DIRECT APPLICATION OF BIOMINERALIZATION TO LIFE …
Transcript of DIRECT APPLICATION OF BIOMINERALIZATION TO LIFE …
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.
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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 Calcium Carbonates …………………........ 6 Cyanobacteria ………………………………………………................... 9
II. Significance of the Project …………………………….…………………. 12
Membrane Based Habitat Water Walls Architectures for Life Support Systems …………………………………...…………...
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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 ……………………………………………………………. …….
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The Anabaena Culture ………………………………………………… 22 The Synechococcus Culture …………………………………............... 22
V. Conclusions and Future Works …………………………………………. 24
References …….……………………………………………………………………..…
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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 ……………………………………………………………..
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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 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
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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.
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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
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).
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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
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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
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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 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.
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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 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.
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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), 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.
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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
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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 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].
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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, 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
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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].
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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
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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
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
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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.
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)
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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.
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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.
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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 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
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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].
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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.
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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 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.
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