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Home , Biomineralization

DIRECT APPLICATION OF BIOMINERALIZATION TO LIFE

SUPPORT SYSTEMS, HABITAT WATER WALL SYSTEM, CARBON

SEQUESTRATION, BIOREMEDIATION AND SOLVING OTHER

VITAL ENVIRONMENTAL PROBLEMS

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A Project Presented to the Faculty of California State University, Chico

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In Partial Fulfillment Of the Requirements for the Degree Master of Science in Environmental Science Professional Science Master Option

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By ©Adane Metaferia 2014 Spring 2014

DIRECT APPLICATION OF BIOMINERALIZATION TO LIFE

SUPPORT SYSTEMS, HABITAT WATER WALL SYSTEM, CARBON

SEQUESTRATION, BIOREMEDIATION AND SOLVING OTHER

VITAL ENVIRONMENTAL PROBLEMS

A Project By Adane Metaferia Spring 2014

APPROVED BY THE DEAN OF GRADUATE STUDIES AND VICE PROVOST FOR RESEARCH:

______Eun K. Park, Ph.D.

APPROVED BY THE GRADUATE ADVISORY COMMITTEE:

______Randy Senock, Ph.D., Chair

______Larry Hanne, Ph.D.

PUBLICATION RIGHTS

No portion of the Project may be reprinted or reproduced in any manner unacceptable to the usual copyright restrictions without the written permission of the author.

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ACKNOWLEDGEMENTS

I am very grateful for Michael Flynn (NASA Ames Research Center) for allowing me to work with him and his teams on this very important and interesting Project. I am very thankful to staff of the Space Biosciences Division, Bioengineering Branch, NASA

Ames Education, and entire NASA Ames Research Center for their valuable support.

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TABLE OF CONTENTS

PAGE

Publication Rights …………………………………………………………………….. iii Acknowledgements …………………………………………………………………… iv List of Tables …………………………………………………………………………. vi List of Figures ………………………………………………………………………… vii List of Abbreviations …………………………………………………………………. viii Abstract ………………………………………………………………………………….. ix

CHAPTER

I. Background Literature Reviews …………………………………….. 1

Bioremediations and Biomineralizations ………………………. ……… 1 The Chemistry of Biogenic …………………...... 6 Cyanobacteria ………………………………………………...... 9

II. Significance of the Project …………………………….…………………. 12

Membrane Based Habitat Water Walls Architectures 12 for Life Support Systems …………………………………...…………... Life Support Systems and the Water Wall Membrane…………….…….. 16 Strategic Objective Goals………………………………….………… …. 17

III. Methodology ……………………………………………………………. 18

Cyanobacteria Cultures and CO2 Fixation …………………………..… 18 Physiochemical and Mechanistic Studies ……………………………… 19

IV. Results ……………………………………………………………. ……. 21

The Anabaena Culture ………………………………………………… 22 The Synechococcus Culture …………………………………...... 22

V. Conclusions and Future Works …………………………………………. 24

References …….……………………………………………………………………..… 27

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LIST OF TABLES

TABLE PAGE

1. Names and Chemical Composition of Biogenic ……………….. 2

2. Summary of the Primary Functions of the Components of the Water Walls System …………………………………………………………….. 13

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LIST OF FIGURES

FIGURE PAGE

1. Biologically Controlled Mineralization ………………………………. 4

2. Biologically Induced Mineralization ………………………………….. 5

3. Model of Carbon Concentrating Mechanism (CCM) …………………. 7

4. Forward Osmosis Treatment Bag, X-Pack TM ……………………….. 14

5. Water Walls Functional Flow of Life Support System Architecture ….. 15

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LIST OF ABBREVIATIONS

AES: Advanced Exploration System

CCM: Carbon Concentrating Mechanism

CSS: Caron Capture Storage

CTB: Cargo Transfer Bag

FO: Forward Osmosis

FOB: Forward Osmosis Bag

FO-CTB: Forward Osmosis-Cargo Transfer Bag

GCDP: Game Changing Development Program

HTI: Hydration Technology Innovations

LLC: Limited Liability Company

NASA: National Aeronautics and Space Administration

NIAC: Innovative Advanced Concepts

STS: Space Transportation System

WW: Water Wall

XANES: X-Ray Absorption Near Edge Structure

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ABSTRACT

DIRECT APPLICATION OF BIOMINERALIZATION TO LIFE SUPPORT

SYSTEMS, HABITAT WATER WALL SYSTEM, CARBON SEQUESTRATION,

BIOREMEDIATION AND SOLVING OTHER VITAL ENVIRONMENTAL

PROBLEMS

By

Adane Metaferia 2014

Master of Science in Environmental Science:

Professional Science Master Option

California State University, Chico

Spring 2014

The main objectives of this research project, is to investigate the efficiency of various in CO2 sequestrations and other waste products during space missions. The report also examines current scientific literature in biomineralization and

CO2 sequestration for the purpose of managing space mission waste products and air revitalization of spacecraft cabin . The management of air pollutants, proper disposal or recycling of waste materials and toxic chemicals are factors in the planning, designing and implementing of space missions. The design and architecture of life support systems in space missions are principally geared towards the removal of toxic substances and revitalization of the habitat with life sustaining materials. Current

ix mechanical and physical technologies of life supporting systems are not only complex and expensive, but are also error prone especially for extended duration missions. Such crucial and massive life support systems need to be augmented or wholly supported by simpler, efficient and reliable advanced technologies. The next generation life support technologies could be developed by the integration of multidisciplinary efforts of wide ranging fields such as Chemistry, Engineering, and Biotechnology.

In recent years, waste recycling and pollution remediation technologies that are integrated with biological systems have become tremendously attractive and a subject of various applied research programs. Biologically mediated recycling of waste materials could be best suited for space missions due to their efficiency, simplicity and most importantly could be reliable and self-sustaining. Hence, the overarching goals of this project are to integrate microalgal with NASA’s life support system and evaluate its usefulness as a sustainable technology. This life support system here after called the Water Wall (WW) system can sequester spacecraft pollutants and convert them into value added products. The WW architecture requires microorganisms that could facilitate the of pollutants and revitalize the spaceship habitat. Hence, initial candidates of suitable microorganisms were selected and optimal growth conditions, critical limiting factors and efficiencies of air revitalization were investigated.

Among the model microorganisms, Myxococcus Xanthus, Brevundimonas Diminuta,

Anabaena (PCC 7120), Synechococcus (BG04351), Chlorella, Spirulina species and etc., have been studied to establish optimum growth conditions. In the preliminary optimization stage of the project variables such as growth media, levels of carbon dioxide, hydrogen ion concentration etc. have been evaluated and optimized.

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CHAPTER I

BACKGROUND LITERATURE REVIEWS

Bioremediations and Biomineralizations

Bioremediation is the use of biological process and systems to facilitate the conversion of harmful chemical contaminants or pollutants into less toxic and environmentally friendly by-products in self-sustaining manner. Bioremediation through biological sequestration and degradation is especially suitable and practical as a point- source carbon capture for confined spaces such as submarines and spaceships [Lackner, et al., 2013]. In the application of bioremediation, selecting a suitable and effective for the specific pollutant plays a key role for its success. In order to fully benefit the application of microorganisms in bioremediation, it is vital to systematically study the nature of the microorganisms optimum growth conditions, and metabolic by- products. Critical studies involving both in situ mineralization and the basic chemical crystallization processes are keys in developing efficient waste recycling technologies.

Biomineralization is a process by which organisms form biominerals through their natural metabolic processes. Many microorganisms biologically sequester organic and inorganic materials and are involved in secretion or precipitation. There are several examples of biogenic minerals as a result of biominerlization processes; these include carbonates, sulfates, oxalates, and mixtures of such minerals with humic substances (Table 1). 1

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Table 1. The Names and Chemical Compositions of Biogenic Minerals [Weiner and Dove, 2003].

