Implementation of a Sustainable Process for Waste Disposal and Omega-3 Fatty Acid

Production

Richard Barton Nielsen

A Thesis in the Field of Biotechnology

for the degree of Master of Liberal Arts in Extension Studies

Harvard University

November 2014

© 2014 Richard Barton Nielsen

Abstract

Omega-3 fatty acids are popular nutritional supplements with a high global demand.

Currently, the primary method for harvesting omega-3’s is by extracting them from fish.

This practice is damaging to marine ecosystems and to the economic and social

infrastructures that support the fishing industry. Additionally, omega-3’s extracted from

fish are more likely to be contaminated with pollutants (e.g., mercury) and are not

suitable for vegetarian diets. Identifying alternative sources for omega-3’s are important

to decrease the negative impacts associated with utilizing fish. Omega-3 fatty acids can

be obtained from a variety of sources, including farmed microalgae. Exploiting

microalgae for omega-3 fatty acids would relieve some pressures placed on global fish

stocks. Farming microalgae also provides a healthier, more pure form of omega-3’s.

The primary objective of this project is to create a biotechnological system that

optimizes the use of microalgae farming for the production of omega-3 with

eicosapentaenoic acid (EPA). Our investigations generated previously unreported data

necessary for selecting a microalgae species, Nannochloropsis oculata, and for optimizing

its ability to produce omega-3 with EPA. Experiments testing optimal growth conditions

(e.g., temperature, CO2 concentration, light availability) and pest control methods were

conducted. Our results directed the construction of an algae farming process that

efficiently produces Nannochloropsis oculata with a high concentration of omega-3 with

EPA.

The second objective of this project is to build a sustainable system to support the

farming of Nannochloropsis oculata. Increasing the concentration of CO2 in the water

used for microalgae farming increases the growth rate of microalgae. Therefore,

identifying a stable source of CO2 is necessary and, as we have reported, widely available

in the form of biological waste. Transforming this waste into CO2 is possible by using

plasma gasification. Numerous benefits are derived through the use of plasma

gasification technology for generating CO2. Air pollutants are destroyed in the process

and landfill disposal is greatly reduced. Negative environmental and human health

impacts are significantly less than those of current waste disposal activities (landfilling

and incineration).

The successful implementation of our proposed waste-algae processing system for

the generation of omega-3 fatty acids shines light on a sustainable process that can

provide vast global benefits for the environment, economy, agriculture, and society.

v

Acknowledgments

My successful journey through the thesis process would not have been possible

without Dr. Ramon Sanchez. Ramon, you were a true guide and teacher who I hope to

continue learning from. You truly opened my eyes allowing me to understand the huge

potential that merging biotechnology and sustainability can have. I’ve learned that

sustainability is synonymous with efficiency. A sustainable system is efficient and the

use of biotechnology is a perfect medium to build such systems. The possibilities are

endless…

Encouragement to perceiver and finish my project came to me from numerous family

and friends. Thank you to dad, Richard, and my mom, Kathryn. Both are educators with

graduate degrees who gave me something to look up to. Dad, I wish you were here to

witness the completion of my project. Mom, I’m afraid to think of where I would be

without your constant encouragement. You never give up on me. You’re always there.

Thank you to the love of my life, Marisa Chattman, for being a perfect partner and

thesis editor.

Thank you to my sister, Laura, for being my closest family and a constant supporter.

To my friend Jason Costigan, thank you for motivating me to finish my project by

finishing your master’s degree program first!

vi

Lastly, special thanks to Carlos, Pedro, and Bamboo, the furry creatures who gave

me constant love and attention through the many late nights spent at my desk and on my

couch reading and writing.

vii

Table of Contents

Acknowledgments............................................................................................................... v

List of Tables ...................................................................................................................... x

List of Figures .................................................................................................................... xi

I. Introduction and Background ....................................................................................... 12

Aquaculture of microalgae to produce omega-3 fatty acids ................................. 12

Overfishing and Omega-3 Supply ........................................................................ 13

Waste in the Life Sciences .................................................................................... 14

Using Waste to Grow a Product............................................................................ 17

The Biology of Microalgae ....................................................................... 18

Health benefits of omega-3 fatty acids ..................................................... 19

Omega-3 Fatty Acid Production ............................................................... 20

Omega-3 Fatty Acid Production in Microalgae ........................................ 21

Industrial Processing of Omega-3 Fatty Acids in Microalgae .................. 22

Harvesting Microalgae .............................................................................. 26

Extraction and Purification of Omega-3 Fatty Acids from Microalgal

Biomass ..................................................................................................... 27

Managing Waste for a Carbon Dioxide Source .................................................... 28

Biological Waste ....................................................................................... 29

Waste Management Technologies ........................................................................ 33

Incineration ............................................................................................... 34

Plasma Gasification .................................................................................. 35

viii

II. Methods ....................................................................................................................... 38

Selecting a disposal technology ............................................................................ 39

Life Cycle Assessment .......................................................................................... 39

Life Cycle Assessment Goal ..................................................................... 39

Functional Unit ......................................................................................... 41

System Boundaries.................................................................................... 41

Determining LCIA .................................................................................... 42

Estimating volume of waste needed to sustain microalgae culturing system ....... 44

Creating a microalgae culturing system ................................................................ 44

Selecting the species of microalgae ...................................................................... 47

III. Results ........................................................................................................................ 49

Biohazardous Waste Composition ........................................................................ 49

Life cycle assessment of waste disposal technologies .......................................... 49

Estimation of carbon dioxide production from plasma gasification ..................... 53

Algae Species Selection and Estimation of Omega-3 Production ........................ 54

IV. Discussion and Conclusions ...................................................................................... 61

Description of the biological waste stream ........................................................... 61

Waste Disposal Technology Selection.................................................................. 65

A Stable Source of Carbon Dioxide ...................................................................... 69

Farming Nannochloropsis oculata ........................................................................ 71

Technology Innovations for Tomorrow ................................................................ 76

Concluding Remarks ............................................................................................. 80

ix

Appendix 1. ....................................................................................................................... 82

Appendix 2 ........................................................................................................................ 83

Appendix 3. ....................................................................................................................... 84

Appendix 4. ....................................................................................................................... 85

Appendix 5. ....................................................................................................................... 86

Appendix 6. ....................................................................................................................... 87

Appendix 7. ....................................................................................................................... 88

Appendix 8. ....................................................................................................................... 89

Appendix 9. ....................................................................................................................... 90

Appendix 10. ..................................................................................................................... 91

Appendix 11. ..................................................................................................................... 92

Appendix 12. ..................................................................................................................... 93

References ......................................................................................................................... 94

x

List of Tables

Table 1. Advantages and limitations of open ponds and photobioreactors.............. 18

Table 2. Summary of PUFA enrichment processes….…...……………………..… 22

Table 3. Classification of Infectious Microorganisms by Risk Group…....……..... 26

Table 4. Estimated Composition of Synthetic Gases from Plasma Gasification of

Biological Waste……………………………………………………..…... 48

xi

List of Figures

Figure 1. Examples of a bioprocess production chain in a microalgal biorefinery.

Apart from omega-3 fatty acids, the product portfolio includes biodiesel

and protein rich animal feed from the remaining

biomass……………………………………………………………...……. 16

Figure 2. Process diagram showing necessary steps for the conversion of waste to

omega-3 fatty acids……………………………………………………..... 32

Figure 3. System boundary of the comparative LCA study………..………...…….. 36

Figure 4. Racetrack design for a micro-algae pond………………………...………. 40

Figure 5. Small open ponds in Southern California used to conduct experiments to

determine optimal conditions for farming Nannochloropsis Oculata for

Omega-3 fatty acids……………...…………………………….....….…… 50

Figure 6. Picture of a “rotifer” micro-organism under the microscope (50X)…...… 52

Figure 7. Images of the inside of biological waste containers showing various

plastic and paper waste materials…………………………...…………..... 58

12

Chapter I

Introduction and Background

The expansion of human activities throughout the world has produced countless

innovations along with a long list of issues that negatively impact the environment and

ultimately human health and well-being. The human drive toward discovering better

technologies allows for the improvement of the quality of life but also for the mitigation

of the harmful effects that growing industries have on the environment locally, regionally

and globally. Measureable environmental impacts include the generation of greenhouse

gases, species endangerment and extinction, and the use of valuable resources such as

arable land and clean water. Various data collection programs and software innovations

allow for a much more comprehensive and detailed ability to measure environmental

impacts from industrial activities. The biotechnology industry thrives on innovation

however in the life sciences there are many activities that can be managed differently to

decrease the negative environmental impacts while achieving the same or better results.

Aquaculture of microalgae to produce omega-3 fatty acids

The industrial applications involving the use of microalgae have rapidly grown

with the expansion of new technologies. A significant push to use more biologically

based tools has been fueled by the rise in environmental issues, such as the abuse of

13

natural resources and global warming. Applications involving microalgae include

(Subashchandrabosea, Ramakrishnan, Megharaj, Venkateswarlu, & Naidu, 2013):

• use as a biofuel,

• use as a product for human nutrition,

• animal or aquaculture feed,

• creation of biochar for use as a biofertilizer,

• to create recombinant proteins that may be used the nutraceuticals, cosmetics, food

and feed industries, and

• as a source of polyunsaturated fatty acids (PUFAs).

Potential environmental benefits that can be harnessed during the algae producing activities

include: carbon sequestration and wastewater processing.

Overfishing and Omega-3 Supply

Commercial fishing has long been an important source of various materials

including protein, vitamins A and D, minerals, beneficial amino acids and long-chain

omega-3 fatty acids (Demars, 2012). Omega-3 fatty acids are not produced by the human

body but are essential for metabolism and must be consumed as part of the diet. They are

a popular supplement that is in high demand for its eicosapentaenoic acid (EPA) which

has been linked to numerous health benefits.

14

The high demand for this supplement has been shown to have a damaging impact

on the New England coastal ecosystem and food chain because species such as alewife,

Atlantic herring, and Atlantic menhaden are harvested in massive quantities. These

species play a vital role at the bottom of the food chain as a food source and as algae

eaters (Pew Environmental Group, 2007).

As the demand for supplements increases with our growing populations the need

for a sustainable source of omega-3 fatty acid oils becomes increasingly important if we

are to maintain a healthy ocean ecosystem. Microalgae farming is a source for omega-3

fatty acids that has great potential for large scale production.

Waste in the Life Sciences

Research and development of therapeutic solutions involves the use of

considerable resources and generates an equally significant amount of waste. Typical Life

Science facilities are involved in research with animals and laboratory scale chemical and

biological activities. There are also various administrative departments and common use

areas, such as cafeterias and office space, where paper, food, and various other related

wastes are generated. The efficient management of this material can become a difficult

task. A person must navigate through a sea of regulations, disposal and treatment options,

new and existing technologies, and vendors to determine the best options for minimizing

cost, maintaining or increasing operational efficiencies and minimizing the company’s

environmental footprint.

15

One of the common waste streams generated at a Life Sciences facility is

biohazardous waste. Biohazardous waste describes waste materials that are a biological

hazard to living organisms. It includes medical waste, which consists of infectious or

potentially infectious waste to humans, as well as plant and animal research wastes that

are potentially dangerous to those organisms, and genetically modified organisms that

may pose a threat to human, animal or environmental health (Mecklem & Neumann,

2003). According to the World Health Organization, biohazardous waste is the waste type

suspected to contain pathogens (bacteria, viruses, parasites, or fungi) in sufficient

concentration or quantity to cause disease in susceptible hosts (Prüss, Giroult, &

Rushbrook, 1999). These waste streams are believed to be high in organic content,

however, the composition of biotechnology biological waste has not been identified.

Regulatory oversight requires biohazardous wastes be destroyed either by thermal

decomposition or chemical treatment. Today, approximately 80% of the medical and

biohazardous waste in the US is disposed via offsite treatment (Forsman, 2013). This

waste is either transported to a medical waste incinerator for destruction or to an

autoclave to render the waste non-infectious. The ash from incineration and the solid

waste that is autoclaved is then landfilled. Currently, a significant portion of these wastes

are incinerated. This generates greenhouse gasses, primarily carbon dioxide, and renders

potentially energy rich wastes unrecoverable. Incineration activities also generate gas

emissions that contain various amounts of acid gas, carbon monoxide, lead, cadmium,

mercury, particulate matter, chlorinated dibenzodioxin, chlorinated dibenzofuran, NOx (a

generic term for mono-nitrogen oxides NO and NO2), and sulfur dioxide (SO2); all of

16

which are deemed hazardous air pollutants by the USEPA (United States Environmental

Protection Agency, n.d.).

Waste incineration is a relevant contributor of emissions that contribute to global

warming potential (GWP). These emissions include carbon dioxide, dinitrogen oxide, and

methane. Human activities have increased the concentration of greenhouse gasses (GHG)

in the atmosphere. This is expected to warm the Earth’s surface leading to climate

change. Efforts to slow the potential for climate change include measures to reduce the

emissions of CO2, reduce emissions of non-CO2 GHG’s and to promote carbon

sequestration.

A popular incineration option is to use a waste- to -energy facility for disposal.

This option uses plasma for the destruction of burnable wastes. Even though a significant

amount of carbon dioxide and ash are still generated, the use of plasma gasification

technology to dispose of wastes is becoming a more attractive option. The high heat of

plasma disintegrates materials to very basic components that can be utilized in the

production of fuels and other commercially viable products. These components are

syngas (a mixture of CO, H2, and CO2,) and slag (a mixture of metal oxides) (Kuo,

Wang, Tsai, & Wang, 2009). Commercial plasma gasification facilities in the US

generate energy from the heat released by plasma but they do not efficiently utilize the

gasification by-products.

