Intrinsic Magnetism of Bacillus Spores: Theoretical Studies and
Potential Applications
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Jianxin Sun
Ohio State Biochemistry Program
The Ohio State University
2010
Dissertation Committee:
Jeffrey J Chalmers, Advisor
Jiyan Ma
Shang-Tian Yang Copyright by
Jianxin Sun
2010 Abstract
Our lab was the first to report that Bacillus atrophaeus spores have intrinsic magnetism, and it was demonstrated that the magnetic susceptibility is sufficient to separate and deposit these spores on glass slides via a magnetic deposition system
(Melnik et al. 2007). Surprisingly, Mn was the only paramagnetic element found in the spores, as indicated by Energy Dispersive Spectroscopy (EDS) analysis; but Mn is lacking in vegetative cells or other nonmagnetic spores. Moreover, magnetic susceptibility of different Mn oxides varies significantly, suggesting that the magnitude of a spore’s paramagnetism can differ based on a number of factors, including level of oxidation and time of sporulation. Experimentally, variation in the magnitude of paramagnetism was observed from batch to batch, and studies were conducted to further understand this variation including elemental quantification, optimization of the culturing, sporulating condition, and genetic studies.
By adding polystyrene microparticles, which are of a very different size and magnetic susceptibility than spore clusters, the accuracy of Cell Track Velocitimetry
(CTV) to measure and calculate magnetic susceptibility was explored and is reported.
With the combination of CTV and X-ray Photoelectron Spectra, the oxidation state of Mn on spores and its imparting magnetic susceptibility to the spores was quantified, and the
ii Mn per spore cluster was calculated. The calculation was consistent with the result from
Inductively Coupled Plasma- Mass Spectrometry independent measurements.
Different metal ion concentrations, oxygen availability and heat shock effects were examined during fermentation and Mn concentration was found to be the only factor that influenced the paramagnetism. Using these optimization studies, the Mn concentration was doubled in the media and the fermentation was scaled-up to 4 L using
Biostat B bioreactor, and reliable magnetic spores were obtained.
Potential applications of paramagnetic spores were also discussed: separation from various liquid food and adsorption of heavy metal ion from waste water. The intrinsic magnetism enabled the spores to be separated from air or liquid food, such as milk and chicken broth. However, spores couldn’t be separated from juice probably due to the low pH, in which the spores lost their paramagnetism. Interesting, the magnetism of spores recovered after adding Mn. Since spores could survive a sterilization process, the effect of sterilization on spores’ magnetism was examined. It turned out that the process didn’t alter the magnetic susceptibility of spores dramatically and surviving spores could be separated through the magnetic deposition system.
iii Dedication
Dedicated to my parents
iv Acknowledgment
I would like to thank Dr. Jeffrey Chalmers, my adviser, for his guidance, support and enthusiasm during the whole development of my research, which made the dissertation possible.
I would like to thank Dr. Zborowski, who helped me with my papers and also theoretical questions during my study.
I would like to thank my committee members for their suggestions on my dissertation.
I would like to express my appreciation for Lisa in Evans Lab helping me with X-ray
Photoelectron Spectrometry.
I am grateful to Dr. Yang Zhao for his help in CTV adjustment and improvement all the time, to Priya and Brian for teaching me how to use fluorescent microscope and associated software.
In addition, I would like to thank my friends, especially Dr. Yali Zhang and Ching-suei
Hsu, for their suggestions, Dr. Wei-lun Chang, for her help in writing, Dr. Mingrui Yu and Derek Lyttle for proofreading.
Finally, I would say thank you to my parents. I couldn’t make it without their encouragement.
v Vita
March, 1982...... Born, Hebei, China
June, 2004 ...... B.S. Biological Science,
Nankai University, China
September, 2004-December, 2009...... M.S. Applied Statistics
The Ohio State University
September, 2004-September, 2010 ...... Graduate Research Associate,
Ohio State Biochemistry Program
The Ohio State University
Publications
Melnik, K., Sun, J., Fleischman, A., Roy, S., Zborowski, M. and Chalmers, J.J. (2007). Quantification of magnetic susceptibility in several strains of Bacillus spores: Implications for separation and detection. Biotechnology and Bioengineering 98(1): 186- 192.
Fields of Study
Major Field: Ohio State Biochemistry Program
Specialization: Biochemical Engineering vi Table of Contents
Abstract...... ii
Dedication...... iv
Acknowledgment ...... v
List of Tables ...... xiv
List of Figures...... xvii
Chapter 1 Introduction ...... 1
1.1 Biological Warfare (BW) and detection challenges...... 1
1.2 Detection and separation of Bacillus atrophaeus spores ...... 4
1.3 Other Bacillus strains in the study ...... 7
1.4 Bacillus spores, Mn and paramagnetism...... 8
1.5 Immunomagnetic Separation (IMS)...... 12
1.5.1 Introduction of IMS...... 12
1.5.2 Commercial Immunomagnetic Separation Technologies...... 14
1.5.3 Magnetic deposition system ...... 15
1.6 Intrinsic magnetism...... 16
vii 1.7 Magnetic susceptibility and magnetophoretic mobility ...... 19
1.7.1 Magnetic susceptibility...... 19
1.7.2 Magnetophoretic Mobility (MM) ...... 21
1.8 Organization of the dissertation ...... 24
Chapter 2 Quantification of magnetic susceptibility in several strains of Bacillus spores27
2.1 Introduction ...... 28
2.2 Materials and methods ...... 31
2.3 Results ...... 37
2.4 Discussion ...... 45
Chapter 3 Improvement of Cell Tracking Velocimetry (CTV) accuracy by the inclusion of internal control in spore samples...... 48
3.1. Motivation ...... 48
3.2. Theory ...... 51
3.2.1 Fundamental forces, velocities of particles in CTV ...... 51