Carbonates CaCO3 Mg-Calcite (MgxCa 1-x)CO3 CaCO3 CaCO3 Monohydrocalcite CaCO3.H2O Protodolomite CaMg(CO3)2 Hydrocerussite Pb3(CO3)2(OH)2 Amorphous Calcium CaCO3 Phosphates Octacalcium Ca8H2(PO4)6 Brushite CaHPO4.2H2O Francolite Ca10(PO4)6F2 Carbonated- (Dahllite) Ca5(PO4CO3)3(OH) 2+ Whitilokite Ca18H2(Mg,Fe)2 (PO4)14 Struvite Mg(NH4)(PO4).6H2O 2+ Vivianite Fe3 (PO4)2.8H2O Amorphous Variable Amorphous Calcium-Pyrophosphate Ca2P2O7.2H2O Sulfates Gypsum CaSO4.2H2O Barite BaSO4 Celestite SrSO4 3+ Jarosite KFe3 (SO4)2(OH)6 Sulfides FeS2 Hydrotroilite FeS.nH2O Sphalerite ZnS Wutzite ZnS PbS Fe3S4 Mackinawite (Fe,Ni)9S8 Amorphous Pyrrhotite Fe 1-xS (x=0-0.17) Acanthite Ag2S Hydrated silica, arsenates, chlorides, fluorides and sulfur Orpiment As2S3 Amorphous silica SiO2.nH2O Atacamite Cu2Cl(OH)3 CaF2 Hieratite K2SiF6 Sulfur Element S Organic crystals Earlandite Ca3(C6H5O2)2.4H2O Whewellite CaC2O4.H2O Weddelite CaC2O4.(2+x)H2O (x,0.5) Glushinskite MgC2O4.4H2O Manganese Oxide (unnamed) Mn2C2O4.2H2O Sodium Urate C5H3N4NaO3 Uric acid C5H4N4O3 Ca tartrate C4H4CaO6 Ca malate C4H4CaO5 Paraffin Hydrocarbon CnH 2n+2 Guanine C5H3(NH2)N4O Oxides, hydroxides and hydrous oxides Fe3O4 2+ Amorphous Ilmwnite Fe TiO3 Amorphous oxide Fe2O3 Amorphous Manganese Oxides Mn3O4 a-FeOOH Lepidocrocite g-FeOOH Ferryhydrite 5Fe2O3.9H2O +2 +4 Todorokite (Mn CaMg)Mn3 O7.H2O Bimessite Na4Mn14O27.9H2O

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The phenomena of biominerlization in both prokaryotes and is driven by evolutionary advantages the organisms gain in order to survive [Gilbert, et al., 2005].

Biominerlizing organisms consume and thrive on nutrients they absorb from their environments. In most cases the biomineral that is produced by the microorganism are benign and less harmful to the environment. The term biomineral refers not only to the minerals produced by these organisms but also composite products of minerals and organic components. Biomineral phases (substrates) have very distinct properties such as physical, chemical morphological, isotopic and trace element composition compared to the synthetically (inorganic) created counterparts [Weiner and Dove, 2003]. Weiner and

Dove also demonstrated the comparison between calcite single crystal formed by a stereo of (biomineral) and synthetic single rhombohedral crystal forms of calcite.

These differences are attributed to the fact that the biomnerals are formed under complex biologically controlled conditions.

In situ biomineralization process is classified either as biologically controlled or induced [Lowenstam and Weiner, 1989; Dupraz et al., 2009]. There are two mechanisms by which organisms undergo biologically controlled biomineralization [Weiner and

Dove, 2003]. The first biologically controlled mineralization mechanism involves active release of cations (positive ions) outside the cytosol and passive gradient diffusion to the organic matrix (Figure 1A). In the second mechanism of biologically controlled mineralization the cations are actively transported into vesicles inside the cytosol (Figure

1B) then transported through passive diffusion towards the nucleation sites. In both cases

(Figures 1A and 1B), controlled biomineralization, takes place under specific metabolic

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and genetic control. In the second biologically induced mineralization, mineral crystallization is guided by changes in metabolic processes such as pH, amount of CO2, concentration of ions and etc. (Figure 2).

Figure 1. Biologically controlled mineralization A) the cations are actively transported to the extracellular organic matrix B) the cations are actively pumped into intracellular vesicles and secreted out towards the organic matrix. The schematic is modified from Weiner and Dove 2003, Overview of Biomineralization Processes and Vital Effect Problem.

The major share of global carbon cycle is due to CO2 sequestration and biomineralization [Ridegewell and Mucci, 2005]. It is a common process in marine, freshwater and terrestrial ecosystems. Therefore, it is worthwhile to clearly understand the mechanism of the process and factors that influence this dynamics.