17

Using Waste to Grow a Product

Currently the option to gasify waste biological waste does not exist. Should this

option become available in the future, a biotechnology company could divert organic

waste for gasification which would minimize emissions of environmentally harmful

pollutants and generate syngas that can be used to create useful products. Instead of

releasing the carbon rich syngas into the atmosphere it can be diverted to a

biotechnological process that can consume greenhouse gases and produce materials that

benefit the environment. This disposal technology could also be a source of negative

emissions because there are options for generating energy during the process.

The use of carbon capture and storage technology avoids the emissions of CO2 at

the generation site. This method is considered at WTE facilities where syngas and heat

are used to generate electricity. With society looking for ways to eliminate GHG

emissions, this option has been shown to be an effective method (Zeman, 2010). The

capture of CO2 from the plasma gasification process can then be transported and stored

for future use.

The carbon rich gasses generated from plasma gasification could be sequestered

to a living system that generates useful products. The culturing of algae has been shown

to use CO2 to enhance plant and microbial growth (Kumar, Dasgupta, Nayak, Lindblad,

& Das, 2011). One such system is the use of aquaculture to grow algae for the production

of omega-3 fatty acids. Enhancing the growth of algae using collected CO2 can have a

substantial impact on decreasing GHG emissions while generating a useful product that

18

successfully sequesters carbon that would otherwise be emitted to the atmosphere

(Adarme-Vega, et al., 2012). Furthermore innovative research can continue to enhance

the ability of algae to sequester CO2 and produce larger amounts of fatty acids. Various

methods to genetically engineer microalgae have been successful at optimizing

photosynthesis, generating higher yields of fatty acids and producing a greater amount of

biomass. Ultimately, a microalgae culturing system that utilizes the syngas from the

gasification process will generate a number of benefits including safe and compliant

disposal of biohazardous wastes, pollution reduction, carbon sequestration, reduced

demand on the fishing industry, and the generation of a health supplement in high

demand.

The Biology of Microalgae

Algae are primitive plants known as the oldest life-forms on earth due to their

lack of roots, stems, and leaves. They also have no sterile covering of cells around the

reproductive cells and have chlorophyll a as their primary photosynthetic pigment

(Brennan & Owende, 2010). Algae are simply evolved to efficiently convert energy

without robust cellular development, allowing them to adapt to changing environmental

conditions. Based on the International Code of Botanical Nomenclature, the phycologists

consider microalgae to be of both eukaryotic and prokaryotic (cyanobacteria) cell types

(Subashchandrabosea, Ramakrishnan, Megharaj, Venkateswarlu, & Naidu, 2013).

19

Algae are either autotrophic, heterotrophic, or mixotrophic. Heterotrophs are non-

photosynthetic and require an external source of organic compounds and nutrients as an

energy source for survival. Autotrophs require only inorganic carbon (e.g., carbon

dioxide), salts, and a light energy source for growth, a process known as photosynthesis

(Brennan & Owende, 2010). Autotrophic algae utilize photosynthesis to convert solar

radiation and carbon dioxide into adenosine triphosphate (ATP) and oxygen which is

used in respiration to produce energy supporting growth and propagation. Mixotrophic

algae have the ability to generate energy from photosynthesis and through acquisition of

exogenous organic nutrients (Brennan & Owende, 2010). Due to the ability of

autotrophic algae to fix atmospheric carbon dioxide during photosynthesis, a process

utilizing this type of algae is optimal for the purposes of this project.

Health benefits of omega-3 fatty acids

Omega-3 fatty acids are polyunsaturated fatty acids (PUFAs) which provide

significant health benefits to humans. The eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) have been found to be the most important fatty acids to

reduce cardiac diseases such as arrhythmia, stroke and high blood pressure as well as

offering beneficial effects to depression, rheumatoid arthritis and asthma (Tong, et al.,

2012) (Robinson & Stone, 2006) (Lee, O'Keefe, Lavie, & Harris, 2009) (Ross, Seguin, &

Sieswerda, 2007). In addition to the cardiovascular benefits, there is evidence indicating

omega-3 fatty acids enhance brain and nervous system function (Simopoulos & Bazan,

20

Omega-3 Fatty Acids, the Brain and Retina, 2009). When used as immunomodulators,

benefits have been observed during treatment of inflammatory diseases such as cystic

fibrosis, asthma, lupus, Crohn’s disease, ulcerative colitis, psoriasis, and rheumatoid

arthritis (Stenson, et al., 1992) (Simopoulos, 2002). These health promoting effects have

increased demand for microalgae in the pharmaceutical and nutraceutical industries.

Microalgal PUFAs and extracts are used in a variety of products including: infant

formulae, face and skin care applicants, anti-aging cream, sun protection cream, and anti-

irritant in peeler treatments (Spolaore, Joannis-Cassan, Duran, & Isambert, 2006).

Omega-3 Fatty Acid Production

Fatty fish, such as salmon, mullet and mackerel, are the primary source for these

fatty acids but utilizing fish as a source has numerous unattractive side effects. Over

fished stocks negatively impact the vital marine food chain (Buchsbaum, Pederson, &

Robinson, 2005). The use of fish also renders the supplements unsuitable for vegetarians

and lends to unattractive odors. Lastly, fish have also long been known to bio-accumulate

chemicals, such as mercury, which are harmful to consumers (Adarme-Vega, et al.,

2012). The benefits of consuming fish derived omega-3 fatty acids are well documented;

however, the negative effects indicate that alternate sources for these supplements should

be exploited (United States Food and Drug Administration).

Bacteria, fungi, plants and microalgae are being explored for commercial

production of omega-3 fatty acids. Fungi require an organic carbon source and have long

21

growth cycles while plants require arable land, have long growth cycles, and must be

genetically engineered to induce the production of PUFAs (Barclay, Meager, & Abril,

1994) (Ursin, 2003). Alternatively, microalgae have faster, natural grown cycles that can

be controlled under a variety of conditions. The growth of microalgae does not have to

depend on seasonal variations due to the technologies available that allow for year round

production. Additionally, microalgae fix carbon dioxide and can be grown on non-arable

land reinforcing its positive environmental impact (Brennan & Owende, 2010). A

comparison shown in Appendix 1 indicates microalgae generate higher concentrations of

PUFAs than other sources.

Omega-3 Fatty Acid Production in Microalgae

The high levels of oils, lipids, and fatty acids generated in marine microalgae are

closely linked to the algal growth stages and environmental conditions. Under poor

environmental conditions or during cell division, omega-3 fatty acids are accumulated

due to their high energy content and to assist with critical cellular functions (Cohen,

Khozin-Goldberg, Adlerstein, & Bigogno, 2000). This accumulation is initiated for

survival in response to growth limiting stresses such as UV radiation, temperature, and

nutrient deprivation (Adarme-Vega, et al., 2012). The production of omega-3 fatty acids

can be controlled by modifying growth conditions. For example, Pavlova lutheri

increased its relative EPA content from 20.3 to 30.3 % when the temperature was

decreased to 15°C (Tatsuzawa & Takizawa, 1995).

22

Another promising approach to increasing the production of omega-3 fatty acids

has been the use of genetic engineering (Schuhmann, Lim, & Schenk, 2012). More

research is needed to gain a better understanding of the mechanisms involved in the fatty

acid biosynthetic pathways in microalgae; however, genes have been identified for

encoding key enzymes in Ostreococcus tauri, Thalassiosira pseudonana, Phaeodactylum

tricornutum, and Chlamydomonas reinhardtii (Adarme-Vega, et al., 2012). Additional

gene-based actions for PUFA degradation inhibition remain exciting options as mutations

in one or more saturates may result in less efficient β-oxidation of PUFA and a higher

percentage of these fatty acids (Adarme-Vega, et al., 2012).

Industrial Processing of Omega-3 Fatty Acids in Microalgae

There is great potential in utilizing autotrophic microalgae for the production of

numerous materials, particularly omega-3 fatty acids, on a large scale. Figure 1 shows the

steps necessary for omega-3 fatty acid production in a microalgae biorefinery. Various

industries invest in microalgae production for the generation of nutraceutical and

pharmaceutical ingredients, biofuels, and protein-rich biomass (Adarme-Vega, et al.,

2012). The large scale production of autotrophic microalgae can be engineered using a

variety of differing technologies that include using open ponds or closed photobioreactors.

The use of hybrid facilities that combine both systems has also been explored with success

(Brennan & Owende, 2010).

23

Figure 1. Examples of a bioprocess production chain in a microalgal biorefinery. Apart

from omega-3 fatty acids, the product portfolio includes biodiesel and protein rich animal

feed from the remaining biomass (Adarme-Vega, et al., 2012).

Cultivation in open pond systems has been used since the 1950’s and can be

installed into natural waters and artificial ponds or containers. Open pond systems are

simpler and cheaper to manage than photobioreactor systems but are found to be less

efficient at producing biomass for a number of reasons (Table 1). Open pond systems are

more susceptible to contamination and pollution, may limit light exposure, can

Microalgae

Culturing

Harvesting

Lipid Extraction

Output

Ʊ-3BiodieselBiomass

Raceway Photobioreactor Open Pond

Solvent

Supercritical fluid extraction Winterization Distillation Transesterification

Filtration Flocculation Centrifugation

24

experience carbon dioxide deficiencies, and experience evaporative loss (Adarme-Vega,

et al., 2012). Also, poor mixing results in poor carbon dioxide transfer rates causing low

biomass productivity (Ugwu, Aoyagi, & Uchiyama, 2008).

Table 1. Advantages and limitations of open ponds and photobioreactors (Brennan &

Owende, 2010)

Production System Advantages Limitations

Raceway Pond

Relatively cheap Poor biomass productivity Easy to clean Large area of land required Utilizes non-agricultural land Limited to a few strains of algae Low energy inputs Poor mixing, light and CO2 utilization Easy Maintenance Cultures are easily contaminated

Tubular photobioreactor

Large illumination surface area Some degree of wall growth Suitable for outdoor cultures Fouling Relatively cheap Requires large land space Good biomass productivities Gradients of pH, dissolved oxygen and CO2 along

the tubes

Flat Plate Photobioreactor

High biomass productivities Difficult to scale-up Easy to sterilize Difficult temperature control Low oxygen build-up Small degree of hydrodynamic stress Readily tempered Some degree of wall growth Good light path Large illumination surface area Suitable for outdoor cultures

Column Photobioreactor

Compact Small illumination area High mass transfer Expensive compared to open ponds Low energy consumption Shear stress Good mixing with low shear

stress Sophisticated construction

Easy to sterilize Reduced photoinhibition and

photo-oxidation

Closed photobioreactor systems are known to generate high yields of algal

biomass with greater efficiency. The closed system allows for better contamination

control and permits the cultivation of microalgae for extended periods of time (Adarme-

Vega, et al., 2012). These systems currently exist as tubular, flat plate and column

25

photobioreactors. These straight glass or plastic tubular arrays capture sunlight and

recirculate algae cultures either with a mechanical pump or airlift system. Column

photobioreactors are arguably the most attractive technology for microalgae production

as they allow for more efficient mixing, offer the highest volumetric mass transfer rates

and the best conditions for cultivation (Eriksen, 2008). They are also low cost, compact

and easy to operate (Adarme-Vega, et al., 2012).

The increased amount of research into the closed bioreactor systems is very likely

due to the greater degree of control that closed systems have over the open systems. This

allows for higher biomass production rates and therefore greater generation of desirable

algae products, such as omega-3 fatty acids (Appendix 2).

Optimizing the photosynthesis of microalgae will allow for a greater yield in algal

oils and biomass. Various methods for enhancing photosynthesis using genetic

engineering have been proposed. One method involves genetically engineering algal

species to produce photosynthetic pigments that would allow for a greater amount of the

light spectrum to be absorbed for energy production. Typical biological systems harness

radiation in the wavelength range of 400-700 nm. By engineering the pigments

Chlorophyll f (706 nm), Chlorophyll d (710 nm), and bacteriochlorophyll (700-1000 nm)

into microalgae a greater range of radiation will be available for photosynthesis (Chew &

Bryant, 2007) (Chen, et al., 2010).

26

Harvesting Microalgae

There are various harvesting technologies available for microalgae producers.

Selecting the proper technology is important to accumulating higher levels of biomass

and is dependent on the species of microalgae being cultivated. The processes available

include flocculation, filtration, flotation, and centrifugal sedimentation and involve the

bulk harvesting of the microalgae followed by thickening of the accumulated slurry

(Adarme-Vega, et al., 2012). The selection of a harvesting technology should attempt to

capture the following features (Uduman, Qi, Danquah, Forde, & Hoadley, 2010):

• Low energy consumption,

• Complete recycling of water and nutrients,

• No addition of harmful chemicals/materials, and

• A compact unit of small-foot print.

