Mixotrophic Magnetosome-Dependent
Magnetoautotrophic Metabolism of Model
Magnetototactic Bacterium Magnetospirillum
magneticum AMB-1
Dissertation
Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Eric Keith Mumper, BA
Graduate Program in the School of Earth Sciences
The Ohio State University
2019
Dissertation Committee:
Steven K. Lower, Adviser
Brian H. Lower
Ratnasingham Sooryakumar
Ann E. Cook Copyright by
Eric Keith Mumper
2019 Abstract
Magnetospirillum magneticum AMB-1 is a member of a phylogenetically diverse group of bacteria characterized by their ability to biomineralize magnetic minerals known collectively as magnetotactic bacteria (MTB).1,2,3 MTB produce chains of membrane- bound intracellular magnetic nanocrystals, collectively known as magnetosomes.1,2,3 The current scientific consensus is that magnetosomes are used by MTB to orient themselves in vertically stratified water columns in order to achieve optimal oxygen concentrations in a process known as magnetoaerotaxis.4,5 Biomineralization of magnetosomes is an energy intensive process which accounts for roughly 33% of the cell's metabolic budget.6
This high metabolic cost seems to contradict with the amount of time MTB cells spend aligned with external magnetic fields.5 Due to this apparent discrepancy, I examined the potential role the magnetosome may play in bacterial metabolism. Through analysis of comparative growth on a variety of media compositions both magnetic, wild type and non-magnetic, mutant strains of AMB-1, I discovered that cells grown under stress conditions exhibit an inversion of growth dynamics which indicates some advantage for magnetic cells. Non-magnetic, mutant cells display a direct relationship between external magnetic field strength and growth, indicating magnetic field dependence. I believe that this represents a novel magnetosome-dependant mixotrophic metabolism. Due to the
i ubiquity of MTB1,2,3 and the diversity of sessile eukaryotes which either produce biogenic magnetite or exhibit magnetosensing,7 this system may be part of a widespread, previously unknown component of global carbon cycling.
ii Dedication
This work is dedicated to Alicia Mumper, my wife of seventeen years and counting, who has supported me through sickness and in health, for richer and for poorer, through better
and through worse, and, most impressively of all, through eleven years of college.
iii Acknowledgments
The completion of this thesis would not have been possible without the help of the friends, family and colleagues listed below:
Thanks to both my current and former committee members: Drs. Steven K Lower,
Brian H. Lower, Ratnasingham Sooryakumar, Ann E. Cook, Michael Barton and Mike J.
Wilkins for their advice, encouragement, and criticism of my research.
Thanks to my children: Padraig, Briona, and Nevin for their patience and understanding as their dad spent ten years in college.
Thanks to my parents: John and Terri Mumper as well as Richard and Roxy
Witmer for their financial support and emotional encouragement through this experience.
Thanks to my colleagues Anne Booker, Kelsey Danner, James Dunn, Mike
Johnston, Dr. Nicola Lorenz, Rowan McLachlan, Dr. Zachery Oestreicher, Chris Pierce,
Casey Saup, Kylienne Shaul, and Max Wheeler for their expertise, advice, and encouragement with various aspects of this research.
Thanks to the Dick, EMSL, Sawyer, Wilkins, and Wrighton labs to access to equipment and materials while pursuing various aspects of this research.
iv Vita
Education BA in Geology / minor in Biology 2007-2012 Ohio Wesleyan University Delaware, OH
Teaching Graduate Teaching Assistant 2012-current School of Earth Sciences, The Ohio State University Facilitator 2016 University Center for the Advancement of Teaching
Research Publications Pierce Chrostopher J, Wijesinghe Hiran, Mumper Eric, Lower Brian H, Lower Steven K, Sooryakumar Ratnasingham. “Hydrodynamic Interactions, Hidden Order, and Emergentt Collective Behavior in an Active Bacterial Suspension.” Phys. Rev. Lett. (submitted 2018) Pierce Christopher J, Mumper Eric, Brangham, Jack T, Lower Brian H, Lower Steven K, Yang Fengyuan Y, Sooryakumar Ratnasingham. “Tuning Bacterial Hydrodynamics with Magnetic Fields” 2017. Phys. Rev. E. 10.1103/PhysRevE.95.062612. Oestreicher Zachery, Mumper Eric, Gassman Carol, Bazylinski Dennis, Lower Steven K, Lower Brian H. “Spatial Localization of Mms6 During Biomineralization of Fe3O4 Nanocrystals in Magnetospirllum magneticum AMB-1”2016. J. Mat. Res. 31(5):527-535.
Conference Posters “Pushed, Poked, and Prodded: Documenting Changes in Magnetotactic Bacteria from Wetland to Pure Culture” Goldschmidt Conference 2015, Prague, Cz. “Iron-sulfide Concretions of the Ohio Shale: Glimpses of Deep Subseafloor Microbial Ecosystems of the Late Devonian” Geologic Society of America 2012, Dayton, OH
v Presentations “Magnetotactic bacterium Magnetosprillum magneticum AMB-1 displays magnetic field dependent growth” American Geophysical Union 2018, Washington, DC. “Biologically Controlled Mineralization of Magnetite Nanocrystals” Goldschmidt Conference 2014, Sacramento, CA.
Awards
School of Earth Sciences Distinguished Teaching Award 2016 Robert E. Shanklin Distinguished Scholar Award in Geology 2012
Fields of Study
Major Field: Earth Sciences Minor Fields: Geomicrobiology, biomineralization, biochemistry, geochemistry
vi Table of Contents
Abstract i
Dedication iii
Acknowledgements iv
Vita v
List of Tables x
List of Figures xii
Chapter 1: Characteristics of magnetotactic bacteria and magnetoaeroaxis 1
1.1: Introduction to magnetotactic bacteria 1
1.2: Magnetoaerotaxis model 3
1.3: MTB diversity and magnetosome formation and diversity 4
1.4: Energetics of magnetosome biomineralization 6
1.5: Interesting MTB characteristics 7
1.6: Highly unusual MTB characteristics 8
1.7: Critiquing magnetoaerotaxis model 11
1.8: Alternative possibilities for the magnetosome 13
Chapter 2: Magnetotactic bacteria grown in various media 17
2.1: Magnetotrophic Model 17
vii 2.2: Media rationale, recipes and experimental design 19
2.3: Autotrophic media version 1 – Modified MSGM 24
2.4: Autotrophic media version - BG11 26
2.5: Autotrophic media version – Bazylinski / Frankel formula 27
2.6: Enhanced growth on mixed media 36
Chapter 3: Magnetotactic bacteria grown on reduced organic carbon media 51
3.1: Dynamics of mixed media growth of M. magneticum AMB-1 51
3.2: Analysis of nitrogen sources 58
3.3: Fixation of radiolabeled bicarbonate 61
3.4: Role of organic carbon 63
3.5: Summary of mixed media growth 65
Chapter 4: Magnetic-field-dependent growth of Magnetospirillum magneticum
AMB-1 66
4.1: Preface 66
4.2: Rationale for alternative to magnetoaerotaxis 66
4.3: Methods 67
4.4: Growth of AMB-1 strains on MSGM and BFM50 68
4.5: Growth in variable strength external magnetic fields 72
4.6: Magnetic-field-dependent biology 73
4.7: Magnetic-field-dependent growth as a novel metabolism 76
References 78
Appendix A: Growth of AMB-1 on MSGM without organic carbon 78
viii Appendix B: AMB-1 growth on MSGM and BG11 79
Appendix C: Growth of AMB-1 in Multivariable Experiment 80
Appendix D: Comparison of Growth of AMB-1 on BFAM of Various Oxygen
Concentrations 86
Appendix E: Mixed Media Growth of Wild-type and Mutant AMB-1 strains 88
Appendix F: Growth of Wild-type and Mutant AMB-1 Strains on Mixed Media 97
Appendix G: AMB-1 growth on NO3 vs NH4 106
Appendix H: Incorporation of Radiolabeled Bicarbonates 107
Appendix I: Organic-carbon Pulse on 100% BFAM 108
ix List of Tables
Table 1: Comparison between MTB and E. coli 9
Table 2: Revised Magnetospirillum Growth Media (MSGM) recipe
(ATCC1653) 20
Table 3: Wolfe's Vitamin Solution 20
Table 4: Wolfe's Mineral Solution 21
Table 5: Ferric Quinate (0.01 M) 21
Table 6: Magnetic Spirillum growth medium organic carbon removed 22
Table 7: Modified BG-11 Media 22
Table 8: Bazylinski / Frankel Autotrophic Media (BFAM) 23
Table 9: Modified Wolfe's Mineral Solution 23
Table 10: Frankel's Vitamin Solution 24
Table 11: Variables, controls, and expected results of multivariable experiments 29
Table 12: Oxygen experiment media preparation 35
Table 13: Modified Magnetospirillum Growth Media (MSGM) recipe 37
Table 14: Mixed media composition 37
Table 15: Bazylinski / Frankel Media (BFM) 50
Table 16: Mixed media composition II 52
x Table 17: Calculated variations in the composition of mixed media recipes 59
xi List of Figures
Figure 1: Image of magnetic AMB-1 cells, under field and no field 2
Figure 2: Diagram of magnetoaerotaxis 4
Figure 3: Magnetic vs non magnetic gammaproteobacteria comparison 13
Figure 4: Iron-oxide precipitate in autotrophic media 25
Figure 5: M. magneticum AMB-1 cells in autotrophic media precipitate 25
Figure 6: Growth of AMB-1 on MSGM without organic carbon 26
Figure 7: AMB-1 growth on MSGM and BG-11 27
Figure 8: Growth of M. magneticum AMB-1 on MSGM 30
Figure 9: Growth of M. magneticum AMB-1 on BFAM in dark 31
Figure 10: Growth of M. magneticum AMB-1 on BFAM in blue light 31
Figure 11: Growth of M. magneticum AMB-1 on BFAM in red light 32
Figure 12: Growth of M. magneticum AMB-1 on BFAM in increased magnetic
field 32
Figure 13: Sterile BFAM in dark 33
Figure 14: Average growoth of M. magneticum AMB-1 in multivariable
experiments 33
Figure 15: Comparison of growth of AMB-1 on BFAM of various oxygen
xii concentrations 35
Figure 16: Growth of magnetic M. magneticum AMB-1
100% MSGM / 0% BFAM 38
Figure 17: Growth of magnetic M. magneticum AMB-1
95% MSGM / 5% BFAM 39
Figure 18: Growth of magnetic M. magneticum AMB-1
90% MSGM / 10% BFAM 39
Figure 19: Growth of magnetic M. magneticum AMB-1
75% MSGM / 25% BFAM 40
Figure 20: Growth of magnetic M. magneticum AMB-1
50% MSGM / 50% BFAM 40
Figure 21: Growth of magnetic M. magneticum AMB-1
25% MSGM / 75% BFAM 41
Figure 22: Growth of magnetic M. magneticum AMB-1
10% MSGM / 90% BFAM 41
Figure 23: Growth of magnetic M. magneticum AMB-1
5% MSGM / 95% BFAM 42
Figure 24: Growth of magnetic M. magneticum AMB-1
0% MSGM / 100% BFAM 42
Figure 25: Growth of magnetic M. magneticum AMB-1 43
Figure 26: Growth of non-magnetic M. magneticum AMB-1
100% MSGM / 0% BFAM 44
xiii Figure 27: Growth of non-magnetic M. magneticum AMB-1
95% MSGM / 5% BFAM 45
Figure 28: Growth of non-magnetic M. magneticum AMB-1
90% MSGM / 10% BFAM 45
Figure 29: Growth of non-magnetic M. magneticum AMB-1
75% MSGM / 25% BFAM 46
Figure 30: Growth of non-magnetic M. magneticum AMB-1
50% MSGM / 50% BFAM 46
Figure 31: Growth of non-magnetic M. magneticum AMB-1
25% MSGM / 75% BFAM 47
Figure 32: Growth of non-magnetic M. magneticum AMB-1
10% MSGM / 90% BFAM 47
Figure 33: Growth of non-magnetic M. magneticum AMB-1
5% MSGM / 95% BFAM 48
Figure 34: Growth of non-magnetic M. magneticum AMB-1
0% MSGM / 100% BFAM 48
Figure 35: Growth of non-magnetic M. magneticum AMB-1 49
Figure 36: Growth of magnetic, wild type M. magneticum AMB-1 on mixed
media 54
Figure 37: Growth of non-magnetic, mutant M. magneticum AMB-1 on mixed
media 54
Figure 38: Relative growth of magnetic, wild type M. magneticum AMB-1
xiv cells on mixed media 56
Figure 39: Relative growth of non-magnetic, mutant M. magneticum AMB-1
cells on mixed media 56
Figure 40: Growth of magnetic, wild type M. magneticum AMB-1 on various
nitrogen sources 60
Figure 41: M. magneticum AMB-1 radiolabeled carbon pulse / chase
experiment 62
Figure 42: Growth of magnetic, wild type M. magneticum AMB-1 on a single
organic carbon source 64
Figure 43: Growth of magnetic and non-magnetic strains of M. magneticum
AMB-1 under various conditions 69
Figure 44: Characteristics of magnetic and non-magnetic strains of
M. magneticum AMB-1 on MSGM and BFM50 71
Figure 45: Schematic of radical base pair mechanism 75
xv Chapter 1: Characteristics of magnetotactic bacteria
and magnetoaeroaxis
1.1: Introduction to Magnetotactic Bacteria
Nearly fifty years ago, Richard P. Blakemore noticed that cells isolated from marsh mud preferentially gathered at one side of the microscope slide.8 Upon further investigation, Blakemore discovered that the cells aligned themselves with Earth's magnetic field and could be manipulated with external magnetic fields in general (Figure
1). Blakemore termed this motility magnetotaxis and called the cells magnetotactic bacteria (MTB). Since the first publication in an English-language journal in 1975,8
MTB have captured the attention and imagination of scientists both for their unique response to magnetic fields and for their capability to biomineralize intracellular magnetic nanocrystals. They have been the subject of study for biochemists, physicists, paleontologists, engineers, and microbiologists in order to unravel the mysteries of how they biomineralize magnetic nanocrystals,9 exploit their swimming for microrobotics,10,11 and utilize the cells for medicinal applications.11
MTB are generally characterized by their unique ability to biomineralize intracellular magnetic nanocrystals. These nanocrystals are produced and contained
1 within membrane-bound psuedo-organelles known as magnetosomes.1,2,3 Magnetosomes are typically arranged in a a chain or multiple chains which align roughly with the cell's axis of motility, providing the cell with the ability to passively align with magnetic field lines in spite of thermal (Brownian) forces. It's this passive alignment that allows the cells to undergo magnetotaxis.4,5
Figure 1: Demonstration of magnetotaxis in AMB-1. MTB cells primarily aerotax (A) when under only geomagnetic conditions (B) yielding a more or less stochastic cell orientation. Cells will align (C) when a strong external magnetic field is applied (D). Red bars are 10 μm.
2 1.2: Magnetoaerotaxis Model
In 1997, Frankel et al noted that MTB respond to oxygen concentrations in addition to magnetic field lines and re-described the unique taxis of MTB as magnetoaerotaxis.12 In the magnetoaerotactic model, MTB utilize a combination of chemotactic responses to oxygen gradients and passive alignment geomagnetic field lines to attain optimal position in vertically stratified sediment or water columns (Figure 2). In many aquatic environments, oxygen gradients are vertically oriented. Across much of the planet, geomagnetic field lines decline at varying angles toward the center of Earth. As most, if not all, MTB are microaerophillic to anaerobic, obtaining optimal microaerophilic conditions is essential for their fitness. With the magnetoaerotaxis model, MTB are able to move up or down in their environment by aligning themselves to the geomagnetic field lines and this up or down movement presumably allows them to attain optimal oxygen concentrations within their environment by reducing a three dimensional search for optimal conditions to a two dimensional search.
3 Figure 2: Model of magnetoaerotaxis. With a vertically stratified oxygen concentration in the water column, the ability to sense declining geomagnetic field lines would allow MTB to limit their walk along a two-dimensional line rather than trying to aerotax in three dimensions as non-magnetic cells must do.
1.3: MTB diversity and magnetosome formation and diversity
MTB share several characteristics. They are universally Gram negative, motile, microaerophillic to anaerobic, and produce magnetic nanocrystals.1,2,3 That is, however, where the similarities end. MTB are a polyphyletic group which includes Alpha, Beta,
Delta, and Gamma- proteobacteria subphylums as well as members of the Nitrospirilla phylum.1,2,3 In spite of the genetic diversity of MTB, the magnetosome is produced by a highly conserved gene island known as the magnetosome island (MAI).13,14 The variations in MTB morphology, crystal structure, genetics and diversity have all been described in great length in previously written reviews so only a summary will be presented here.1,2,3,14,15,16
4 The polyphyletic distribution of the MAI is believed to be the result of horizontal gene transfer among members of the MTB group. The origin of the MAI is still somewhat contentious. In dueling letters in PNAS in 2017,17 Wang and Chen estimate the age of the origin of MTB at about 2.0 Ga18 whereas Lin et al assign the origin of MTB to about 3.0 Ga.19 This discrepancy is dependent upon the interpretation of horizontal vs vertical gene transfer with regard to the origination of the MAI. Regardless of the exact specifics of its origin, the success of MTB is indisputable.
MTB are ubiquitous to both freshwater and marine aquatic systems. Nearly every aquatic system from pristine lakes to drainage ditches harbor some species of MTB.1,2,3
Many aquatic systems host multiple species of MTB simultaneously and even thermophilic and akaliphilic extremophile MTB have been discovered.20 MTB vary greatly in morphology with representatives which are coccoid, spirochete, vibrio, bacillus and even some multicellular prokaryotic MTB.15,21
While individual genes within the MAI are conserved, conservation of the MAI does not predicate that all MTB produce the same type of magnetic nanocrystals.13,14
Quite the contrary, crystal morphology, size, shape, and quantity are as varied as the species of MTB themselves.16,22 Composition of nanocrystals is commonly magnetite
22,23,24,25 (Fe3O4) but can also be gregite (Fe3S4). Some MTB produce a combination of magnetite and gregite crystals while still others produce gregite and non-magnetic pyrite
23 (FeS2).