3.2.2 Algorithm of CTV and classification by CTV associated software ...... 54
3.2.3 Classification by Support Vector Machines (SVMs) ...... 54
3.3 Materials and methods ...... 54
3.3.1 Experimental apparatus ...... 54
3.3.2 Spores and particles...... 56
viii 3.4 Result...... 60
3.4.1 Classification by 2 methods...... 60
3.4.2 Magnetic susceptibility measurements of Polystyrene Microspheres (PSM) .. 61
3.4.3 Magnetic susceptibility of measurements and calculations of the spores ...... 65
3.5 Conclusion...... 73
Chapter 4 Quantification of both the presence, and oxidation state, of Mn in Bacillus atrophaeus spores and its imparting of magnetic susceptibility to the spores...... 74
4.1 Introduction ...... 75
4.2 Theory ...... 78
4.2.1 Magnetic properties ...... 78
4.2.2 Bulk magnetic susceptibility of a mixture...... 78
4.2.3 Calculated magnetic force and magnetically induced velocity as a result of Mn
composition ...... 79
4.3 Materials and methods ...... 83
4.3.1 Strains and sporulation ...... 83
4.3.2 Mn valence state determination by X-Ray Photoelectron Spectroscopy ...... 83
4.3.3 Mn and Fe concentration determination by Inductively Coupled Plasma-Mass
Spectroscopy (ICP-MS)...... 84
4.3.4 Magnetophoretic mobility (MM) of treated spores from Cell Tracking
Velocimetry ...... 85 ix 4.4. Results ...... 86
4.4.1 Magnetophoretic mobilities of PSM and spores...... 86
4.4.3 XPS analysis of the spores...... 87
4.4.4 Quantitative estimates of the amount of Mn on the spores ...... 92
4.4.5 Estimates of the amount of Mn per spore...... 96
4.4.6 Total Mn mass balance...... 96
4.5 Discussion ...... 96
Chapter 5 Factors affecting the intrinsic magnetism of Bacillus spores using spectra and fermentation studies...... 100
5.1 Motivation ...... 100
5.2Materials and methods ...... 102
5.2.1 Strains, growth curve and sporulation ...... 102
5.2.2 Sporulating using the Applikon bioreactor...... 103
5.2.3 X-Ray Photoelectron Spectroscopy...... 104
5.2.4 Cell Tracking Velocimetry ...... 104
5.3 Results and discussion...... 104
5.3.1 Paramagnetism of Bacillus spores...... 104
5.3.2 EDS spectra for Bacillus subtilis and Bacillus magaterium spores...... 106
5.3.3 Surface Scan for Bacillus strains using XPS...... 108
x 5.3.4 Selection of culturing and sporulating media...... 113
5.3.5 Modifications of sporulation using Mod G media...... 114
5.3.6 Ion effects during fermentation ...... 118
5.3.7 Iron effects during fermentation ...... 119
5.3.8 Heat shock after sporulation doesn’t increase the intrinsic magnetism...... 120
5.3.9 Response surface analysis ...... 122
5.3.10 Fermentation by bioreactors ...... 122
Chapter 6 Genetic studies of Bacillus atrophaeus...... 124
6.1 Introduction ...... 124
6.2 Materials and methods ...... 126
6.2.1 Strains ...... 126
6.2.2 Validation of mnxG homolog using PCR and sequencing ...... 126
6.3 Result and discussion ...... 126
Chapter 7 Spore separation from the liquid food via magnetic deposition system ...... 128
7.1 Motivation ...... 128
7.2 Experimental scope ...... 131
7.3 Materials and methods ...... 133
7.3.1 Strains and treatment ...... 133
7.3.2 Cell Tracking Velocimetry ...... 133
xi 7.3.3 Separation of spores...... 134
7.4 Result...... 135
7.4.1 Magnetic deposition before treatment ...... 135
7.4.2 Magnetophoretic mobility and magnetic deposition after sterilization treatment
...... 137
7.4.4 Magnetism recovery after adding Mn back in to the spores’ suspension...... 142
7.5 Discussion and conclusion ...... 144
Chapter 8 Potential application of Bacillus atrophaeus for contaminated water with heavy metal ion...... 146
8.1 Introduction ...... 146
8.1.1 Heavy metal contamination source...... 146
8.1.2 Lead effect ...... 147
8.1.3 Nowadays techniques for contamination...... 148
8.1.4 Recyclable biogenic Mn oxides on Bacillus atrophaeus spores ...... 148
8.2 Materials and methods ...... 150
8.2.1 Strains and sporulation ...... 150
8.2.2 Pb sorption experiments ...... 150
8.2.3 Mn recovery...... 151
8.2.4 Cell Tracking Velocimetry ...... 151
xii 8.3 Result and discussion ...... 153
8.3.1 Magnetism recovery by increasing the pH after acid treatment...... 153
8.3.2 Pb effect on magnetophoretic mobility of spores...... 153
8.3.3 Addition of Mn after Pb treatment ...... 154
8.3.4 Spore recycling process simulation ...... 155
Chapter 9 Conclusions and future work...... 157
9.1 Conclusions ...... 157
9.2 Future work ...... 161
9.2.1 Other paramagnetic strains ...... 161
9.2.2 Optimized media...... 161
9.2.3 Continuous study for decontamination...... 162
9.2.4 Mn oxidase expression level...... 163
References...... 164
Appendix A: R code and result for classification of spores and PSM...... 179
Appendix B: Result from the R code in appendix A ...... 182
xiii List of Tables
Table 1.1 Comparison between common biological detection and separation methods .... 3
Table 1.2 Magnetic susceptibility of Mn and Mn compounds ...... 11
Table 1.3 Summary of IMS technology (Chalmers et al., 1998)...... 13
Table 1.4 Mn(II)-oxidizing and non-Mn(II)-oxidizing Bacillus strains ...... 20
Table 2.1 Media Recipes of Sporulation of Bacillus spores...... 33
Table 2.2 Media recipes for the sporulation of Bacillus species ...... 40
Table 2.3 Sporulated Bacillus strains and magnetophoretic mobility measurements ...... 44
Table 3.1 Current and its corresponding magnetic energy gradient. The current and its corresponding magnetic energy in red are used in the this chapter...... 57
Table 3.2 CTV analysis of PSM ...... 62
Table 3.3 Mean, and standard deviation, of settling (A) and magnetic velocity (B) of the
PSM and spores when analyzed as a mixture in the CTV system ...... 68
Table 3.4 Spore settling and magnetic velocity when not mixed with PSM ...... 70
Table 4.1 Spore settling and magnetic velocity when not mixed with PSM ...... 80
Table 4.2 Experimentally reported average values of magnetic susceptibility of selected cells and polystyrene microspheres...... 84
Table 4.3 CTV analysis of PSM and spores ...... 88
xiv Table 4.4 Quantification of Mn in different chemical state from XPS...... 95
