DIRECT APPLICATION OF BIOMINERALIZATION TO LIFE

SUPPORT SYSTEMS, HABITAT WATER WALL SYSTEM, CARBON

SEQUESTRATION, BIOREMEDIATION AND SOLVING OTHER

VITAL ENVIRONMENTAL PROBLEMS

_________________

A Project

Presented

to the Faculty of

California State University, Chico

_________________

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

in

Environmental Science

Professional Science Master Option

__________________

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.

iii

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.

iv

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.

v

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 Calcium Carbonates …………………........ 6 Cyanobacteria ………………………………………………................... 9

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

Membrane Based Habitat Water Walls Architectures for Life Support Systems …………………………………...…………...

12

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

vi

LIST OF TABLES

TABLE PAGE

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

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

13

vii

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

viii

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

ix

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 microorganisms 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 atmospheres. 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

x

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 organisms 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 biodegradation 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.

1

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

microorganism 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 mineral secretion or precipitation. There are

several examples of biogenic minerals as a result of biominerlization processes; these

include carbonates, sulfates, oxalates, phosphates and mixtures of such minerals with

humic substances (Table 1).

2

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

Carbonates Calcite Mg-Calcite Aragonite Vaterite Monohydrocalcite Protodolomite Hydrocerussite Amorphous Calcium Carbonate

CaCO3 (MgxCa 1-x)CO3 CaCO3 CaCO3 CaCO3.H2O CaMg(CO3)2 Pb3(CO3)2(OH)2 CaCO3

Phosphates Octacalcium Phosphate Brushite Francolite Carbonated-Hydroxyapatite (Dahllite) Whitilokite Struvite Vivianite Amorphous Calcium Phosphate Amorphous Calcium-Pyrophosphate

Ca8H2(PO4)6 CaHPO4.2H2O Ca10(PO4)6F2 Ca5(PO4CO3)3(OH) Ca18H2(Mg,Fe)2

2+(PO4)14 Mg(NH4)(PO4).6H2O Fe3

2+(PO4)2.8H2O Variable Ca2P2O7.2H2O

Sulfates Gypsum Barite Celestite Jarosite

CaSO4.2H2O BaSO4 SrSO4 KFe3

3+(SO4)2(OH)6 Sulfides

Pyrite Hydrotroilite Sphalerite Wutzite Galena Greigite Mackinawite Amorphous Pyrrhotite Acanthite

FeS2 FeS.nH2O ZnS ZnS PbS Fe3S4 (Fe,Ni)9S8 Fe 1-xS (x=0-0.17) Ag2S

Hydrated silica, arsenates, chlorides, fluorides and sulfur Orpiment Amorphous silica Atacamite Fluorite Hieratite Sulfur

As2S3 SiO2.nH2O Cu2Cl(OH)3 CaF2 K2SiF6 Element S

Organic crystals Earlandite Whewellite Weddelite Glushinskite Manganese Oxide (unnamed) Sodium Urate Uric acid Ca tartrate Ca malate Paraffin Hydrocarbon Guanine

Ca3(C6H5O2)2.4H2O CaC2O4.H2O CaC2O4.(2+x)H2O (x,0.5) MgC2O4.4H2O Mn2C2O4.2H2O C5H3N4NaO3 C5H4N4O3 C4H4CaO6 C4H4CaO5 CnH 2n+2 C5H3(NH2)N4O

Oxides, hydroxides and hydrous oxides Magnetite Amorphous Ilmwnite Amorphous Iron oxide Amorphous Manganese Oxides Goethite Lepidocrocite Ferryhydrite Todorokite Bimessite

Fe3O4 Fe 2+TiO3 Fe2O3 Mn3O4 a-FeOOH g-FeOOH 5Fe2O3.9H2O (Mn+2CaMg)Mn3

+4O7.H2O Na4Mn14O27.9H2O

3

The phenomena of biominerlization in both prokaryotes and eukaryotes 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 echinoderm (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

4

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.

5

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 calcification 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.