Crystal size, morphology, and quantity seem to be closely controlled by the cell and varies by species.21,22 Examples of crystal mophologies include bullet-shaped, tooth
5 shaped, cubo-octehedral, and rounded.2,21,22 Some MTB produce only two or three large crystals while others produce hundreds of small crystals.2,22
1.4: Energetics of magnetosome biomineralilzation
Regardless of cell size, shape, crystal morphology, or any other variable aspect of
MTB, production of the magnetosome is a complicated, energy-intensive process which has been described in great detail elsewhere. For the purposes of this discussion, several aspects of magnetosome formation must be reviewed here. The first such aspect is the construction of the magnetosome membrane. The magnetosome membrane is a lipid bilayer membrane which is formed from the invagination of the inner membrane of the cell. The magnetosome membrane has its own cytoskeleton proteins which help the magnetosome membrane maintain its shape, alignment and rigidity. 26 The magnetosome membrane contains a number of magnetosome membrane-specific proteins which facilitate the import of iron and the precipitation of mineral crystals within the magnetosome membrane many of which have been the subject of extensive study in an effort to elucidate their role in magnetic nanocrystal production.27
The process of importing iron for the magnetic crystals and the production of magnetic crystals on an atomic level is a particular note. How MTB uptake iron is still something of a mystery.27 However, what is known is that MTB are unable to produce magnetosome crystals in iron-starved conditions and that MTB are unable to survive in iron-saturated considerations.27 Soluble iron ions taken up into the cells must pass through three hydrophobic membranes in order to occupy the magnetosome.27 Once within the magnetosome, the cell controls the oxidation state and ratio of Fe2+ and Fe3+
6 ions in order to ensure the development of magnetic domains within the crystals.27
Magnetite, for example, must have a ratio of one Fe(II) ion for every two Fe(III) ions in order to provide the crystal with its ferromagnetic properties.27
1.5: Interesting MTB characteristics
As if having the ability to produce magnetic crystals within pseudo-organelles was not exceptional enough, MTB have a variety of other characteristics that set them apart from many other bacteria. These characteristics are notable in light of attempting to test the veracity of the magnetoaerotaxis model.
While MTB utilize magnetoaerotaxis to sense and respond to oxygen concentrations, they are not limited to magnetoaerotaxis as their only form of chemotaxis.
In fact, MTB have unusually high numbers of methyl-accepting chemotaxis protein homologs.2,16,27 The numbers of chemosensing genes for MTB range by species from 60-
65 while other non-magnetic proteobacteria such as Escherichhia coli produce only 1/12th of that number.16 Not only are MTB capable of chemosensing and chemotaxis, MTB are also known to be able to sense light and heat and respond with appropriate taxis.2,27 At this point the purpose of negative taxis responses toward light and heat are unknown, but are indicative of the general quality of MTB possessing many potentially competing forms of taxis. The high number of chemosensing proteins and the wide variety of potential taxis responses raises the question: just how much do MTB magnetoaerotax?
The answer seems to be “very little”. While magnetoaerotaxis is apparently energetically beneficial to obtaining optimal cell position, it is not necessary and MTB cells are more than capable of obtaining optimal cell position through non-magnetic means.5
7 Many, if not all, MTB are capable of growing either chemoautotrophically or mixotrophically using one of several carbon fixation pathways.2,18 Known MTB pathways include the Calvin-Benson-Bassham cycle, the reverse TCA cycle and the
Reductive Acetyl CoA pathway.2,18 Still other MTB posses a variety of genes associated with photosynthesis, although none are known to fix carbon in that manner. With so many proteins for taxis, potential metabolic pathways, and their various MAI proteins,
MTB stand out from their environmental neighbors as multi-taskers to the extreme.
1.6: Highly unusual MTB characteristics
MTB are unusual enough due to the formation of magnetosomes. However, there are several other unexpected characteristics of MTB which may be relevant to understanding the purpose of the magnetosome. These characteristics are particularly interesting because they encompass behaviors or aspects of magnetosome formation or existence which seem counter-intuitive to the magnetoaerotaxis model.
The first characteristic of magnetosome formation which must be considered is the metabolic cost. Formation of the magnetosome requires an estimated 80+ proteins, the construction of a lipid bilayer, the transport of iron ions across three membranes, and the biologically controlled precipitation of magnetic crystals. The metabolic burden of this process cannot be understated.
In a 2010 study, by comparing growth of Magnetospirillum gryphiswaldense
(DSM6361) grown on media associated with intracellular magnetosomes with media which is not associated with the production of magnetosomes, Naresh et al calculated the cost of magnetosome formation. Non-magnetic cell growth requires an estimated
8 ~187nJ/cell while magnetosome formation requires an estimated ~5nJ/magnetosome.28 If we assume, as the researchers did, an average of 20 magnetosomes/cell, the total cost of the magnetosome per magnetic cell comes to ~100nJ. This suggests that producing magnetosomes requires about 1/3rd of the overall metabolism of each cell or, put another way, that the magnetosome-producing cells require 150% more energy than non- magnetosome-producing cells. This imbalance is even more exaggerated when comparing MTB to non-MTB Protobacteria such as E. coli which has an energy burden of between 0.6 to 3nJ/cell depending on the calculation (Table 1).
Table 1: Comparison between MTB and E. coli Bacterium Energy per cell Chemosensing Proteins Doubling Time MTB ~290 nJ ~60 proteins ~400 minutes E. coli ~3 nJ 5 proteins ~20 minutes
The cost of magnetosome formation is particularly interesting given the fact that
MTB seem to produce more magnets than needed. If magnetosomes are present in the cells in order to allow the cells to passively align with magnetic field lines in spite of thermal forces, it stands to reason that they would produce only enough magnetosomes to obtain this advantage. The number of magnetosomes is beyond even the necessary quantity to ensure a sufficient magnetic moment for both daughter cells, and, in some cases, the number of magnetosomes confers a magnetic moment to the cell which is 250x greater than that needed for simple magnetoaerotaxis.29 As has already been established,
MTB incur a significant metabolic burden for creating magnetosomes and MTB growth is stunted by magnetosome formation. Why then would cells waste energy producing more magnetosomes than needed?
9 Just as interesting as the cost of magnetosome formation is the behavior of MTB in the presence of higher than natural magnetic fields. That notion that MTB would undergo any substantive changes in higher than geomagnetic field is in itself remarkable.
How do MTB sense changes in magnetic field strength? What evolutionary pathways lead to the biochemical mechanisms needed to sense and respond to magnetic field changes? Nothing we know about Earth's magnetic field suggests that MTB would ever have been exposed to magnetic fields as strong as those used in lab settings or exposed to changes in magnetic field strength on time-scales needed to produce evolutionary responses.
Nevertheless, MTB seem to not only be able to sense changes in magnetic field strength but also to alter their gene regulation based on these changes. Furthermore,
MTB seem to up-regulate magnetosome-related genes in the presence of higher than natural magnetic fields. Wang et al in 2008 examined M. magneticum in a 1mT sinusoidal magnetic field.30 While cell growth was limited in the higher-then-normal magnetic field, the magnetic moment of the cells was enhanced. The magnetosome membrane-bound protein mms6 was up-regulated in enhanced field culture. Mms6 is a key protein in the synthesis of magnetic nanocrystals. In iron-starved aerobic cultures, attempting to grow non-magnetic cells, magA, mamA, and mms6 were all up-regulated in the enhanced magnetic field.30
Setting aside the biochemistry needed to tie gene-regulation to magnetic fields,
M. magneticum seem to form more magnetosomes in enhanced magnetic fields in spite of needing fewer magnetosomes to continue to align to the field lines. From a purely
10 metabolic point of view, it would seem as if the already burdened cells would limit magnetosome formation when fewer magnetosomes are required. In the very least, the number should be unchanged. However, the cells further decrease their growth rates by producing more magnetite.30
1.7: Critiquing Magnetoaerotaxis Model
It should be clear at this point that production of the magnetosome is both an incredible feat of bacterial engineering and an incredible burden upon MTB cells. As with Blakemore's initial description of MTB, we must once again revisit the question: why do MTB produce magnetosomes? With their complex MAI, over-abundance of chemosensing mechanisms, and multiple metabolic pathways, one might be inclined to label MTB as the genetic “hoarders” of the bacterial domain. This genetic “hoarding” seems to inhibit their growth rates and while such genetic proclivities may be beneficial or tolerable in niche habitats, MTB are ubiquitous.
In fact, one of the most obvious arguments against magnetoaerotaxis being the primary driver for magnetosome formation is the ubiquity of MTB. MTB regularly inhabit environments which are shared with myriad non-magnetic, motile microaerophiles. These organisms manage to live in these environments without the aid of a magnetic tether and without the metabolic burden that the magnetosome imposes.
While the magnetosome obviously imparts some competitive advantage, attested to by its ubiquity and polyphyletic distribution, it seems unlikely that directed magnetoaerotaxis alone is a significant evolutionary advantage. At its most basic, this is a question about whether the cost of producing a magnetosome is sufficiently offset by the benefit of being
11 able to magnetoaerotax.
In environmental conditions, magnetoaerotaxis seems limited at best. As mentioned previously, MTB have a plethora of chemosensing proteins. Each of these provides a competing taxis response within the cell. Moving toward food and away from oxygen might pull the cell in two different directions. The over-riding taxis is not always oxygen-sensitive and, contrary to simplistic models of vertically-stratified environments, oxic is not always up and anoxic is not always down.
It has been estimated that MTB only engage in magnetoaerotaxis <1% of the time in their natural environment.5 In the environment, allowing for sedimentary particles, currents, and heterogeneity of natural aquatic systems, actual magnetoaerotaxis is apparently much less significant than in conventional lab settings. Furthermore, as mentioned earlier, non-magnetic MTB are entirely capable of chemotaxing without magnetosomes with no apparent negative impact on their fitness.
It is indisputable that MTB engage in magnetoaerotaxis and that the magnetosome plays a crucial role in that process. However, it seems unlikely that it is beneficial to the fitness of MTB to spend 33% of their metabolisms to produce 100x more magnetic moment needed so that they can magnetoaerotax <1% of the time (Figure 3). It should be apparent that magnetotaxis cannot account for the polyphyletic proliferation of the MAI in light of the metabolic burden of the magnetosome. The probability is high that there could be some yet undiscovered alternative competitive advantage to possessing a magnetosome.
12 Figure 3: Comparison of inputs and outputs between a generic MTB and a non- magnetic cell like E. Coli. MTB require considerably more energy to acheive less growth and similar types of taxis.
1.8: Alternative Possibilities for the Magnetosome
If we suspend for a moment, the idea that the primary purpose of the magnetosome is primarily for or was initially evolved for magneto-aerotaxis, we must return to the initial question: why do MTB produce the magnetosome-crystal complex?
We know that MTB are capable of multiple metabolic pathways and can grow heterotrophically, autotrophically and mixotrophically. We know that they have
13 exceptionally high numbers of chemosensing proteins which suggests that maintaining optimal environmental conditions is especially important to MTB. We know that MTB have been extremely successful in passing on the highly-conserved MAI through both horizontal and vertical gene transfer. And we know that MTB are highly varied in their phyla, cell morphologies, and crystal morphologies and compositions. This suggests that being in the MTB “club” requires enhanced chemosensing and access to hetero-and autotrophic pathways but that many other features are readily varied.
Given the metabolic cost and complexity of magnetosome production, it can be assumed that there is some substantive evolutionary advantage conferred to MTB by the presence of the magnetosome. Exactly what that advantage might be is still a matter of question. However, given the current understanding of MTB and bacteria in general it is possible to speculate as to some potential advantages.
The first and simplest possibility is that the magnetosome functions as mobile electron acceptor with which the bacteria can travel. There are many species of bacteria including, Geobacter and Shewenella, which make use of specialized cytochrome proteins in order to deposit electrons onto extracellular mineral surfaces. For motile organisms such as MTB, staying in constant contact with a mineral surface is counter- intuitive. Therefore, the most obvious solution is for the bacteria to take the mineral crystal along with the cell in its travels. The magnetosome membrane contains specialized magnetosome-membrane specific cytochrome proteins. These proteins, accompanied by H+ translocation proteins could function similarly to the MTR proteins found in Geobacter and enable MTB to transport spent electrons directly onto the
14 magnetosome crystal. Given the microaerophillic nature of MTB, carrying a terminal electron acceptor along with the cell might be a sufficient way to bypass the need to compete for alternative terminal electron acceptors.
The idea of the magnetosome as the storage place for spent electrons can be taken a step further. A recent Nature publication by Byrne et al, documents the use of a magnetite “battery” by co-cultured organism. In this paper, co-cultured Geobacter sulfurreducens and Rhodopseudomonas palustris use magnetite as an electron acceptor and donor respectively based on the day and night cycles of the metabolisms of each organism. If MTB, which, as we have discussed, can carry out both heterotrophic and chemoautotrophic metabolic pathways could they use the magnetosome as not only a terminal electron acceptor but also as an electron donor. The magnetosome could function as a sort crystalline fat source for the cell with the cell storing up energy in times of plenty and living off of that energy in times of need.
The final possibility, and admittedly the most far-fetched option, to be discussed here involves a family of photolyases known as cryptochromes. While magnetic fields lack the energy potential to influence biochemical reactions in the conventional sense, many migratory animals and even many plants, fungi and algae contain magnetite, are magnetoreceptive, or possess cryptochrome proteins.7,31,32,33,34 By using the magnetic field of earth to bias the spin states of key electrons in a biochemical reaction, cryptochrome will react with specific wavelengths of light to all for the reduction of FAD. 35,36 While energy yield from this reaction is relatively small when considering the size and energy demand of a eukaryotic cell, it is sufficient to allow the cell to power biochemical
15 signaling responses enabling the organism to sense Earth's magnetic field and has even been speculated to be involved in bacterial photosynthesis.38
If MTB were able to utilize a homologous protein to cryptochrome, the reduction of FAD could be sufficient to power the reductive half of the electron transport chain and enable, quite at odds with conventional biophysics, the magnetically-induced harvesting of energy from the magnetosome. As it happens, the magnetosome contains membrane- bound constituents of the electron transport chain. For example, Magnetospirillum magneticum AMB-1 has homologous magnetosome-specific proteins for complexes I-IV of the electron transport chain.38
While the answer to the question of why MTB produce magnets has been considered settled science for decades now, I hope that this discussion has re-opened that debate. Magnetoaerotaxis is certainly a piece to the puzzle of the purpose of the magnetosome, the benefits conferred to a cell by magnetoaerotaxis seems insufficient to justify the incredible metabolic burden of magnetosome formation.
16 Chapter 2: Magnetotactic bacteria grown in various
media
2.1: Magnetotrophic Model
If the formation of the magnetosome is not primarily for the purpose of magnetoaerotaxis, then what purpose might such a complex and metabolically costly feature serve? While, at first glance, there might be a variety of purposes, the overall magnetic field strength of the magnetosome complex is much too weak to do real work.
Possibilities such as the preferential uptake of ions or magnetic coordination between cells simply require a much stronger magnetic field than can be found anywhere on
Earth.
In the 1970s, a similar problem was vexing scientists studying migratory birds.35
It was known that birds seemed to be capable of sensing Earth's magnetic field.
However, there was no known biochemical pathway which could be influenced by the relatively weak geomagnetic field. How could birds sense Earth's magnetic field when the geomagnetic field is far too weak to influence any biochemical reactions?
In 1976, Dr. Klaus Schulten proposed that a forbidden state of polyenes, which can be biased in the presence of a magnetic field, would alter the energy needed for specific chemical reactions to proceed.35 This altered state could essentially bypass the
17 energy needed to carry out oxidation/reduction reactions. Schulten hypothesized that such an oxidation/reduction reaction could be utilized to enable birds to visualize Earth's magnetic field when migrating. While his hypothesis was met with speculation, ultimately his discovery of the specialized protein cryptochrome proved his hypothesis correct.36 What is perhaps more remarkable is Schulten's speculation that such a mechanism was also at work with regard to bacterial photosynthesis and electron transport.37
Based on Schulten's work, it was determined that crypochrome, a photolyase, facilitates the reduction of FAD when exposed to photons of green and blue
31,32,33,34,36 wavelengths. The energy yield from from this FADH2 molecule allows for the signaling and visualizaiton of the geomagnetic field. While in a eukaryote, such as a bird, which might have an inconsequential ratio of magnetite to cells, in a MTB, which might have a magnetite to cell ratio of 30:1, the production of FADH2 by a cryptochrome is much more significant.
The production of FADH2 alone isn't enough to justify the existence of the magnetosome in MTB. However, the role of FADH2 in the electron transport chain might explain how MTB could harness such a reaction to generate energy for the cell. MTB posses magnetosome-specific proteins homologous to those found in the electron transport chain. If MTB are capable of utilizing a similar reaction to crypochrome in their own cells, they could use the FADH2 product within the magnetosome to generate energy in a secondary electron transport chain similar to the mechanism proposed by
Schulten for bacterial photosynthesis. Such a metabolism coule utilize magnetosome-
18 specific proteins and the magnetic field of the cell.
2.2: Media rationale, recipes and experimental design
Under the assumption that any magnetically-mediated metabolism would be autotrophic in nature and in an effort to examine whether the MTB magnetosome is tied to growth, M. magneticum AMB-1 was grown on variety of media, beginning with autotrophic formulations in the absence of direct light and in the absence of a chemoautotrophic electron donor. Even though no MTB are known to be capable of photosynthesis, exclusion of direct light was intended to eliminate any possibility for photosynthetic reactions. The reactions described by Schulten require only a few photons in order to proceed.32,35,36 Likewise, exclusion of an electron donor was intended to eliminate any possibility for chemoautotrophic reactions which have been observed in many species of MTB. As various media were tested and revised, the final formulation in which organic carbon was limited but not absent.
AMB-1 is typically grown on Magneospirillum Growth Medium (MSGM, Table
2). This media provides the cells with abundant organic carbon which is used for both energy and biomass as well as providing essential vitamins and minerals (Tables 3 & 4) for basic cellular functions. MSGM contains abundant soluble iron (Table 5) in order to facilitate magnetosome formation.