Table 4.5 ICP-MS anlaysis of Mn content in spores ...... 99
Table 5.1 Comparison of magnetic velocity, settling velocity, MM and magnetic susceptibility of different strains of Bacillus ...... 105
Table 5.2 The effect of culturing media (LB, NB) and sporulating media (SG, Mod G) to the MM of B. atrophaeus...... 114
Table 5.3 Magnetic susceptibility and thermodynamic parameters of Mn compounds . 115
Table 5.4 The effect of Mn concentration, sporulation time and working volume during the sporulation on MM of B. atrophaeus...... 116
Table 5.5 The effect of ions ...... 119
Table 5.6 The effect of iron ...... 120
Table 5.7 The effect of heat shock after sporulation on MM ...... 121
Table 5.8 MM of spores from bioreactors ...... 123
Table 7.1 The colony counts on TSA plates before and after the spores going through the magnetic deposition system (The suspensions were diluted to 10-4, and for the suspension after separation, 10-2) ...... 135
Table 7.2 Magnetophoretic mobility (×104) (mm3/T•A•s) of B. atrophaeus before and after sterilization ...... 137
Table 7.3 Sizes of spore clumps before and after treatment ...... 143
Table 7.4 Magnetophoretic mobility (mm3/T•A•s) of spores (without any treatment) before and after adding extra Mn...... 143
xv Table 8.1 Predominant anthropogenic ources of Pb to Atmosphere, tons per year (Shotyk and Le Roux, 2005)...... 147
Table 8.2 Magnetophoretic mobility (mm3/T•A•s) of spores after acid treatment and after adding base followed the acid treatment. Mn was also added to examine effect on the magnetism recovery...... 152
Table 8.3 The Magnetophoretic Mobility of spores after days of Pb treatment...... 154
Table 8.4 MM (×104) of spore streated with Pb after 2 days and 4 days. Mn was added after 4 days, and the last column is MM after the addition of Mn...... 155
Table 8.5 Magnetophoretic mobility ((mm3/T•A•s)×105) of spores followed the treatment mentioned in Figure 8.1 ...... 156
xvi List of Figures
Figure 1.1 The Mn cycle of oxidation states found in nature...... 10
Figure 1.2 The principle of immunomagnetic separation of target microorganisms...... 13
Figure 1.3 Schematic diagram of the magnetic deposition system...... 15
Figure 1.4 The magnetic field around the interpoler gap ...... 18
Figure 1.5 Magnetophoretic mobility of spores and other organisms ...... 19
Figure 1.6 Magnetic balance schematic...... 21
Figure 2.1 A diagram of Cell Tracking Velocimetry (A). A an enlargement in the microscopic analysis region (B)...... 34
Figure 2.2 Schematic diagram of the open gradient, magnetic deposition system...... 35
Figure 2.3 An enlargement of the thin channel near the open gradient, deposition region
(A) and a photograph of the deposited, magnetic spores (B) The circled regions correspond to the characteristic deposition bands obtained with this instrument...... 36
Figure 2.4 A histogram of the magnetophoretic mobility of Bacillus globigii spores. A second x-axis provides the actual velocity of the spores in the CTV instrument...... 37
Figure 2.5 A histogram, on a linear scale, of immunomagnetically labeled (anti-CD3-
MACS) human lymphocytes from Zhang et al...... 39
xvii Figure 2.6 Spectra of the Energy Dispersive Spectroscopy analysis of the Bacillus globigii spores (A), and the vegetative cells (B). Note the lack of Mn peaks in the vegetative cells...... 40
Figure 2.7 EDS spectra of B. globiggi spores sporulated in SG media (A), B. cereus (B), and B. thuringiensis (C)...... 42
Figure 3.1 Schematic illustration of forces on paramagnetic particles in solution in the magnetic field...... 52
Figure 3.2 SMV classification schmatic picture ...... 55
Figure 3.3 Electric coils and channel for the electromagnetic CTV...... 57
Figure 3.4 An example of the computer screen output of the CTV software indicating the settling trajectories (vertical traces) and magnetically induced trajectories (horizontal traces) of particles in ROI tracked by the CTV system...... 58
Figure 3.5 An example of digital image analysis by “image view” software. The set, frame and timing information defines the pictures taken in one set; image information defines the area of interest...... 59
Figure 3.6 Scatter plot (A), and histograms of settling velocity (B) and magnetic velocity
(C), for the PSM. In addition, the mean, one and three standard deviations from the mean are presented as dashed lines for the settling and magnetic velocity...... 63
Figure 3.7 Dot plots of a full scale (A) CTV measurement of Bacillus spores mixed with
15.3 µm PSM (A) and an enlarged view (B) ...... 66
Figure 3.8 Representative dot plot (A), histogram of the magnetic velocity (B), and histogram of the settling velocity of the spores not mixed with PSM (C)...... 71
xviii Figure 4.1 Possible mechanisms of Bacterial Mn (II) oxidation. The numbers 1, 2, and 3 in the lower panel indicate different possible pathways for Mn(II) oxidation ...... 76
Figure 4.2 Settling and magnetic velocity of the spores only in distilled water (A) and in distilled water containing EDTA (B)...... 89
Figure 4.3 Setting and magnetic velocity of the spores in distilled water and after being suspended in a solution of pH 1.0 for 14 hours (B). Control (A) ...... 90
Figure 4.4 Full spectra from XPS analysis (A). The box framed spectra contains Mn peaks, which is enlarged in (B)...... 93
Figure 5.1 Mn oxides equations under different combinations of pH and Mn concentration (Hastings and Emerson, 1986) ...... 101
Figure 5.2 EDS spectra for B. sp SG-1 (A), B. magaterium (B), B. subtilis (C)...... 106
Figure 5.3 (A) Bacillus atrophaeus (B) B. SG-1 The peaks circled in (A) and (B) represent Mn. (C) B. cereus surface scan (D) the comparison of Mn peak area between
B. atrophaeus (yellow), B. SG-1 (blue), B. cereus (red), B. subtilis (light blue) and B. megaterium (violet) (from top to bottom)...... 109
Figure 5.4 One way analysis of MM by Mn concentration (A), Volume (B), Sporulating days (C)...... 117
Figure 6.1 Six homologous domains to other multicopper oxidases in MnxG in B. SG-1.
