

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® fertilizer 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 flocculating 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 location 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 calculate 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 electricity 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

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 require 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 has 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 process. While the

Evodos is a more expensive process when the fully burdened cost of the machinery is

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 intensive. 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.

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)

Na2EDTA •2H2O 750 2 FeCl3 •6H2O 97 0.36 MnCl2 •4H2O 41 0.21 ZnCl2 5 0.037 CoCl2 •6H2O 0.2 0.0084 Na2MoO4 •2H2O 0.4 0.017

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

40

Figure 2-2. Base flocculating process flow diagram

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

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))

43

Figure 2-5. Centrifuge process flow diagram

44

Figure 2-6. Evodos water color comparison. (Photo courtesy of Emily Morton).

Inlet water

Outlet water

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

46

CHAPTER 3 ANAEROBIC DIGESTION

Anaerobic digestion (AD) is the conversion of organic materials to methane gas

through symbiotic relationships of microorganisms’ metabolic pathways. This 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

waste. 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 Energy 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. Table 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 conversion 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 it a viable option for algal biomass conversion. AD of algae

can be problematic because of the high proportion of proteins in the cells, causing a low

Carbon/Nitrogn (C/N) ratio. These high protein levels also may cause an increased

47

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 would have to be culitvated from the sediment of brackish swamps or

bogs (Brennan & Owende, 2010). Specific methane yields for carbohydrates, lipids, and

proteins 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 different species of microalgae based on the process parameters. This

table shows a significantly reduced yield when compared with the theoretical potential.

48

Objective

The United States Air Force is currently looking for alternative energy resources

that could 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 availability 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 into 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

49

liquid chromatography (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 reactor

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 was

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 reactor 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 release 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 from the University of

Florida, and has proven to be a reliable measurement tool (Koppar &

Pullammanappallil, 2008).

50

Microbial Inoculum

The anaerobic digestion inoculum was received from the University of Florida.

This thermophilic 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 bacterial consortium was received after an extended period of reduced

feeding. 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 source 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, the gas production

would stop and another dose of molasses 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 whether 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 sealed jar and oxygen scrubbing

packets.

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 composition 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

media in the form of GNC women’s mega® 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 liquid 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 pump. Figure 3-6 is the feeding

schedule of the molasses and algae paste weights.

Once the production rate 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 introduce 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 meter kept a count of each 0.67 L of biogas produced. Each ‘click’

count of the meter was noted daily.

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 would pump biogas

through the meter for 90 seconds. This tool measured CH4, O2, CO2, CO, and H2S 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 μL gas

sample was used a Supelco Analytical Scotty Gas biogas mix of 1%: H2, O2, CO, CH4,

CO2 with a 95% balance of N2 (Cat No. 22561) calibration gas was used.

Liquid characteristics

A YSI probe was used to measure the liquid characteristics of the digester. 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 readings 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 H2SO4 isocratic method with a flow rate of 0.5 mL min-1 and an

injection volume of 50 μL. This had a run time of 25 minutes. A 2 g L-1 sucrose solution

was used as a sugar baseline and comparison.

53

Results and Discussion

Gas Production

The gas production 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 Concentration

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 CO2 and CH4 display a healthy and

productive anaerobic system. The highest methane concentration was 66% the

correlating CO2 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 oxygen (DO), and pH were recorded. The changes in

pH are proportional to the CO2 in the system as seen in figure 3-8. The pH of the

system began at 8.8 and with the increased concentration of CO2, the pH decreased to

an average 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.

54

HPLC

The HPLC was able to show a transitioning sugar to alcohol pattern. The figures

3-10 through 3-16 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 values

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

changing 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 concentration 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 concentrations 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 pretreatment requirements, and ability to pump into the

digester. Anaerobic digestion is a plausible option for energy generation in remote

environments.

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)

56

Table 3-2. Specific methane yield for organic compounds in Chlorella vulgaris

Substrate Composition L CH4 g-1 VS

Proteins CH2.18O0.17N0.08 0.851 Lipids CH1.82O.011 1.014

Carbohydrates (CH1.67O0.83)n/6 0.4515

(Angelidaki and Sanders, 2004), (Becker, 2007)

57

Table 3-3. Gross composition of microalgae species and theoretical methane potential during anaerobic digestion of the total biomass

Species Proteins (%) Lipids (%) Carbs (%) CH4 (L CH4 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 26 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)

58

Table 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 CH4 g VS-1)

CH4

(%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

59

Table 3-5. Chemical composition of P-IV metal solution.

Component Amount (mg L-1 deionized water)

Final Concentration (mM) Na2EDTA •2H2O 750 2

FeCl3 •6H2O 97 0.36

MnCl2 •4H2O 41 0.21

ZnCl2 5 0.037

CoCl2 •6H2O 0.2 0.0084

Na2MoO4 •2H2O 0.4 0.017

60

Table 3-7. Trace vitamins and minerals found in multi vitamin.