19 Table 2: Revised Magnetospirillium Growth Media (MSGM) recipe (ATCC1653) Reagent Quantity Distilled Water 1000 mL Wolfe's Vitamin Solution (Table 3) 10 mL Wolfe's Mineral Solution (Table 4) 5 mL Ferric Quinate (0.01 M, Table 5) 2 mL Resazurin (0.1%) 0.45 mL KH2PO4 0.68 g NaNO3 0.12 g Ascorbic Acid .035 g Tartaric Acid 0.37 g Succinic Acid 0.37 g Sodium Acetate 0.05 g
Table 3: Wolfe's Vitamin Solution Reagent Quantity Biotin 0.002 g Folic Acid 0.002 g Pyridoxine Hydrochloride 0.01 g Thiamine·HCl 0.005 g Riboflavin 0.005 g Nicotinic Acid 0.005 g Calcium D-(+)-pantothenate 0.005 g Vitamin B12 0.0001 g P-Aminobenzoic Acid 0.005 g Thioctic Acid 0.005 g Distilled Water 1000 mL
20 Table 4: Wolfe's Mineral Solution Reagent Quantity Nitrilotriacetic Acid 1.5 g MgSO4·7H2O 3 g MnSO4·H2O 0.5 g NaCl 1 g
FeSO4·7H2O 0.1 g CoCl2·6H2O 0.1 g CaCl2 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g AlK(SO4)2·12H2O 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g Distilled Water 1000 mL
Table 5: Ferric Quinate (0.01 M) Reagent Quantity
FeCl3 0.27 g Qunic Acid 0.19 g Distilled Water 100 mL
In order to force AMB-1 to grow solely using the energy derived through the magnetosome, all other potential energy sources were eliminated. A new medium was formulated by eliminating all of the organic carbon sources in MSGM (table 6) and growing the cells in the dark to rule out any potential photosynthetic interactions. While iron(II) can, in theory, be used for chemoautotrophy, it could not be eliminated due to its role in magnetosome formation. Blakemore calculated that the magnetosome, however, does not contain sufficient energy reserves for chemoautotrophy.39
21 Table 6: Magnetic Spirillium growth medium organic carbon removed Reagent Quantity Distilled Water 1000 mL Wolfe's Vitamin Solution (Table 3) 10 mL Wolfe's Mineral Solution (Table 4) 5 mL Ferric Quinate (0.01 M, Table 5) 2 mL Resazurin (0.1%) 0.45 mL KH2PO4 0.68 g NaNO3 0.12 g
The second media attempt took into consideration that whatever role the magnetosome played in the growth of AMB-1 might be tied to a photosynthetic-like system and that a medium more closely related to the environments in which AMB-1 was discovered might be more appropriate. As such, a modified version of the BG-11 media used to grow cyanobacteria was devised (Table 7).
Table 7: Modified BG-11 Media Reagent Quantity
NaNO3 1.5 g K2HPO4 0.04 g MgSO4·7H2O 0.008 g CaCl2·2H2O 0.036 g Citric Acid·H2O 0.006 g (NH4)2Fe(SO4)2·6H2O 0.006 g Na2EDTA·2H2O 0.001 g NaHCO3 0.02 g Wolfe's Mineral Solution (Table 4) 2 mL Distilled Water 1000 mL
Building upon the idea that the ideal media for evaluating the metabolic role of the magnetosome should be something similar to the environment in which MTB are found, a third medium recipe was devised based upon a formulation by Bazylinski et al.40
The initial formula was created to facilitate the growth of chemoautotrophic MTB strains
MV1 and MV2. For these experiments the Bazylinski/Frankel (Table 8) media was
22 modified by eliminating the sodium thiosulfate (a chemoautotrophic electron donor) and increasing the KH2PO4 after it was discovered that AMB-1 grown on the original
MV1/MV2 concentration were hypotonic.
Table 8: Bazylinski / Frankel Autotrophic Media (BFAM) Reagent Quantity Modified Wolfe's Mineral Solution (Table 9) 5 mL Frankel's Vitamin Solution (Table 10) 0.5 mL Ferric Quinate (0.01 M, Table 5) 2 mL 0.1% Rezazurin 0.45 mL
KH2PO4 9.68 g NH4Cl 0.25 g NaHCO3 0.18 g Cysteine HCl 0.2 g Distilled Water 1000 mL
Table 9: Modified Wolfe's Mineral Solution Reagent Quantity Nitrilotriacetic Acid 1.5 g MgSO4·7H2O 3 g MnSO4·H2O 0.5 g NaCl 1 g
FeSO4·7H2O 0.1 g CoCl2·6H2O 0.1 g CaCl2 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.02 g AlK(SO4)2·12H2O 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.4 g NiCl2 0.01 g Distilled Water 1000 mL
23 Table 10: Frankel's Vitamin Solution Reagent Quantity Biotin 0.1 g Folic Acid 0.04 g Pyridoxine Hydrochloride 4 g Thiamine . HCl 90 g Calcium D-(+)-pantothenate 4 g Vitamin B12 5 g p-Aminobenzoic Acid 5 g Inositol 40 g Niacin 4 g Distilled Water 100 mL
2.3: Autotrophic media version 1 – Modified MSGM
The first attempt to formulate an appropriate media to test the magnetotrophic
reaction was a variation of the standard growth media for AMB-1: MSGM (Table 2). By
taking the MSGM and removing all organic carbon from the recipe (Table 6), it was
thought that such a media could allow for autotrophic growth. This media was simply the
standard media for AMB-1 with the exception of ascorbic acid, tartaric acid, sodium
acetate, and succinic acid.
Samples were examined through optical microscopy to confirm cell growth. The
media formed a precipitate (Figure 4). Sparse cells are visible within the precipitate
(Figure 5), and optical cell counts demonstrated limited growth (Figure 6), the raw data
for the cell counts can be found in Appendix A. One method to determine growth is the
use of a direct cell count and subsequent calculation of the cell estimate. This method
can be useful when cell densities are low and alternative methods might not accurately
capture the cell densities. Cell counts were estimated by manually counting SYBR-gold
labeled cells. SYBR-gold is a fluorescent label (Figure 5) which targets DNA and labels
living cells. This not only identifies living vs dead cells but also aids in identifying cells
24 in optical microscopy. Once the cells are counted, these counts were multiplied by a dilution factor (11,250) and averaged. The premise of the autotrophic media formulation
(Table 6) was predicated on the idea that MSGM contains all of the necessary ingredients needed for magnetotrophic growth which was likely incorrect.
Figure 4: Iron-oxide precipitate (orange) visible in the media. AMB-1 cells are visible within the precipitate (Figure 5).
Figure 5: AMB-1 grown on MSGM without organic carbon and stained with SYBR Gold. (A) Is an optical image taken in normal light. (B) Is a fluorescent image. Cells are visible in the precipitate (indicated with red circles).
25 140000
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L 100000 m
/
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l 80000 e C
d 60000 e t a m i
t 40000 s E 20000
0 10/24/16 10/31/16 11/07/16 11/14/16 11/21/16 11/28/16
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Figure 6: Compilation of cell count data from growth of AMB-1 on MSGM without organic carbon (Table 6 recipe)
2.4: Autotrophic Media Version 2 – BG11
With the understanding that the modified MSGM media recipe may have lacked the necessary components needed to facilitated magnetotrophic growth, growth of AMB-
1 on modified BG11, Table 7, a media for the autotrophic growth of cyanobacteria, was tested. Cultures were grown in the dark and in the absences of a chemoautotrophic electron donor.
Cultures were examined with absorbance at 565nm using a spectrophotometer
(ThermoSpectronic Genesys 10uv) in order to compare cell growth. Seven test tubes containing 13mL of modified BG11 were prepared. Six tubes were inoculated with 1mL of AMB-1 at log phase growth. The seventh, uninnoculated, tube and one of the inoculated tubes were set aside as controls and refrigerated at 4ºC for the duration of the experiment. An identical set of test tubes were prepared with MSGM in order to
26 compare autotrophic and heterotrophic growth of AMB-1 on the two media recipes.
Absorbance of each sample was measured after 7 days.
The results of this comparative analysis can be found in Figure 7 and the raw data is compiled in Appendix B. While growth on modified BG11 is clearly lower than growth MSGM, growth in the absence of organic carbon, direct light, and a chemoautotrophic electron donor is still apparent but not sufficient to conduct further analysis because proteomic or metagenomic analysis requires higher cell densities.
MSGM-CTRL
MSGM-AMB1-CTRL
MSGM-AMB1
Abs 600nm BG11-CTRL
BG11-AMB1-CTRL
BG11-AMB1
0 0 0 0.01 0.01 0.01 0.01 0.01 0.02 0.02
Figure 7: Comparison of the growth of AMB-1 on MSGM vs BG11. MSGM-CTRL and BG11-CTRL are sterile controls. MSGM-AMB-1-CTRL and BG11-AMB1-CTRL are inoculated controls but refrigerated. MSGM-AMB1 and BG11-AMB1 are inoculated samples. Growth on BG11 is clearly lower than that on MSGM but is still apparent.
2.5: Autotrophic media version 3– Bazylinski / Frankel formula
In a further attempt to refine and optimize magnetotrophic media recipes, a media formulation created by Dr. Dennis Bazylinski for the growth of chemolithoautotrophic
27 MTB strains MV1 & MV2, Table 8.40 This formulation was specifically designed to facilitated the growth of a known chemoautotrophic strain of MTB and, with the absence of an electron donor, meets all of the conditions desired for a magnetotrophic test by eliminating all possible metabolic inputs with the exception of magnetic field.
Initial tests of this Bazylinski / Frankel media (BFAM) formula produced less than optimal results with AMB-1 sells displaying swollen, hypotonic morphologies. The media (Table 8) was supplemented with 0.68g/L of K2HPO4 as a buffer. This addition seemed to resolve the hypotonic condition of the cells.
With this modified BFAM, attempts were made to enhance the magnetotrophic growth of AMB-1 by introducing conditions known to facilitate Schultzen's radical pair mechanism. AMB-1 was inoculated into 50mL volumes of both MSGM and the modified BFAM The bottles of the modified BFAM were split between four variables: dark (no light), red light, blue light, and an enhanced magnetic field. According to the current understanding of the radical pair mechanism, cultures grown in blue light should have a more biased reaction (i.e. more growth) than cells grown in the dark. Red light represents a control that should demonstrate that it's not simply light, but specifically blue light that enhances the reaction. Cells grown in an enhanced magnetic field should also experience enhanced growth through a more strongly biased radical pair mechanism. If
AMB-1 was utilizing system similar to that found in birds' eyes, blue light and an enhanced magnetic field should further bias that reaction while red light should have no effect (Table 11).36
28 Table 11: Variables, controls, and expected results of multivariable experiments. Experimental Purpose Expected result Rationale set-up AMB-1 on MSGM Control to establish N/A AMB-1 is a model “normal” growth organism and MSGM is the standard media AMB-1 on Control to establish N/A Establishing a baseline autotrophic media in “normal” autotrophic growth on autotrophic dark growth media for comparison AMB-1 on Testing effects of Autotrophic growth Analogous systems in autotrophic media in blue light should be amplified eukaryotes use photolyase homologues blue light wavelengths on in blue light which are catalyzed by autotrophic growth blue wavelengths of light. In the overall reactions, photons provide the energy so increasing photons should increase the rate of reaction, amplifying the cells' access to energy AMB-1 on Testing effects of red Autotrophic growth Photolyases (as above) autotrophic media in light wavelengths on should not be are catalyzed by blue wavelengths. Red red light autotrophic growth amplified in red light wavelengths of light should have little or no effect on reaction rates AMB-1 on Testing effects of Autotrophic growth The hypothesized autotrophic media in increased magnetic should be amplified process used to make energy in this autotrophic increased magnetic field on autotrophic in an increased system is dependent field growth magnetic field upon an external magnetic field. By using a magnetic field stronger than Earth's magnetic field, the energy yield should be greater and growth should be amplified
29 These multivariable experiments were carried out multiple times with several variations including combining light and an enhanced magnetic field. Absorbance at
565nm was measured for each sample periodically over the duration of the experiments.
The raw data from these experiments can be found in Appendix C and tables of the results can be found in Figures 8-14. Figure 14 shows the compilation of the various iterations of the multivariable experiments. It is clear that is no enhancement of growth on the modified BFAM.
0.25
0.2 m n 5
6 0.15 5
@
e c n
a 0.1 b r o s b A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 8: AMB-1 grown on MSGM and in the dark. Each line is a separate bottle. 50mL of media in each serum bottle were inoculated with 1mL of innoculum. 1 mL samples were acquired at each timepoint and transferred to sterile cuvettes for spetroscopy.
30 0.25
0.2 m n 5
6 0.15 5
@
e c n
a 0.1 b r o s b
A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 9: AMB-1 grown on BFAM and in the dark. Each line is a separate bottle. 50mL of media in each serum bottle were inoculated with 1mL of innoculum. 1 mL samples were aquired at each timepoint and transferred to sterile cuvettes for spetroscopy.
0.25
0.2 m n 5
6 0.15 5
@
e c n
a 0.1 b r o s b
A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
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Figure 10: AMB-1 grown on BFAM and in blue light. Each line is a separate bottle. 50mL of media in each serum bottle were inoculated with 1mL of innoculum. 1 mL samples were aquired at each timepoint and transferred to sterile cuvettes for spetroscopy.
31 0.25
0.2 m n 5
6 0.15 5
@
e c n
a 0.1 b r o s b
A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 11: AMB-1 grown on BFAM and in the red light. Each line is a separate bottle. 50mL of media in each serum bottle were inoculated with 1mL of innoculum. 1 mL samples were aquired at each timepoint and transferred to sterile cuvettes for spetroscopy.
0.25
0.2 m n 5 6
5 0.15
@
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n 0.1 a b r o s b
A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 12: AMB-1 grown on BFAM and in an increased magnetic field. Each line is a separate bottle. 50mL of media in each serum bottle were inoculated with 1mL of innoculum. 1 mL samples were aquired at each timepoint and transferred to sterile cuvettes for spetroscopy.
32 0.25
0.2 m n 5 6
5 0.15
@
e c
n 0.1 a b r o s b
A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 13: Sterile BFAM was stored and in the dark. Each line is a separate bottle. 50mL of media in each serum bottle were not inoculated. 1 mL samples were aquired at each timepoint and transferred to sterile cuvettes for spetroscopy.
0.25
0.2 m n 5
6 0.15 5
@
e c n
a 0.1 b r o s b
A 0.05
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 14: Compilation of all of the multivariable experiments. Pink = MSGM, Light blue = dark, Dark blue = blue light, Red = red light, Green = enhanced magnetic field, Yellow = sterile. This demonstrates that there is very little, if any, amplification of autotrophic growth due to exposure to blue light or increased magnetic fields.
33 As can be seen in several of the above graphs, variability of growth within a single group of samples (Figure 10, for example) could be quite extreme with some samples showing marginal growth and others seeming only to decrease in cell density over time. While attempting to minimize the variability in growth among the samples grown in these experiments, it was noticed that some accounts of autotrophy among MTB require anaerobic conditions. Because the modified Bazylinski / Frankel media requires the purging of the serum bottles and media with nitrogen gas and the addition of a reductant (Cysteine HCl) in the media, it was assumed that the bottles were more or less anaerobic. However, an experiment investigating the growth of AMB-1 on modified
BFAM prepared four different ways demonstrated that the assumption of anaerobism being necessary was likely incorrect.
In order to determine whether oxygen concentration influenced the variability seen in the previous experiments, samples were prepared with four variations in order to create four separate oxygen concentrations. These variations included: purged with N2 & with reductant, purged with N2 & no reductant, unpurged & reductant, unpurged & no reductant (Table 12). Ten serum bottles containing 50mL of Bazylinski/Frankel media of each of the variations on oxygen concentration were inoculated with 1mL of AMB-1 grown to log phase on MSGM. The absorbance of each sample at 565nm was measured using a spectrophotometer periodically for a duration of nine days. While the growth over these nine days was minimal, it is apparent that unpurged media containing a reductant produced the most substantial growth (Figure 17, raw data found in Appendix
D).
34 Table 12: Oxygen Experiment Media Preparation Description Preparation High Oxygen No Cysteine-HCl, Unpurged Moderate-high Oxygen With Cysteine HCl, Unpurged
Moderate-low Oxygen No Cysteine HCl, Purged with N2
Low Oxygen With Cysteine Hcl, Purged with N2
0.05 0.05 0.04
m 0.04 n 5
6 0.03 5
No Cysteine / Unpurged @
0.03 e No Cysteine / Purged c n
a 0.02
b With Cysteine / Unpurged r o
s 0.02
b With Cysteine / Purged
A 0.01 0.01 0 0 1 2 3 4 5 6 7 8 9 10
Time (days)
Figure 15: Comparing AMB-1 grown on BFAM with various oxygen concentrations. If growth rates are equalized by comparing to each initial absorbance measurement, samples with Cysteine HCl but which are unpurged perform the best.
On its own, these data seems to suggest that whatever minimal growth can be achieved on the various autotrophic media recipes attempted, this growth is not able to be enhanced by conditions associated with the cryptochrome reaction. This would seem like the natural end of this line of research were it not for an anecdotal observation made in attempting to ensure that the cell cultures were viable before subculturing for the multivariable experiments (described in Table 11). It was noted through both optical
35 microscopy and optical density observations that cells grown in bottles containing both
MSGM and the Bazylinski / Frankel media seemed to grow at least as well as those grown on only MSGM and the cells appeared to be more strongly magnetic and faster swimmers than those grown on only MSGM. This observation stood in stark contrast to the condition of cells grown on only the autotrophic media recipes. Those cells seemed to be sparse, weakly magnetic and lethargic. The enhancement on mixed media deserved additional attention.
2.6: Enhanced growth on mixed media
Growth of AMB-1 on the mixture of MSGM and Bazylinski-Frankel media was the most observable of any media formulation to date. Initial trials were conducted with very low concentrations of MSGM (~10% of total media volume) and yielded cell densities which far outperformed any previous experiment. However, subsequent subcultures of these cells onto pure Bazylinski-Frankel media produced the sparse, lethargic, and poorly magnetic cell populations previously observed. Some aspect of growing the cells on mixed media was allowing them to thrive. Furthermore, it appeared that the cells grown on mixed media might actually be more magnetic and swim faster than cells grown on pure MSGM.
Because of the subtle differences between the recipes for MSGM and the
Bazylinski-Frankel media, a modified MSMG recipe had to be formulated (Table 13).
This recipe utilized the Bazylinski-Frankel mineral and vitamin solutions as well as cysteine-HCl in an attempt to minimize as much variability in the formulations as possible. Since previous cell enhancement occurred on media comprised of roughly 10%
36 MSGM and 90% Bazylinski-Frankel media, a set of variations ranging from 0% MSGM and 100% Bazylinski-Frankel media to 100% MSGM and 0% Bazylski-Frankel media were formulated (Table 14).
Table 13: Modified Magnetospirillium Growth Media (MSGM) recipe Reagent Quantity Distilled Water 1000 mL ModifiedWolfe's Vitamin Solution (Table 8) 10 mL Frankel's Mineral Solution (Table 9) 0.5 mL Ferric Quinate (0.01 M, Table 5) 2 mL Resazurin (0.1%) 0.45 mL
KH2PO4 0.68 g
NaNO3 0.12 g Ascorbic Acid .035 g Tartaric Acid 0.37 g Succinic Acid 0.37 g Sodium Acetate 0.05 g
Table 14: Mixed media composition MSGM BFAM 100% MSGM / 0% BFAM 100% 0% 95% MSGM / 5% BFAM 95% 5% 90% MSGM / 10% BFAM 90% 10% 75% MSGM / 25% BFAM 75% 25% 50% MSGM / 50% BFAM 50% 50% 25% MSGM / 75% BFAM 25% 75% 10% MSGM / 90% BFAM 10% 90% 5% MSGM / 95% BFAM 5% 95% 0% MSGM / 100% BFAM 0% 100%
37 For most of these experiments, five samples of each of the above formulations were prepared and inoculated with 1mL of AMB-1 inoculum. In a few cases, due to bottle breakage or sample contamination, only four samples were measured. Absorbance at 565nm was measured at time zero, daily for fourteen days, and periodically to thirty- five days. These results can be found in Appendix E and are represented in figures 16-25.