The start and end of each domain are shown above each domain ...... 124
Figure 6.2 DNA gel for PCR product. Conservative sequence in mnxG was amplified for
B. atrophaeus (lane 1) and B.SG-1 (lane 4). Lane 2 is a positive control for the PCR, approximate 850 bp. Lane 3 is the marker...... 127
xix Figure 7.1 The slide with spores deposited. The circled regions correspond to the characteristic deposition bands obtained with this instrument...... 136
Figure 7.2 The suspension of spores before and after magnetic deposition system ...... 136
Figure 7.3 Scatterplots of spores settling velocity vs magnetic velocity in DI water before(A) and after sterilization (B). C showed the scatterplot of the spores in chicken broth after sterilization. Long dash lines stand for three standard deviation from the average velocity; while dotted lines refer to the mean...... 138
Figure 7.4 Scatter plot of magnetic velocity vs settling velocity of spores treated in pH
2.7 (B) overnight and control (A). After that, Mn was added into the same sample (C) 140
Figure 8.1 Schematic picture of the recyclable manganese oxides on spores ...... 149
Figure 9.1 Thermodynamic stability of manganese oxides phases...... 162
xx Chapter 1 Introduction
1.1 Biological Warfare (BW) and detection challenges
Hazardous biological materials have been used as weapons and for homicide intent since prehistoric times (Szinicz, 2005). The first documented attempt to use biological warfare (BW) pathogens traced back to the 14th century when the Mongols catapulted plague-infected victims into a city for which they had laid siege to spread the disease (Szinicz, 2005; Saikaly et al., 2007; Lim et al., 2005). While chemicals were used in World War I as weapons, the development and use of pathogens, such as Yersinia pestis (plague), Bacillus anthracis (anthrax), and Francisella tularensis (tularemia), as
BW agents intensified in World War II (Oyston et al., 2004; Szinicz, 2005). BW agents, along with chemical agents, have been classified as weapons of mass destruction
(Szinicz, 2005). Compared to conventional weapons, relatively small amount of pathogens can cause a large number of casualties and last longer. Gruinard Island at the northwest coast of Scotland remained contaminated for more than 50 years after anthrax spores were field tested by the British, since the spores in soil are very stable and resistant to harsh conditions (Szinicz, 2005).
1 Due to the potential of a strong, negative impact of BW agents to the civilian world, it is desirable to develop rapid, specific and sensitive detection assays to track the delivery and the fate of those bio-threats. Ideally, the instrument or detection platform should be portable, user-friendly and can detect multiple agents simultaneously (Lim et al, 2005). The majority of the common, rapid detection and separation technologies for biological particles are either based on their intrinsic properties (size and/or density), or properties obtained by binding to detection reporter conjugated particles (Melnik et al.,
2007). Examples of such reporters are fluorescent molecules, radioactive atoms, or magnetic micro or nanoparticles. There is a summary in Table 1.1. Of these methods, however, few satisfy all the criteria.
Culture and staining techniques are currently standard for isolation, detection and identification of BW agents. The selective and differential media allow the growth of healthy bacterial cells of target while inhibiting the growth of other organisms. For the food industry, all currently approved rapid assays can be used only to screen for definitive negative results, while positive results have to be confirmed by standard methodologies (Swaminathan and Feng, 1994). These methods involve enrichment and
(or) selection, with subsequent growth, which will take days for the preliminary results.
In general, nucleic acid-based detection systems are more sensitive, but they require clean samples without interference (Lim et al, 2005). In other words, the high sensitivity of
PCR can result in unacceptably high false-positives. Such false-positive results are particularly unacceptably on a battlefield or potential battlefield. Thus, to identify the
2 Method Principle Advantage Disadvantage Comments Reference FTA® filters have been used Easy, fast, for detection of Bacillus Filtration Size difference cheap Nonspecific spores using nested PCR 1; 2 Buoyant Density Gradient Centrifugation can separate Easy, fast, Yersinia enterocolitica in Centrifugation Density difference cheap Nonspecific meat fluids from pork 1; 2 Inhibit other strains’ Selective and growth; presence of Easy, cheap, Differential Plating dye or chemicals enumerative Lengthy Specific bacterial enzymatic activity on In conjunction of high
Enzyme /substrate chromogenic or sensitivity fluorescence 3 method fluorogenic substrates Sensitive detection instruments Specific binding Like Immunomagnetic Antibody (Ab) - affinities of Abs to /Immunofluorescent based specific antigens Sensitive Separation Clean sample; Fast unable to In vitro nucleic High detect protein acid amplification sensitivity toxins Table 1.1 Comparison between common biological detection and separation methods 1.(Lim et al., 2005) 2.(Bernhardt et al., 1991)
3 pathogenic bacteria in a more complex environment, like food matrix, efficient and inexpensive methods must be developed to sequester the target bacteria.
The potential to recover live organisms for culture and further studies is one of the advantages of antibody-based technologies. Even though they are widely used in detection and separation, the detection or separation instruments such as luminometers and spectrophotometers can be expensive and limited in mobility. In contrast, using immunomagnetic labels, and subsequent magnetic separation or detection has become a significant preparative technology in clinical and research applications; the main reason is relatively low cost and simiplicity. Magnetic based separation and detection technology for biological applications has grown in use during the last couple of decades for a number of reasons, including the availability of magnetic micro and nanoparticles and the improvement in the power of permanent (Zborowski and Chalmers, 2005). Since most biological entities are diamagnetic, the biological entities to be detected and or separated must first be labeled with a magnetic tag. The magnetically labeled particles can be captured and released by applying and subsequently removing the magnets. However, there are also some notable exceptions like magnetic bacteria and deoxygentated hemoglobin (Zborowski et al., 2003; Hergt et al., 2005). Immunomagnetic Separation
(IMS) technology not only reduces the cost but also speeds up the processing. The isolated cells are also amenable for further molecular characterization (Faye et al., 2004).