6

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 calcium carbonate. 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

Ar, the formation of calcium carbonate from Ca2+ 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.

7

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), Geobiology, 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.

8

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

calcium carbonates. The presence of divalent Mg2+ ion, other anions such as SO42-, NO3-

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.

Ksp ≥ [Ca2+][CO32-] no crystallization

Ksp ≤ [Ca2+][CO32-] crystallization

9

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 organic mineral

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 bacteria 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 sedimentation, which consequently has

major role in geological formations since the archaen era [Power et al., 2007]. In

comparison to algae, cyanobacteria are much more photosynthetically efficient organisms

and require lower light intensity. Half of the global photosynthesis is accomplished by

phytoplankton, 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].

10

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, proteins, 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

11

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].

12

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 atmosphere producing O2 and supporting food growth using green algae

and other edible microorganisms, controlling humidity and ambient temperature, and

13

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

Water Wall Primary Functions Algae Growth Bag

Black Water

Solid Bag

PEM Fuel Cell

Urine/H2O Bag

Humidity &

Thermal 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 Production

X

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

14

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.

    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

A)  

B)  

15

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

16

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. .

17

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.

18

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 test

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.

19

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 total organic carbon 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

extracellular matrix 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

20

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].

21

CHAPTER IV

RESULTS

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

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

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

equaling 250 mg CO2 fixed L-1 hr-1. 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.

22

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 organism 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:

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

CO2 fixed/L/hr.

• 5.36 x 10-5 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:

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

CO2 fixed/L/hr.

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

23

• 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.

24

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 calcifications 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.

25

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.

26

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.

 

 

REFERENCES

27

REFERENCES

Benemann, J. (1997), Energy conversion and management, 38:S475-S479.

Benfatto, M, M., S. Della Longa, J. Synchro, and C.R. Natoli (2003), The MXAN procedure: a new method for analyzing the XANES spectra of Metalloprotieins to obtain structural quantitative information, Radiat 10:51-57.

Cao, X., X. Ying, , and L. Janelle (2013) The effects of micro-algae characteristics on the bioremediation rate of Deepwater Horizon crude oil. Journal of Emerging Investigators, 1-7

Del Campo, G. Gonzalez, and M. Guerrero. (2007)., Outdoor Cultivation of Microalgae for Carotenoid Production: Current State and Perspective. Appl. Microbiol, Botechnol. 74:1163-1174

Flynn, M., M. Soler, K. Howard, S. Shull, J. Broyan, J. Chambliss, S. Howe, S. Gormly, M.Hammoudeh, and H. Shaw (2012), 42nd International Conference on Environmental Systems, 15 - 19 July 2012, San Diego, California

Fuhrman, J. (2003), Genome sequences from the se, Nature, 424:1001-1002

Gebauer D., A. Volkel and H. Cofen (2008), Stable Prenucleation Calicium Carbonate Clusters. Science, 322: 1819-1822.

Gilbert, P.U.P.A., M. Abrecht, and B. Frazer (2005), the organic-mineral interface in biominerals, Reviews in Mineralogy and Geochemistry, 59:157-185

Jacob-Lopes, E. (2008), Biochemical Engineering Journal, 40:27-34

Jacob-Lopes, E., C. Scoparo, T. Franco (2007), Rates of CO2 removal by Aphanothecemicroscopica N¨ageli in tubular photobioreactors, Chem. Eng. Process.,doi:10.1016/j.cep.2007.06.004

28

Jeng F. A., and J. Handford (2011), An architecture study for mars transit flight and surface missions from radiation exposure perspective, NASA JSC Report ESCG-4470-11-TEAN-DOC-0018, 2011

Kamennaya, N.A., C, C. M. Ako-Franklin, T. Northen and C. Jansson (2012), Cyanobacteria as biocatalysts for carbonate mineralization. Minerals, 2:338-346.

Lopes, E. J., C.H.G. Scoparo, L.M.C.F. Lacerda, and T. T. Franco (2009) Chemical Engineering and Processing: Process Intensification, 48: 306-310.