0.3
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0.2 m n 5 6 5
0.15
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n 0.1 a b r o
s 0.05 b A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 16: Growth of M. magneticum AMB-1 on 100% MSGM / 0 % BFAM. Each line is a separate bottle. This can be considered a "baseline" for additional growth comparisons.
38 0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s 0.05 b A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 17: Growth of M. magneticum AMB-1 on 95% MSGM / 0% BFAM. Each line is a separate bottle. Interestingly, in spite of growing with a slightly lower amount of available organic carbon, the growth of AMB-1 is greatly increased.
0.3
0.25
0.2 m n 5 6 5
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n 0.1 a b r o s 0.05 b A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 18: Growth of M. magneticum AMB-1 on 90% MSGM / 10% BFAM. Each line is a separate bottle. As in figure 17, cells grown on reduced organic carbon attain higher cell densities than those grown on pure MSGM (figure 16).
39 0.3
0.25
0.2 m n 5 6 5
0.15 @
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n 0.1 a b r o s 0.05 b A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 19: Growth of M. magneticum AMB-1 on 75% MSGM / 25% BFAM. Each line is a separate bottle.
0.3
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0.2 m n 5 6 5
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n 0.1 a b r o
s 0.05 b A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 20: Growth of M. magneticum AMB-1 on 50% MSGM / 50% BFAM. Each line is a separate bottle. While initial growth is slightly lower than that of cells grown on 100% MSGM, cells grown on 50% MSGM attain roughly equal growth by twenty days in spite of having only half the available organic carbon.
40 0.3
0.25
0.2 m n 5 6 5
0.15 @
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n 0.1 a b r o s 0.05 b A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 21: Growth of M. magneticum AMB-1 on 25% MSGM / 75% BFAM. While growth is reduced when compared to cells grown on 100% MSGM, the reduction in growth is not proportional to the reduction in organic carbon.
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 22: Growth of M. magneticum AMB-1 on 10% MSGM / 90% BFAM. Each line is a separate bottle.
41 0.3
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0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 23: Growth of M. magneticum AMB-1 on 5% MSGM / 95% BFAM. Each line is a separate bottle.
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 24: Growth of M. magneticum AMB-1 on 0% MSGM / 100% BFAM. Each line is a separate bottle.
42 0.3
0.25 100% MSGM / 0% BFAM 0.2 m 95% MSGM / 5% BFAM n 5
6 90% MSGM / 10% BFAM 5
0.15
@ 75% MSGM / 25% BFAM
e c
n 0.1 50% MSGM / 50% BFAM a b r 25% MSGM / 75% BFAM o s 0.05 b 10% MSGM / 90% BFAM A 0 5% MSGM / 95% BFAM 0% MSGM / 100% BFAM -0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 25: Comparative growth of M. magneticum AMB-1 on various mixed media. Of note is the enhancement of the growth of cells on media with less available organic carbon (95%-75% MSGM) when compared to cells grown on 100% MSGM. This trend is confirmed by the fact that growth on 50% & 25% MSGM is not reduced as much as might be expected.
As indicated by previous observations, cells grown on mixed media with a composition between pure MSGM and pure BFAM grew to substantially higher densities than cells grown on BFAM. While it may seem obvious to attribute the growth to the fact that MSGM contains organic carbon and must be stimulating heterotrophic growth, two aspects of these growth curves must be discussed. The first is that increase in growth from cells grown on 10%, 25%, and 50% MSGM is disproportional to the percentage of
MSGM added. That is to say that adding 25% MSGM to the media increases growth to nearly that of cells grown on 100% MSGM. The second aspect to consider is that media containing concentrations of MSGM near but less than 100% perform better than cells grown on 100% MSGM. If AMB-1 is growing heterotrophically, why do cells grown on
43 less organic carbon grow to greater cell densities?
In order to test the apparent enhanced growth of AMB-1 on mixed media, the above experiment was repeated with a non-magnetic mutant strain of AMB-1. The wild- type strain of AMB-1 produces intracellular magnetic crystals within magnetosomes which give the cells an overall magnetic moment. The non-magnetic mutant cells do not produce intracellular magnetite and do not have an innate magnetic moment. This allows us to test whether the magnetosome is involved in the enhanced mixed media growth. If the magnetosome is in some way enhancing growth of wild-type cells on mixed media, we would expect the growth enhancement of non-magnetic mutant cells on mixed media.
The results of these experiments can be found in Appendix E and are represented in figures 26-35.
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 26: Growth of non-magnetic mutant M. magneticum AMB-1 on 100% MSGM / 0% BFAM. Each line represents a separate bottle. The growth curve of non-magnetic mutants outperform wild-type cells (Figure 16). This is likely due to the fact that the non-magnetic, mutant cells don't have the energy demand of producing magnetosomes.
44 0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 27: Growth of non-magnetic, mutant M. magneticum AMB-1 on 95% MSGM / 5% BFAM. Each line represents a separate bottle. Growth of the non-magnetic, mutant cells is comparable to that of wild-type cells (Figure 17).
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A
0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 28: Growth of non-magnetic, mutant M. magneticum AMB-1 on 90% MSGM / 10% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is slightly less than that of wild-type cells (Figure 18).
45 0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 29: Growth of non-magnetic, mutant M. magneticum AMB-1 on 75% MSGM / 25% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is significantly less than that of wild-type cells (Figure 19).
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 30: Growth of non-magnetic, mutant M. magneticum AMB-1 on 50% MSGM / 50% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is significantly less than that of wild-type cells (Figure 20).
46 0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 31: Growth of non-magnetic, mutant M. magneticum AMB-1 on 25% MSGM / 75% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is significantly less than that of wild-type cells (Figure 21).
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 32: Growth of non-magnetic, mutant M. magneticum AMB-1 on 10% MSGM / 90% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is slightly less than that of wild-type cells (Figure 22).
47 0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 33: Growth of non-magnetic, mutant M. magneticum AMB-1 on 5% MSGM / 95% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is slightly less than that of wild-type cells (Figure 23).
0.3
0.25
0.2 m n 5 6 5
0.15 @
e c
n 0.1 a b r o s
b 0.05 A 0
-0.05 0 5 10 15 20 25 30 35
Time (days)
Figure 34: Growth of non-magnetic, mutant M. magneticum AMB-1 on 0% MSGM / 100% BFAM. Each line represents a separate bottle. Growth of non-magnetic, mutant cells is slightly less than that of wild-type cells (Figure 24).
48 0.3
0.25 100% MSGM / 0% BFAM
m 0.2 95% MSGM / 5% BFAM n 5
6 90% MSGM / 10% BFAM 5 0.15
@ 75% MSGM / 25% BFAM
e c
n 50% MSGM / 50% BFAM a
b 0.1 r 25% MSGM / 75% BFAM o s b 10% MSGM / 90% BFAM A 0.05 5% MSGM / 95% BFAM 0 0% MSGM / 100% BFAM 0 5 10 15 20 25 30 35 -0.05
Time (days)
Figure 35: Comparative growth of non-magnetic M. magneticum AMB-1 on various mixed media. Of note is that while the 90% and 95% MSGM samples perform similarly to magnetic cells, the 25%-75% MSGM samples perform significantly worse. This indicates that as the growth of AMB-1 becomes less reliant on organic carbon, it becomes more dependent on the magnetism provided the cell.
Accounting for the differences in lag phases, the 90% MSGM / 10% BFAM and
95% MSGM / 5% BFAM non-magnetic cells followed the same enhancement observed in magnetic cell growth. However, the non-magnetic cells grown on media containing
25%-75% MSGM / 75%-25% BFAM respectively performed significant worse than the magnetic cells. Essentially, on media of ~50% MSGM / 50% BFAM, when the cells have less available energy from organic carbon, the cells with the highest metabolic demand6 perform the best. This indicates that there may be some magnetosome- dependent aspect of the enhancement of magnetic cells grown on mixed media of the above concentrations.
While the exact nature of the enhancement of mixed media growth and whether it
49 is definitively tied to the magnetosome or cell magnetism cannot be elucidated with these experiments. It is clear that there is some metabolic system at work in addition to heterotrophy and that this metabolic system is dependent upon the magnetism of the cell.
However, several of the bottles of interest in question developed a whitish precipitate which led to relatively large error bars. After some experimentation, it was determined that this precipitate was related in some way to the cysteine-HCl in the media and a new media formulation was developed without cysteine-HCl (Table 15). Additional experiments (Chapter 3), were conducted with this new formulation of mixed media in the effort to elucidate the growth enhancement that the magnetosome imparts.
Table 15: Bazylinski / Frankel Media (BFM) Reagent Quantity Modified Wolfe's Mineral Solution (Table 9) 5 mL Frankel's Vitamin Solution (Table 10) 0.5 mL Ferric Quinate (0.01 M, Table 5) 2 mL 0.1% Rezazurin 0.45 mL
KH2PO4 9.68 g
NH4Cl 0.25 g
NaHCO3 0.18 g Distilled Water 1000 mL
50 Chapter 3: Magnetotactic bacteria grown on reduced
organic carbon media
3.1: Dynamics of mixed media growth of M. magneticum AMB-1
While many species of MTB have been documented to grow heterotrophically, autotrophically, and mixotrophically, M. magneticum AMB-1 has, to date, only been shown to be capable of heterotrophic growth. Therefore, the capacity for the growth of
AMB-1 should be tied to the concentration of organic carbon in the media. As organic carbon becomes more limited, the growth of AMB-1 should also become more limited.
By mixing modified MSGM (Table 13) which contains organic carbon and BFAM (Table
15) which does not contain organic carbon, nine concentrations of media with varying concentrations of organic carbon were created (Table 16). These concentrations range from 100% MSGM and 0% BFAM to 0% MSGM and 100% BFAM while keeping most ingredients constant with the exception of those in Table 17.
For these experiments, five 100mL serum bottles of each formulation were filled with 100mL of media and inoculated with 1mL of inoculum. Samples were measured for growth at 565nm daily for ten days. Samples were then measured weekly at 14, 21, 28, and 35 days. These experiments were conducted for both the magnetic wild type AMB-1
51 strain as well as a non-magnetic mutant AMB-1 strain.
Table 16: Mixed media composition MSGM BFAM 100% MSGM / 0% BFAM 100% 0% 95% MSGM / 5% BFAM 95% 5% 90% MSGM / 10% BFAM 90% 10% 75% MSGM / 25% BFAM 75% 25% 50% MSGM / 50% BFAM 50% 50% 25% MSGM / 75% BFAM 25% 75% 10% MSGM / 90% BFAM 10% 90% 5% MSGM / 95% BFAM 5% 95% 0% MSGM / 100% BFAM 0% 100%
Growth curves for the magnetic wild type AMB-1 strain (Figure 36, Appendix F) reveal growth that is not proportional to the concentration of organic carbon in the media.
It would be expected that maximal rate and extent of growth of AMB-1 would occur in the 100% MSGM / 0% BFAM media and that growth would decrease as the concentration of MSGM in the mixture decreases. Instead, while the fastest growth rates do appear to occur with 90-100% MSGM recipes, the maximal growth extents occur in the 50% & 75% MSGM recipes (dark red and green curves, Fig. 36). The shape of the growth curves may be telling as well with the 90%-100% MSGM subset following a very similar pattern of growth and the 25%-75% MSGM subset following a different pattern of growth.
Growth of non-magnetic, mutant AMB-1 strain on mixed media recipes produced similar but less extreme results (Figure 37, Appendix F). While maximal growth is still attained in the 75% MSGM media, the rate of growth in both 50% and 75% MSGM is
52 slower than observed in the wild type cells. However, non-magnetic cells attain higher maximal growth on 100% MSGM than magnetic cells. This is interesting because it indicates that non-magnetic cells grow better than magnetic cells when organic carbon is plentiful but that magnetic cells grow better than non-magnetic cells when organic carbon is more limited. This result seems to be in conflict with the notion that the cost of magnetosome formation accounts for up to 33% of the cell's overall metabolism.28 Why then do magnetic cells, which have to divert a significant amount of available energy to their magnetosomes, outperform non-magnetic cells when energy in the form of organic carbon becomes more limited? This descrepency in growth and organic carbon concentration suggests that AMB-1 may have some magnetosome-dependent metabolic system in place for when organic carbon is limited in their environment.
53 0.18 0.16
0.14 100% MSGM / 0 % BFAM
m 0.12 95% MSGM / 5% BFAM n 5
6 90% MSGM / 10% BFAM 5 0.1
@ 75% MSGM / 25% BFAM
e c 0.08 n 50% MSGM / 50% BFAM a b r 25% MSGM 75% BFAM o 0.06 s b 10% MSGM / 90% BFAM A 0.04 5% MSGM / 95% BFAM 0.02 0% MSGM / 100% BFAM 0 0 5 10 15 20 25 30 35
Time (days)
Figure 36: The growth of magnetic, wild type AMB-1 strain cultures through time on various media compositions. Interestingly, the highest organic carbon concentrations (blue, red, yellow) do not represent the greatest extent of growth.
0.18
0.16
0.14 100% MSGM / 0 % BFAM
m 0.12 95% MSGM / 5% BFAM n 5
6 90% MSGM / 10% BFAM 5
0.1
@ 75% MSGM / 25% BFAM
e c
n 0.08 50% MSGM / 50% BFAM a b r 25% MSGM 75% BFAM o
s 0.06 b 10% MSGM / 90% BFAM A 0.04 5% MSGM / 95% BFAM 0% MSGM / 100% BFAM 0.02
0 0 5 10 15 20 25 30 35
Time (days)
Figure 37: Growth of non-magnetic, mutant AMB-1 on various media through time. As in Figure 36, non-magnetic, mutant AMB-1 displays a similar trend of growth disproportional to organic carbon concentration albeit to a lesser extent.
54 At this point, there are three moving pieces to this puzzle. The first piece is cell magnetism. This is a binary variation between the magnetic, wild type strain and the non-magnetic, mutant strain. The second piece is the nine different media compositions which ranges from the highest organic carbon 100% MSGM recipe to the 0% MSGM which contains no organic carbon. The third piece is the time which ranges from 0 to 35 days. Over the course of the first three days, growth of both magnetic and non-magnetic cells is greatest in the highest organic carbon recipes. Beyond day three, magnetic and non-magnetic cells grown on moderate organic carbon media begins to exceed the extant of high organic carbon growth. This change in growth dynamics through time and composition is both curious and unexpected.
In order to investigate this change in growth dynamics better, we can observe relative growth through time. Because the initial assumption is that either strain of AMB-
1 should grow best on media of 100% MSGM composition, growth on that composition is set that as our default “maximum”. Each average measurement for each specific time point will be divided by the 100% MSGM measurement which will yield a series of relative growth measurements. Measurements <1 will indicate growth that is less than cells grown on our “optimal” media composition while measurements >1 will indicate growth that exceeds the “optimal” media. Relative growth is be represented on the y- axis and media composition is represented on the x-axis. Time is represented by the respective line colors with the black line representing anticipated growth if growth is perfectly proportional to the concentration of organic carbon in the media.
55 Figure 38: Growth of magnetic, wild-type AMB-1 on various media over time. Organic carbon concentration increases along the X-axis from left to right. Relative growth is calculated by dividing measured growth by growth on MSGM.
Figure 39: Growth of non-magnetic, mutant AMB-1 on various media over time. Organic carbon concentration increases along the X-axis from left to right. Relative growth is calculated by dividing measured growth by growth on MSGM.
56 For both magnetic and non-magnetic cells the growth most well-aligned to organic carbon concentration occurs on days 3 & 4. Beyond that, the growth of both strains on 25%-75% MSGM evolves away from the the baseline. This effect is greatest with magnetic, wild type cells which attain maximal growth by day 14 and then regress to the maximal growth of non-magnetic, mutant cells by day 35.
Growth of both strains on 90%-95% MSGM by day 35 is actually worse than expected if we assume that growth is proportional to organic carbon concentration.
Interestingly, this result is strongest in the non-magnetic, mutant cells while magnetic, wild type cells display more expected growth on the higher organic carbon media until day 14.
The fact that magnetic, wild type AMB-1 strain cells grow to a greater extent when grown on media that contains less organic carbon, and thus, less available energy for growth, is perplexing. The juxtaposition of enhancement of growth in media of moderate organic carbon media and depression of growth in high organic carbon media as well as the inversion of growth dynamics between magnetic and non-magnetic cells gives us valuable clues as to what might be going on. It's clear that both strains of AMB-
1 possess some metabolic system which enhances long term (20-35 days) growth when organic carbon sources are decreased. This system has a lower limit (10% MSGM and below) beyond which there appears to be no enhancement. Due to the fact that very low concentrations of organic carbon seem to have little or no enhancement effect, it seems reasonable to assume that this metabolic system requires some organic carbon component to proceed. If this is the case, the repression of growth in 90-95% MSGM cultures makes
57 more sense. Organic carbon is needed for both this alternative metabolic system in addition to heterotrophy. If cells are attempting to grow primarily through heterotrophy but some organic carbon is proceeding down the alternative metabolic system's pathways, growth would then be limited rather than enhanced when organic carbon concentrations are not low enough to fully drive the alternative metabolic system.
The greater enhancement on magnetic, wild-type cells suggests that this metabolic system is related in some way to the presence of a magnetosome. Because the modified
MSGM contains the same mineral and vitamin mixtures as the BFAM, there are only a few ingredients that vary between the two media recipes. MSGM has NaNO3 as a nitrogen source while BFAM uses NH4Cl. BFAM contains NaHCO3 as a carbon source while MSGM has sodium acetate as well as succinic and tartaric acids as carbon sources.
The effects of each of these components is examined further in sections 3.2-3.4.
3.2: Analysis of nitrogen sources
One of the key ingredients which varies between the composition of MSGM and
BFAM is nitrogen. MSGM contains 0.12g/L NaNO3 while BFAM contains 0.25 g/L
NH4Cl. This discrepancy in nitrogen sources varies among each of the mixed media
+ composition as represented in Table F. Because NH4 can be a chemoautotrophic electron
- donor and NO3 can be an electron acceptor, the unexpected changes in the growth of
AMB-1 on mixed media could be due to variation in the nitrogen source.