1.2 Detection and separation of Bacillus atrophaeus spores
Since it is not always ethically permissible, economically feasible, or politically palatable to use actual BW agents (O’Connell et al., 2006); research on the corresponding
4 surrogates is indispensible. Nonpathogenic Bacillus species, such as B. atrophaeus, B. subtilis, and B. cereus have been used in place of B. anthracis (Arakawa et al., 2003; O'
Connel et al., 2006). Specifically, Bacillus atrophaeus has been used as a stimulant for B. anthracis to develop methods: to detect and separate B. atrophaeus (Stratis-Cullum et al.,
2003; Turnbough, 2003; Czerwieniec et al., 2005); to develop methods for viability assessment of Bacillus spores using fluorescence dyes (Laflamme et al., 2004); to investigate the effectiveness of decontamination methods against B. atrophaeus spores by surface sampling (Raber and McGuire.R., 2002; Buttner et al., 2004); and to study inactivation of Bacillus spores (Robinson et al., 2010; Talbot et al., 2010). Recent uses and threats of B. anthracis in the United State in 2001 (Saikaly et al., 2007) have lead to increased research on the development of rapid and specific detection technology for BW agents such as Bacillus anthracis spores.
Bacillus atrophaeus, also called Bacillus subtilis var. niger, Bacillus niger, or earlier as Bacillus globigii (bg) is a rod-shaped, Gram-positive, aerobic and endosporeforming bacterium (Bargar et al., 2000). The shape may vary depending on growth conditions, but has typical dimensions of 0.5 to 1.0 μm wide by 2.0 to 4.0 μm long in the vegetative state (Nakamura, 1989). In the endospore state, B. atrophaeus
(ATCC #9372) is approximately 0.7 µm in width and 1.8 µm in length when measured in a dry state using Electronic Microscopic (Plomp et al., 2005). B. atrophaeus is not as well studied as other Bacillus, such as B. subtilis, partially as a result of B. atrophaeus only becoming a new species in 1989 (Nakamura, 1989). In the same paper, Nakamura reported that the DNA relatedness of B. atrophaeus to B. subtilis ranges from 20% to
5 34%, and the G+C content is approximately 41-43%, determined by CsCl buoyant density. Further reclassifications have been conducted by analyzing 16S rRNA (Fritze and Pukall, 2001) and amplified fragment length polymorphism (Burke et al., 2004).
Though it is more closely related to B. subtilis, B. atrophaeus has routinely been used as a B. anthracis surrogate due to its lack of pathogenicity and unique colonial characteristics. Thus, detection and separation of Bacillus atrophaeus has grown in interest (Saenz et al., 1999; Johnson et al., 2000; Lighthart et al., 2004; Czerwieniec et al., 2005; Tobias et al., 2006). As to the detection, the production of pigment on plates containing an organic nitrogen source can be used as an indicator for the presence of B. atrophaeus (Nakamura, 1989). However, there are some other pigmented Bacillus variants, which are believed significant different in DNA-DNA re-association measurements, multilocus enzyme electrophoresis, and pigment production (Nakamura,
1989). Amplified fragment length polymorphism (AFLP) analysis combined with 16S rRNA is a discriminatory genetic fingerprinting technique, which can detect molecular diversity within strains, usually used as a tool for bacterial taxonomy (Burke et al., 2004).
Microcalorimetric spectroscopy improved the sensitivity of the detection compared with conventional infrared and Fourier-transform Infrared Microscopic spectroscopy techniques, but with less detectors (Arakawa et al., 2003). ‘Marker peaks’ were found using Bioaerosol Mass Spectrometry (Srivastava et al., 2005). Q-PCR assays described in
2007 have high specificity and sensitivity and the detective limit for B. atrophaeus is only 7.5pg (Saikaly et al., 2007). Improved fluorescence in situ hybridization (FISH) allows the detection of Bacillus spores within 1 hour (Filion et al., 2009). Even though
6 these methods have improved the detection dramatically, some are complex, expensive or require lots of trainings for operators (like spectroscopy). In this situation, as
Swaminathan and Feng described (Swaminathan and Feng, 1994), IMS is a very promising method to achieve the goal of “efficient and inexpensive”.
1.3 Other Bacillus strains in the study
Even though B. atrophaeus is the primary strain used in my PhD research, there are several other strains used as well. B. subtilis is one of the well-studied Bacillus strains, commonly found in soil (Madigan and Martinko, 2005). B. subtilis produces the proteolytic enzyme subtilisin. B. subtilis can also form spores to survive high heat conditions. It has been shown to have a high amenity to genetic manipulation, and has therefore become widely adopted as a model organism as the Gram-positive equivalent of
Escherichia coli, an extensively studied Gram-negative rod, for laboratory studies
(Harwood, 1990). The common plasmids used in Bacillus, transformation and the process of sporulation are also well studied (Harwood, 1990).
B. subtilis is not regarded as a pathogen. It may contaminate food but rarely causes food poisoning (Ryan and Ray, 2004). However, B. cereus is considered to cause
2-5% foodborne illnesses, including severe nausea, vomiting and diarrhea (Kotiranta,
2000). The problem will get worse when food is improperly refrigerated, allowing the endospores to germinate. B. cereus is a facultative anaerobic and bacterial growth can produce enterotoxin, highly resistant to heat and to pH between 2 and 11.
Bacillus thuringiensis, referred to as Bt, occurs naturally in the environment, like soil, insects and plant surfaces (Schnepf et al., 1998; Gould et al., 2002). Bacillus
7 thuringiensis (Bt) produces insecticidal proteins (d-endotoxins) during the sporulation phase as parasporal crystals. These crystals are predominantly comprised of one or more proteins, called Cry and Cyt toxins (Bravo et al., 2007). Bt. is a microorganism that produces chemicals only toxic to insects, so that Bt. genes such as Cyt are put in some plants, like cotton and tomatoes, to give the plants insect resistance (Bravo et al., 2007;
Soberon et al., 2007). It was also registered in the United States for use as a pesticide
(Mcclintock et al., 1995) There are 3 major applications of Bt. achieved: 1) the control of defoliator pests, 2) the control of mosquitoes that are vectors of human diseases and 3) in the development of transgenic insect resistant plants (Bravo et al., 2007).