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Atmospheric CO2 is sequestered largely in the form of carbonates of metal ions such as Ca2+, Mg2+, Mn2+, Fe2+, and Sr2+. However, calcium carbonates are the most biomineralized and ubiquitous in many terrestrial, marine and lacustrine organisms

[Lowenstam and Weiner, 1989]. The term is widely used due to this high abundance of calcium-rich biominerals [Weiner and Dove, 2003]. Calcium containing minerals comprise more than 50% of known biominerals [Lowenstam and Weiner, 1989].

This high abundance of calcium containing minerals correlates with calcium being the most important cellular messenger in microorganisms and a highly controlled equilibrium. As a result, we can suggest that biomineralization of Ca2+ is a tightly controlled cellular mechanisms [Morse, et al.,].

Figure 2. Biologically induced mineralization. Mineral crystallization form due to metabolic processes such as pH changes, amount of CO2 ions and etc. Schematic modified from Weiner and Dove 2003 overview of Biomineralization Process and Vital Effect Problem.

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High ionic strength, thermodynamics and solubility factors favor calcium ion to form diverse groups of biominerals (Table 1). Mechanisms and the factors that affect its crystal growth (polymorphism) are not well understood especially in the context of the recently identified amorphous . Regardless of the actual crystal growth mechanism, biomineralization processes will indefinitely capture and sequester significant amount of CO2 and convert it to various solid carbonates.

The Chemistry of Biogenic Calcium Carbonates

Due to the high water solubility of CO2 compared to other gases such N2, O2 and

2+ Ar, the formation of calcium carbonate from Ca and CO2 is thermodynamically favorable [Gebauer, et al., 2009]. However, the mechanism of this reaction is complicated by several equilibrium that takes place in the formation of calcium carbonate. Consequently the rate of formation (kinetics) of this phenomenon is quite complex. The carbon concentrating mechanism (CCM) shown in Figure 3, underscores the intricacy of the process that goes through the progression of carbon concentration and the final fate of carbon in the calcification process.

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Figure 3. Model of carbon concentrating mechanism (CCM) and calcification inside a cyanobacteria cell. The diagram shows the complex chemical reactions taken place inside a cyanobacteria cell to achieve the end result of calcium carbonate. Modified from Riding, R.,(2006), , 4:299-316

Briefly, dissolved CO2 initially reacts with water to form less stable carbonic acid, which triggers a cascade of equilibrium reaction depending upon the acidity and alkalinity of the medium as shown below. In each one of these cascading equilibriums, the pH has a direct influence as to which side of the equilibrium will be favored. The effective concentration or ionic strength of the CO2 is highly dependent on the pH of the medium where low acidity or higher alkalinity favors the dissociation into the carbonate anion.

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Therefore, the ionic strength becomes higher and results in the crystallization of

2+ 2- 3- calcium carbonates. The presence of divalent Mg ion, other anions such as SO4 , NO and Cl- and organic macromolecules have been shown to influence the morphology, crystal size distribution and composition of the growing crystals [Ren, et al., 2013]. The effect of these materials before nucleation happens is inhibition, however once the seed of nucleation is formed they contribute by facilitating the crystallization process. Such effects were more pronounced in aragonite than the other polymorphs. Peptides rich in negatively charge residues such as aspartate and glutamate electrostatically attract the positive ions from solution to initiate nucleation and crystallization [Weiner and Dove,

2003].

The various polymorphs are characterized by different solubility product constants, which is a measure of the saturation or increase in effective ionic strength of both the cation and the anion that form the solid phase crystal.

2+ 2- Ksp ≥ [Ca ][CO3 ] no crystallization

2+ 2- Ksp ≤ [Ca ][CO3 ] crystallization

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Based on the solubility product constants (Ksp) values of the three most common polymorphs of natural CaCO3 (Ksp calcite < Ksp aragonite < Ksp vaterite), calcite is the most stable polymorph while vaterite is the least stable carbonate [Kamennaya et. al.,

2012]. It is important to understand the reaction mechanism at the interface in adequate details to design future carbon recycling methods and technologies.

The applications of x-ray spectromicroscopy and biological methods are necessary in the elucidation of the chemistry of nucleation and the organic-mineral interface [Gilber, et al., 2005].