58 Table 17: Calculated variations in the composition of mixed media recipes (in grams/liter)
NaNO3 NH4Cl NaHCO3 Acetate Tartaric Succinic 100% MSGM / 0% BFAM 0.120 0.000 0.000 0.050 0.370 0.370 95% MSGM / 5% BFAM 0.114 0.013 0.009 0.048 0.352 0.352 90% MSGM / 10% BFAM 0.108 0.025 0.018 0.045 0.333 0.333 75% MSGM / 25% BFAM 0.090 0.063 0.045 0.038 0.278 0.278 50% MSGM / 50% BFAM 0.060 0.125 0.090 0.025 0.185 0.185 25% MSGM / 75% BFAM 0.030 0.188 0.135 0.013 0.093 0.093 10% MSGM / 90% BFAM 0.012 0.225 0.162 0.005 0.037 0.037 5% MSGM / 95% BFAM 0.006 0.238 0.171 0.003 0.019 0.019 0% MSGM / 100% BFAM 0.000 0.250 0.180 0.000 0.000 0.000
In order to test the role of the nitrogen source in the growth of AMB-1, 100mL serum bottles containing media of 50% MSGM / 50% BFAM was inoculated with wild type, magnetic cells. One version of the media contained only NaNO3 while the other contained only NH4Cl. Three bottles of each variation were inoculated and growth was measured at time zero and daily for ten days at 565nm.
The results of this experiment can be found in Figure 40 (raw data can be found in
Appendix G. From these results, it appears that NaNO3 is essential for mixed media growth of AMB-1 while NH4Cl is a less efficient nitrogen source. This result is simultaneously expected and unexpected. AMB-1 possesses the gene for ammonium monooxygenase (Amb0163) which is used in ammonium oxidation but not the gene for hydroxylamine oxireductase which is needed for the second step of the reaction. In contrast, AMB-1 does possess the full suite genes for nitrate reduction. This result is
59 unexpected, though, because our media deliberately lacks a known electron donor for
AMB-1. It was possible that AMB-1 was utilizing ammonium in a previously unknown pathway and would have provided a relatively simple explanation as to the enhanced growth of AMB-1 on mixed media. However, that does not appear to be the case.
0.16
0.14
0.12 m n
5 0.1 6 5
@
0.08 e NaNO3 c n a NH4Cl b
r 0.06 o s b
A 0.04
0.02
0 0 1 2 3 4 5 6 7 8 9 10
Time (days)
Figure 40: Magnetic, wild type AMB-1 grown on a 50%/50% mixture of MSGM and BFAM with separate nitrogen sources indicates that nitrate (blue) is essential for growth on this media. Cultures grown on only ammonium (red) demonstrate a significantly slower rate of growth than those grown on only nitrate. The rate and extent of growth in the nitrate curve most closely matches the growth curve of magnetic, wild type AMB-1 on 50% MSGM/50% BFAM in Figure 36.
While it appears that NaNO3 is the more important nitrogen source for the mixed media growth of AMB-1, this only provides a clue for a possible electron acceptor in whatever metabolic system is at work here. It does not explain why the AMB-1 grows better when provided with less organic carbon and less of this apparently essential nitrogen source.
60 3.3: Fixation of radiolabeled bicarbonate
The enhanced growth of AMB-1 on media containing lower concentrations of available organic immediately suggests that there might be some type of carbon fixation metabolism at work. Because cells are cultured in the dark and in the absence of any known chemoautotrophic electron donors, this appears, on the surface, to possibly be a previously unknown type of carbon fixation. In order to test the capacity for carbon fixation of AMB-1 grown on mixed media, cultures of both wild type and mutant cells were grown in triplicate on mixed media compositions of 100% MSGM / 0% BFAM,
50% MSGM / 50% BFAM, and 25% MSGM / 75% BFAM. After three days of growth,
13 each sample was pulsed with NaH CO3. Control cultures were pulsed with normal
NaHCO3 instead. At day 5, cultures were centrifuged to pelletize cells and the dried pellets were analyzed for incorporation of 13C. The raw data from this experiment can be found in Appendix H and the results are represented in Figure 41.
61 62 While both magnetic and non-magnetic AMB-1 appear to be capable of incorporating bicarbonate into their biomass, the difference in the extent growth between magnetic and non-magnetic should not be overlooked. The measurement of incorporation of bicarbonate (δ13C/12C) is a normalized ratio of 13C vs 12C. Therefore, while extent of growth in non-magnetic, mutant AMB-1 grown on 100% MSGM / 0%
MSGM (Figure 37) was greater than the that of magnetic, wild type AMB-1 (Figure 36), the amount of 13C incorporated ends up being significantly less.
It is important to draw a distinction between incorporation of inorganic carbon and the claim of autotrophy. While AMB-1 is clearly incorporating inorganic carbon, we cannot say at this point that AMB-1 is using an autotrophic pathway to do so. What can be said from this data is the following: inorganic carbon incorporation into the biomass of AMB-1 seems to be slightly enhanced as organic carbon availability is limited and seems to be slightly enhanced for magnetic, wild type strain cells (see blue color line in
Figure 36).
3.4: Role of organic carbon
The enhanced growth of AMB-1 on mixed media is limited to intermediate formulations of MSGM and BFAM (25%-75% MSGM, Figure 38). This limitation suggests that there is come essential organic carbon component to this enhanced growth.
MSGM has three main carbon sources: succinic acid, tartaric acid, and sodium acetate.
Quntuplicate cultures of 100% BFAM were prepared and inoculated with magnetic, wild type AMB-1 strain cells. These cultures were grown for three days, measuring
63 absorbance at 565nm at time zero and each subsequent day. At day three, cultures were pulsed in 0.003M/L of a single carbon source into each bottle so that there were five bottles of succinic acid, five of tartaric acid, and five of sodium acetate. Three bottles were left un-pulsed as controls. Samples were measured for growth daily at 565nm to ten days total. The raw data from this experiment can be found in Appendix I and are represented in Figure 42. The only samples which demonstrated any growth were those pulsed with sodium acetate. These results indicate that the growth of magnetic, wild type
AMB-1 cells grown on otherwise entirely BFAM media is dependent upon sodium acetate.
0.18 0.16 0.14
m 0.12 n
5 6 5
0.1 No Pulse @
e Succinic Acid c 0.08 n
a Tartaric Acid b r
o 0.06 s Acetate b
A 0.04 0.02 0 0 1 2 3 4 5 6 7 8 9 10
Time (days)
Figure 42: Cultures of magnetic, wild type AMB-1 strain cells on each of the various organic carbon sources found in MSGM, indicate that the most essential organic carbon for cells grown on otherwise 100% BFAM media.
64 3.5: Summary of mixed media growth
These experiments indicate that when AMB-1 is grown on media with limited organic carbon, the cells will utilize some yet unknown form of metabolism which increases the fixation of inorganic carbon and enhances growth. Curiously, this enhanced growth can even exceed the growth of cells exposed to higher concentrations of organic carbon which suggests that this blend of heterotrophic metabolism and carbon fixation is more efficient than heterotrophy alone. Furthermore, it's evident that this mixed media metabolism is dependent upon four key components: the presence of a magnetosome
(Figure 36 vs Figure 37), sodium nitrate (Figure 40), reduced availability of organic carbon (Figure 41), sodium acetate (Figure 42)
At this point, it seems reasonable to term this system: a magnetosome-dependent, mixotrophic metabolism which likely utilizes sodium acetate as a primary organic carbon sources and nitrate as a terminal electron acceptor.
65 Chapter 4: Magnetic-field-dependent growth of
Magnetospirillum magneticum AMB-1
4.1: Preface
This chapter was written with the following co-authors: Christopher J. Pierce,
Rhea H. Mehta, Nathan R. Lee, Michael J. Wilkins, Ratnasingham Sooryakumar, Michael
Barton, Dennis A. Bazylinski, Brian H. Lower and Steven K. Lower.
4.2: Rationale for alternative to magnetoaerotaxis
Magnetotactic bacteria (MTB) are a phylogenetically diverse group of prokaryotes ubiquitous in nearly all marine and freshwater systems from drainage ditches to estuaries.1,2,3 Despite their diversity, all MTB use specific enzymes to biomineralize intracellular, magnetic crystals of either magnetite (Fe3O4) or greigite (Fe3S4) within a membrane-bounded compartment known as the magnetosome1,2,3. It is widely accepted that the magnetosome enables the cell to engage in magnetically-mediated aerotaxis by aligning along the declination of Earth's magnetic field lines in vertically-stratified oxygen environments4,5. However, MTB in sedimentary environments may align with the geomagnetic field as little as 1% of the time5. This is remarkable because the process of biomineralization accounts for approximately 33% of the cell's metabolic budget6. In this
66 study, we investigate a potential metabolic role for magnetosomes in Magnetospirillum magneticum AMB-1.
4.3: Methods
AMB-1 strains were grown in two different defined liquid media. MSGM consisted of medium 1653 from the American Type Culture Collection with the substitution of Frankel's vitamin solution and modified Wolfe's mineral solution4. The composition of BFM medium was similar to the MSGM except that it contained half of the following components: NaNO3, ascorbic acid, tartaric acid, succinic acid, and sodium acetate. NaH4Cl (2 mM) and NaHCO3 (1 mM) were also added to BFM medium. For growth experiments, serum bottles containing 100 mL of medium were inoculated with 1 mL of inoculum. Cultures were sampled daily for 10 days. Growth was determined by measuring the optical density at 565 nm using a ThermoSpectronic Genesys 10-UV spectrophotometer. All growth experiments were performed in quintuplicate.
A portion of dried microbial material was weighed in Sn capsules and combusted using a Carlo Erba 1108 CHN elemental analyzer. Samples were exposed to a stream of oxygen at a temperature of 1020°C. The evolved CO2 and nitrogen oxides (NOx) were passed over copper to remove the excess oxygen and to reduce the NOx to nitrogen (N2).
The resulting gas mixture was separated and eluted as CO2 and N2 using a chromatographic column (Porapak PQS). Subsequently, the CO2 and N2 were passed to a
Delta V Advantage mass spectrometer (Thermo Fisher Scientific, Waltham, MA) for determination of the 13C/12C ratio.
Subsamples of cell cultures were placed in a fluid chamber constructed from two
67 cover slips (size 1, 18x18mm, Fisher Scientific) attached to one another by a double- sided tape O-ring, which contained the fluid. An integrated three-axes electromagnet41 was used to apply fields (0-100 G) as the cells were imaged on a conventional reflected mode brightfield microscope (Leica) with a 20x objective. Microscopy images were recorded to a digital camera (QImaging Retiga EXI) and the cell swimming trajectories were tracked using the TrackMate Plugin in ImageJ42. The trajectories were analyzed in
MATLAB, to calculate the average velocity and mean swimming direction of each trajectory. For each measurement, a fixed magnetic field (50 G) was applied in a single direction and video recorded for 30s. The videos were then analyzed as described above.
This process was repeated each day during growth with each sample.
4.4: Growth of AMB-1 strains on MSGM and BFM50
AMB-1 is typically grown in medium termed MSGM (magnetic spirillum growth medium), which contains a high concentration of organic carbon. Heterotrophic growth in this media provides cells with carbon for biomass and energy for metabolic activity.
When grown in MSGM, the magnetic, wild-type strain of AMB-1 is out performed by a non-magnetic, mutant strain missing genes associated with the formation of the magnetosome43 (Figure 1A). Given the high energetic burden of magnetosome biomineralization, it is not surprising to see enhanced growth in mutant cells unencumbered by the cost of biomineralizing magnetite. Indeed, similar results have been previously reported in another species of MTB6.
68 Figure 43: (A) Growth of magnetic wild-type (red) and nonmagnetic mutant (green) strains of M. magneticum AMB-1 on organic carbon-rich MSGM medium. Non- magnetic mutant cells display more rapid log phase growth (days 0-3) and achieve a higher population density during stationary phase (days 3-10) than wild-type cells. Greater growth observed for the mutant strain is expected given the high metabolic cost of producing intracellular magnetite. (B) Growth of the same two strains of AMB-1 on organic carbon-poor, BFM medium. While nonmagnetic mutant cells (blue) grow more rapidly during the log phase, magnetic wild-type cells (orange) obtain higher overall cell growth. It is surprising that the magnetic cells outperform the non-magnetic cells since BFM medium has half the organic carbon, and therefore less available energy compared to MSGM medium. (C) Growth of the non-magnetic mutant strain on BFM medium in different magnetic fields. Cell growth increases from a relatively low magnetic field strength (0.01 Gauss; light blue, dash) to geomagnetic strength (0.5 G; blue, solid) to an enhanced magnetic field strength (7 G; dark blue, dotted). These results on the low organic carbon media demonstrate magnetic-field dependent cell growth of AMB-1.
69 However, in a modified growth medium containing significantly less organic carbon (medium termed BFM, Bazylinski-Frankel media40), growth of magnetic, wild- type AMB-1 outpaces the non-magnetic, mutant strain (Figure 1B). This result was unexpected because BFM provides cells with less energy from organic carbon while wild-type cells, with their intracellular magnetite, are expected to have a higher metabolic demand than the non-magnetic, mutant strain. Indeed, the magnetism of magnetite within cells of wild-type AMB-1 did not decrease over time (Figures 2A & 2B) indicating that
these cells were actively synthesizing magnetosomes rather than harvesting energy from
Fe2+ ions within the magnetite in some form of a “battery”44. This inversion in the growth dynamics of AMB-1 strains suggests that there is some direct metabolic advantage in possessing intracellular, biogenic magnetite when access to organic carbon is reduced.
A number of phenotypic changes in AMB-1 were observed with growth in media containing a lower concentration of organic carbon. The polarity of wild-type cells grown in low organic-carbon BFM was equally divided between aligned (south-seeking) and anti-aligned (north-seeking) phenotypes (Figures 2A & 2B). Conversely, wild-type cells grown in high organic-carbon MSGM exhibited predominately a south-seeking, aligned phenotype (Figures 2C & 2D). Wild-type AMB-1 cells grown in BFM swam at higher velocities than cells grown in MSGM (Figure 2E). This significant change in motility indicates a higher metabolic budget for BFM-grown cells in spite of having less energy available from organic carbon. When grown in BFM, both the magnetic, wild-type and the non-magnetic, mutant strains of AMB-1 incorporated more inorganic carbon from
NaH13CO3 compared to growth on MSGM (Figure 2F); although, magnetic wild-type
70 cells generally incorporate inorganic carbon more readily than non-magnetic, mutant cells. These phenotypic changes suggest some measurable metabolic advantage to magnetic over non-magnetic cells of AMB-1 when grown in media containing low concentrations of organic carbon.
Figure 44: Alignment of magnetic, wild-type AMB-1 cells on low-organic carbon BFM medium (panels A and B) vs. high-organic MSGM medium (panels C and D) at days 3 and 10. Arrows in panel A show swimming direction. The magnetic moment remains constant (or slightly increases) over time for cells in the BFM medium, which confirms biomineralization of magnetite despite the energetic cost. The magnetic moment of AMB-1 cells on MSGM medium follows a similar pattern with time. Unlike the predominately south-seeking cells observed on MSGM, the cells grown on BFM are roughly evenly distributed between south- and north-seeking magnetic alignments. (E) The magnetic, wild-type AMB-1 cells grown on BFM (orange) display a faster swimming speed than the same magnetic strain grown on MSGM (red) indicating that the BFM grown cells have a higher energy budget. (F) Incorporation of NaH13CO3 is greater for both wild-type and mutant strains when grown on BFM (with lower organic carbon) as compared to MSGM.
71 4.5: Growth in variable strength external magnetic fields
The magnetic moment of a single wild-type AMB-1 cell is estimated to be roughly 10-16 Am2.45 Magnetic dipole fields decay spatially as 1/r3, where r is the distance from the magnetic dipole. If we take r to be the cell radius (500 nm) then the field strength on the outer membrane resulting from the magnetosome chain is estimated to be
~10 gauss (G). As such, any experiment attempting to reduce or amplify the external magnetic field for a culture of wild-type AMB-1 is irrelevant unless the field strength exceeds 10 G as the wild-type cells possess their own innate magnetic field of approximately 10 G across the entire cell.
Therefore, we tested the effects of external magnetic fields on the growth of
AMB-1 by growing the non-magnetic, mutant strain in both an increased and decreased magnetic field relative to the ambient geomagnetic field (~0.5 G). Increased magnetic field conditions were achieved by growing the cells within a large solenoid supplied with a current that resulted in a magnetic field of approximately ~7 G or roughly 1400% of the geomagnetic field. Decreased magnetic field conditions were achieved by growing bacteria within a highly permeable shielding unit, which reduced the magnetic field to approximately ~0.01 G or roughly 2% of the geomagnetic field. Cells in both devices were sampled daily for growth measurements with minimal exposure to external conditions outside of their respective devices.
We observed significantly greater growth of the non-magnetic mutant cells within the ~7 G field compared to cells held under local geomagnetic conditions (Figure 1 C).
When grown in a ~0.01 G field, the non-magnetic, mutant strain grew slower and to a
72 lesser extent than cells grown under local geomagnetic conditions (Figure 1C). These observations reveal a clear link between magnetic field strength and growth of the non- magnetic mutant strain of AMB-1.
As described above, magnetosomes generate an internal stray field of ~10 G across the cell in wild-type AMB-1. Therefore, an external field of comparable strength applied to non-magnetic, mutant cells should effectively restore the field effects associated with the presence of magnetosomes. This can be seen in the comparable growth characteristics of the non-magnetic, mutant cells in the ~7 G field (Figure 1C, dark-blue, dotted line) and the magnetic, wild-type AMB-1 cells grown under local geomagnetic conditions and in the same medium (Figure 1B, orange line). The metabolic advantage observed in the non-magnetic, mutant grown at ~7 G is constantly available on
Earth to magnetic, wild-type AMB-1, which essentially carry their own ~10 G field. This apparent field-dependent growth of AMB-1 represents the first time that such a metabolic system has been observed and presents us with a question of explaining how a magnetic field might influence the metabolism of a living organism.
4.6: Magnetic-field-dependent biology
Magnetic fields do not generally affect the outcome of chemical reactions due to the fact that thermal energies within the system are far greater than any shift in the energy levels of molecular orbitals due to a magnetic field. However, magnetic field effects are large enough to cause changes to spin states that may influence possible relaxation pathways of excited electrons within a molecule, as pointed out by Schulten et al. in
1976.35 While not implicated in any metabolic system, Schulten's35 mechanism underlies
73 several cases in which magnetic fields directly influence biochemical outcomes (e.g., signaling). Magnetic-field-dependent biochemical reactions are found in plants31, insects32,33, and birds34,36. The products of these previously described reactions are not involved in any known eukaryotic metabolism. They do, however, involve the reduction of flavin adenine dinucleotide (FAD) by photosensitive molecules known as cryptochromes31,32,33,34,36,44,45. FAD is a key metabolic currency. If magnetic fields are enabling the reduction of metabolic currencies in MTB, this could explain the metabolic advantage observed in the magnetic, wild-type AMB-1 cells grown in media with low organic carbon as well as the non-magnetic mutant cells grown in the presence of enhanced magnetic fields.