Flocculation is a step taken prior to the algae harvesting that is necessary to

concentrate the algae. Algae, which carry a negative charge, do not aggregate naturally in

suspension and by adding flocculants the charge repulsion is overcome. Flocculants are

multivalent cations and cationic polymers that neutralize this charge enhancing the ability

of the algae to aggregate (Brennan & Owende, 2010). Several flocculation harvesting

methods have been tested and shown to be efficient, however there is a lack of

information and comparative studies for micro-algae (Uduman, Qi, Danquah, Forde, &

Hoadley, 2010). Centrifugation is a rapid and energy intensive harvesting technology

(Uduman, Qi, Danquah, Forde, & Hoadley, 2010). It is considered an efficient and

27

reliable method but higher energy and maintenance costs persist. Biomass filtration may

be used for larger (>70µm) or smaller (<30µm) microalgae. Larger microalgae may be

harvested by conventional filtration, which operates under pressure or suction. Membrane

microfiltration and ultra-filtration methods used for smaller microalgae have been found

to be more cost effective than centrifugation when processing low volumes (<2m3)

(Uduman, Qi, Danquah, Forde, & Hoadley, 2010). The cost of membrane replacement

and pumping required for large scale operations (>20m3) indicate that centrifugation may

be a more cost effective method for algal biomass harvesting (Uduman, Qi, Danquah,

Forde, & Hoadley, 2010).

Extraction and Purification of Omega-3 Fatty Acids from Microalgal Biomass

Prior to lipid extraction the harvested microalgal biomass must be dewatered and

dried (Adarme-Vega, et al., 2012). Methods used include sun drying, low-pressure shelf

drying, spray drying, drum drying, fluidized bed drying, freeze drying and Refractance

Window technology drying (Brennan & Owende, 2010). After dehydrating the biomass a

solvent based extraction method is used. The solvent used varies depending on the scale

of the extraction. Smaller operations typically use mixtures of methanol and chloroform

for lysing cells and lipid extraction, while larger scale extractions typically use hexane

(Adarme-Vega, et al., 2012). This is followed by separation of the unsaturated fatty acids

from the total lipids by fractional distillation or winterization. Additional technologies

28

(Table 2) are used to further enrich and purify the PUFA, particularly when used to

produce products intended for human consumption (Adarme-Vega, et al., 2012).

Table 2. Summary of PUFA enrichment processes (Adarme-Vega, et al., 2012)

Method Procedure Molecular distillation (Fractional distillation) Purification of fatty acid esters in a vacuum

system based on the different boiling points of different fatty acids.

Molecular sieves Separation via membrane permeability and selectivity.

PUFA transformations Esterification of PUFA and free fatty acids to produce esters (ethyl-, glyceryl-, sugar-, other). Inter-esterification to enrich lowly unsaturated fatty acids with PUFA.

Super Critical Fluid Extraction Optimization of lipid solubility and fractionation in supercritical CO2.

Urea Complexation Solubilization of fatty acids, adding urea and ethanol to saturation point exposing it to heat. Recovery of product by filtration.

Winterization Temperature reduction to render more saturated fats insoluble.

Managing Waste for a Carbon Dioxide Source

The first step of waste management is to complete waste identifications. This

crucial first step is necessary to determine the components contained in waste and

subsequently, how those components can be managed and disposed. This process must be

in compliance with various regulatory entities and utilize the best demonstrated available

technology to decrease the environmental impact of disposal. A very broad way of

initially segregating waste types is to categorize by radioactive, chemical, biological, or

non-hazardous characteristics.

29

Biological Waste

Biological waste is known by numerous terms. It is common to hear it referred to

as medical waste, hazardous medical waste, healthcare waste, and biohazardous waste.

For the purposes of this project we will use the term “biological waste” which includes

wastes that are infectious (samples or cultures known to be infectious in healthy human

adults), potentially infectious (uncharacterized human or non-human primate tissue or

body fluid samples), and non-infectious (samples known to not be infectious in healthy

human adults). The management of biological wastes in the life sciences typically results

in offsite disposal of such wastes via incineration. There an estimated 33 medical waste

incinerators in the US, all which utilize fossil fuels to power their destruction activities

(Hambrick, 2013). State authorities usually regulate biological waste. The EPA has

released a guidance document to assist states in the implementation of their biological

waste regulations, though it has not been updated since its original publication in 1992.

The Center for Disease Control and the National Institutes of Health have guidelines for

the management of a biological safety program which includes the management of

biological wastes. The state of Massachusetts defines biological waste as (Department of

Public Health, 2007):

Waste that because of its characteristics may cause, or significantly contribute to,

an increase in mortality or an increase in serious irreversible or incapacitating

reversible illness; or pose a substantial present potential hazard to human health or

the environment when improperly treated, stored, transported, disposed of, or

otherwise managed.

30

Massachusetts also identifies and defines the following types of waste as biological waste

(Department of Public Health, 2007):

(1) Blood and Blood Products. Discarded bulk human blood and blood products in free

draining, liquid state; body fluids contaminated with visible blood; and materials

saturated/dripping with blood. Blood Products shall not include; feminine hygiene

products.

(2) Pathological Waste. Human anatomical parts, organs, tissues and body fluids

removed and discarded during surgery, autopsy, or other medical or diagnostic

procedures; specimens of body fluids and their containers; and discarded material

saturated with body fluids other than urine. Pathological waste shall not include:

Teeth and contiguous structures of bone without visible tissue, nasal secretions,

sweat, sputum, vomit, urine, or fecal materials that do not contain visible blood or

involve confirmed diagnosis of infectious disease.

(3) Cultures and Stocks of Infectious Agents and Associated Biologicals. All discarded

cultures and stocks of infectious agents and associated biologicals, including

culture dishes and devices used to transfer, inoculate, and mix cultures, as well as

discarded live and attenuated vaccines intended for human use, that are generated

in:

(a) Laboratories involved in basic and applied research;

(b) Laboratories intended for educational instruction; or

(c) Clinical laboratories

31

(4) Contaminated Animal Waste. Contaminated carcasses, body parts, body fluids,

blood or bedding from animals known to be:

(a) Infected with agents of the following specific zoonotic diseases that are

reportable to the Massachusetts Department of Agricultural Resources, Bureau

of Animal Health pursuant to 105 CMR 300.140: African swine fever, Anthrax,

Avian influenza – H5 and H7 strains and any highly pathogenic strain, Bovine

spongiform encephalopathy (BSE), Brucellosis, Chronic wasting disease of

cervids, Foot and mouth disease, Glanders, Exotic Newcastle disease, Plague

(Yersinia pestis), Q Fever (Coxiella burnetti), Scrapie, Tuberculosis, Tularemia

(Francisella tularensis); or

(b) Infected with diseases designated by the State Epidemiologist and the State

Public Health Veterinarian as presenting a risk to human health; or

(c) Inoculated with infectious agents for purposes including, but not limited to, the

production of biologicals or pharmaceutical testing.

(5) Sharps. Discarded medical articles that may cause puncture or cuts, including, but

not limited to, all needles, syringes, lancets, pen needles, Pasteur pipettes, broken

medical glassware/plasticware, scalpel blades, suture needles, dental wires, and

disposable razors used in connection with a medical procedure.

(6) Biotechnology By-product Effluents. Any discarded preparations, liquids, cultures,

contaminated solutions made from microorganisms and their products including

genetically altered living microorganisms and their products.

32

The last step in characterizing medical waste is to evaluate the level of risk posed

by the known biological agent in the waste materials. The NIH and WHO ranks all

biological agents into risk groups (Table 3).

Table 3. Classification of Infectious Microorganisms by Risk Group

Risk Group

Classification

NIH Guidelines for Research involving

Recombinant DNA Molecules 20022

World Health Organization Laboratory Biosafety

Manual 3rd Edition 20041

Risk Group 1 Agents not associated with disease in

healthy adult humans.

(No or low individual and community risk) A

microorganism unlikely to cause human or animal

disease.

Risk Group 2 Agents associated with human disease that

is rarely serious and for which preventive or

therapeutic interventions are often available.

(Moderate individual risk; low community risk) A

pathogen that can cause human or animal disease

but is unlikely to be a serious hazard to laboratory

workers, the community, livestock or the

environment. Laboratory exposures may cause

serious infection, but effective treatment and

preventive measures are available and the risk of

spread of infection is limited.

Risk Group 3 Agents associated with serious or lethal

human disease for which preventive or

therapeutic interventions may be available

(high individual risk but low community

risk).

(High individual risk; low community risk) A

pathogen that usually causes serious human or

animal disease but does not ordinarily spread from

one infected individual to another. Effective

treatment and preventive measures are available.

Risk Group 4 Agents likely to cause serious or lethal

human disease for which preventive or

therapeutic interventions are not usually

available (high individual risk and high

community risk).

(High individual and community risk) A pathogen

that usually causes serious human or animal disease

and can be readily transmitted from one individual

to another, directly or indirectly. Effective treatment

and preventive measures are not usually available.

1. World Health Organization. Laboratory biosafety manual. 3rd ed. Geneva; 2004.

2. The National Institutes of Health (US), Office of Biotechnology Activities. NIH guidelines for research involving

recombinant DNA molecules. Bethesda; 2002, April.

All wastes determined to contain Risk Group 2 – 4 materials must be disinfected

to render the materials non-infectious or non-biohazardous. Movements to have validated

33

procedures for the inactivation of biological agents are growing, as seen in current

agreements such as the CEN Workshop Agreement (CWA) 158793. Disinfection

typically occurs via thermal steam and pressure treatment (autoclave), chemical

treatment, or incineration. Some disinfection activities take place at the site of the waste

generation but much of the waste is shipped to disposal facilities for incineration.

Incineration facilities generate gas emissions that contain various amounts of acid gas,

carbon monoxide, lead, cadmium, mercury, particulate matter, chlorinated dibenzodioxin,

chlorinated dibenzofuran, NOx, and sulfur dioxide (SO2) (Vergara & Tchobanoglous,

2012). These emissions products are common during incineration activities that involve

the use of fossil fuels and are tracked and controlled by the US EPA.

Waste Management Technologies

There are a number of different ways to manage waste after collection. Various

technologies can be harnessed to transform waste into useful products. These methods

can reduce the amount of waste requiring disposal and can recover resources and energy.

One method uses biological systems to convert the organic fraction of waste (biogenic

wastes) into energy and soil amendments. Soil amendments are known to be material

such as lime, gypsum, sawdust, compost, animal manures, crop residue or synthetic soil

conditioners that are worked into the soil or applied on the surface to enhance plant

growth. Amendments may contain important fertilizer elements but the term commonly

refers to added materials other than those used primarily as fertilizers. The degradation of

34

organic wastes occurs naturally and a thorough understanding of this microbiological

process can allow for the extraction of useful resources and the diversion of materials that

may be harmful to human and environmental health. A second method utilizes non-

biological processes to recover materials or energy. Also known as non-biogenic waste

transformation, this method includes incineration, pyrolysis, plasma gasification, and

recycling.

Incineration

Incineration is the thermal treatment of organic wastes using carbon-based fuels

that results in the generation of ash, air emissions (NOX, CO, CO2, SO2, PM, dioxins,

furans, and others), heat, and energy (Vergara & Tchobanoglous, 2012). This process

reduces the volume of solid waste by 80-85% and allows for energy recovery when the

proper technologies are in place (Quina, Bordado, & Quinta-Ferreira, 2008). However,

the ash and air pollutants emitted represent an environmental burden. Modern

incinerators have pollution controls that can lower the pollutant emissions to meet

regulatory standards. Cyclones, electrostatic precipitators, and fabric filters remove

particulate matter from the flue gas; scrubbers remove acid gases; catalytic reduction and

temperature control minimize NOX emissions; and activated carbon removes dioxins,

furans, and heavy metals from the flue gas (Quina, Bordado, & Quinta-Ferreira, 2008).

The ash consists of fly ash and bottom ash. The fly ash constitutes more of a health

hazard than does the bottom ash because the fly ash often contains high concentrations of

35

heavy metals such as lead, cadmium, copper and zinc as well as small amounts of dioxins

and furans (Chan & Kirk, 1999).

Plasma Gasification

Plasma gasification technology is not new but it is emerging as a disposal option

that can provide solutions to numerous environmental, social, and economic issues. A

growing world population means increased demands for more energy and resources.

Landfills continue to be dead end dumping grounds for solid waste. The use of plasma

gasification can alleviate the burden placed on landfills since such wastes can be utilized

as fuel. The gasification process can utilize a variety of carbon based materials such as

garbage, plant material, hazardous, and biological wastes.

Plasma is known as the 4th state of matter after solid, liquid and gas. It is created

when gases are superheated allowing them to become electrically conductive, such as in

lightening or on the surface of the sun (Prüss, Giroult, & Rushbrook, 1999). Plasma

technology involves passing electrical current through a gas generating heat due to

electrical resistivity. This process generates plasma, an ionized gas stream that has a

liquid-like viscosity and conductivity that can approach those of metals (Auciello &

Flamm, 1989). The temperatures of a plasma arc can reach 10,000 °F creating a system

capable of destroying any substance found on Earth with the exception of radioactive

materials.

36

There are various applications that use plasma technologies including (Auciello &

Flamm, 1989):

a) Coating techniques, such as plasma spraying, wire arc spraying and thermal

plasma chemical vapor deposition (TPCVD);

b) Synthesis of fine powders, in the nanometer size range;

c) Metallurgy, including clean melting and re-melting applications in large furnaces;

d) Extractive metallurgy including smelting operations;

e) Destruction and treatment of hazardous and non-hazardous waste materials.

The use of plasma gasification for waste disposal is very attractive due to its

ability to destroy the solid matter and transform it into basic components: syngas and

slag. Slag is a glass-like solid material composed of the inorganic elements present in

plasma treated wastes. The composition of slag changes depending on the nature of the

treated waste however it is usually composed of metals and various oxides such as

aluminum oxide (Al2O3), calcium oxide (CaO), silicon oxide (SiO2), iron oxide (Fe2O3),

sodium oxide (Na2O) and magnesium oxide (MgO) (Demars, 2012) (Byun, et al., 2010).