Component Amount (g L-1 water)

nitrilotriacetic acid 12.8

FeCl3•6H2O 1.35

MnCl4•H2O 0.1

CoCl2 •6H2O 0.024

CaCl2 •2H2O 0.1

ZnCl2 0.1

CuCl2 •2H2O 0.025

H3BO3 0.01

Na2MoO4 •4H2O 0.024

NaCl 1.0

NiCl2 •6H2O 0.12

Na2-SeO3 •5H2O 4.0 mg

Na2WO4 •2H2O 4.0mg

(Suwannoppadol et al., 2012)

61

Figure 3-1. Pathway for the conversion of organic biomass to biogas through anaerobic

digestion processes.

62

Figure 3-2. Process Flow diagram.

63

Figure 3-3. Bench scale anaerobic digestion flasks successfully producing biogas. (Photo courtesy of Emily Morton)

64

Figure 3-4. Pilot scale reactor (Photo courtesy of Emily Morton)

65

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

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)

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

Cumulativemethane

Cumulative Methane (L)

68

Figure 3-8. Dissolved oxygen concentration 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

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

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

mA

U

time (d)

8.5 min (Fructose)

Digestate

Sucrose (2 g/L)

71

Figure 3-11. Liquid 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

mA

U

time (d)

10.0 minutes (Glucose)

Digestate

Sucrose (2 g/L)

72

Figure 3-12. 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

mA

U

time (d)

13.2 minutes (Lactic Acid)

Digestate

73

Figure 3-13. Liquid samples over time, HPLC retention time 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

mA

U

time (d)

15.4 minutes (Glycerol)

Digestate

Sucrose (2 g/L)

74

Figure 3-14. 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

mA

U

time (d)

19.36 minutes (acetate)

Digestate

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Figure 3-15. 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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Figure 3-16. Liquid 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

mA

U

time (d)

22.8 minutes (Ethanol)

Digestate

Sucrose (2 g/L)

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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 atmosphere 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 CO2 that is being released in the form of flue gas (United

States Department of State, 2007). Flue gas is primarily between 3% and 15% CO2,

noting that coal-fired power plants produce a higher percentage of CO2. Coal

combustion power plants account for 0.95 kg CO2 per 1kWh of electricity produced

(DOE, 2000).

Currently, the processes for collecting CO2 are limited by the high cost of

capture, the difficulty of storage, and the low efficiency of current technologies (Chi,

O'Fallon, & Chen, 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. petroleum industry has injected over 600 million tons of CO2 into wells, producing

245,000 barrels of oil per day (Meyer, 2010). This process uses the CO2 for the

enhanced recovery of oil, but it is released back into the environment after the liquid

petrol has been collected.

Algae

There has been a high interest in the use of microorganisms as a biofuel

resource because of the potentially high productivity and the possibility for CO2 fixation.

Specifically, both marine and freshwater species of microalgae are being utilized to

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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

2007). Photobioreactor production systems resulted 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). Flue gases contain NOx and SOx, which have proven not to be

harmful to the growth of the algae themselves, but they can cause a harmful change in

the pH of the aqueous media, resulting in slow growth or death (Packer, 2009). Packer

also states that an increased CO2 in bubbling gas from 0.038% to 1.0% is responsible

for doubling the algae biomass produced, which is why CO2 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 objective 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 in 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 CO2 decreased.

Materials and Methods

Four 500mL side-arm flasks were used as bioreactors to 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 for a 12 hour automatic

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 reactor; flask 2 had a

sealed balloon filled with the lab’s 1.5% CO2:98.5% air mix; flask 3 was just stopped

with a sponge to reduce evaporation; and flask 4 was a control flask where the 1.5%

CO2:98.5% 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 with

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 250μL gas sample was

used and a Supelco Analytical Scotty Gas mix of 1%: H2, O2, CO, CH4, CO2 with a 95%

balance of N2 (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 CO2/air mix.

.

80

Results and Discussion

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

figure 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 the 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 considered for any future work.

Flask 2, the flask with sealed CO2/Air mix, had the worst least amount of cellular

growth. The low initial concentration of 13% CO2 was did not provide enough inorganic

carbon to sustain the culture for the duration of the experiment.

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 systems

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.

81

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

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

H2 O2 CO N2 CH4 CO2

83

Figure 4-2. GC results from flask 2: CO2/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

H2 O2 CO N2 CH4 CO2

H2 O2 CO N2 CH4 CO2

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

85

Figure 4-4. Changing Optical density 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

86

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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 Science 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 to continue her research in the field of alternative energy

with an emphasis on biomass to renewable fuels.