Figure 3A illustrates the prototypical example of a radical-pair-based reaction36, which enable magnetic-field-dependent biochemical reactions found in avian magneto- reception systems45. Beginning with a pair of molecules, an electron donor (D) and acceptor (A), an energy input of some kind, such as a photon, excites an electron in molecule A to an excited state 1A*. The system relaxes via electron transfer creating a radical pair 1(2A·-+2D·+). If each molecule is initially in a spin singlet state, the radical pair, taken collectively, must in turn be in a spin singlet state, with each component molecule in a doublet spin state. In the absence of a magnetic field, this radical pair will relax back to the ground state through the back-transfer of the electron with no net energy change (see blue track in Figure 3A).
74 Figure 45: (A) A simple schematic of the radical-pair-based reaction, which enables magnetic-field dependent biochemical reactions in migratory animals and which could be at work within the magnetic field-dependent metabolism of AMB-1. With energy input, 1A+1D can be elevated to the higher energy state 1A*+1D. Under normal circumstances 1A*+1D relaxes to 1(2A·-+2D·+) and eventually to the ground state of 1A+1D. Under a magnetic field, 1(2A·-+2D·+) can be transformed to 3(2A·-+2D·+), which can then relax to 3A*+1D. This higher energy product can facilitate biochemical redox reactions downstream. Here the superscripts, 1, 2, and 3 refer to singlet, doublet and triplet spin configurations respectively. The * indicates an electron in an excited state, while the · denotes a radical with the indicated charge. (B) The relatively well understood radical-pair mechanism in the eyes of birds. Using an energy input from photons, cryptochrome facilitates the transfer of electrons from tryptophan to FAD+ in the presence of a magnetic field. Relaxation of the excited 1FAD* through singlet states back to the ground state occurs via ~1 μs relaxation times; however production of the triplet state through magnetic effects enhances the population of the long lived FADH intermediate state (~10 ms) making it available to the organism. While diffusion and subsequent collision of radical pair fragments can also lead to conversion of singlets into the triplet states, this occurs on a longer timescale (~10 μs) relative to the magnetic mechanism (~1 ns). Hence the ultimate population of the stable FADH intermediate relies on the magnetic field. While AMB-1 is unlikely to be utilizing this exact reaction, the reduction of FAD+ demonstrates that the radical-pair mechanism can produce products involved in the metabolism of a cell.
While seemingly trivial compared to thermal energy, a small external magnetic field perturbation is sufficiently large to change the spin configuration, thereby magnetically polarizing the radical pair and converting it to a triplet spin state, which may only relax to a triplet spin state upon electron back transfer (3A*) or along a spin- independent relaxation pathway (see purple track in Figure 3A). In effect this “freezes” the electron in a state with higher total spin. More importantly, it forbids direct relaxation back to the ground state. In the case of cryptochrome36, this enhances the ability of
75 electrons to relax into the spin-independent, higher energy state (Figure 3B) when the magnetic field is applied, whereas, in the absence of the field, the electron simply back- transfers into the ground state directly (see blue track in Figure 3B). In the case of AMB-
1, it appears that the presence of a magnetic field biases cell energetics thereby allowing for increased growth.
Because the magnetic field biases the reaction, stronger fields may yield a greater chance that the reaction will result in a redox reaction while weaker fields will result in more electron relaxations with no net change. In the case of non-magnetic, mutant AMB-
1 grown in 7 G fields (Figure 1C), we attribute the observed increase in growth to such a mechanism (e.g., described in Figure 3A), wherein the magnetic field perturbs the spin configuration of a pair of molecules, changing the possible relaxation pathways and resulting in the trapping of energy in an intermediate state with a sufficient lifetime to be useful to the organism. Conversely, the mechanism is less likely to influence the relaxation of electrons in weaker than local geomagnetic fields and should yield no net observable change as demonstrated in Figure 1C for the non-magnetic, mutant strain of
AMB-1 grown in conditions where the local geomagnetic field was suppressed (~0.01
G).
4.7: Magnetic-field-dependent growth as a novel metabolism
At this time we cannot implicate a specific pair of molecular fragments in the trapping of energy in elevated states via magnetic perturbations to the electron transfer process. However, we posit that the relatively generic physical principles illustrated in
Figure 3A are the basis for the magnetic-field-dependent growth of the MTB presented
76 here. Due to the relatively small energy scale of magnetic field perturbations, few alternative explanations regarding the coupling of chemical reactions to magnetic fields have been presented34,36. While the exact bio-machinery and pathways utilized by MTB to exploit the radical-pair mechanism for growth are still unknown, Schulten and colleagues37 speculated that radical-pair-based interactions could be at the core of reaction centers in photosynthetic bacteria.
We suggest that this magnetic-field-dependent system of magnetically-enhanced trophism represents a newly discovered mechanism for harnessing energy for life on
Earth. Perhaps MTB evolved the magnetosome pseudo-organelle to maximize energy uptake and/or sustain cells in the organic-carbon depleted environments that these microorganisms often occupy in nature. Such a metabolic system enables access to energy inputs otherwise unavailable to competing organisms, making it incredibly efficient. Additionally the deep evolutionary history of MTB suggests that such a system has been active on Earth for at least 2.2 billion years19. The ubiquity of MTB on Earth ensures that this magnetic-field-dependent system plays a previously unknown and perhaps significant role in the global carbon cycle. The abundance of sessile organisms capable of magnetoreception (e.g. fungi, plants, and algae)7, suggests that this radical- pair-based metabolic system may be widespread and perhaps involved in global primary production.
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80 Appendix A: Growth of AMB-1 on MSGM without organic carbon (Table 6)
Date Cell Counts 10/24/16 14 37 17 19 30 0 (cont.) 9 11 30 26 10/31/16 2 25 14 7 26 18 11/07/16 24 25 78 97 114 11/14/16 173 126 53 74 58 32 11/21/16 102 124 97 94 52 91 11/28/16 89 52 200 179 68 36 12/05/16 169 269 117 83 107 91
78 Appendix B: AMB-1 growth on MSGM (Table 2) and BG11 (Table 7)
Sample Name Absorbance @ 600nm MSGM-CTRL 0.002 0.002 0.002 MSGM-AMB1-CTRL 0.004 0.004 0.004 MSGM-AMB1-1 0.009 0.005 0.006 0.005 MSGM-AMB1-2 0.016 0.017 0.015 0.012 MSGM-AMB1-3 0.014 0.012 0.015 0.01 MSGM-AMB1-4 0.023 0.025 0.028 0.019 MSGM-AMB1-5 0.025 0.024 0.026 0.021 BG11-CTRL 0 0 0 BG11-AMB1-CTRL 0 0 0 BG11-AMB1-1 0.006 0.006 0.006 0.006 BG11-AMB1-2 0.006 0.006 0.006 0.006 BG11-AMB1-3 0.012 0.012 0.012 0.012 BG11-AMB1-4 0.011 0.011 0.011 0.011 BG11-AMB1-5 0.001 0.001 0.001 0.001
79 Appendix C: Growth of AMB-1 in Multivariable Experiment (Table 11) AMB-1 on MSGM Day Trial 1.1 Trial 1.2 Trial 1.3 Trial 1.4 Trial 1.5 Trial 2.1 Trial 2.2 Trial 2.3 0 0.045 0.048 0.035 0.047 0.038 0.057 0.049 0.055 1 0.103 0.091 0.054 0.063 0.06 0.068 0.09 0.074 2 0.204 0.198 0.072 0.096 0.086 0.151 0.16 0.167 3 0.17 0.159 0.102 0.118 0.108 0.125 0.128 0.149 4 0.165 0.146 0.196 0.115 0.172 0.14 0.134 0.159 5 6 7 0.155 0.14 0.166 0.131 0.174 0.125 0.118 0.165 8 9 10 0.159 0.136 0.142 0.104 0.15 0.13 0.123 0.161 11 12 13 0.169 0.146 0.144 0.085 0.138 0.12 0.126 0.151 AMB-1 on MSGM Day Trial 2.4 Trial 2.5 Trial 3.1 Trial 3.2 Trial 3.3 Trial 3.4 0 0.053 0.053 0.051 0.05 0.049 0.056 1 0.076 0.095 0.078 0.086 0.085 0.07 2 0.138 0.165 0.137 0.13 0.149 0.127 3 0.103 0.138 0.109 0.113 0.12 0.098 4 0.107 0.143 0.115 0.106 0.114 0.096 5 6 7 0.086 0.138 0.097 0.086 0.113 0.092 8 9 10 0.077 0.146 0.087 0.091 0.12 0.092 11 12 13 0.074 0.146 0.08 0.08 0.0116 0.073
80 AMB-1 on autotrophic media in dark Day Trial 1.1 Trial 1.2 Trial 1.3 Trial 1.4 Trial 1.5 Trial 2.1 Trial 2.2 Trial 2.3 0 0.032 0.038 0.038 0.048 0.048 0.027 0.053 0.041 1 0.04 0.05 0.046 0.055 0.059 0.036 0.064 0.046 2 0.043 0.051 0.045 0.053 0.062 0.034 0.06 0.047 3 0.042 0.047 0.045 0.049 0.067 0.034 0.043 0.032 4 0.037 0.051 0.04 0.044 0.059 0.034 0.056 0.044 5 6 7 0.039 0.052 0.04 0.044 0.061 0.032 0.061 0.057 8 9 10 0.036 0.037 0.04 0.04 0.061 0.03 0.059 0.046 11 12 13 0.039 0.037 0.039 0.039 0.063 0.031 0.066 0.046 AMB-1 on autotrophic media in dark Day Trial 2.4 Trial 2.5 Trial 2.6 Trial3.1 Trial 3.2 Trial 3.3 Trial 3.4 Trial 3.5 0 0.036 0.049 0.051 0.027 0.032 0.03 0.032 0.031 1 0.04 0.05 0.054 0.028 0.028 0.026 0.028 0.027 2 0.039 0.055 0.054 0.016 0.012 0.014 0.012 0.013 3 0.022 0.037 0.041 0.011 0.006 0.009 0.008 0.011 4 0.042 0.055 0.051 0.032 0.029 0.028 0.027 0.032 5 0.019 0.014 0.02 0.014 0.022 6 0.025 0.023 0.023 0.022 0.025 7 0.042 0.053 0.056 0.023 0.031 0.035 0.021 0.022 8 0.025 0.039 0.025 0.03 0.027 9 0.033 0.03 0.03 0.032 0.027 10 0.04 0.051 0.054 0.024 0.03 0.028 0.024 0.026 11 0.025 0.019 0.022 0.021 0.028 12 13 0.034 0.052 0.054
81 AMB-1 on autotrophic media in blue light Day Trial 1.1 Trial 1.2 Trial 1.3 Trial 1.4 Trial 1.5 Trial 2.1 Trial 2.2 Trial 2.3 0 0.029 0.038 0.043 0.038 0.04 0.025 0.051 0.041 1 0.039 0.057 0.061 0.051 0.055 0.047 0.056 0.038 2 0.04 0.056 0.062 0.049 0.057 0.039 0.059 0.034 3 0.043 0.058 0.062 0.046 0.058 0.037 0.041 0.012 4 0.043 0.058 0.06 0.043 0.054 0.035 0.055 0.03 5 6 7 0.041 0.056 0.059 0.044 0.053 0.034 0.06 0.032 8 9 10 0.032 0.06 0.069 0.037 0.055 0.03 0.057 0.032 11 12 13 0.032 0.061 0.064 0.037 0.058 0.032 0.055 0.031 AMB-1 on autotrophic media in blue light Day Trial 2.4 Trial 2.5 Trial 2.6 Trial 3.1 Trial 3.2 Trial 3.3 Trial 3.4 Trial 3.5 0 0.041 0.051 0.05 0.033 0.031 0.027 0.029 0.027 1 0.045 0.055 0.057 0.019 0.015 0.016 0.016 0.011 2 0.043 0.057 0.058 0 0.004 0 0 0 3 0.022 0.045 0.04 0 0 0 0 0 4 0.04 0.056 0.059 0.011 0.01 0.017 0.014 0.012 5 0 0 0 0 0 6 0.002 0.001 0.003 0.005 0.002 7 0.051 0.058 0.055 0.004 0.009 0.003 0.01 0.009 8 0.012 0.007 0.013 0.008 0.002 9 0.006 0.004 0.006 0.0021 10 0.06 0.059 0.052 0 0.009 0.009 0.003 11 0.008 0.002 0 0.001 12 13 0.05 0.057 0.043
82 AMB-1 on autotrophic media in red light Day Trial 1.1 Trial 1.2 Trial 1.3 Trial 1.4 Trial 1.5 Trial 2.1 Trial 2.2 Trial 2.3 0 0.028 0.038 0.038 0.039 0.039 0.043 0.029 0.053 1 0.034 0.062 0.058 0.059 0.062 0.063 0.042 0.046 2 0.037 0.059 0.053 0.057 0.053 0.061 0.03 0.05 3 0.036 0.057 0.061 0.053 0.048 0.061 0.017 0.03 4 0.034 0.06 0.042 0.054 0.044 0.06 0.035 0.046 5 6 7 0.033 0.057 0.043 0.054 0.043 0.06 0.051 0.049 8 9 10 0.03 0.055 0.038 0.052 0.04 0.061 0.031 0.042 11 12 13 0.031 0.056 0.044 0.052 0.037 0.064 0.026 0.037 AMB-1 on autotrophic media in red light Day Trial 2.4 Trial 2.5 Trial 2.6 Trial 3.1 Trial 3.2 Trial 3.3 Trial 3.4 Trial 3.5 0 0.051 0.051 0.054 0.032 0.029 0.03 0.029 0.028 1 0.056 0.053 0.059 0.018 0.016 0.017 0.017 0.022 2 0.055 0.058 0.065 0.001 0.003 0 0 0.001 3 0.041 0.039 0.043 0 0 0 0 0 4 0.054 0.056 0.068 0.016 0.018 0.018 0.013 0.009 5 0.004 0 0 0 0 6 0.01 0.003 0.006 0.006 0.005 7 0.054 0.052 0.059 0.006 0.005 0.005 0.005 0.005 8 0.01 0.008 0.016 0.004 0.014 9 0.01 0.008 0.009 0.005 0.005 10 0.053 0.049 0.054 0.007 0.005 0.012 0.005 0.007 11 0.011 0.002 0.005 0.007 0 12 13 0.052 0.048 0.054
83 AMB-1 on autotrophic media in increased magnetic field Day Trial 1.1 Trial 1.2 Trial 1.3 Trial 1.4 Trial 1.5 Trial 2.1 Trial 2.2 Trial 2.3 0 0.025 0.042 0.04 0.044 0.036 0.023 0.05 0.055 1 0.036 0.055 0.053 0.048 0.054 0.035 0.055 0.062 2 0.037 0.063 0.058 0.048 0.057 0.035 0.052 0.078 3 0.037 0.059 0.059 0.047 0.055 0.035 0.038 0.043 4 0.035 0.059 0.055 0.043 0.07 0.038 0.047 0.058 5 6 7 0.036 0.06 0.056 0.039 0.053 0.032 0.042 0.063 8 9 10 0.032 0.059 0.056 0.035 0.051 0.043 0.041 0.06 11 12 13 0.032 0.061 0.063 0.035 0.051 0.051 0.039 0.064 AMB-1 on autotrophic media in increased magnetic field Day Trial 2.4 Trial 2.5 Trial 2.6 Trial 3.1 Trial 3.2 Trial 3.3 Trial 3.4 Trial 3.5 0 0.031 0.05 0.055 0.028 0.031 0.029 0.031 0.031 1 0.032 0.051 0.054 0.026 0.025 0.028 0.026 0.026 2 0.029 0.053 0.064 0.012 0.01 0.01 0.012 0.01 3 0.011 0.035 0.046 0.012 0.011 0.008 0.009 0.008 4 0.031 0.052 0.059 0.031 0.032 0.031 0.027 0.027 5 0.014 0.012 0.013 0.016 0.011 6 0.03 0.02 0.02 0.02 0.018 7 0.03 0.053 0.06 0.024 0.028 0.018 0.026 0.022 8 0.032 0.022 0.025 0.022 0.022 9 0.022 0.026 0.031 0.025 0.022 10 0.027 0.053 0.063 0.026 0.029 0.027 0.022 0.021 11 0.021 0.025 0.024 0.023 0.014 12 13 0.031 0.053 0.061