The volumetric reduction of waste to slag from plasma gasification is up to 99%, a

significantly greater proportion than a conventional waste incinerator utilizing a fuel

burning system which reduces the volume of waste by 90% (Bie, Li, & Wang, 2007).

Another benefit of generating slag containing heavy metals and other contaminants is that

the hazardous components are effectively immobilized thus keeping them from leaching

out into the environment (Bie, Li, & Wang, 2007).

37

Syngas consists of carbon monoxide (CO), hydrogen gas (H2), and carbon dioxide

(CO2). Recent advances in plasma gasification technologies allow for the generation of a

much cleaner syngas allowing it to be used safely to produce fuels and other products.

The typical plasma gasification process (Appendix 3) utilizes a plasma gasifier,

the chamber where organic waste is fed to the plasma torches. In this gasifier the plasma

jets are located at the bottom where they generate sufficient heat for the gasification of

waste to occur. As the waste descends through the chamber it is converted to gas and

liquid slag. The gas generated is also known as syngas and consists of CO, H2, and small

amounts of CO2. The syngas is then passed through a secondary combustion chamber

where it is converted to CO2 and water.

38

Chapter II

Methods

The primary objective of this project was to create a process to sequester carbon

rich gas emissions into algae cultivated with the intention of producing omega-3 fatty

acids in Southern California. This process combines microalgae production and

biohazardous waste disposal to create a more sustainable and productive system.

To build an efficient system for converting biohazardous waste into omega-3 fatty

acids by utilizing plasma gasification and algae farming technologies the process shown

in Figure 2 was created.

Figure 2. Process diagram showing necessary steps for the conversion of waste to

omega-3 fatty acids.

• Collection• Transportation

Biohazardous Waste

CO2 Capture and Storage

Plasma Gasification

• Carbon Sequestration

• Role of Biotechnology

• Culturing & Harvesting

Algae Farming

• Extraction from algae biomass

• Purification

Omega-3 Fatty Acids

39

Selecting a disposal technology

Biological waste disposal technologies have not experienced much change since

the EPA released its guidance document for biohazardous waste management to states in

1992. Current methods involve incineration, chemical disinfection and thermal and

pressure treatment. Plasma gasification is not used for the incineration of biological waste

however if used efficiently such a technology could be a valuable option for waste

generators.

Using life cycle assessments (LCAs) municipal incineration, plasma gasification

and autoclaving/landfilling were compared to measure their environmental impacts.

Life Cycle Assessment

Life Cycle Assessment Goal

The process known as life cycle assessment (LCA) is used to evaluate the GHG

emissions throughout the full product or service life cycle. The International Standards

Organization (ISO) has developed standards for conducting LCAs that can be applied to

industrial activities and associated GHG emissions, capture, and sequestration. LCA is a

methodology that provides certain principles and framework to analyze the

transformation processes and infrastructure required to produce the main products and

co-products for an energy production operation in order to estimate its environmental

impacts. The use of this technique for assessing environmental impacts involves: 1)

generating an inventory of linked energy and material inputs and environmental releases,

40

2) Evaluating the possible environmental impacts associated with the inputs and releases,

and 3) Interpreting the results to support more informed decision making (Scientific

Applications International Corporation, 2006).

The LCA analysis of the environmental performance of a waste disposal system should

include emissions from the system being evaluated, emissions from other sources

indirectly linked with the system under evaluation, emissions from production processes

of electricity used in the system being evaluated, all emissions avoided, and emissions

from processes of recycling and benefits, or emissions avoided because of replaced virgin

production of materials by the recycling processes (Pikoń & Gaska, 2012). The

production of one material often involves production of byproducts or waste. When a

byproduct is used to replace another product from a different process, a portion of

environmental damage can be avoided. The values for the avoided damage can be

calculated using the LCA approach and are termed “displacement credits” (Pikoń &

Gaska, 2012). LCA has several sequential steps in which the goal and scope of the

project are defined, then a Life-Cycle Inventory (LCI) is performed. This LCI is used to

assess impacts using a set of environmental damage indicators. Results are compiled and

interpreted to compare environmental impacts of different projects in an objective way

(Bauman & Tillman, 2004). A LCIA measures the link between the process and its

potential environmental impacts. This should address ecological and human effects as

well as resource depletion (Scientific Applications International Corporation, 2006).

Using these systems we will measure the environmental damage mitigated by using the

alternative technology, plasma gasification.

41

Functional Unit

The use of a functional unit in the LCA is necessary to compare two or more

products or services. The functional unit should describe the function of the services

being compared (Scientific Applications International Corporation, 2006). The LCA for

this project will compare the disposal of biohazardous waste by plasma gasification,

autoclave/landfill, and incineration. The functional unit for this study has been defined as:

“The disposal of 8129.3 Kg of biohazardous waste.”1

The use of the functional unit has allowed for the calculation the amount of

carbon rich gas that can be sequestered during the algae culturing process.

System Boundaries

The geographical boundaries of this study will be limited to Southern California.

The boundaries of the technical system are shown in Figure 3. Within the boundary the 3

options include cradle-to-grave analysis of the collection and disposal of waste and the

impact of gas capture. Options B and C allow for the direct comparison between existing

waste disposal processes while Option A is the proposed alternative process which was

used to demonstrate the impact of gas capture on the process LCA.

1 This unit was derived from disposal metrics of a medium sized (~300 employees and ~100,000

ft2 laboratory space) biotechnology company located in Cambridge, MA, throughout 2012.

42

Determining LCIA

The results of an LCIA should show the relative differences in potential impacts

for each option shown in Figure 3 and will provide an estimation of health and

environmental effects of omega-3 fatty acid production using carbon from plasma treated

waste to farm algae.

Figure 3. System boundary of the comparative LCA study

Life-Cycle Inventories (LCI) for pollutant emissions from regular (business-as-

usual) operations were estimated using libraries of average pollution emissions waste

processing and disposal contained in the EcoInvent 2.2 database within the OpenLCA V.

1.4 software. Average values come from the inventories of several thousands of

operations which are recorded in these libraries. Then, a life cycle inventory was made

Option A

100% Plasma Gasification

Collection of BHW

Plasma Gasification

Gas capture via microalgae culturing

Option B

100% Regular Incineration

Collection of BHW

Regular Incineration

Gas capture via microalgae culturing

Option C

Landfill

Collection of BHW

Autoclave

Landfill

No gas capture

43

using plasma gasification and municipal incineration as a source of carbon dioxide. Life-

cycle inventories come from all pollutant emissions registered in LCI libraries for each of

these components. The end result of this first part of the analysis is a set of life-cycle

pollutant emissions (midpoint indicators) per functional unit of waste and microalgae

omega-3.

The life-cycle emissions per functional unit were used to perform an end-point

environmental damage assessment using global settings with the ReCiPe Method to

estimate overall damages to human health, ecosystem diversity and resources cost from

each operation. ReCiPe is an assessment method for environmental impacts created in

2008 that integrates a comprehensive set of damage functions and calculation methods

into a structured non-software based methodology. This LCA methodology has open

source information and values that can be modified to function in a regional scale. The

use of the ReCiPe method with world normalization provides preliminary values of

comparison between production projects. The units for these endpoint indicators are

Disability Adjusted Life Years (DALYs) for Human Health, Species.Year for Ecosystem

Diversity and $2010 USD for Resources Cost of depleting resources for future

generations. In this way these units are easily transferable for other health and

environmental analyses that require information about environmental impacts of waste

disposal and omega-3 fatty acid production operations.

44

Estimating volume of waste needed to sustain microalgae culturing system

It will be important to calculate the volume of synthetic gas generated as this will

be a factor in calculating the biomass production potential of the microalgae that will

sequester the CO2. Gas volume from the incineration process will be estimated by

creating 1 ton of a mix of biohazardous waste with the same percentage composition to

the one estimated for the company in the case study. This biohazardous waste will be

gasified using a bench plasma torch built by using regular welding electrodes attached to

a ceramic container. Parameters from a Westinghouse Plasma incinerator in a poor

oxygen atmosphere will be used to emulate conditions of a full size prototype. Samples

of the resulting synthetic gas will be collected and analyzed with a gas chromatographer.

All gases and compounds with a 0.1% by weight or more of the total weight of the

sample will be listed as the main components of the synthetic gas. This gas composition

will be reproduced in the lab and used in a bench gas turbine in a pure oxygen

atmosphere in order to determine the amount of carbon dioxide produced by the process.

We will determine total CO2 generated from the functional unit by using this information.

Creating a microalgae culturing system

The fact that algae are potential sources for various oils and supplements is well

known. However, to create a sustainable process that generates sufficient amounts of

algae, cultivation and harvesting must be designed in a very specific manner. Microalgae

production is enhanced using a source of CO2. Conventional electric power generation

45

with fossil fuels is a common source as well as waste incineration activities that utilize

either fossil fuels or plasma for waste destruction. Carbon rich emissions, known as

syngas, are generated by plasma gasification of biological wastes. CO2 is captured from

post-combustion gases using a monoethanolamine (MEA) plant where flue gases and

solvent (30% MEA solution) are mixed. MEA reacts with and captures CO2 so that the

gases can be transported for use at a microalgae production facility. In order to efficiently

utilize the trapped gas, the gasification facility should be near or part of the facility that

will cultivate and process the algae for the production of omega-3 fatty acids to minimize

costs associated with gas capture activities and transportation. In order to release the CO2

the MEA is heated which liberates the trapped gas which can then be fed directly to

algae. The CO2 recovery efficiency is approximately 91.2% (Al-Juaied & Whitmore,

2009) (Gonzalez-Diaz, et al., 2010).

Prior to adding CO2, microalgae ponds are built with a racetrack design and a

depth of 0.4 m (Figure 4), covered with a plastic liner, filled with pre-treated saltwater

and supplied with a water mixing system. A water mixing system is necessary to

maintain microalgae cells in suspension, to prevent thermal stratification, provide

uniform sunlight absorption for all microalgae cells in the pond and to disperse nutrients

(Natural Resources Defense Council, 2009). Water is pre-treated before entering the

system in order to avoid the formation of parasitic bio-mass that might use this rich

nutrient medium to overwhelm useful algae.

46

Figure 4. Racetrack design for a micro-algae pond.

Microalgae strains are isolated from water samples in nature and cultured in

laboratory conditions. The algae ponds are inoculated with this algae culture and

exposed to sunlight. The mixing system is activated after inoculation and carbon dioxide

is added to the saltwater in a gaseous form as the final step of water treatment.

Algae biomass is harvested daily by extracting 30 to 35 % of the water with

microalgae from the pond (1000 to 1200 m3/Hectare*day). Saltwater restocking is

continuous to compensate for water losses during harvesting and daily evaporation

(Benemann & Oswalk, 1996). Fresh or brackish water is treated and mixed with

saltwater to compensate for increases in salt concentration due to daily evaporation

(Energy and Environmental Research Center, 2002).

47

Production yields are highly dependent daily fluctuations in physical conditions

and potential parasite contamination. Biomass will be separated from water by using a

centrifuge and electroflocculation (water passes through electrodes and polarizes the cell

wall of algae which tends to agglomerate so it is easy to extract it).

Selecting the species of microalgae

Considerations must also be made for the species of algae utilized. The ideal

species of autotrophic algae will efficiently harness light and carbon enriched media.

Experiments were conducted to identify the algal species that would most efficiently

produce EPA. Small scale experimentation to determine optimal species and growth

conditions has shown that cold water algae is likely to be an ideal candidate for growth

under controlled conditions for the production of omega-3 fatty acids (Fang, Wei, Zhao-

Ling, & Fan, 2004). For that reason, water samples will be taken from the sea in close

proximity to the coasts of California and Baja California and reviewed under the

microscope. Algae species will be isolated and identified. Then their fat, protein and ash

content will be estimated. Algae with the highest lipid content will be cultured in a

laboratory and types of lipids will be characterized to determine the species with the

highest concentration of unsaturated fats, particularly omega-3 fatty acids with EPA.

The algae species with the highest omega-3 content will be cultured in a lab, its species

and/or family will be recorded if identified. However, if the algae species is a new strain

it will be catalogued and assigned a number, then a sample will be sent to a specialized

48

phycology (algae science) research laboratory for further family identification using

DNA sequencing. A 5,000 –liter open pond will be inoculated with the selected species.

Operating conditions will be continuously recorded and production rates will be

estimated daily for at least 15 days. The most likely conditions that influence algae

biomass production are nutrient quality and quantity, carbon dioxide concentration in

water, solar irradiation, culture media pH, turbulence, salinity, and water temperature.

Variations in these factors are likely to affect algae lipid production differently depending

on the algae species being cultured. For that reason, the significance of the most relevant

operating parameters and practices will be determined and characterized in the

production of omega-3 fatty acids with EPA.

49

Chapter III

Results

Biohazardous Waste Composition

A biotechnology research company that employs approximately 300 people and

that has approximately 100,000 ft2 of laboratory space generates an annual average of

1129 biohazardous waste containers for disposal. The maximum weight that is permitted

per container is 50 pounds (22.7 kilograms). The average weight of each biohazardous

waste container was determined to be 15.86 pounds (7.20 kilograms).

Appendix 4 shows the results from the sampling of biohazardous waste containers

from a biotechnology research facility.

The calculation of the total annual average biohazardous waste (Appendix 4) is

the Functional Unit for all further LCA activities. Average composition for waste in the

Functional Unit is maintained in all calculations.

Life cycle assessment of waste disposal technologies

Using LCA software waste disposal technologies were evaluated to determine the

LCIA for each disposal option: regular incineration, landfill and plasma gasification.