Bacillus sp. SG-1 is a marine strain that has been studied for manganese oxidation
(Tebo et al., 2004). Manganese is oxidized much more slowly without microbes
(Hastings and Emerson, 1986). The mature spores suspended in natural seawater can bind and oxidize Mn under dormant conditions. It was suggested that Mn2+ was chelated or compounded by a spore component, perhaps an exosporium or spore coat proteins. Once bounded, the manganese was rapidly oxidized as long as it was bound to spores (Rosson and Nealson, 1982). In many environments, manganese-oxidizing bacteria, both Gram- positive and Gram-negative, are thought to be geochemically important because they initiate or accelerate the deposition of minerals and ores (Marshall, 1979).
1.4 Bacillus spores, Mn and paramagnetism
Bacillus, including but not only including the strains discussed above, can form dormant and highly resistant dormant cell types, called spores, in response to severe conditions like starvation. The spore is composed of a series of concentrically arranged
8 structures, each of which contributes in a different way to resist against environmental stress. Mn plays an important role in the developmental cycle of Bacillus. Mn homeostasis is essential for efficient initiation of sporulation in the vegetative cell.
Moreover, the septum formation, forespore development, spore coat production and germination all require the presence of Mn (Nicholson and Setlow, 1990). Mn is also required for the growth and survival of many organisms, and within bacterial cells Mn metalloenzymes have many diverse functions (Jakubovics and Jenkinson, 2001). The concentrations of Mn2+ in most cells are within micro molar range, but some bacteria like
Lactobacillus plantarum have a much higher intracellular Mn2+ concentration of 0.025M
(Crowley et al., 2000). Some bacteria, like E. coli, L. plantarum and Staphylococcus aureus can take in Mn by transporters (Jakubovics and Jenkinson, 2001).
Manganese has various valence states in the environment and 2+, 3+, 4+ are the most common forms in nature. Since Mn is the Earth’s second most abundant transition metal and Mn oxides can scavenge the toxic metal ions, it is considered geochemically important to the ocean (Tebo et al., 2004). Figure 1.1 demonstrates the Mn cycle and also the thermodynamically favored conditions for each state (Tebo et al., 2004). In low pH solution or in the absence of oxygen, Mn(II) is thermodynamically favored; on the other hand, Mn (III) and Mn(IV) will be preferably formed at high pH and presence of oxygen
(Tebo et al., 2004). The abiotic oxidation of Mn(II) is kinetically inhibited below pH 9 even though thermodynamically favorable.
Microorganisms like bacteria and fungi, which can oxidize Mn from state 2+ to
3+, 4+ with a much faster rate, are widespread in nature (Tebo et al., 2004). There are
9 three model organisms for Mn oxidation: Leptothrix. discophora SS1, Pseudomonas. putida strains MnB1 and GB-1, which represent β-proteobacteria, γ-proteobacteria and low-GC Gram-positive bacteria respectively (Tebo et al., 2004). For instance, L. discophora SS1 can also oxidize Mn at a maximum rate at pH 7.5 and temperature of 30o
C (Nelson and Lion, 2003). All three organisms have genes that are involved in Mn(II) oxidation, sharing sequence similarity with multicopper oxidase enzymes. The bacteria often become encrusted with Mn oxides usually at stationary phase or after sporulation.
Figure 1.1 The Mn cycle of oxidation states found in nature. Mn(II) is thermodynamically stable in the absence of O2 and at low pH, whereas in the presence of O2, Mn(III) and Mn(IV), which occur primarily as insoluble Mn (oxyhydr)oxides, are favored (Tebo et al., 2004).
10 Molar magnetic susceptibility
6 3 + Compound m x 10 (cm /mol)
Mn 529
β Mn 293
MnO (II) 4850
MnO2 (IV) 2280
Mn2O3 (III) 14100
Mn3O4 (III, IV) 12400
MnCl2 (II) 14,350
MnSO4-4H2O 14,600
H2O -13.0
Table 1.2 Magnetic susceptibility of Mn and Mn compounds
+ using CGS units
For different chemical states of Mn, the magnetic susceptibility of each varies
(Table 1.2). The Mn(III) oxide is six times more magnetic than Mn(IV) oxide. Mn oxides were also reported to be produced by Bacillus spores (Tebo et al., 2004). More interestingly, paramagnetism of Bacillus spores was found (Melnik et al, 2007), and further studies indicate the paramagnetism could be related to the oxidation of Mn bound on the surface (Chapter 4). Since the oxidation of Mn is strongly dependent on the
11 environmental or culturing condition, it is very helpful that we understand how the magnetism of Bacillus spores correlate oxidation state. Furthermore, that multiple strains can oxidize manganese (Francis and Tebo, 2002) makes it highly possible to obtain more strains paramagnetic with optimizing the oxidation process.
1.5 Immunomagnetic Separation (IMS)
1.5.1 Introduction of IMS
As described in section 1.1 and 1.2, IMS is considered very promising in bacteria detection. Normally most particles like cells are slightly diamagnetic with respect to biological buffer (mainly water), so magnetic label is required to separate the diamagnetic particles. Traditionally, the sample was mixed with corresponding antibody- coated paramagnetic beads in a centrifuge tube. After the antibodies bound the sample, a magnet was applied to the side of the tube to hold the magnetic beads while the rest was removed from the tube (Nou et al., 2006). The bacteria were resuspended after washing several times and a small volume was plated onto selective or differential agar media for the detection of the bacteria of interest (Nou et al., 2006) (Figure 1.2).
Though this procedure is not technically complicated, traditional IMS is labor- intensive, and not amenable to high throughput. Poor sensitivity is another issue for the target organism, when the background microorganisms are not effectively washed away after separation. Many modifications of the standard IMS procedure have been made, including modifications in apparatus, bead sizes, and washing procedures (Nou et al.,
2006). i.e. BioNobile (Turku, Finland) has developed PickPen, which has primarily been
12 used for rapidly transferring DNA, RNA, and protein molecules linked to magnetic particles.