Lowenstam, H.A., and S. Weiner (1989), On biomineralization, Oxford University Press, New York

Martins, S., T. Caetano, and M. Mata (2010), Microalgae for biodiesel production and other applications 4200-072

Marvasi, M., P.T. Visscher, B. Perito, G. Mastromei, and L. Casillas-Martine (2009), Physiological requirements for carbonate precipitation during biofilm development of Bacillus subtilis etf A mutant, FEMS Microbial Ecology Volume71, Issue 3 Pages 327–479

Morse, D.E., M. A. Cariolou,, G. D. Stucky,. C.M. Zaremba,.;P.K. Hansma (1999), Genetic Coding in biomineralization of Mcrolaminate composities. MRS proceedings Symposium: Biomolecular Materials: 292.

Murakami, M. and M. Ikenouchi (1997), Energy conversion and management. 38:S493-S497.

Northen, T., and C. Jansson (2010) Calcifying Cyanobacteria. Curr. Opin. In Botechnol. 21: 1-7.

Ono, E. and J.L. Cuello (2006), Biosystems Engineering 95:597-606.

Power, I.M., Wilson, S.A., J.M. Dipple and G. Sotham (2007), Bilologically induced mineralization of dypingite by cyanobacteria from an alkaline wetland. Geochemical Transactions; 813 doi: 1186/1467-4866-8-13

Ren, D., Q. Feng, and X. Bourrat (2013) The coeffect of Organic Matrix from Carpotolith and Microenvironment on Calcium Carbonate Mineralization. Mater. Sci. Eng. C Mater. Biol. Appl. 33.3440-3449

29

Rice, W., R. Baird, A. Eaton, and L. Clesceri (2012), Standard Methods for the Examination of Water and Wastewater, 22nd Ed., American Public Health Association/Port City Press, Md. Pages 5-23 to 5-25.

Ridgewell, A. and A. Mucci (1993), Calcite precipitation in seawater using a constant addition technique- a new overall reaction kinetic expression, Geochemical Et Cosmochmica Acta 57:1409-1417.

Riding, R.; (2006) Cyanobacterial Calcification, Carbon Dioxide concentrating mechanisms, and Proterozoic- Cambarian Changes in atmospheric cmoposition. geobiology, 4:299-316

Ryu, H. J., K.K. Oh, and Y. S. Kim (2009) Journal of Industrial Engineering Chemistry, 15:471-475.

Rodriguez-Navarro, C., F. Jroundi, M. Schiro, E. Ruiz-Agudo, and M.T. González-Muñoz (2012), Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation, Applied and Environmental Microbiology, 78(11) 4017-4029

Rohwer, F. (2009), Thurber RV: Viruses manipulate the marine environment, Nature. 459:207-212

Sankar, V., D.K. Danie,and A. Krastanov (2011), Carbon dioxide fixation by Chlorella Minutissima batch ultures in a stirred tank bioreactor. Biotechnology & Biotechnology Equipment. 25: 2468-2476.

Semple, K. T., R.B. Cain and S. Schmidt (1999), Biodegradation of Aromatic Compounds by Microalgae.; FEMS Microbiology Letters. 170: 291-300

Sung, K. D., J. S. Lee, C. S. Shin and S. C. Park (1999), Renewable Energy, 16: 1019-1022.

Weiner, S., and P. M. Dove, An Overview of biomineralization processes and the problem of the vital effect (2003)

Wiley, P., L. Harris, S. Reinshch, S. Tozzi, T. Embaye, k. Clark, B. McKuin, Z. Kolber, C. Beal, R. Adams, H. Kagawa, T. J. Richardson, J. Malinowski, M.A. Claxton, E. Geiger, J. Rask, J. E. Campbell, and J.D. Trent,(2013), “Microalgae cultivation using offshore membrane enclosures for growing algae (OMEGA),” Journal of Sustainable Bioenergy Systems, Vol. 3, pp. 18-32

Yue, L. and W. Chen (2005), Energy Conversion and Management, 46: 1868-1876.