Environmental impacts’ estimations include the process of incinerating and disposing of

8129.3 Kg of biohazardous waste, it does not include transportation and “legacy”

50

environmental impacts, therefore all environmental impacts for raw materials extraction,

supply activities, manufacturing, warehousing, distribution and use of these items are not

considered in this analysis because pre-waste damages should be assigned to the useful

life of the products. This analysis only considers the disposal phase for the product, there

is no recycling involved because that activity would involve unnecessary health risks for

the general population as biological waste materials might be a source of potential

contagions for waste processing workers. All scenarios will use the same assumptions

dealing only with safe disposal activities for biological waste, so they can be compared

objectively.

The Landfill Scenario requires neutralization of biological threats before sending

biological waste to the landfill. Potential biological contamination is treated using an

autoclave that uses pressured steam at temperatures above 100 °C to kill any potential

pathogens attached to biological waste. The autoclave cycle assumed for the LCA is:

• Start at room temperature and increase temperature from 25 °C to 100 °C

• Increase pressure relative to the atmosphere from 0 to 1.7kg/cm2 at 100 °C

• Maintain sterilization at 121 °C and 1.7kg/cm2 for 15 minutes

• Bring materials back to atmospheric pressure and drop temperature to 100 °C

• Reduce temperature further to 50 °C

These biologically neutral materials can be disposed in a landfill after the autoclave

treatment.

51

Disposal of biological waste using plasma gasification includes the molecular

disintegration of waste and the generation of inert slag and syngas emissions. A bench

plasma gasification device using a comparable composition of biohazardous waste

(Appendix 4) is used to simulate real working conditions for a large-scale plasma

gasification system. Exhaust gases from plasma gasification are analyzed to determine

their composition and caloric content to estimate potential electricity production using a

combined cycle gas turbine coupled with a thermoelectric power plant. Calculations for

electricity production are in the range of 580 to 640 KWh per metric ton of biohazardous

waste (average production of 620 KWh per metric ton). Environmental impacts of

substituting energy from the regular electricity mix are not considered because only

overall direct emissions are used to estimate environmental impacts.

Life cycle inventory assessments for incineration (Appendix 5), autoclave plus

waste landfill (Appendix 6) and plasma gasification (Appendix 7) for the functional unit

of 8129.3 Kg of biological waste are processed into Endpoint Environmental Impacts by

using ReCiPe damage factors with a Hierarchist (H) approach with world normalization

that considers a 100 year horizon for damages as this is the operating practice for the

United Nations Framework for Climate Change (Appendix 8).

Endpoint damages for Human Health are expressed in Disability Adjusted Life

Years (DALYs) which indicate the sum of years of potential life lost due to premature

mortality and the years of productive life lost due to disability (ReCiPe, 2008). Endpoint

damages to ecosystem diversity are expressed in Species.years which is a way to measure

52

extinction rate. There is approximately one extinction caused per million Species.years

(ReCiPe, 2008). The disposal of 8129.3 Kg of biological waste using incineration causes

approximately 0.083 DALYs. Health damages from using an autoclave and landfilling

for the same amount of waste is 0.031 DALYs. When using a plasma gasification process

0.001 DALYs are caused. This is a 62.6% and 98.7% reduction respectively when

comparing gasification to regular incineration and autoclaving/landfilling of biological

waste (Appendix 9). Ecosystem damages are reduced by 86% when using autoclave and

landfilling of biohazardous waste and by 98.6% when using plasma gasification

(Appendix 9).

Resource depletion costs are expressed in 2008 US Dollars per year that future

generations would need to spend for additional exploration, extraction and processing

costs for non-renewable materials and oil due to increased scarcity. Resource depletion

costs due to disposing of 8129.3 Kg of biological waste using regular incineration are

$3931, $5593 when using an autoclave and landfilling process and $1686 if the plasma

gasification process is used (Appendix 10).

Therefore, environmental damages for all categories are the lowest when plasma

gasification is used to dispose of biological waste. Environmental impacts for the other

processes show mixed results. The process of autoclave and landfilling is better than

regular incineration for Human Health and Ecosystem Damages, and worse in resource

depletion costs.

53

Estimation of carbon dioxide production from plasma gasification

Comprehensive analysis of exhaust gases from the plasma gasification process

shows that effluent gases are composed mainly by carbon monoxide, water and carbon

dioxide as these 3 gases represent 89% of the total mass of plasma incineration exhaust

gases when processing biohazardous waste (Table 4).

Table 4. Estimated composition of synthetic gases from the plasma gasification of

biological waste.

Effluent gas Wt % Vol % CO 45.10% 35.40% H2O 23.95% 29.28% CO2 20.95% 10.49% N2 4.32% 3.40% H2 1.67% 18.26%

CH4 0.90% 1.24% C2H6 0.67% 0.50% C4H10 0.66% 0.25% C3H8 0.49% 0.24% C2H4 0.31% 0.24% H2S 0.14% 0.09%

By using the estimated composition of synthetic gas generation from plasma

gasification, stoichiometric reactions for adding pure oxygen in the combustion process,

and information of the combined cycle operation it was estimated that 8129.3 Kg of

biological waste will generate 6958 kg of carbon dioxide, 1610 Kg of water, 980 Kg of

54

nitrogen oxides and 18.21 Kg of sulfur dioxide. This reaction requires the addition of

2656 Kg of pure oxygen in order to reduce nitrogen oxides formation.

Algae Species Selection and Estimation of Omega-3 Production

Algae species selection was made by reviewing lipid content for 50 cold water

species. After reviewing the lipid contents, the 10 species with the largest amount of

lipids were isolated and cultured in a laboratory. A comprehensive analysis to determine

the type of prevailing lipids was conducted for these 10 species and after a careful

examination, the 3 species with the highest concentration of omega-3 fatty acids were

selected (Appendix 11).

The algae species with the highest omega-3 content is Nannochloropsis oculata

with an average of 40% of EPA (20:5 n-3) which is a form of high-value omega-3. This

species is found in all oceans globally, so there is no risk of becoming an invasive species

if there is an accidental spill of this microalgae to the sea or coastal bodies of water. This

species has also shown high tolerance to chloride which is used to clean algae production

ponds in case there is biological contamination.

Numerous factors affect algae growth, such as pest contamination, temperature,

CO2 availability, solar irradiation, rain, and wind. Attempting to control these factors

result in elevated operating costs. The use of coastal waters and technologies helps

maintain optimal culture conditions allowing for increased production of microalgae and

their oils. For that reason, Nannochloropsis oculata was farmed in 24 small racetrack

55

ponds of 1000 liters each (approximately 1/3,000 of a hectare of a large scale microalgae

farm) to test different concentrations of carbon dioxide, nutrients, fresh and saltwater,

water levels and chemical pest control parameters (Figure 5).

Figure 5. Small open ponds used to conduct experiments to determine optimal conditions

for farming Nannochloropsis oculata for omega-3 fatty acids.

Growth rates and omega-3 content of biomass were assessed daily as the outcome

variables. These information were used to derive lessons learned and recommendations to

enhance biomass growth. Some of the most important discoveries for enhancing

microalgae growth are:

56

• Water temperatures should never go above 30 °C or below 5 °C. This is

important to be able to get meaningful amounts of lipids from algae. Any

temperature above 30 °C increases cell mortality and reduces biomass production.

Any temperature below 5 °C reduces algae metabolism to a minimum which

reduces reproduction rates and biomass production.

• Water mixing has to be constant during daylight hours in order to eliminate

microalgae stratification which leads to overexposure to solar irradiation to algae

close to the water surface and underexposure to algae below this upper layer.

Overexposure to solar irradiation causes a “light saturation” effect where all

microalgae light receptors are filled. When this occurs algae start to produce

pigments for protection from over-exposure to the sunlight. This consumes

energy that would have been used in reproduction. Pigment producing survival

mechanisms take over and production yields for biomass decrease and no omega-

3 is generated. Underexposure of algae to the sun simply reduces photosynthesis

and drastically reduces microalgae’s ability to reproduce. Water mixing can also

be used as a cooling mechanism by increasing mixing rate.

• Optimum water pH should be 6.8 - 7.2. This is controlled by regulating the

amount of CO2 that enters a microalgae pond. Higher concentrations of CO2

lower the pH which increases the acidity of the ponds.

• Black liners should not be used in open microalgae ponds because they increase

water temperature and reduce reflectivity of the pond’s bottom. These 2

conditions reduce biomass production yields.

57

• An open pond microalgae farm should be located in a region with access to

saltwater, between the 30° North and South parallels for sufficient solar radiation,

and with very little or no rain precipitation. Rain has inhibitory effects which

reduces omega-3 production. Rain events reduce solar irradiation and therefore

biomass production. Rain also adds nitrogen containing freshwater to the ponds

which directly inhibits omega-3 formation. A cloudy or rainy day reduces

production yields between 30 and 50%.

The production yields in conventional open pond systems are predictable until

biological contamination occurs. One prevalent biological contaminant is a family of

aquatic micro-organisms called “rotifers”. Rotifers are medium sized multicellular

microorganisms (about 1000 cells) living mostly in freshwater or coastal habitats (Figure

6). Daily observations showed that contamination occurred when a new source of water

was used to compensate for pond evaporation (fresh or brinish water is added daily to the

system in order to maintain an appropriate salinity level). These contamination episodes

reduce useful biomass production by 50 to 80 %. Rotifers were suspected when the

lipid/protein composition of the biomass was analyzed after every event. Observations

under the microscope confirmed this suspicion.

58

Figure 6. Picture of a “rotifer” micro-organism under the microscope (50X)

Water could be treated chemically or with ozone in order to get rid of rotifers, but

that will also kill the microalgae. Then the pond would have to be emptied, filled with

treated water and re-inoculated. This process takes 3 to 4 working days and production to

pre-contamination levels is re-established in 5 to 7 days. That is a big loss in overall

yield for the operation, considering that microalgae is harvested every day with a yield

ranging from 36 to 102 liters of omega-3 oil with 40 % EPA per hectare per day

(depending on the time of the year and weather).

Another way to kill the rotifers without killing the microalgae was developed by

understanding the physiology of this microorganism:

59

• Rotifers have a life span of 3 to 4 days at 25°C, the same temperature that

optimizes algae production. Temperature could not be changed without

decreasing microalgae production levels.

• Rotifers require dissolved oxygen to survive and females lay eggs

approximately every 4 hours. Larvae become adult after 0.5 to 1.5 days in

order to re-start the reproduction cycle. This was a key factor used to develop

a system to eliminate rotifers without affecting the production yields for

microalgae.

Water for open ponds will be treated with ozone or other non-invasive systems

like ultraviolet radiation (UV rays). Then water in open ponds will be inoculated with

microalgae. Production will normally occur for a few days (5 or 6) and then the water

from that pond will be introduced in a photobioreactor (PBR). Carbon dioxide will not be

added so algae will reduce their metabolic rate and stop producing oxygen. The gas

exchange system will remove all the oxygen from the water inside the PBR, thus

eliminating all adult rotifers and their larvae. Water will remain anoxic for 5 to 12 hours

which eliminates all rotifer eggs. Meanwhile, the empty pond where contamination

occurred is cleaned with a low concentration chloride solution and exposed to the sun in

order to kill any potential rotifer residues. After less than a working day, water is

pumped out of the PBR and returned to the clean pond to resume regular production

operations. Overall yield losses are minimal or non-existent due to the fact that

microalgae growth is accelerated in a PBR, so all of the available CO2 dissolved in the

water was consumed and biomass was formed even with low metabolic rates.

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Dinoflagellates are marine plankton that also contaminate open pond systems.

They are mixotrophic but prefer to behave like algae predators. This predatory behavior

reduces biomass production rates and omega-3 production. A way to eliminate a

dinoflagellate outbreak is to increase water pH to 8.4 by adding sodium hydroxide and

stop all inputs of CO2 and nutrient into the pond. Due to a lack of nutrients (phosphates)

to feed on, the bacteria in their digestive tracks begin “eat” dinoflagellates from the inside

out.

Sustained production rates are approximately 15 to 20 grams/m2. Plasma

gasification of 8129.3 Kg of biological waste produce 6958 Kg of CO2 which produce in

average 3760 Kg of algae biomass and 1500 Kg of omega-3 with EPA. Between 587 and

785 m2 of land are required to process the daily CO2 inputs from gasifying 8129.3 Kg of

biological waste annually.

61

Chapter IV

Discussion and Conclusions

Description of the biological waste stream

The fact that biological waste contains infectious or potentially infectious

materials is well documented; however, the composition of biological waste is not well

characterized. On the other hand, municipal and chemical wastes have been well studied.

Municipal waste is variable due to the vast amount of materials that end up in trash. Even

with variation in composition studies have discovered that typical municipal wastes from

2010 contain 28% food waste, 18% yard waste, 24% paper, 22% plastics, 4% glass, and

4% metals (Habib, Schmidt, & Christensen, 2013). U.S. regulations require that chemical

wastes be characterized so that percentages of all components are known at all times.

Important decisions regarding the treatment of municipal or chemical waste are better

made knowing the composition of the waste that is collected. This allows for more

efficient management of resources to minimize environmental, social and financial

impacts. Biological waste streams contains a mixture of paper, plastic, metal, glass and

biogenic materials believed or known to be contaminated with infectious materials.

However, there is no information available which accurately describes the composition of

biological waste. The term biological waste describes a type of waste commonly known

by various other names such as biohazardous waste, medical waste or infectious waste.