FigureFigure1.2 The 1.2 principle. The principle of immunomagnetic of immunomagnetic separseparationation of of target target microorganisms microorganisms (Safarik(Safarik and Safarikova, 1999) and Safarikova, 1999)
Techniques Mode Throughput Purity Recovery (cells/s) (%) (%) Mini MACS® Batch 103 - 104 >70 >40 Dynabeads, Batch 106 >70 60 MPC® Quadrupole, Flow 103 99 60-86 Immunomagnetic separation Dipole, Flow 102 – 103 98 37 Immunomagneitc Separator Table 1.3 Summary of IMS technology (Chalmers et al., 1998)
13 The immunomagnetic cell separation systems can be either continuous or batch
(Table 1.3), positive selection or negative selection. For positive selection, cells targeted are the cells of interest; while for the negative selection, cells targeted will be discarded.
Some researchers prefer negative selection as cells obtained are not modified or attached to labels that may influence future study.
1.5.2 Commercial Immunomagnetic Separation Technologies
Commercially available magnetic particles to conjugate target cells have a wide size range, from nanometers to micrometers. The magnetic strength is, in general, proportional to the particle size in a magnetic field and they have no residue magnetic charge left when the magnetic field is removed. BD™ Imag streptavidin beads (BD
Pharmingen, CA, USA) and other three particles studied (Zhang et al., 2005) were reported to be in a range of 50 to 200 nm in diameter. Since the magnetic force generated on magnetic particle is directly proportional to the product of the volume of magnetic material and the material’s magnetic susceptibility relative to suspending fluid, micro- magnetic particles create orders of magnitude more force than nanoparticles, assuming they have similar material composition. Due to the low magnetic moment the smaller magnetic particles can produce, high gradient magnetic fields are required for separation.
Though not discussed, more immunomagnetic separation technologies have been commercialized (i.e. MPC separator series, Dynal, Trondheim, Norway; EasySep®, Stem
Cell Techonlogies, Canada; Immunicon, Huntington Valley, PA).
14 1.5.3 Magnetic deposition system
The immunomagnetic separation apparatus, magnetic deposition system, was developed in Cleveland Clinic Foundation (CCF) and our lab for positive selection of particles (Figure 1.3). Magnetically labeled particles deposited on the slide can be directly examined under microscope using this instrument. The high gradient magnetic field is generated by a magnetic interpolar gap of a permanent magnet (Figure 1.4). The magnitude of the magnetic energy gradient at the deposition surface, in the middle of the interpolar gap was reported to be 1,870 T-A/mm2 (Melnik et al., 2007). The flow chamber is composed of a top and a bottom cover, and a silicon rubber spacer, which is
Figure 1.3 Schematic diagram of the magnetic deposition system (Yang, 2008)
15 tightly sandwiched between the top and bottom covers (Yang, 2008). The flow channel cross-section is 6.4 mm × 0.25 mm. Inlet tubing is connected through top plate and outlet flow tubing is directed to centrifuge tube for collection. A 150 μm thick microscopic glass coverslip is placed behind rubber spacer for deposition. The deposition forms a well-defined band visible by an unaided eye, defined by the breadth of the flow channel and the width of the interpolar gap.
Magnetic cell separation depends highly on the difference of magnetism (either labeling or intrinsic) between the cells of interest and cells that requires depletion (or even suspension fluid). The magnetic force on the magnetic cells has to be high enough to hold the cells or particles, against the flow. Moreover, for labeling, nonspecific binding is a big challenge and for intrinsic magnetism, reliable and strong magnetism of spores should be obtained for further studies and related applications like separation from food.
IMS will be only discussed a little in Chapter 2, all the rest study of the dissertation focuses on the next topic: intrinsic magnetism.
1.6 Intrinsic magnetism
Before the discovery of paramagnetism of Bacillus spores, only erythrocytes, which can be either diamagnetic or paramagnetic, depending on the oxygenation state of their hemoglobin (Zborowski et al., 2003) and magnetotactic bacteria, which contain small magnetic particles within the cell (Blakemore, 1975) are reported for their intrinsic magnetism.
The unpaired electrons in the four heme groups of deoxy- and met-hemoglobin
(metHb) make the erythrocytes paramagnetic compared to oxyhemoglobin (Zborowski et
16 al., 2003). The magnetophoretic mobility of deoxygenated erythrocytes is slightly positive. The magnetotactic bacteria contain either magnetite (Fe3O4) or greigite (Fe3S4) which is responsible for the magnetic properties (Bazylinski et al., 1995). The formation of magnetosome requires strict pH and reduction potential, and involves a very complex process; it is predominately found in α-proteobacteria (Schuler, 1999).
In contrast, Melnik et al (2007) reported non-labeled Bacillus atrophaeus spores are magnetic; the magnitude of this magnetic susceptibility is comparable to that of lymphocytes labeled with commercial magnetic reagents. Figure 1.5 shows the comparison of the magnetism between oxy- (left) and deoxy- erythrocyte (right); and the comparison of the paramagnetism between Bacillus spores and other magnetic organisms. Clearly, Bacillus atrophaeus spores are much more magnetic than other species.
Energy dispersive X-ray spectroscopy, EDS, (Melnik et al, 2007) indicated that
Mn is the only paramagnetic element in the Bacillus atrophaeus spores. The fact that paramagnetic Mn oxides were found bound on Bacillus spore surface indicates that the oxidation of Mn can lead to intrinsic magnetism. Not only were the strains listed below in table 1.3 found that can oxidize Mn, three more strains have been used as model bacteria for Mn oxidation. Since many organisms can oxidize Mn, further understanding about the culturing condition, surface chemistry can be applied to more strains.
17 Figure 1.4 The magnetic field around the interpoler gap (Zborowski et al., 1995)
18 oxy RBC
0.05 0.25 oxy RBC myeloma SP2/0 deoxy RBC 0.04 0.20 B. globigii spores carcinoma MCF7 0.03 0.15
0.02 0.10
frequency frequency
frequency frequency
0.01 0.05
0.00 0.00 -4 -2 0 2 4 0.0001 0.001 0.01 0.1 1 10 m x106 (mm3/T.A.s) m x 103 (mm3/T.A.s)
Figure 1.5 Magnetophoretic mobility of spores and other organisms
1.7 Magnetic susceptibility and magnetophoretic mobility
1.7.1 Magnetic susceptibility
Magnetic susceptibility is a material property used to characterize the degree of magnetization of a material in response to an applied magnetic field (Zborowski, 2008).