84 Control – sterile Day Trial 1.1 Trial 1.2 Trial 1.3 Trial 1.4 Trial 1.5 0 0.031 0.032 0.032 0.029 0.03 1 0.03 0.03 0.03 0.026 0.029 2 0.013 0.015 0.013 0.012 0.014 3 0.016 0.012 0.01 0.012 0.008 4 0.039 0.03 0.031 0.033 0.038 5 0.025 0.011 0.014 0.017 0.012 6 0.019 0.025 0.019 0.025 0.021 7 0.026 0.025 0.025 0.024 0.022 8 0.027 0.028 0.028 0.02 0.022 9 0.024 0.021 0.02 0.025 0.023 10 0.02 0.031 0.024 0.033 0.02 11 0.02 0.029 0.019 0.018 0.025
85 Appendix D: Comparison of Growth of AMB-1 on BFAM of Various Oxygen Concentrations
Autotrophic Media: Without cysteine-HCl / Not purged with nitrogen 1 2 3 4 5 6 7 8 9 10 Day 0 0.032 0.026 0.03 0.029 0.026 0.025 0.023 0.024 0.023 0.028 Day 1 0.023 0.026 0.026 0.026 0.021 0.026 0.024 0.027 0.027 0.025 Day 2 0.028 0.021 0.025 0.024 0.022 0.027 0.021 0.026 0.025 0.026 Day 3 0.027 0.027 0.025 0.026 0.024 0.026 0.025 0.029 0.025 0.027 Day 4 0.037 0.026 0.03 0.03 0.025 0.032 0.025 0.033 0.028 0.03 Day 5 0.033 0.028 0.03 0.033 0.031 0.031 0.031 0.032 0.03 0.031 Day 7 0.035 0.029 0.032 0.034 0.033 0.036 0.028 0.037 0.033 0.033 Day 9 0.034 0.03 0.036 0.033 0.028 0.032 0.028 0.036 0.032 0.035 Autotrophic Media: Without cysteine-HCl / Purged with nitrogen 1 2 3 4 5 6 7 8 9 10 Day 0 0.032 0.042 0.039 0.058 0.048 0.04 0.052 0.054 0.031 0.041 Day 1 0.032 0.036 0.035 0.047 0.047 0.036 0.054 0.053 0.035 0.041 Day 2 0.026 0.035 0.036 0.05 0.046 0.035 0.05 0.048 0.034 0.039 Day 3 0.028 0.039 0.039 0.05 0.047 0.036 0.05 0.049 0.041 0.042 Day 4 0.03 0.037 0.038 0.053 0.046 0.04 0.054 0.05 0.038 0.041 Day 5 0.031 0.04 0.038 0.051 0.049 0.041 0.05 0.053 0.04 0.044 Day 7 0.032 0.038 0.038 0.048 0.05 0.039 0.051 0.05 0.038 0.044 Day 9 0.033 0.04 0.041 0.05 0.049 0.037 0.049 0.052 0.039 0.043 Autotrophic Media: With cysteine-HCl / Not purged with nitrogen 1 2 3 4 5 6 7 8 9 10 Day 0 0.01 0.008 0.011 0.007 0.011 0.011 0.009 0.01 0.015 0.008 Day 1 0.01 0.01 0.016 0.006 0.012 0.013 0.012 0.008 0.013 0.013 Day 2 0.01 0.011 0.012 0.009 0.01 0.009 0.011 0.008 0.013 0.01 Day 3 0.014 0.015 0.013 0.014 0.012 0.013 0.016 0.016 0.015 0.018 Day 4 0.012 0.023 0.011 0.017 0.015 0.013 0.015 0.012 0.024 0.013 Day 5 0.018 0.014 0.014 0.011 0.011 0.013 0.013 0.013 0.017 0.016 Day 7 0.013 0.011 0.014 0.012 0.012 0.009 0.014 0.014 0.011 0.011 Day 9 0.014 0.009 0.01 0.01 0.012 0.012 0.011 0.02 0.017 0.01
86 Autotrophic Media: With cysteine-HCl / purged with nitrogen 1 2 3 4 5 6 7 8 9 10 Day 0 0.019 0.029 0.021 0.019 0.025 0.023 0.016 0.024 0.021 0.021 Day 1 0.018 0.025 0.02 0.02 0.021 0.026 0.015 0.019 0.026 0.022 Day 2 0.019 0.023 0.021 0.018 0.02 0.022 0.014 0.02 0.021 0.02 Day 3 0.018 0.022 0.02 0.021 0.021 0.019 0.019 0.023 0.02 0.018 Day 4 0.023 0.022 0.021 0.023 0.024 0.023 0.017 0.019 0.025 0.02 Day 5 0.02 0.023 0.02 0.025 0.029 0.025 0.023 0.023 0.024 0.023 Day 7 0.018 0.018 0.018 0.03 0.022 0.02 0.019 0.019 0.021 0.019 Day 9 0.018 0.018 0.018 0.022 0.02 0.02 0.017 0.022 0.02 0.019
87 Appendix E: Mixed Media Growth of Wild-type and Mutant AMB-1 Strains 100% MSGM / 0% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.006 -0.003 -0.001 -0.002 0.002 1 0.004 -0.006 0.006 -0.003 -0.004 2 0.019 0.011 0.009 0.014 0.014 3 0.017 0.022 0.022 0.014 0.014 4 0.066 0.096 0.079 0.085 0.076 5 0.121 0.117 0.118 0.109 0.119 6 0.122 0.117 0.124 0.115 0.116 7 0.103 0.105 0.111 0.097 0.101 8 0.108 0.103 0.104 0.104 0.106 9 0.1 0.103 0.103 0.098 0.096 10 0.104 0.103 0.111 0.094 0.102 14 0.1 0.104 0.12 0.106 0.1 21 0.124 0.14 0.137 0.124 0.144 28 0.133 0.13 0.14 0.134 0.149 35 0.112 0.126 0.125 0.124 95% MSGM / 5% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.001 0.003 0.002 0.001 1 0.002 0.003 0.004 0.004 0.002 2 0.007 0.007 0.01 0.008 0.006 3 0.015 0.019 0.025 0.022 0.012 4 0.045 0.056 0.067 0.06 0.032 5 0.126 0.156 0.185 0.176 0.084 6 0.196 0.187 0.188 0.179 0.233 7 0.19 0.195 0.188 0.18 0.211 8 0.196 0.19 0.193 0.189 0.208 9 0.196 0.21 0.196 0.207 0.185 10 0.208 0.205 0.195 0.19 0.194 14 0.184 0.179 0.182 0.177 0.205 21 0.185 0.187 0.162 0.17 0.182 28 0.203 0.183 0.184 0.21 0.2 35
88 90% MSGM / 10% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0 0.001 0.002 1 0.001 0.001 0.001 0.001 0.001 2 0.003 0.004 0.003 0.002 0.002 3 0.003 0.003 0.004 0.004 0.003 4 0.004 0.004 0.004 0.005 0.005 5 0.007 0.007 0.006 0.008 0.006 6 0.014 0.015 0.012 0.016 0.014 7 0.022 0.022 0.017 0.027 0.023 8 0.043 0.048 0.034 0.052 0.042 9 0.089 0.105 0.079 0.103 0.092 10 0.253 0.255 0.276 0.25 0.274 14 0.198 0.206 0.202 0.198 0.212 21 0.195 0.234 0.185 0.184 0.248 28 0.201 0.236 0.166 0.184 0.228 35 75% MSGM / 25% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.001 0.002 0.002 1 0.003 0.002 0.003 0.003 2 0.009 0.01 0.009 0.008 3 0.028 0.029 0.03 0.028 4 0.077 0.077 0.084 0.077 5 0.175 0.168 0.151 0.157 6 0.167 0.142 0.139 0.136 7 0.168 0.152 0.143 0.146 8 0.179 0.165 0.149 0.15 9 0.185 0.164 0.159 0.165 10 0.199 0.171 0.172 0.177 14 0.208 0.19 0.174 0.184 21 0.253 0.228 0.191 0.182 28 0.27 0.261 0.223 0.214 35
89 50% MSGM / 50% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.005 -0.001 -0.004 0.001 -0.005 1 -0.004 -0.001 -0.002 -0.003 -0.003 2 0.017 0.017 0.012 0.014 0.014 3 0.028 0.032 0.015 0.033 0.018 4 0.065 0.069 0.055 0.071 0.053 5 0.088 0.095 0.065 0.087 0.077 6 0.092 0.101 0.096 0.101 0.101 7 0.094 0.09 0.096 0.091 0.099 8 0.097 0.099 0.098 0.097 0.099 9 0.096 0.1 0.097 0.097 0.098 10 0.099 0.104 0.102 0.1 0.104 14 0.111 0.117 0.103 0.107 0.111 21 0.138 0.139 0.137 0.139 0.132 28 0.144 0.149 0.149 0.164 0.149 35 0.133 0.141 0.129 0.129 0.12 25% MSGM / 75% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 -0.002 0.007 0 0 -0.002 1 0.002 0.006 0.001 0.002 -0.002 2 0.023 0.013 0.017 0.009 0.015 3 0.035 0.033 0.036 0.016 0.02 4 0.091 0.066 0.075 0.061 0.062 5 0.123 0.118 0.092 0.089 0.103 6 0.124 0.118 0.107 0.104 0.113 7 0.122 0.126 0.106 0.101 0.106 8 0.106 0.111 0.096 0.097 0.103 9 0.107 0.092 0.083 0.097 0.097 10 0.095 0.095 0.076 0.082 0.092 14 0.078 0.079 0.068 0.075 0.093 21 0.084 0.096 0.07 0.078 0.071 28 0.087 0.075 0.062 0.068 0.079 35 0.064 0.07 0.046 0.057 0.057
90 10% MSGM / 90% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.005 -0.002 -0.001 0.002 1 0.001 0.007 0.007 0.003 0.008 2 0.02 0.013 0.01 0.014 0.012 3 0.02 0.018 0.021 0.028 0.023 4 0.043 0.045 0.042 0.047 0.034 5 0.054 0.044 0.055 0.048 0.047 6 0.048 0.042 0.041 0.041 0.036 7 0.042 0.036 0.036 0.041 0.038 8 0.04 0.033 0.042 0.038 0.035 9 0.033 0.031 0.029 0.03 0.034 10 0.024 0.027 0.028 0.023 0.032 14 0.024 0.029 0.024 0.024 0.025 21 0.022 0.032 0.04 0.031 0.029 28 0.029 0.043 0.031 0.039 0.023 35 0.013 0.017 0.015 0.017 0.016 5% MSGM / 95% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.005 0 0.002 0 0.002 1 0.004 0.005 0.002 0.007 0.005 2 0.012 0.013 0.009 0.012 0.014 3 0.014 0.013 0.013 0.015 0.011 4 0.023 0.023 0.021 0.026 0.026 5 0.025 0.032 0.028 0.028 0.027 6 0.033 0.029 0.026 0.024 0.035 7 0.023 0.021 0.024 0.025 0.02 8 0.028 0.027 0.028 0.024 0.019 9 0.019 0.022 0.017 0.012 0.01 10 0.014 0.013 0.013 0.024 0.019 14 0.009 0.016 0.012 0.011 0.015 21 0.029 0.015 0.011 0.014 0.017 28 0.022 0.018 0.02 0.028 0.023 35 0.008 0.009 0.004 0.006 0.005
91 0% MSGM / 100% BFAM Wild Type Magnetic AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.006 0.005 0.003 0.002 0.003 1 0.004 0.004 0.001 0.007 0 2 0.007 0.008 0.01 0.012 0.012 3 0.002 0.003 0.001 0.004 0.003 4 0.012 0.015 0.009 0.011 0.011 5 0.013 0.008 0.013 0.014 0.01 6 0.016 0.01 0.011 0.013 0.013 7 0.012 0.007 0.012 0.011 0.008 8 0.018 0.014 0.013 0.013 0.012 9 0.007 0.004 0.007 0.003 0.006 10 0.003 0.005 0.011 0 0.003 14 0.003 0.006 0.002 0.005 0.001 21 0.005 0.002 0.004 0.006 0.006 28 0.009 0.018 0.009 0.014 0.012 35 -0.002 -0.002 -0.007 -0.002 -0.004 100% MSGM / 0% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.002 0.002 0.003 0.003 1 0.003 0.003 0.004 0.004 0.004 2 0.004 0.003 0.004 0.004 0.004 3 0.004 0.004 0.004 0.005 0.005 4 0.005 0.004 0.006 0.006 0.005 5 0.006 0.005 0.008 0.009 0.007 6 0.008 0.006 0.013 0.014 0.011 7 0.01 0.013 0.023 0.025 0.017 8 0.02 0.015 0.057 0.054 0.039 9 0.041 0.028 0.129 0.119 0.077 10 0.092 0.069 0.167 0.165 0.172 11 0.184 0.14 0.175 0.173 0.173 12 0.178 0.174 0.186 0.179 0.177 13 0.184 0.18 0.195 0.19 0.188 14 0.205 0.191 0.201 0.204 0.193 17 0.183 0.168 0.175 0.167 0.172 21 0.198 0.184 0.158 0.17 0.172 28 0.178 0.17 0.206 0.201 0.199 35 0.179 0.143 0.126 0.149 0.159
92 95% MSGM / 5% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.002 0.001 0.004 1 0.002 0.003 0.003 0.005 2 0.007 0.008 0.008 0.015 3 0.02 0.022 0.022 0.043 4 0.051 0.051 0.055 0.11 5 0.149 0.158 0.168 0.192 6 0.209 0.205 0.203 0.199 7 0.219 0.222 0.22 0.217 8 0.234 0.236 0.233 0.23 9 0.242 0.245 0.245 0.242 10 0.232 0.234 0.237 0.227 14 0.226 0.23 0.243 0.238 21 0.28 0.129 0.217 0.208 28 35 90% MSGM / 10% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.001 1 0.004 0.001 0.002 0.002 2 0.016 0.005 0.004 0.002 3 0.039 0.004 0.005 0.002 4 0.114 0.005 0.004 0.004 5 0.185 0.009 0.006 0.005 6 0.183 0.013 0.009 0.008 7 0.197 0.025 0.014 0.016 8 0.205 0.052 0.029 0.026 9 0.231 0.104 0.066 0.059 10 0.221 0.257 0.229 0.216 14 0.202 0.251 0.25 0.257 21 0.139 0.305 0.208 0.292 28 35
93 75% MSGM / 25% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.008 0.003 0.004 1 0.008 0.005 0.004 0.011 2 0.016 0.016 0.017 0.036 3 0.038 0.042 0.042 0.084 4 0.127 0.129 0.132 0.145 5 0.154 0.152 0.146 0.137 6 0.141 0.137 0.133 0.135 7 0.148 0.14 0.144 0.14 8 0.147 0.144 0.147 0.145 9 0.153 0.147 0.15 0.147 10 0.155 0.149 0.147 0.146 14 0.156 0.146 0.147 0.143 21 0.201 0.137 0.211 0.12 28 35 50% MSGM / 50% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.003 0.003 0.002 0.003 1 0.003 0.004 0.004 0.004 0.004 2 0.004 0.003 0.004 0.004 0.004 3 0.004 0.004 0.004 0.004 0.005 4 0.005 0.004 0.005 0.005 0.005 5 0.006 0.005 0.006 0.006 0.006 6 0.008 0.007 0.008 0.008 0.008 7 0.012 0.011 0.013 0.015 0.012 8 0.024 0.017 0.025 0.024 0.022 9 0.042 0.032 0.049 0.045 0.041 10 0.09 0.067 0.099 0.096 0.089 11 0.13 0.11 0.121 0.119 0.118 12 0.12 0.115 0.114 0.111 0.11 13 0.115 0.111 0.113 0.11 0.109 14 0.115 0.121 0.114 0.112 0.109 17 0.118 0.111 0.114 0.124 0.111 21 0.141 0.131 0.133 0.132 0.126 28 0.166 0.155 0.151 0.151 0.143 35 0.126 0.125 0.136 0.136 0.159
94 25% MSGM / 75% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.003 0.004 0.003 0.003 1 0.004 0.004 0.004 0.004 0.004 2 0.004 0.004 0.004 0.004 0.004 3 0.004 0.005 0.005 0.007 0.005 4 0.005 0.007 0.005 0.006 0.005 5 0.007 0.009 0.008 0.007 0.007 6 0.01 0.014 0.01 0.011 0.011 7 0.018 0.024 0.018 0.02 0.018 8 0.037 0.037 0.037 0.043 0.037 9 0.053 0.056 0.054 0.072 0.055 10 0.071 0.068 0.065 0.086 0.078 11 0.083 0.077 0.07 0.08 0.085 12 0.079 0.076 0.07 0.082 0.081 13 0.077 0.074 0.072 0.08 0.081 14 0.085 0.085 0.075 0.078 0.084 17 0.063 0.071 0.066 0.081 0.075 21 0.071 0.073 0.076 0.075 0.073 28 0.061 0.066 0.063 0.07 0.07 35 0.048 0.056 0.055 0.06 0.063 10% MSGM / 90% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.003 0.002 0.002 0.003 1 0.004 0.004 0.004 0.004 0.005 2 0.004 0.004 0.004 0.004 0.004 3 0.005 0.005 0.005 0.004 0.005 4 0.007 0.007 0.006 0.006 0.006 5 0.01 0.01 0.01 0.008 0.009 6 0.015 0.014 0.016 0.012 0.012 7 0.023 0.025 0.022 0.017 0.018 8 0.033 0.023 0.028 0.023 0.025 9 0.045 0.026 0.025 0.029 0.026 10 0.041 0.024 0.028 0.029 0.028 11 0.045 0.026 0.028 0.029 0.031 12 0.044 0.024 0.026 0.027 0.026 13 0.042 0.022 0.025 0.026 0.026 14 0.041 0.022 0.025 0.025 0.025 17 0.037 0.021 0.023 0.023 0.024 21 0.035 0.02 0.022 0.022 0.023 28 0.029 0.019 0.019 0.03 0.026 35 0.024 0.015 0.014 0.037 0.02
95 5% MSGM / 95% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.002 0.003 0.002 0.002 1 0.004 0.003 0.004 0.003 0.004 2 0.004 0.004 0.004 0.004 0.004 3 0.005 0.005 0.005 0.005 0.005 4 0.006 0.007 0.007 0.006 0.007 5 0.009 0.01 0.01 0.009 0.01 6 0.012 0.015 0.013 0.014 0.014 7 0.015 0.02 0.015 0.015 0.018 8 0.015 0.017 0.014 0.015 0.016 9 0.014 0.016 0.012 0.014 0.014 10 0.012 0.015 0.012 0.012 0.013 11 0.013 0.019 0.012 0.012 0.013 12 0.013 0.014 0.011 0.012 0.012 13 0.011 0.013 0.01 0.013 0.013 14 0.011 0.013 0.011 0.012 0.014 17 0.011 0.013 0.01 0.011 0.012 21 0.011 0.014 0.011 0.011 0.012 28 0.013 0.028 0.016 0.014 0.016 35 0.009 0.012 0.009 0.013 0.012 0% MSGM / 100% BFAM Non-Magnetic Mutant AMB-1 Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.002 0.002 0.003 0.003 1 0.003 0.003 0.005 0.004 0.003 2 0.003 0.003 0.003 0.004 0.004 3 0.005 0.004 0.003 0.004 0.004 4 0.004 0.004 0.004 0.004 0.004 5 0.005 0.004 0.004 0.005 0.005 6 0.007 0.005 0.004 0.004 0.005 7 0.012 0.005 0.004 0.007 0.004 8 0.026 0.005 0.005 0.005 0.005 9 0.038 0.006 0.004 0.004 0.005 10 0.084 0.004 0.004 0.005 0.004 11 0.081 0.005 0.005 0.004 0.004 12 0.084 0.004 0.004 0.004 0.004 13 0.084 0.004 0.005 0.004 0.004 14 0.084 0.007 0.006 0.004 0.004 17 0.083 0.058 0.027 0.007 0.013 21 0.113 0.108 0.091 0.005 0.052 28 0.106 0.09 0.118 0.005 0.135 35 0.051 0.084 0.108 0.012 0.083