Cyanobacteria

Cyanobacteria are class of Gram-negative that use aerobic photosynthesis and live in a wide-ranging habitat such as marine, fresh water, terrestrial and extreme environments such as hot springs, deserts and bare rocks. They have played significant role in Calcium Carbonate precipitation and , which consequently has major role in geological formations since the archaen era [Power et al., 2007]. In comparison to , cyanobacteria are much more photosynthetically efficient organisms and require lower light intensity. Half of the global photosynthesis is accomplished by , which mainly comprised of cyanobacteria [Fuhrman, 2003] and 25% of the global photosynthesis is carried out by two marine genera of cyanobacteria namely

Synechococcus and Prochlorococcus [Rohwer, 2009].

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Cyanobacteria thrive in high level CO2 environment and they are considered the most lucrative systems for CO2 capture from flue gas [Ono and Cuello, 2007]. The carbon concentrating mechanism (CCM) by Cyanobacteria is very complex and varies between cyanobacteria [Northen and Jansson, 2010]. However, majority of the cyanobacteria share CCM depicted on Figure 3 above.

Algae or cyanobacteria produce life supporting pure O2 while CO2 is being absorbed from the environment by photosynthetic process. With high content of nitrogen and other trace nutrients, one can expect algae biomass to utilize up to 50% of the CO2 available and likely return more than 10% by weight as biomass production [Wiley, et al.,

2013]. Initially such a system can provide an immediate dividend potentially as odor control and trace gas management tools, while optimizing the larger objectives of maximizing O2 over CO2 production.

Microalgal organisms are also very efficient in converting CO2 into a biomass via a process of photosynthesis [Ono and Cuello, 2006; Ryu, et al., 2009]. There are also added advantages of microbial sequestration; the metabolites or biomass generated by microalgal fixation of CO2 and other pollutants are full of energy rich products such as carbohydrates, , and lipids which are good sources of nutrients and renewable energy [Del Campo, et al., 2007].

Most importantly microalgal systems tolerate highly alkaline media, saline environments, and varying light intensities which are important traits necessary for such

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system to be interfaced with various technological processes [Benemann, 1997;

Murakami and Ikenouchi, 1997].

Biofixation of CO2 by cyanobacteria in photo bioreactor systems is a sustainable strategy, since CO2 can be incorporated into the molecular structure of cells in the form of proteins, carbohydrates and Lipids by way of photosynthetic reactions.

The advantages of these processes are related to the greater photosynthetic efficiency of cyanobacteria compared to eukaryotic algae and higher plants, as well as the resistance of these microorganisms to high CO2 concentrations, and the possibility of better controlling the culture growth conditions. Microalgae systems are advantageous because of their tolerance of high salt concentration, pH and CO2 concentration, and temperature variation and light intensity. [Benneman, et al., 1997].

CHAPTER II

SIGNIFICANCE OF THE PROJECT

Membrane Based Habitat Water Walls Architectures

for Life Support Systems

Reliable and sustainable life support systems are crucial for long-term human space exploration missions. Replenishing these life support necessities in the open loop case to crewmembers continuously requires tremendous resources. To leverage these challenges, it is important to find cheaper, reliable, and sustainable closed loop life support systems. Engineering designs that incorporate microorganisms or biological processes are among the best-sought strategies in mitigating the effects of crew waste products. The WW system will significantly reduce the cost and other issues related to the use of the mechanical and error prone life support technologies. Hence, by significantly reducing payload mass and cost, one can extend space exploration from low-earth orbit to the deep space missions. The proposed WW system is designed to have significant contribution in sustaining and revitalizing the different compartments of the spacecraft habitat. It will be useful in processing and purifying black water, removing

CO2 from the producing O2 and supporting food growth using green algae and other edible microorganisms, controlling humidity and ambient temperature, and 12

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providing radiation protection for the crews The matrix of WW subsystems and the processes they perform are described in Table 2.

The approach provides novel and potentially game changing mass reduction and structural advantages over current mechanical life support systems [Flynn, et al., 2011]

Table 2. Summary of the primary functions of the components of the Water Wall System

Humidity Black Algae PEM Urine/H & Water Wall Primary Functions Water Growth Bag Fuel Cell O Bag Thermal Solid Bag 2 Bag O2 Revitalization X

CO2 Removal X

Denitrification/N2 Liberation X X X

Clean Water Production X X

Urine &Gray Water Processing X

Semi-Volatile Removal X

Black Water Processing X X

Humidity & Thermal Control X

Nutritional Supplement X Production

Electrical Power Production X

The fundamental technology behind the WW system rests on simple but yet powerful forward osmosis (FO). FO has been proven as an ideal technology to remove

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contaminated organic matter and water, which provides clean input for a biotic system such as an algal culture. A commercially available FO technology called X-Pack™ from

Hydration Technologies Innovations (HTI) is currently used as water purification system

(Figure 4A.). A similar purification technology with minor modification known as a

Forward Osmosis Bag (FOB) has been tested in microgravity on the STS 135 space shuttle mission.