This type of waste contains or potentially contains materials that may be infectious to

62

healthy humans and therefore must be kept secure until final destruction renders the

waste non-infectious. Various industries, such as healthcare, higher education and the

life sciences, generate biological waste. The composition of biological waste changes

depending on the activities being performed at the site of waste generation. Healthcare

institutions generate significant amounts of: contaminated materials that have come into

contact with patients, disposable materials such as IV bags, and materials contaminated

with drugs such as chemotherapeutics and pain medications excreted or unused by

patients. Biotechnology research generates much less waste containing tissue or body

fluids and more waste contaminated with laboratory scale cell culture. Biotechnology

research is focused on the manipulation of a biological system to achieve a predictable

outcome. Many innovations involving human immune modulations have advanced

healthcare therapies targeting Severe Combined Immune Deficiency, Chronic

Granulomatous Disorder, Hemophilia, and various cancers and neurodegenerative

diseases. Human therapeutic research involves using human blood, cell lines and tissue

samples for laboratory scale manipulation. Successful research programs involve animal

research which generates various animal tissue and body fluid samples as waste. The

various activities involved in research with human and animal materials generates a large

amount of contaminated single use equipment. Also known as consumables, this

equipment includes: pipette tips, serological pipettes, cell culture flasks, and nitrile work

gloves.

To properly conduct a life cycle assessment comparing regular incineration and

plasma gasification, an accurate description of the make-up of biological waste is

63

necessary. Studies indicate that sharps materials such as syringes, needles, scalpels, glass

tubes, microscope slides, and broken glass are present in the biological waste stream

(Mecklem & Neumann, 2003). The United States Environmental Protection Agency

characterizes biological waste to include: cultures and stocks of infectious agents, human

blood and blood products, human pathological wastes (including those from surgery and

autopsy), contaminated animal carcasses from medical research, wastes from patients

isolated with highly communicable diseases, and all used sharp implements (such as

needles and scalpels) and certain unused sharps (U.S. Environmental Protection Agency,

1989). Sampling of biological waste containers from a biotechnology research company

in Cambridge, MA, indicated that 80% percentage of biological waste is composed of

single use plastic materials (Appendix 4). The remaining materials are paper, cardboard,

and biogenic. It is important to note that glass and metals (e.g., needles) are present in

very small percentages but they are not captured in our assessment since we are focusing

on organic materials that are combusted during disposal. The maximum allowable weight

of one full biological waste container due to Department of Transportation shipping

container requirements is 22.7 kg (50 lbs). The average weight of a full container in our

study was approximately 7.2 kg (15.9 lbs). The fact that the average weight was less than

half the maximum allowable weight indicates that there is a very low density of materials

in the biological waste containers. Our observations indicated that a large amount of

empty but contaminated plastic containers were disposed of into the biological waste

stream. Additionally, a significant amount of paper and plastic packaging materials were

also observed in the biological waste containers (Figure 7).

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Figure 7. Images of the inside of biological waste containers showing various plastic and

paper waste materials.

Knowing the basic composition of biological waste is important if a waste

manager is expected to run an efficient disposal program that minimizes waste. Poor

laboratory practices and training may contribute to an increase in the collection of non-

contaminated materials as regulated biological waste. For example, Figure 7 shows

empty media and buffer solution bottles in a waste container. Proper lab practices should

have allowed these bottles to remain contamination free. This indicates that there is a

high percentage of laboratory waste that should be recycled or reused.

65

Waste Disposal Technology Selection

While incineration is a readily available and accepted disposal option for the

destruction of waste materials, it adversely impacts human health and the environment.

These harmful impacts have driven our waste management industry to identify and

implement numerous technologies that are much less damaging than incineration. Some

biogenic waste disposal activities include composting, anaerobic digestion, biochar

production, and conversion technologies that can generate ethanol or biodiesel (Vergara

& Tchobanoglous, 2012). These methods are effective at managing organic waste

materials with minimal inputs and minimal environmental and human health impacts.

However, they require well segregated organic wastes and specialized facilities that are

not available on a commercial scale (Vergara & Tchobanoglous, 2012). In a fast paced

biotechnology research facility, managers must make decisions about how to organize

waste streams and communicate the waste management strategy to employees. This

requires additional floor space and containers as well as time for additional training

which can become a burden that company leaders may find unacceptable. Therefore more

manageable, non-biogenic strategies seem to be popular for biotechnology waste

management. Some non-biogenic activities include incineration, plasma gasification,

pyrolysis, and recycling (Vergara & Tchobanoglous, 2012).

Recycling facilities are not qualified to destroy infectious materials. Pyrolysis

technology is not helpful since it requires biomass waste which typical biological waste

does not contain at a high level (Figure 7). Depending on the actual contents of biological

waste, a waste manager may decide that the waste should be disinfected via an autoclave

66

prior to final disposal in a landfill. Autoclaving is effective at sterilizing most biologicals

but the contaminated waste debris are not destroyed and continue to take up a larger

volume of space. Alternatives to landfills have been pursued since 1903, when Denmark

built the first incineration plant due to a lack of landfill space (Habib, Schmidt, &

Christensen, 2013). Since that time we have learned that landfills are a primary source of

methane, a harmful and powerful greenhouse gas (Vergara & Tchobanoglous, 2012). The

United States Department of Health and Human Services requires that pathogenic

materials, such as prions, be destroyed by using incineration only (United States

Department of Health and Human Services, 2009). Standard disinfection techniques, such

as autoclaving, irradiation, boiling, dry heat and chemicals (formalin, alcohols,

Betapropiolactone), are not effective at inactivating prions (United States Department of

Health and Human Services, 2009). Incineration is effective at destroying all biological

materials, including prions, due to high furnace temperatures. The resulting ash is

landfilled as the final step of incineration.

Incineration is a commonly used technology that effectively destroys biologicals

and minimizes the volume of landfill waste. Modern incinerators can be designed to

process varying types of waste, are characterized by emissions abatement and pollution

prevention systems and use various types of combustion technologies (Marchettini,

Ridolfi, & Rustici, 2006). The practice of using incineration as a disposal method has

been heavily scrutinized for its harmful impacts on human health and the environment.

Health issues are directly (via occupations in the industry) and indirectly (e.g., via

ingestion of contaminated food, water, and soil) associated with all steps of waste

67

management (Giusti, 2009). The environmental impacts of incineration can be wide

ranging but generally impact water fall-out of atmospheric pollutants, air (SO2, NOx,

N2O, HCl, HF, CO, CO2, dioxins, furans, PAHs, VOCs, odor, noise), soil (fly ash and

slags), landscape (visual effect) and the climate (generation of greenhouse gases) (Giusti,

2009). As indicated by Appendix 9, human health and the environment is more

negatively impacted by the use of incineration. Incineration has been shown to adversely

impact human health by increasing the incidence of cancer and congenital birth defects

(Forastiere, et al., 2011). A review of health effects related to incineration indicated that

studies conducted from 1983 and 2008 provided evidence of increased risk for various

types of cancer (stomach, colorectal, lung, liver, soft-tissue carcinoma, non-Hodgkin’s

lymphoma) and congenital malformations (facial cleft, renal dysplasia) (Porta, Milani,

Perucci, & Forastiere, 2009).

Additionally, there are documented health effects linking landfills to an increased

risk for congenital malformations (neural tube defects, hypospadias, epispadias,

abdominal wall defects, gastroschisis, exomphalos) and very low birth weight (Porta,

Milani, Perucci, & Forastiere, 2009). While specific linkages between pollutants and

cancer or congenital malformations were not identified, exposure to dioxins has been

suggested as a primary causative agent (Porta, Milani, Perucci, & Forastiere, 2009). It has

also been established that landfills generate a significant amount of methane, a powerful

greenhouse gas estimated to be 21 times more potent than CO2, which is the leading

contributor from the waste management industry to climate change and global warming.

In Europe, methane from landfills accounts for 1/3 of the anthropogenic emissions while

68

CO2 and N2O, both significant contributors to global climate change, account for only 1%

and <0.5% of emissions respectively (Pikoń & Gaska, 2012). Landfills remain the most

widely used waste disposal activity however political and financial factors are beginning

to require that landfilling be the last step of waste disposal, after all possible material and

energy recovery has taken place. Landfilling has been identified as a disposal method that

is significantly more costly due to high and prolonged operational costs and low energy

and materials recovery (Marchettini, Ridolfi, & Rustici, 2006).

We show that incineration and landfilling significantly contribute to an increase in

human disease and negative environmental impacts, whereas plasma gasification

combined with gas capture and sequestration has been shown to mitigate these

destructive factors (Appendix 9). The extremely high temperature of plasma gasification

obliterates all chemical pollutants leaving syngas, a valuable material that will be

recovered. This process also generates slag, the inorganic, inert, glasslike material that

can be either reused or harmlessly disposed on in an inert landfill due to its low

leachability (Gomez, et al., 2009). The LCIA results for the incineration (Appendix 5)

and autoclaving/landfilling (Appendix 6) of biological waste show a greater negative

impact on climate change and human health than those from plasma gasification

(Appendix 7). Landfilling of biological waste (Appendix 4) does not significantly

contribute to these negative effects since much of the waste is relatively inert. However,

since biological waste is disinfected via an autoclave prior to landfilling, a high amount

of energy is consumed to generate the temperatures and pressures necessary for

disinfection. The energy needed for this activity is generated by fossil fuel burning power

69

plants. The fossil fuel combustion combined with the inherent risks created by depositing

waste into landfills are the main contributors to the LCIA categories for climate change,

human toxicity, freshwater ecotoxicity, and marine ecotoxicity (Appendix 6). LCIA

results from plasma incineration of biological waste show that the categories related to

human health and environmental impacts are minimal and indicate that the complete

destruction of organic pollutants and the capture of inorganic pollutants in slag mitigate

most of the harmful impacts created when incinerating or landfilling/autoclaving

biological waste (Appendix 9).

A Stable Source of Carbon Dioxide

The amount of CO2 generated by the plasma gasification of biological waste

produced by a biotechnology research company with 300 employees and approximately

100,000 ft2 of lab space is sufficient for the production of about 3760 Kg of algae

biomass and 1500 Kg of omega-3 with EPA. As of August 20, 2014, the state of

Massachusetts has 738 biotechnology and pharmaceutical establishments which accounts

for approximately 21 million square feet of lab space and 57,000 employees

(Massachusetts Biotechnology Council, 2014). When square footage of lab space is used

to virtually scale up microalgae production, 789,600 Kg of algae biomass and 315,000

Kg omega-3 with EPA is generated. It is difficult to verify these figures by using such a

correlation since each company differs in the type of research activities and materials that

are utilized. However, it is relevant for the purposes of establishing the potential impact

of using biological waste for the large scale production of microalgae.

70

California is a leading state in the biotechnology industry with employment at

approximately 24,000 for research and development and 45,000 for manufacturing

(Massachusetts Biotechnology Council, 2014). The selection of Southern California is

helpful due to the concentration of biotechnology research institutions in and near San

Diego and Los Angeles. Southern California counties, consisting of Los Angeles, San

Diego, Orange, Riverside, and San Bernardino, account for 59% of the total

biotechnology employment in the State of California (Gollaher & Claude, 2014). Not

only is Southern California is an attractive location for our proposed system due to the

abundance of biological waste sources but also for its proximity to 30° North latitude and

seawater, its lack of rain, and the availability of non-arable land.

The characterization of biological waste from a biotechnology research company

had not been previously established. After characterizing biological waste it became clear

that there are many similarities in basic composition to municipal solid waste. General

categories of waste materials commonly found in municipal solid waste are plastic, metal,

glass, paper, and organic. While the composition of municipal solid waste differs by

location it continues to be composed of the same basic materials. On a global level

developing nations have more organic materials and developed nations have more

complex waste compositions (Appendix 12). The waste fractions that we are targeting in

our gasification process are organic and the abundance of organic materials that exist in

municipal solid waste indicate that this waste stream would be a suitable candidate from

which CO2 can be collected for algae farming. One study calculated that greenhouse gas

emissions from solid waste disposal in Europe contributes <0.5% CO2, 33% CH4, and

71

1% N2O of the total European GHGs (Giusti, 2009). While solid waste disposal accounts

for a large amount of emissions in Europe, there are other activities generating greater

amounts greenhouse gases. Facilities engaged in producing power, manufacturing

fertilizer, and a variety of additional activities may also be valuable sources of CO2

emissions.

Farming Nannochloropsis oculata

The use of algae for the production of oils and fatty acids has increased as the

technologies for the extraction of these high value products has improved. As strains of

microalgae continue to be researched and analyzed, we are better able to select for

species that will efficiently produce specific oils or fatty acids based on logistical and

environmental factors. For the large scale cultivation that is necessary to produce

sufficient quantities of fatty acids, an open pond system is used. The use of a large open

pond system is relatively cheap, easy to maintain and clean, utilizes non-arable land, and

requires low energy inputs. However, it is limited by the number of algae strains that can

be cultivated and is susceptible to pest contamination (Brennan & Owende, 2010).

Nannochloropsis oculata has been a popular microalgae species to study for oils

and fatty acid production. This species is cultivated on an industrial scale for use as an

aquaculture feed (Islam, et al., 2013). Commercial scale cultivation can be cheaply

achieved using open pond systems that operate year round (Islam, et al., 2013). In one

study, a profile of 9 algae strains showed that Nannochloropsis oculata is a favorable

microalgae species for the production of high yields of total lipid and fatty acid content

(Islam, et al., 2013). Our comparison of microalgae fatty acid content (Appendix 11)

72

indicate that Nannochloropsis oculata contains higher concentrations of EPA than the

Mexican diatom CIBNOR 2008, Phaeodactylum tricornutum and 47 other species. This

determination is welcomed due to the wide availability of the Nannochloropsis oculata

around the world. As a non-invasive species, a more sustainable and environmentally

friendly process can be achieved. Additionally, high growth rates, resistance to mixing

and contamination, high nutritional values and high fatty acid contents fit the needs of the

algaculture industry well.