96 Appendix F: Growth of Wild-Type and Mutant AMB-1 Strains on Mixed Media
Magnetic 100% MSGM / 0% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.002 0.001 0.001 0.002 1 0.006 0.006 0.005 0.008 0.005 2 0.03 0.029 0.029 0.031 0.03 3 0.124 0.125 0.12 0.123 0.12 4 0.101 0.104 0.104 0.105 0.106 5 0.102 0.104 0.107 0.108 0.109 6 0.106 0.108 0.108 0.111 0.116 7 0.106 0.11 0.109 0.109 0.119 8 0.102 0.104 0.107 0.106 0.118 9 0.103 0.103 0.105 0.106 0.116 10 0.101 0.098 0.1 0.101 0.113 14 0.089 0.097 0.094 0.098 0.106 21 0.088 0.099 0.093 0.099 0.102 28 0.089 0.099 0.099 0.102 0.102 35 0.081 0.095 0.091 0.092 0.1 Magnetic 95% MSGM / 5% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.002 0.001 0.001 0.002 1 0.006 0.006 0.006 0.006 0.005 2 0.034 0.034 0.033 0.032 0.033 3 0.112 0.115 0.111 0.118 0.111 4 0.1 0.097 0.093 0.099 0.091 5 0.103 0.096 0.095 0.102 0.091 6 0.105 0.106 0.097 0.102 0.093 7 0.111 0.108 0.099 0.104 0.091 8 0.113 0.106 0.096 0.102 0.09 9 0.117 0.106 0.096 0.102 0.09 10 0.108 0.1 0.091 0.099 0.087 14 0.086 0.081 0.072 0.082 0.082 21 0.084 0.084 0.075 0.079 0.068 28 0.079 0.082 0.079 0.085 0.063 35 0.078 0.082 0.078 0.086 0.058
97 Magnetic 90% MSGM / 10% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.002 0.002 0.002 0.001 1 0.039 0.006 0.006 0.006 0.007 2 0.068 0.035 0.033 0.034 0.029 3 0.128 0.12 0.121 0.119 0.127 4 0.12 0.099 0.101 0.099 0.102 5 0.114 0.096 0.097 0.101 0.109 6 0.126 0.1 0.101 0.102 0.11 7 0.115 0.099 0.101 0.096 0.108 8 0.115 0.098 0.101 0.095 0.107 9 0.114 0.094 0.091 0.088 0.102 10 0.107 0.085 0.083 0.083 0.098 14 0.105 0.079 0.082 0.077 0.082 21 0.082 0.078 0.079 0.079 0.08 28 0.076 0.066 0.076 0.079 0.092 35 0.068 0.067 0.07 0.073 0.079 Magnetic 75% MSGM / 25% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.001 0.001 0.001 0.001 1 0.006 0.006 0.007 0.006 0.006 2 0.034 0.035 0.037 0.036 0.038 3 0.099 0.096 0.091 0.096 0.101 4 0.091 0.096 0.091 0.09 0.108 5 0.094 0.118 0.11 0.118 0.124 6 0.105 0.139 0.125 0.129 0.134 7 0.114 0.163 0.139 0.143 0.152 8 0.115 0.159 0.14 0.146 0.155 9 0.123 0.166 0.145 0.142 0.168 10 0.12 0.165 0.154 0.152 0.172 14 0.143 0.149 0.145 0.167 0.148 21 0.124 0.127 0.104 0.15 0.124 28 0.113 0.101 0.095 0.119 0.104 35 0.103 0.093 0.084 0.093 0.088
98 Magnetic 50% MSGM / 50% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.002 0.003 1 0.007 0.007 0.007 0.007 0.0044 2 0.04 0.039 0.037 0.039 0.092 3 0.04 0.076 0.076 0.075 0.125 4 0.074 0.076 0.078 0.075 0.13 5 0.089 0.095 0.112 0.103 0.139 6 0.096 0.103 0.117 0.11 0.147 7 0.105 0.117 0.128 0.122 0.152 8 0.113 0.125 0.139 0.129 0.15 9 0.113 0.133 0.138 0.135 0.147 10 0.12 0.132 0.123 0.129 0.142 14 0.138 0.139 0.132 0.128 0.144 21 0.127 0.137 0.117 0.112 0.128 28 0.113 0.125 0.103 0.094 0.098 35 0.103 0.129 0.099 0.097 0.082 Magnetic 25% MSGM / 75% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.002 0.002 1 0.008 0.008 0.008 0.008 0.007 2 0.045 0.044 0.042 0.038 0.041 3 0.062 0.058 0.058 0.061 0.059 4 0.062 0.066 0.063 0.062 0.063 5 0.07 0.084 0.082 0.078 0.076 6 0.088 0.089 0.086 0.089 0.087 7 0.088 0.094 0.086 0.089 0.087 8 0.094 0.089 0.091 0.095 0.092 9 0.1 0.079 0.085 0.098 0.085 10 0.091 0.076 0.076 0.09 0.078 14 0.077 0.071 0.068 0.076 0.068 21 0.07 0.066 0.06 0.069 0.061 28 0.066 0.055 0.057 0.063 0.055 35 0.061 0.052 0.051 0.059 0.051
99 Magnetic 10% MSGM / 90% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0.001 0.001 0.001 1 0.007 0.007 0.007 0.007 0.007 2 0.039 0.04 0.036 0.033 0.035 3 0.049 0.044 0.048 0.047 0.047 4 0.047 0.045 0.047 0.048 0.047 5 0.04 0.036 0.037 0.037 0.041 6 0.04 0.039 0.04 0.04 0.042 7 0.038 0.038 0.039 0.039 0.042 8 0.037 0.037 0.039 0.036 0.04 9 0.036 0.036 0.036 0.036 0.039 10 0.036 0.035 0.036 0.035 0.038 14 0.036 0.033 0.034 0.033 0.036 21 0.033 0.032 0.034 0.033 0.035 28 0.031 0.029 0.031 0.03 0.032 35 0.03 0.029 0.028 0.028 0.03 Magnetic 5% MSGM / 95% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.003 0.002 1 0.007 0.007 0.008 0.008 0.007 2 0.032 0.031 0.03 0.03 0.029 3 0.025 0.024 0.026 0.025 0.024 4 0.023 0.023 0.025 0.024 0.023 5 0.022 0.022 0.024 0.024 0.023 6 0.022 0.022 0.025 0.023 0.023 7 0.022 0.023 0.025 0.023 0.025 8 0.022 0.02 0.024 0.021 0.023 9 0.021 0.021 0.023 0.022 0.023 10 0.021 0.021 0.023 0.021 0.021 14 0.021 0.021 0.026 0.021 0.021 21 0.02 0.02 0.021 0.02 0.02 28 0.018 0.018 0.022 0.018 0.019 35 0.017 0.017 0.021 0.018 0.018
100 Magnetic 0% MSGM / 100% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.002 0.001 0.001 0.001 1 0.005 0.005 0.005 0.005 0.007 2 0.005 0.005 0.005 0.005 0.008 3 0.006 0.006 0.006 0.006 0.008 4 0.005 0.005 0.005 0.005 0.007 5 0.006 0.005 0.005 0.006 0.007 6 0.006 0.005 0.006 0.006 0.008 7 0.006 0.006 0.006 0.006 0.009 8 0.005 0.005 0.006 0.005 0.008 9 0.006 0.006 0.006 0.006 0.011 10 0.004 0.005 0.005 0.006 0.006 14 0.005 0.006 0.007 0.006 0.008 21 0.006 0.006 0.006 0.006 0.007 28 0.005 0.005 0.005 0.005 0.007 35 0.005 0.005 0.004 0.008 0.008 Non-magnetic 100% MSGM / 0% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0.001 0.001 0.001 1 0.007 0.008 0.008 0.008 0.008 2 0.041 0.045 0.048 0.047 0.046 3 0.134 0.132 0.13 0.128 0.128 4 0.121 0.121 0.123 0.119 0.121 5 0.128 0.125 0.126 0.127 0.133 6 0.122 0.12 0.12 0.121 0.121 7 0.125 0.123 0.126 0.125 0.124 8 0.118 0.119 0.122 0.115 0.12 9 0.119 0.124 0.117 0.114 0.123 10 0.117 0.117 0.116 0.117 0.115 14 0.118 0.102 0.112 0.116 0.118 21 0.107 0.106 0.115 0.1 0.107 28 0.113 0.108 0.114 0.108 0.115 35 0.104 0.103 0.114 0.106 0.108
101 Non-magnetic 95% MSGM / 5% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0.001 0.001 0.001 1 0.008 0.008 0.008 0.008 0.008 2 0.05 0.051 0.05 0.054 0.052 3 0.111 0.109 0.114 0.11 0.109 4 0.104 0.102 0.106 0.103 0.103 5 0.103 0.101 0.109 0.103 0.105 6 0.103 0.099 0.102 0.098 0.098 7 0.1 0.097 0.103 0.095 0.102 8 0.096 0.094 0.095 0.091 0.092 9 0.095 0.094 0.099 0.094 0.094 10 0.092 0.091 0.095 0.092 0.098 14 0.094 0.084 0.092 0.085 0.093 21 0.093 0.09 0.095 0.091 0.089 28 0.084 0.082 0.092 0.096 0.095 35 0.085 0.081 0.089 0.084 0.098 Non-magnetic 90% MSGM / 10% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0.001 0.001 0.001 1 0.007 0.008 0.008 0.008 0.007 2 0.046 0.043 0.053 0.048 0.052 3 0.125 0.124 0.121 0.127 0.124 4 0.108 0.107 0.106 0.108 0.112 5 0.103 0.099 0.098 0.108 0.107 6 0.096 0.099 0.101 0.102 0.104 7 0.098 0.094 0.101 0.101 0.103 8 0.095 0.093 0.095 0.099 0.099 9 0.092 0.089 0.092 0.098 0.098 10 0.088 0.086 0.086 0.093 0.096 14 0.081 0.086 0.081 0.095 0.089 21 0.082 0.08 0.081 0.092 0.087 28 0.081 0.079 0.077 0.098 0.09 35 0.076 0.073 0.065 0.092 0.086
102 Non-magnetic 75% MSGM / 25% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0.001 0.002 0.001 1 0.009 0.008 0.008 0.039 0.008 2 0.06 0.056 0.057 0.077 0.06 3 0.102 0.104 0.106 0.11 0.1 4 0.097 0.096 0.099 0.104 0.099 5 0.111 0.11 0.105 0.111 0.102 6 0.109 0.11 0.108 0.111 0.106 7 0.115 0.113 0.115 0.117 0.11 8 0.118 0.113 0.115 0.117 0.116 9 0.121 0.118 0.121 0.129 0.123 10 0.123 0.123 0.122 0.128 0.126 14 0.119 0.122 0.121 0.146 0.167 21 0.124 0.122 0.12 0.141 0.131 28 0.123 0.134 0.127 0.135 0.11 35 0.122 0.125 0.113 0.124 0.097 Non-magnetic 50% MSGM / 50% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.002 0.002 1 0.009 0.009 0.009 0.012 0.009 2 0.061 0.059 0.061 0.081 0.059 3 0.085 0.083 0.093 0.08 0.085 4 0.081 0.08 0.084 0.082 0.08 5 0.092 0.083 0.091 0.096 0.097 6 0.092 0.091 0.096 0.098 0.096 7 0.101 0.099 0.106 0.102 0.102 8 0.101 0.099 0.104 0.103 0.109 9 0.106 0.105 0.114 0.11 0.116 10 0.109 0.107 0.117 0.112 0.121 14 0.107 0.108 0.109 0.121 0.143 21 0.109 0.109 0.112 0.118 0.127 28 0.132 0.133 0.14 0.109 0.112 35 0.136 0.135 0.142 0.097 0.101
103 Non-magnetic 25% MSGM / 75% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.003 0.002 1 0.01 0.01 0.009 0.01 0.009 2 0.054 0.055 0.057 0.071 0.057 3 0.069 0.068 0.068 0.063 0.07 4 0.068 0.064 0.067 0.066 0.069 5 0.086 0.078 0.078 0.08 0.082 6 0.095 0.091 0.09 0.082 0.092 7 0.082 0.091 0.09 0.084 0.085 8 0.078 0.091 0.083 0.086 0.065 9 0.081 0.083 0.087 0.084 0.083 10 0.08 0.086 0.079 0.088 0.087 14 0.072 0.075 0.073 0.073 0.078 21 0.059 0.067 0.064 0.069 0.074 28 0.059 0.063 0.059 0.067 0.071 35 0.057 0.054 0.054 0.065 0.063 Non-magnetic 10% MSGM / 90% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.001 0.001 1 0.009 0.01 0.009 0.009 0.009 2 0.046 0.041 0.04 0.048 0.042 3 0.04 0.042 0.043 0.046 0.043 4 0.042 0.041 0.04 0.049 0.041 5 0.04 0.039 0.037 0.045 0.037 6 0.04 0.038 0.037 0.044 0.037 7 0.04 0.037 0.037 0.043 0.042 8 0.038 0.037 0.037 0.041 0.04 9 0.035 0.034 0.035 0.038 0.038 10 0.035 0.034 0.034 0.039 0.036 14 0.033 0.034 0.033 0.037 0.035 21 0.029 0.031 0.031 0.035 0.033 28 0.028 0.029 0.028 0.034 0.031 35 0.022 0.025 0.029 0.032 0.026
104 Non-magnetic 5% MSGM / 95% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.002 0.002 1 0.009 0.011 0.01 0.01 0.01 2 0.027 0.026 0.025 0.029 0.026 3 0.022 0.024 0.021 0.028 0.024 4 0.021 0.025 0.021 0.027 0.024 5 0.021 0.023 0.02 0.026 0.025 6 0.02 0.022 0.021 0.025 0.025 7 0.022 0.022 0.022 0.025 0.025 8 0.018 0.02 0.021 0.024 0.025 9 0.018 0.025 0.02 0.024 0.023 10 0.017 0.021 0.019 0.023 0.023 14 0.017 0.02 0.019 0.023 0.022 21 0.015 0.017 0.019 0.021 0.021 28 0.014 0.017 0.021 0.02 0.02 35 0.013 0.013 0.012 0.013 0.016 Non-magnetic 0% MSGM / 100% BFAM Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.001 0.001 0.001 0.001 0.001 1 0.005 0.005 0.005 0.006 0.005 2 0.005 0.003 0.005 0.007 0.004 3 0.004 0.003 0.003 0.007 0.004 4 0.005 0.005 0.004 0.007 0.007 5 0.004 0.004 0.003 0.006 0.007 6 0.004 0.005 0.003 0.007 0.007 7 0.004 0.005 0.005 0.007 0.009 8 0.004 0.004 0.003 0.006 0.007 9 0.005 0.005 0.005 0.006 0.007 10 0.003 0.004 0.003 0.006 0.006 14 0.005 0.005 0.005 0.006 0.007 21 0.004 0.004 0.004 0.007 0.007 28 0.004 0.004 0.007 0.006 0.006 35 0.004 0.003 0.007 0.004 0.004
105 Appendix G: AMB-1 growth on NO3 vs NH4
NaNO3 Bottle 1 Bottle 2 Bottle 3 Bottle 4 0 0.001 0.003 0.002 0.002 1 0.007 0.007 0.008 0.005 2 0.03 0.04 0.031 0.039 3 0.114 0.115 0.114 0.106 4 0.1 0.102 0.098 0.1 5 0.097 0.098 0.101 0.107 6 0.106 0.114 0.106 0.106 7 0.108 0.11 0.107 0.108 8 0.119 0.118 0.111 0.109 9 0.129 0.161 0.138 0.112 10 0.124 0.122 0.116 0.12
NH4Cl Bottle 1 Bottle 2 Bottle 3 Bottle 4 0 0.001 0.002 0.002 0.001 1 0.003 0.007 0.008 0.007 2 0.032 0.037 0.036 0.041 3 0.05 0.051 0.049 0.04 4 0.05 0.06 0.057 0.055 5 0.062 0.063 0.063 0.065 6 0.08 0.075 0.075 0.074 7 0.08 0.086 0.079 0.082 8 0.099 0.093 0.096 0.093 9 0.101 0.105 0.107 0.102 10 0.113 0.113 0.119 0.11
106 Appendix H: Incorporation of Radiolabeled Bicarbonate
Magnetospirillum magneticum AMB-1 magnetic cells Carbon 12 pulse Carbon 13 pulse 100/0 50/50 25/75 100/0 50/50 25/75 -20.948 -21.386 -21.462 88.324 124.874 126.286 -21.683 -21.246 -21.942 94.091 99.573 133.455 -21.176 -22.358 -21.854 93.31 124.916 130.272
Magnetospirillum magneticum AMB-1 non-magnetic mutant cells Carbon 12 pulse Carbon 13 pulse 100/0 50/50 25/75 100/0 50/50 25/75 -21.345 -19.68 -21.158 80.794 130.949 122.015 -21.612 -21.431 -21.25 85.611 118.122 123.482 -20.734 -21.194 -21.161 92.958 112.582 123.085
107 Appendix I: Organic-carbon Pulse on 100% BFAM
Succinic Acid Pulse Day Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.002 0.002 0.002 0.002 0.002 1 0.01 0.011 0.01 0.009 0.01 2 0.012 0.012 0.012 0.012 0.012 3 0.009 0.009 0.01 0.009 0.01 4 0.007 0.007 0.007 0.007 0.008 5 0.007 0.006 0.007 0.007 0.007 6 0.006 0.006 0.006 0.006 0.007 7 0.007 0.006 0.007 0.009 0.008 8 0.006 0.006 0.006 0.006 0.007 9 0.006 0.006 0.005 0.006 0.006 10 0.005 0.006 0.007 0.005 0.006 Tartaric Acid Pulse Day Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.002 0.003 0.002 0.002 1 0.01 0.009 0.009 0.009 0.01 2 0.012 0.012 0.012 0.012 0.012 3 0.009 0.009 0.009 0.01 0.01 4 0.009 0.009 0.008 0.008 0.009 5 0.008 0.008 0.007 0.007 0.007 6 0.006 0.006 0.008 0.005 0.005 7 0.006 0.006 0.005 0.005 0.005 8 0.005 0.005 0.005 0.005 0.005 9 0.005 0.005 0.005 0.004 0.004 10 0.004 0.004 0.004 0.005 0.004 Sodium Acetate Pulse Day Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 0 0.003 0.002 0.002 0.002 0.002 1 0.009 0.01 0.01 0.009 0.009 2 0.012 0.012 0.012 0.013 0.012 3 0.01 0.009 0.009 0.009 0.009 4 0.039 0.034 0.029 0.029 0.032 5 0.051 0.048 0.053 0.061 0.053 6 0.071 0.068 0.078 0.083 0.087 7 0.088 0.085 0.09 0.082 0.096 8 0.092 0.103 0.106 0.092 0.095 9 0.071 0.082 0.081 0.074 0.078 10 0.069 0.075 0.078 0.071 0.075
108 No Pulse Day Bottle 1 Bottle 2 Bottle 3 0 0.002 0.002 0.002 1 0.009 0.01 0.009 2 0.012 0.012 0.013 3 0.01 0.01 0.01 4 0.01 0.012 0.012 5 0.01 0.011 0.011 6 0.009 0.01 0.01 7 0.01 0.011 0.012 8 0.01 0.009 0.01 9 0.012 0.01 0.01 10 0.01 0.01 0.01
109