A B ) )

Figure 4. A) Forward Osmosis Treatment Bag, X-Pack TM, Commercially available through Hydration Technology Innovations, LLC. B) The same Forward Osmosis Bag slightly modified for flight experiment.

A membrane WW system shown in Figure 5, utilizing a forward osmosis process is proposed as an integrated system that could efficiently and reliably removes toxic materials and replenishes the spacecraft with critical life support systems. As an example, the orange colored box represents the FEED where black water, organic fuels, solids are stored temporarily and the PERMEATES from this compartments filled with fertilizers dissolved in clean water as soluble salts transferred to the green box where algae growth takes place. Oxygen regeneration as well as power production can also be anticipated, and algae-cyanobacteria reactors can provide vital back up to many aspects

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of a fully developed system. Much of the secondary metabolites of these microorganisms are scarce and valuable medicinal and nutritional products that can have potential as nutrient resources.

Figure 5. Water Walls Functional Flow Life Support System Architecture (Courtesy: NASA AMES Research Center)

Air vitalization efforts are not only concerned with CO2 and H2O. Urine and other waste materials contribute to the production of high levels of toxic nitrogenous gases. Particular emphasis should be given for ammonia gas in the confined habitat and it is crucial to address it through an integrated approach within the WW system.

Bioavailable nitrogen is found to be a limiting nutrient in algal growth under different

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growth conditions, however in the case of cyanobacteria, it is not a limiting nutrient due to their nitrogen fixing capability. Algae utilize nitrogen in the form of ammonia and can take a substantial amount of nitrogen in their biomass.

Life Support Systems and the Water Wall Membrane

Space exploration plays an important role in advancing science and technology, the discoveries from human space missions can greatly contribute to better understand the universe and potentially lead us to innovations that are not within our reach at present.

Despite the high cost of such explorations, significant technological advances have been made in space exploration. Human space missions are particularly expensive mainly due to the complexity and challenges of life support systems. Life support systems need to be highly efficient, reliable, safe and self-sustaining. One of the most critical area is air quality and toxic effluents produced in the space station and within the confined manned spacecraft. The current life support systems are dependent on mechanical systems, they are material intensive, and require transportation of pay-load to and from the space station. To address these challenges and develop advanced life support systems, wide ranging research programs are underway by the private sector and governmental agencies. This Project proposes a water wall (WW) membrane system that can recycle toxic effluents, gases and human waste in an integrated format through biological sequestration and biomineralization processes. .

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The WW could adequately provide life support systems efficiently, reliably and cost effectively. The WW system is designed to recycle undesirable toxic materials and revitalize the enclosed habitat with critical life supporting oxygen, clean water and nutrients.

Furthermore, the engineering and by products of such WW system could serve as shields from dangerous radiation, humidity and temperature control. As part of the overall goal of the WW project which aims to develop reliable, less expensive and renewable life support systems and expand current low-earth orbits programs to the deep space explorations, this particular project report focuses on the application of biomineralizing microorganisms for CO2 sequesterations, air vitalization and the selection of suitable organisms for these purposes

Strategic Objective Goals

• Develop membrane WW system by integrating skills and knowledge acquired

from disciplines such as Biology, Chemistry, Architecture, Engineering and etc.

• Enhance the membrane WW system by interfacing it with efficient

biomineralizing organisms that can metabolize CO2 and other toxic waste

products and convert them to useful biomasses.

• Investigate metabolic limiting factors that affect in-situ biomineralization

processes.

• Explore and apply advanced biotechnology and genomics principles to facilitate

biomineralization.

CHAPTER III

METHODOLOGY

Cyanobacteria Cultures and CO2 Fixation

Pure cultures of the freshwater Anabaena (PCC 7120) were obtained from the provosolli-guillard culture collection and the marine Synechococcus (BG04351) was obtained from the Hawaii culture collection.