Optimizing the production of omega-3 with EPA from Nannochloropsis oculata

involves a thorough understanding of necessary environmental conditions. As we have

indicated, Nannochloropsis oculata can grow around the world but the microalgae must

be grown in conditions that favor an increase in fatty acid production. Stressful

conditions or seasonal variations will change how the microalgae grows and reproduces.

During winter and spring seasons, algal biomass production decreases, presumably due to

a decrease in photosynthesis and temperature which slows reproduction and growth

(Olofsson, et al., 2012). As we have indicated, over exposure to light is also detrimental

to the efficient production of fatty acids. It is important to ensure that a farming system

be designed to allow for uniform light exposure in the outdoor ponds. In order to protect

themselves from over irradiance, microalgae produce pigments. Therefore it is essential

to have adequate water mixing during daylight hours in outdoor ponds. Over exposure to

light and a nutrient deficient environment have been shown to decrease the accumulation

of omega-3 with EPA in Nannochloropsis oculata (Srinivas & Ochs, 2012). When

temperatures are too cold (below 5 °C), the microalgae reproduce more slowly due to a

73

decrease in metabolism. This stressful condition forces the microalgae into a stationary

phase in which the algae begin to accumulate oils instead of remaining in a growth phase.

The continuous reproduction of the microalgae is important for the accumulation of

omega-3 with EPA. If the microalgae moves into a stationary phase 68% of the

accumulated EPA is converted into triglycerides as opposed to 8% when it remains in the

exponential growth phase (Guschina & Harwood, 2006). During our studies, when

Nannochloropsis oculata is exposed to temperatures above 30 °C the algae mortality is

increased leading to a decline in biomass production. Various other studies have shown

similar results for Nannochloropsis species concluding that at temperatures above 35 °C

no growth is observed (Van Wagenen, et al., 2012). Our studies also show that it is

important to utilize materials that enable better temperature control. Better control is

achieved by avoiding the use of black pond liners which increase the water temperature

and reduce reflectivity. Both of these factors reduce microalgae biomass accumulation.

Nannochloropsis oculata is found naturally in cold waters so the large scale

culturing of this species is optimal in areas such as Southern California. The open pond

system is vulnerable to bacterial and rotifer contamination which can be devastating to a

microalgae farm. A common problem associated with the large scale cultivation of

microalgae is contamination with herbivorous rotifers. Rotifers are difficult to control as

they can survive in extreme environments and reproduce rapidly. Additionally, their

consumption of microalgae can quickly reduce algal biomass (Huang, Li, Liu, & Lin,

2014). Various methods exist for removing rotifers from algae cultures. Physical removal

by disinfecting equipment and the micropore filtration of microalgal seed inocula are

74

commonly used methods but they are only effective when used on laboratory or pilot

scale cultures, not mass culture (Huang, Li, Liu, & Lin, 2014). Preventing rotifer

contamination with chemical controls is also possible but has limitations. The chemicals

xylene, benzene, toluene, and hexane were shown to have toxic effects on rotifers as well

as having harmful effects on microalgae and other aquatic organisms (Ferrando &

Andreu-Moliner, 1992). Such chemicals have also been proven to be human health

hazards and introduce occupational safety and environmental health challenges to the

cultivation process. Synthetic chemical pesticides are proven to be toxic to rotifers by

inhibiting reproduction and egg production in various rotifer species. However, the use of

chemical pesticides is problematic and has been shown to harm non-target organisms, the

environment and human health (Jeyaratnam, 1990). A benefit to using Nannochloropsis

oculata is that it has a high tolerance to chloride, so a chemical treatment of the water to

remove parasitic phytoplankton and rotifers (zooplankton) is possible. In most

contamination cases the algae growth ponds must be emptied, cleaned, refilled and

reinoculated. This process decreases the yield of biomass considerably therefore we

devised a solution for removing rotifers from the microalgae ponds without harming the

microalgae.

Understanding the rotifer reproduction cycle was essential for creating a

decontamination system. Since rotifers require oxygen to survive a controlled system

using a photobioreactor was used to induce an oxygen deficient environment. When

contamination in an open pond is discovered the algae are removed and pumped into a

photobioreactor where all oxygen is removed. The algae remain in this anoxic

75

environment long enough for all eggs and larvae to die (5-15 hours). Microalgae

production is minimally impacted when using this PBR system (< 1 day) compared to a

complete cleansing process (5-7 days).

The use of botanical pesticides has been shown to maintain microalgal production

rates while reducing the rotifer density and fecundity. These pesticides include

celangulin, matrine, and toosendanin which are widely used as crop protectants due to

their selective toxicity. Celangulin targets the digestive system and neuromuscular

synapse resulting in cell death (Qi, Shi, Hu, Zhang, & Wu, 2011). Matrine exposure leads

to death by suffocation caused by central nervous system paralysis (Sanchez-Fortun &

Barahona, 2005). Toosendanin also causes paralysis by blocking the pre-synaptic

transmission in the neuromuscular system (Zhou, Liner, Bai, & Jing, 1987) and acts as a

feeding deterrent (Chen, Isman, & Chiu, 1995). In addition to the selectivity of these

botanical pesticides for rotifers, they are also significantly less toxic to aquatic organisms

and do not persist in the environment (Huang, Li, Liu, & Lin, 2014). The potential

benefits of botanical pesticides in algaculture are exciting due to the fact that logistical

needs will be minimal and operational downtime needed for decontamination may be

eliminated. More research is required to better understand the potential of using botanical

pesticides in large scale operations.

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Technology Innovations for Tomorrow

The successful implementation of our proposed waste-algae processing system for

the generation of omega-3 fatty acids will shine light on a sustainable process that can

provide vast global benefits for the environment, economy, agriculture, and society. Our

process creates a method that can be used to mine landfills on a global scale to remove

organic materials for the generation of CO2 via plasma incineration. Landfills are a

primary source of methane, a powerful greenhouse gas, which is generated by the

decomposition of organic matter. The elimination of this fraction would be a substantial

step towards reducing the generation of greenhouse gases. The mining of landfills would

also generate vast amounts of recyclable materials such as metals and plastics which

would be collected as slag. Slag materials have numerous applications, depending on

their composition, including being used in asphalt and concrete (Gomez, et al., 2009).

The recycling and reclamation of materials are the best strategies for managing solid

wastes in urban areas (United Nations Human Settlements Programme, 2010) and

landfills are readily available sources of such materials. It is estimated that if in 2025

everyone in the world generated waste at the current rate, the total annual world

generation would be 5.9 billion tons – nearly three times the current estimate. This

equates to about 59 billion cubic meters of municipal solid waste each year which is

enough to cover a country the size of Costa Rica or Ireland to a depth of one meter

(United Nations Human Settlements Programme, 2010). The implementation of plasma

gasification on a global scale for the management of solid wastes would help greatly by

eliminating or minimizing the impact that the immense volume of waste has on our

77

planet. By using plasma incineration the elimination of new landfill waste and the

recovery of current landfill space is possible. This would have a tremendous impact on

reducing the generation of greenhouse gases, minimizing environmental and human

exposures to toxic byproducts, and increasing the availability of arable and inhabitable

land.

Our gasification and algae farming process also assists in decreasing the harmful

impacts that the commercial fishing industry has on the sustainability of fisheries on a

global scale. For every 100 grams of Atlantic herring there are 2.2 grams of omega-3

with EPA (Food and Agricultural Organization of the United Nations, Fisheries Division,

1986). In order to generate 1500 Kg of omega-3 with EPA, the amount of omega-3 with

EPA that our proposed system can generate in 1 year, approximately 68,000 kg of herring

would need to be harvested. Commercial fisheries are primarily responsible for providing

a source of protein for human consumption and the harvesting of fish on a global scale

was estimated to be 77,000,000 metric tons in 2010 (Pontecorvo, 2014). Over fishing has

contributed to the collapse of various fish stocks along with other factors including illegal

fishing, environmental shocks, and the modification of regulations to ease economic

effects of quota cutbacks (Pontecorvo, 2014). The decline and potential collapse of fish

stocks can have devastating impacts on regional and global social and economic viability.

Fish prices will increase and ecosystems will lose diversity which breaks the natural food

chain, causing a decline in all marine life. Alternate sources for omega-3 with EPA, such

as our proposed solution, would assist in sustaining healthy marine ecosystems as well as

78

minimizing the impacts of declining fisheries on social and economic infrastructure

around the world.

The technologies described in this project will become more efficient and better

understood over time allowing them to have a greater global role. Biotechnological

research on microalgae strains should continue to identify and improve the abilities of

microalgae to produce biomass, oils and omega-3 fatty acids. The desired product (e.g.,

omega-3 fatty acids for this project) dictates how the cultivation and processing of

microalgae should be completed. Producing a maximal amount of omega-3 fatty acids

while minimizing production costs requires careful management of light intensity,

temperature, CO2 availability, and pH during microalgae culturing. The balancing needed

to achieve maximum efficiency can be best controlled via the use of photobioreactors

where growth conditions can be measured and manipulated. The thorough understanding

of how to cultivate a specific species of microalgae directs research on how bioreactors

can be designed to grow algae and produce the desired product. The study of

computational fluid dynamics (CFD) makes it possible to predict the hydrodynamics and

related characteristics of photobioreactors. This tool uses computers and numerical

techniques to solve problems by modeling fluid movements and light penetration (Bitog,

et al., 2011). Companies are already developing unique technologies for low-cost

production of microalgae biomass in a closed environment. Technology that utilizes a

flexible, floating, multi-compartment photobioreactor that can be deployed either on land

in ponds or in the ocean is being developed. Another new technology under development

is an extended surface area culture system that incorporates specialized photobioreactor

79

panels. The system consists of vertical panels suspended in a water basin which

increases the surface area illuminated by both direct and diffuse light. Additionally, the

water basin provides structural support and improved temperature control to

optimize algal growth. Common benefits promoted by companies developing new

technologies for efficiently producing microalgae on an industrial scale include: higher

productivity, superior crop protection, improved process control, more rapid scale-up,

and being applicable to a broad range of microalgae.

Focusing on manipulating the genetic makeup of microalgae is another factor that

will become instrumental in creating an efficient production system. To date only a few

marine algae species have been genetically manipulated successfully, such as Dunaliella,

Porphyridium, Nannochloropsis, Laminaria, Undaria, Porphyra, and Gracilaria (Qin,

Lin, & Jiang, 2012). The continued research on microalgae genetics and photobioreactor

design will expand our ability to implement these systems on a larger scale resulting in

environmental, economic, social and regulatory benefits (Ziolkowska & Simon, 2014).

80

Concluding Remarks

The major findings of this project indicate that a new, cross functional process

should be used to generate our desired product, omega-3 with EPA. This system was

reverse engineered by first identifying the biotechnologically derived product desired and

then creating an efficient and sustainable process to produce this product. Our more

sustainable steps make this process efficient because they utilize existing resources to

deliver a better, more pure form of omega-3 with EPA. We have shown that omega-3

with EPA is an increasingly popular supplement that is in high demand around the world

and that it is currently delivered to the commercial market at a high cost to the

environment. Harvesting the world’s fish for the extraction of omega-3’s is damaging to

marine ecosystems, increases the vulnerability of our commercial fishing industry to the

collapse of fish stocks, and is unnecessary. Research shows that the extraction of valuable

omega-3’s from the microalgae, Nannochloropsis oculata, are beneficial for numerous

reasons including being free of pollutants (e.g., mercury) and being able to utilize non-

arable land or the oceans for algae farming. Our review of literature highlights the fact

that algae are able to produce a variety of additional valuable products such as biodiesel

and biomass which provides even greater support for expanding the use of microalgae

globally. The publication of our previously unreported results allows for a more efficient

algae farming system to be harnessed for omega-3 with EPA production. Since algae

production is enhanced by increasing the concentration of CO2 in the algae growth media,

we are compelled to take advantage of secure, CO2-rich sources, such as carbon rich

emissions from waste incineration and power generation. The management of waste is a

81

complex system that is influenced by a variety of factors including geography,

economics, regulatory oversight and available technology. Regardless of how waste can

be managed, if a method to reduce the amount of waste accumulation is not implemented,

the world's growing human population will only exacerbate the management of the

continuously increasing volumes of waste. Therefore, we deemed the inclusion of a waste

elimination technology, plasma gasification, for the generation of CO2 necessary. Not

only does plasma gasification virtually eliminate high volumes of solid waste, it also

eliminates harmful pollutants that would be generated via landfills (e.g., methane) and

regular incineration (e.g., dioxins).

The field of biotechnology has become an increasingly effective medium for

discovery in many industries. The manipulation of a biological system to achieve a

desired outcome is an efficient way to create less invasive and more efficient process. We

should put our energy towards harvesting alternate, abundant and sustainable resources,

like algae, instead of destroying a limited and fragile resource such as fish. The issues

and solutions identified in our project show that we have a sustainable path forward

which has the potential to deliver a better product and to greatly improve environmental

and human health. Further studies should focus on design and implementation of these

sustainable systems on a global scale.

82

Appendix 1.