Anabaena cultures were maintained and grown in BG-11 medium (Sigma-

Aldrich) and Synechococcus cultures were maintained on BG-11, to which 30 g/L of commercial sea salts (Sigma-Aldrich) were added. Growth phases were monitored by optical density measurements. Temperature and pH changes in the growth medium were monitored periodically.

The 30 g/L salt concentration is used as a baseline osmotic agent reference to the performance of the FO membrane. This value is obtained from prior experimental testing and development of the FO bag. And thus function as a convenient benchmark for assessing competing FO membranes and their derived elements.

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Ten mL of mid log-phase cultures of Anabaena and the marine Synechococcus were used to inoculate 500 mL Erlenmeyer flasks containing 100 mL of either BG-11 medium (Anabaena) or BG-11 to which 30 g/L of commercial sea salts (Sigma-Aldrich) were added (Synechococcus). In addition one mL of mid-log-phase cultures were used to inoculate 9 mL of medium contained inside a gas permeable biological canisters called

OptiCellsTM membrane systems.

The flasks and OpticellTM systems were incubated at room temperature (220C) under ambient room fluorescent lights (16 hrs on 8 hrs off) for 7 to 14 days. After incubation the content of each culture was determined by combustion compatible with total organic carbon, high-temperature combustion method

5310 B. Briefly, combustion samples were dried overnight at 800C. The dried samples were then weighed and heated for three hours at 6000C and re-weighed and resulting mass, volume, and reactor area were analyzed.

Physiochemical and Mechanistic Studies

After optimization of CO2 sequestration, a closer look at the intracellular and changes during crystallization process will be investigated by a scanning electron microscopy. Accurate measurements of variables such as temperature, salinity and dissolved oxygen would be carried out by a Multiparameter meter (Thermo

Scientific). The effect of pH in the growth rate of the organisms will be studied in ranges

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between pH 4 to 8. Since biomass production depends on the degree of exposure to light

[Lopes, 2009], systematic studies are also necessary.

Different intensities of light (2000, 4000, 6000 and 10000 lx) will be used to determine the optimal intensity in relation to biomass production. Provided there could be access to x-ray absorption, a near edge structure (XANES) microscopy, the nucleation mechanism at the interface of solid minerals and growth medium can be elucidated

[Benfatto et al., 2003].

CHAPTER IV

RESULTS

-5 -2 -1 The overall rate of CO2 fixed by Anabaena was 5.36 x 10 g CO2 fixed cm hr .

-1 -1 This equals 53.6 mg CO2 fixed L hr . The overall rate of CO2 fixed by the marine

-5 -2 -1 Synechococcus was greater by about 4.7 times, equaling 25 x 10 g CO2 fixed cm hr ,

-1 -1 equaling 250 mg CO2 fixed L hr . The reasons for the difference in results between the freshwater and marine cyanobacteria are under further investigation. Ongoing tests include conducting similar experiments using species of the green alga, chlorella, and the edible cyanobacteria spirulina.

The chlorella, spirulina, aphanothece and scenedesmus species have become attractive in CO2 fixation studies due to the high level of tolerance of CO2 concentration

[Sung, 1999; Yue, 2005] and also the value added nutrients they produce [Sankar, et al.,

2011].

The next major step was to examine CO2 fixation rates in the WW candidate bags.

The size of the WW bag that can support a single crewmember per day was determined based on the efficiency of the CO2 fixation rates for the two organisms.

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The Anabaena Culture

Sizing parameters from CO2 sequestration results for freshwater cultures of the

Anabaena (PCC 7120) were determined by calculating the daily fixation rate of CO2 by the and the daily amount of CO2 exhaled by a crewmember. The result indicates about 800 L of culture is required to support a single crew member per day and the WW bag need to be 16 m2 with 5 cm thickness (with double side illumination).

The calculations are as follows:

-5 • CO2 When scrubbing ambe = 53.6 mg CO2 fixed/L/hr. or 5.36 x 10 kg

CO2 fixed/L/hr.

-5 • 5.36 x 10 kg/L/hr. x (24) hrs = 1.286 x10-3 kg/L/day CO2 could be

fixed, and

• 1 kg CO2 produced per crew member/day

The volume of culture = 1 kg/day CO2/1.286 x 10-3 kg/L/day = 777.3 L, which is about 800L of Anabaena culture required for sequestration. This volume is the same as 0.8 m3. Therefore, the design of the WW bag will be 5 cm thick with an area of 16 m2.

The Synechococcus Culture

For CO2 sequestration results for marine (i.e. salt water/OA compatible)

Synechococcus (BG 04351) cultures:

-4 • CO2 When scrubbing ambe = 250 mg CO2 fixed/L/hr. or 2.50 x 10 kg

CO2 fixed/L/hr.

• 2.50 x 10-4 kg/L/hr. x (24) hrs. = 6.0 x10-3 kg/L/day

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• 1 kg CO2 produced per crew member/day =1 kg/day CO2 exhaled =166.7

L synechococcus required per crewmember per day for CO2 fixation is

6.00 x10-3 kg/L/day.

So, we will get 170L (rounded up four significant Figures) of synechococcus/water solution. With a 5cm depth of synechococcus bags if illuminated on both sides, the bag will have a 3.4 m2 size.

Based on the above results, it is worth mentioning that the variation in the type of cyanobacteria used can produce significant difference in the performance of the WW system as air revitalization and carbon sequestration integrated systems. It is encouraged to further characterize various organisms and study the variables that maximize CO2 fixation. By identifying more efficient species of cultures, a robust and sustainable WW membrane system can be designed. Unfortunately, due to drastic NASA’s budget cut

(sequestration), the project has been discontinued. Several limiting factors that were planned to be investigated have not been performed because of limited financial resources. The natural extension of the identification and characterization of suitable cultures was to systematically optimize the growth condition and include more for screening.

CHAPTER V

CONCLUSIONS AND FUTURE WORKS

One of the objectives of the research project was the optimization of growth conditions of microalgal organisms and the determination of the amount of CO2 fixed.

Accordingly, the overall rate of CO2 fixed by the fresh water Anabaena and the marine

Synocococcus is determined. This result has provided the foundation necessary for baseline air revitalization parameters in the WW concept and have been published on

NASA Innovative Advanced Concepts (NIAC) [Flynn et al., 2012]. Based on the final volume of cultures required for CO2 sequestration for the freshwater and marine species, the size of the membrane WW bag that can support a single crewmember for optimum air revitalization requirement is successfully determined.

This paper also attempted to explain the intricacy of the kinetics and thermodynamics of biomineralizations and processes from both in-situ and complex physicochemical reaction perspectives. Though, the project ultimate goal is to interface the biomineralization processes with the WW membrane, more rigorous research is essential to achieve this goal. This research has demonstrated the practicality of cyanobacteria and algal cultures for CO2 sequestration and application for spaceship air revitalization purposes.

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Biomineralization and calcification processes have tremendous benefits in terms of augmenting and improving the WW membrane based life support system. Efficient and tailored biomineralizing microorganisms that can be fully integrated to the WW system play critical role in waste recycling, air revitalization, waste products sequestration, radiation shield, and etc. The research proposal in the use of uniquely nanostructured biomaterials derived from microalgal metabolism and biomineralization process deserves rigorous and deeper investigation.

These materials due to their unique architecture that could not be imparted by artificial synthesis could have an important application in radiation protection and shielding. To fully explore and utilize the technological and environmental applications of microorganisms, there is a greater need for a multidisciplinary approach and adequate financial resources. In the future, the hope is to pursue with much more focused research and development strategies on the applications of biomineralizing microorganisms to life support systems and carbon capture and sequestration technologies. We are currently working on a follow up proposal for submission to the NASA Game Changing

Development Program (GCDP) to fund this research project.

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Even though the progress of the project was adversely affected by the lack of funding due to budgetary constraints, progress has been made to select and cultivate certain species of algae, cyanobacteria and other microorganisms and their growth conditions were optimized. Provided the budgetary situations improve, the project could continue to study the rate of CO2 consumption as a function of O2 production, the fixation of biogenic ammonia and its conversion rates will be optimized. Biomineralization as CO2 scrubbing strategy and the final fate of the solid carbonate will be investigated. Finally, the laboratory data will be extrapolated to build a scaled up WW membrane system to study its performance to support real time space missions.

Lessons learned from the integrated WW system are also important in advancing currently existing knowledge about point-source carbon capture sequestration methodologies.

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