Comparison of EPA and DHA fatty acid contents as percentage from total lipids in

examples of bacteria, fungi, fish, transgenic plants and microalgae (Adarme-Vega et al.,

2012)

Organism % EPA and/or DHA production

Bacteria

Shewanella putrefaciens 40.0 EPAAlteromonas putrefaciens 24.0 EPAPneumatophorus japonicas 36.3 EPAPhotobacterium 4.6 EPA

Fungi

Thraustochytrium aureum 62.9 EPA + DHA Mortierella 13.0 - 20.0 EPA Pythium 12.0 EPAPythium irregular 8.2 EPA

Fish

Merluccius productus 34.99 EPA + DHA Theragra chalcogramma 41.35 EPA + DHA Hypomesus pretiosus 33.61 EPA + DHA Sebastes pinniger 29.8 EPA + DHA Oncorhynchus gorbusha 27.5 EPA + DHA Mallotus villosus 17.8 EPA + DHA Sardinops sagax 44.08 EPA + DHA Clupea harengus pallasi 17.32 EPA + DHA

Plant (transgenic)

Soybean 20.0 EPABrassica carinata 25.0 EPANicotiana benthamiana 26.0 EPA

Microalgae

Nannochloropsis sp. 26.7 EPA + DHA Nannochloropsis oceanica 23.4 EPANannochloropsis salina ~28 EPAPinguiococcus pyrenoidosus 22.03 EPA + DHA Thraustochytrium sp. 45.1 EPA + DHA Chlorella minutissima 39.9 EPADunaliella salina 21.4 EPAPavlova viridis 36.0 EPA + DHA Pavlova lutheri 27.7 - 41.5 EPA + DHA Isocrysis galbana ~28.0 EPA + DHA

83

Appendix 2

Biomass productivity figures (Adarme-Vega, et al., 2012)

Algae species Reactor Type Volume (l) Xmax (g l-1)

Paerial (gm-2 day-1)

Pvolume (g l-1 day-1)

PE (%)

Chlorella sp.

Open Pond

10 25 - -

Spirulina platensis - - 0.18 -

Spirulina platensis 0.47 14 0.05 -

Haematococcus pluvialis 0.202 15.1 - -

Spirulina 1.24 69.16 - -

Spirulina platensis 0.9 12.2 0.15 -

Spirulina platensis 1.6 19.4 0.32 -

Anabaena sp. 0.23 23.5 0.24 >2

Chlorella sp. 40 23.5 - 6.48

Chlorella sp. 40 11.1 - 5.98

Chlorella sp. 40 32.2 - 5.42

Chlorella sp. 40 18.1 - 6.07

Porphyridium cruentum Airlift tubular 200 3 - 1.5 -

Phaeodactylum tricornutum Airlift tubular 200 - 20 1.2 -

Phaeodactylum tricornutum Airlift tubular 200 - 32 1.9 2.3

Chlorella sorokiniana Inclined tubular 6 1.5 - 1.47 -

Arthrospira platensis Undular row tubular 11 6 47.7 2.7 -

Phaeodactylum tricornutum Outdoor helical tubular 75 - - 1.4 15

Haematococcus pluvialis Parallel tubular (AGM) 25,000 - 13 0.05 -

Haematococcus pluvialis Bubble Column 55 1.4 - 0.06 -

Haematococcus pluvialis Airlift tubular 55 7 - 0.41 -

Nannochloropsis sp. Flat plate 440 - - 0.27 -

Haematococcus pluvialis Flat plate 25,000 - 10.2 - -

Spirulina platensis Tubular 5.5 - - 0.42 8.1

Arthrospira Tubular 146 2.37 25.4 1.15 4.7

Chlorella Flat plate 400 - 22.8 3.8 5.6

Chlorella Flat plate 400 - 19.4 3.2 6.9

Tetraselmis Column ca. 1,000 1.7 38.2 0.42 9.6

Chlorococcum Parabola 70 1.5 14.9 0.09 -

Chlorococcum Dome 130 1.5 11 0.1 -

84

Appendix 3.

Plasma Gasification Process Diagram

Plasma Gasification Chamber

Secondary Combustion

Chamber

Generation of

Synthesis Gas (a.k.a.

Syngas)

Propane + Air

Quench Vessel

Scrubber

Induced Draft

Blower

Combustion Air

Fresh Water

Fresh Water Make-up

Caustic Soda Filtered Water to

Drain

Air & Steam

Biohazardous Waste

Syngas to

Algae Farm

85

Appendix 4.

Biohazardous Waste Composition in a typical biotechnology research company in

Cambridge Massachusetts

Description Example

Average weight per waste container (kg)

Total average weight per year (kg)

Percentage

Polypropylene Centrifuge tubes, pipette tips 0.9613 1085.3 13.4%

Polystyrene Serological pipettes, weighing dishes

0.9613 1085.3 13.4%

Polyvinylchloride Pipet basins 0.9613 1085.3 13.4% Polyethylene terephthalate

Cell culture media bottles 0.9613 1085.3 13.4%

Polyethylene Pipettes, 0.9613 1085.3 13.4% Mixed plastics Nitrile gloves, culturing flasks 0.9613 1085.3 13.4% Cardboard Packaging materials 0.9000 1016.2 12.5%

Paper Packaging materials, paper towels

0.1727 203.23 2.50%

Biogenic materials

Animal carcass, tissue samples 0.3600 406.46 5.00%

Biohazardous Waste 7.2004 8129.3 100%

86

Appendix 5.

Life Cycle Inventory Assessment of Regular Incineration for 8129.3 Kg of Biological

Waste in New England

Impact category Unit Total Damages of Regular Incineration of Biohazardous Waste

Climate change kg CO2 eq 19178.064 Ozone depletion kg CFC-11 eq 0.000 Human toxicity kg 1,4-DB eq 1291.309 Photochemical oxid. formation kg NMVOC 4.456 Particulate matter formation kg PM10 eq 1.631 Ionizing radiation kg U235 eq 446.495 Terrestrial acidification kg SO2 eq 4.969 Freshwater eutrophication kg P eq 0.593 Marine eutrophication kg N eq 0.416 Terrestrial ecotoxicity kg 1,4-DB eq 0.151 Freshwater ecotoxicity kg 1,4-DB eq 60.363 Marine ecotoxicity kg 1,4-DB eq 57.550 Agricultural land occupation m2a 20.396 Urban land occupation m2a 5.430 Natural land transformation m2 0.094 Water depletion m3 41.443 Metal depletion kg Fe eq 54.359 Fossil depletion kg oil eq 244.377

87

Appendix 6.

LCIA Biological Waste Landfill of 8129.3 Kg in a New England Landfill

Impact category Unit

Total Damages Business as Usual Damages from Biological Waste Disposal (Autoclave + Landfill Disposal)

Climate change kg CO2 eq 2720.342 Ozone depletion kg CFC-11 eq 0.000 Human toxicity kg 1,4-DB eq 862.621 Photochemical oxid. formation kg NMVOC 4.161 Particulate matter formation kg PM10 eq 3.422 Ionizing radiation kg U235 eq 36.098 Terrestrial acidification kg SO2 eq 6.461 Freshwater eutrophication kg P eq 0.829 Marine eutrophication kg N eq 5.464 Terrestrial ecotoxicity kg 1,4-DB eq 0.077 Freshwater ecotoxicity kg 1,4-DB eq 81.519 Marine ecotoxicity kg 1,4-DB eq 77.493 Agricultural land occupation m2a 1.102 Urban land occupation m2a 12.710 Natural land transformation m2 -0.148 Water depletion m3 1.587 Metal depletion kg Fe eq 9.231 Fossil depletion kg oil eq 348.026

88

Appendix 7.

LCIA Biological Waste Plasma Incineration

Impact category Unit

Total Damages Plasma Gasification of Biohazardous Waste

Climate change kg CO2 eq 245.698 Ozone depletion kg CFC-11 eq 0.000 Human toxicity kg 1,4-DB eq 6.674 Photochemical oxidant formation kg NMVOC 203.040 Particulate matter formation kg PM10 eq 1.383 Ionizing radiation kg U235 eq 0.468 Terrestrial acidification kg SO2 eq 3.948 Freshwater eutrophication kg P eq 0.107 Marine eutrophication kg N eq 0.184 Terrestrial ecotoxicity kg 1,4-DB eq 0.000 Freshwater ecotoxicity kg 1,4-DB eq 0.043 Marine ecotoxicity kg 1,4-DB eq 0.045 Agricultural land occupation m2a 0.000 Urban land occupation m2a 0.000 Natural land transformation m2 0.000 Water depletion m3 0.018 Metal depletion kg Fe eq 0.675 Fossil depletion kg oil eq 8.880

89

Appendix 8.

Endpoint damage factors for ReCiPe 2008 with a Hierarchist (H) approach with world

normalization.

Impact Category Units HH(yr) ED (yr) RC ($/yr)

Climate Change KgCO2 3.51E-06 1.88E-05 0 Ozone Depletion KgCFC-11 0 0 0 Human Toxicity Kg DCB air 7.00E-07 0 0 Photochemical Oxidant Formation

Kg NMVOC air 3.90E-08 0 0

Particulate Matter Formation

Kg PM10 2.60E-04 0 0

Ionizing Radiation Kg U235 air 1.64E-08 0 0 Terrestrial Acidification

KgSO2 0 1.42E-08 0

Freshwater Eutrophication

KgP freshwater 0 4.40E-08 0

Marine Eutrophication

Kg Neq 0 0 0

Terrestrial Ecotoxicity

KgDCB Soil 0 0 0

Freshwater Ecotoxicity

KgDCB freshwater

0 2.60E-10 0

Marine Ecotoxicity Kg DCB marine water

0 4.20E-14 0

Agricultural Land Occupation

m2a 0 0 0

Urban land occupation

m2a 0 0 0

Natural land transformation

m2 0 0 0

Water depletion m3 0 0 0 Metal depletion KgFe 0 0 0.0715 Fossil depletion KgOil 0 0 16.07

90

Appendix 9.

Human health and ecosystem damages of disposing of 8129.3 Kg of biological waste

using regular incineration, autoclave and landfilling, and plasma gasification.

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

Total Damages of RegularIncineration of

Biohazardous Waste

Total Damages Autoclaveplus landfilling of

Biohazardous Waste

Total Damages PlasmaGasification of

Biohazardous Waste

Years

Human Health inDisability AdjustedLife Years (DALYs)

Ecosystem Damagesin Species.Years(Sp.yr)

91

Appendix 10.

Depletion costs for future generations for resources used to dispose of 8129.3 Kg of

biological waste with regular incineration, autoclave and landfilling and plasma

gasification.

$0

$1,000

$2,000

$3,000

$4,000

$5,000

$6,000

Total Damages of RegularIncineration of Biohazardous

Waste

Total Damages Autoclave pluslandfilling of Biohazardous

Waste

Total Damages PlasmaGasification of Biohazardous

Waste

($/y

r)

92

Appendix 11.

Relative % of total fatty acids from the strains CIBNOR 2008, Phaeodactylum

tricornutum (UTEX 646) and Nannochloropsis oculata (UTEX LB 2164) in 100L

columns.

CIBNOR 2008 Phaeodactylum tricornutum Nannochloropsis oculata

Fatty acids T3 T4 T3 T4 T5 T5 T6 T7 T8 12:0 -- -- -- -- -- 0.47 0.21 0.26 0.18 14:0 2.99 3.98 5.97 4.18 3.98 5.99 4.27 4.92 3.53 15:0 0.81 0.6 0.57 1.32 0.98 0.75 0.84 0.79 0.8 16:0 16.13 16.82 13.69 10.87 16.82 19.2 14.34 17.05 18.71 16:1 n-9 2.04 2.04 1.87 2.74 2.04 -- -- -- -- 16:1 n-7 6.73 11.35 16.88 11.96 11.35 21.68 19.88 20.28 19.85 16:2 n-4 1.58 1.92 8.22 4.62 1.92 0.33 0.24 0.25 0.55 16:3 1.9 3.03 6.61 6.28 3.03 -- -- -- -- 16:4 0.82 1.11 1.5 1.5 1.11 -- -- -- -- 17:0 -- -- -- -- -- 0.58 0.59 0.5 0.66 18:0 6.27 1.6 0.93 1.98 1.9 0.45 0.46 0.52 -- 18:1 n-9t 7.33 6.03 0.69 1.15 1.3 0.35 0.35 0.42 0.47 18:1 n-9c 3.9 0.96 0.35 0.38 0.96 2.68 2.28 2.32 1.53 18:2 n-6 0.53 0.92 2.07 1.64 0.92 0.32 0.52 0.46 0.35 18:3 n-6 0.22 0.48 0.45 0.23 0.47 1.51 2.37 2.27 2.17 18:3 n-3 1.28 1.54 -- 5.42 1.54 0.15 0.11 0.11 0.11 18:4n-3 -- -- 0.61 0.61 0.64 -- -- -- -- 20:4 n-6 0.26 0.35 -- -- -- 4.42 4.8 5.15 4.88 20:5 n-3 26.84 27.95 35.51 35.9 36.27 36.79 44.44 44.22 37.4 22:0 5.16 4.61 2.78 2.7 4.61 -- -- -- -- 22:6 n-3 1.89 1.1 0.99 0.78 1.06 -- -- -- -- 24:0 2.45 1.9 1.16 1.16 1.9 -- -- -- -- Not Identified 10.9 11.7 0.5 4.6 7.2 4.33 4.3 0.48 8.8

93

Appendix 12.

Waste composition for 20 selected cities around the world (Vergara & Tchobanoglous, 2012).

94

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