Experimentally, it is measured by the strength of force on the target substance by a well defined magnetic field. Some substances having weak response to the magnetic field are ‐ considered “nonmagnetic”. Even though the nonmagnetic materials can become strongly responsive to high magnetic field, it will not be discussed here. This study is limited to static or quasistatic magnetic field effects.
19 Organism Strain Origin of Mn oxidizing isolates Mn (II) oxidation Bacillus sp. SG-1 Scripps Pier sediments, La Jolla, CA + MB-1 Mission Bay sediments, San Diego, CA + MB-3 Mission Bay sediments, San Diego, CA + MB-5 Mission Bay sediments, San Diego, CA + MB-7 Mission Bay sediments, San Diego, CA + MB-9 Mission Bay sediments, San Diego, CA + MB-12 Mission Bay sediments, San Diego, CA + PL-7 Point Loma sediments, San Diego, CA + PL12 Point Loma sediments, San Diego, CA + PL-16 Point Loma sediments, San Diego, CA + PL-21 Point Loma sediments, San Diego, CA + PL-26 Point Loma sediments, San Diego, CA + PL-30 Point Loma sediments, San Diego, CA + SD-18 San Diego Bay sediments, CA + B. cereus ATCC 10876 ATCC - Table 1.4 Mn(II)-oxidizing and non-Mn(II)-oxidizing Bacillus strains
The simplest experiment to measure magnetic susceptibility is via magnetic balance (Figure 1.6). One arm of the balance holds a sample exposed to an inhomogeneous magnetic field at a point where the product of the magnetic field and the field gradient reaches maximum. The other arm of the balance (not within the magnetic field) holds a weight that keeps the balance at equilibrium. For paramagnetic and ferromagnetic substances, the weight balances the magnetic force, acting on the sample
(Figure 1.6, left). For diamagnetic substances, the sample is pushed out from the magnet and therefore the weight (magnetically neutral) has to be added to the sample to keep the balance (Figure 1.6, right). The diamagnetic effects are much weaker than the para- and ferromagnetic ones (Zborowski, 2008). Paramagnetic substances have magnetic susceptibly χ > 0, while diamagnetic substances have susceptibility χ < 0.
20 Figure 1.6 Magnetic balance schematic (Zborowski, 2008)
There are some other methods used to determine magnetic susceptibility, including Evans Balance, superconducting quantum interference devices, SQUID, or
Nuclear magnetic resonance (NMR) for liquid samples. These methods are limited to liquid samples or bulk average magnetic susceptibility measurements. To quantify how magnetic cells are after immunomagnetic labeling or how magnetic the cells or spores can be after fermentation or treatment, further parameters will be discussed below.
1.7.2 Magnetophoretic Mobility (MM)
The magnetic force exerted on a volume V of substance in a diamagnetic fluid medium is expressed using the volume magnetic susceptibility by Eq. (1.1):
dB F VH 0 (1.1) dx
21 where H and B0 are magnitudes of the applied magnetic field vectors, H is defined as a modification of B due to magnetic fields produced by material media, V is the volume of the particle, and ∆χ is the difference of magnetic susceptibility between the solution and the particles. The drag force, counteracting the magnetic force, is represented by:
F F d (1.2)
In the limit of a creeping flow, the Stokes formula applies
F 6Rv d (1.3) where R is the radius of the particle, η is the viscosity of the medium and v is the moving speed of the particle. Combined the equations above, the induced magnetic velocity can be expressed :
2 V dB0 V B0 v H 6Rv dx 6Rv 20 (1.4) 2 V B0 It is noted that the two parts: and are independent of each other. The 6Rv 20 term magnetophoresis is defined as follows, and has been proposed in an analogous manner to “electrophoresis”.
V m 6R (1.5)
Magnetophoresis is demonstrated to be a sensitive, small sample volume, low cost, live- cell based and quantitative measure of the magnetic susceptibility of cells and particles.
The magnetic energy gradient can be further defined as:
B2 0 Sm 20
22 Combining the definition of m and Sm gives
v mS m v Rearrangement of the equation above gives: m Sm
Experimental measurements of a magnetically induced velocity of a cell, or particle, in known value of Sm, allow the determination of magnetophoretic mobility.
Magnetophoretic mobility, which has analogies to electrophoretic mobility, is a measure of the transport of the magnetic entity as a result of an imposed magnetic field in a viscous medium. The Chalmers and Zborowski laboratory have been using magnetophoretic mobility as a measure of the effectiveness of antibody-magnetic nanoparticle conjugated to label cells of interest as well as to design, optimize and operate magnetic cell separation technology for over 10 years (Zborowski et al., 2002). It is very important to point out that magnetophoretic mobility is independent of the magnetic field for paramagnetic and diamagnetic particles, which is actually the case for the spores. In other words, the magnetophoretic mobility depends only on the spore itself.
The magnetophoretic mobility of particles can be experimentally measured by
Cell Tracking Velocimetry (CTV) developed in our lab on a particle by particle basis
(Chalmers et al., 1999; McCloskey et al., 2001; Nakamura et al., 2001). This instrument includes microscope, CCD camera, light system, computer, and a cell-flow-through channel between two magnets. A well designed, and a constant magnetic gradient of 142
T∙A/mm2 was developed in the microscopic field of view. The motion of the cells is recorded in a computer and the images are analyzed using associated software. CTV will be used and demonstrated as a convenient and accurate tool of the characterization of the
23 Bacillus spores for both chemical analysis and application in food science and decontamination. More details will be discussed in Chapter 3.
1.8 Organization of the dissertation
The outline of the dissertation from Chapter 2 to Chapter 7 is as follows:
In Chapter 2, three strains of Bacillus: Bacillus atrophaeus (formally Bacillus globigii), Bacillus thuringiensis and Bacillus cereus were tested for their intrinsic magnetic susceptibility. Of those strains, B. atrophaeus was demonstrated to have significant magnetic susceptibility using an instrument referred to as Cell Tracking
Velocimetry (CTV). Energy dispersive spectroscopy confirmed the presence of paramagnetic elements, Mn, in the B. atrophaeus spores. It was demonstrated that this magnetic susceptibility is sufficient to separate and deposit these spores on glass slides in a magnetic deposition system, which indicates the potential to separate spores directly out of water or air samples without label.
Given the desire to increase the accuracy of the analysis of the magnetophoretic mobility of Bacillus spores, internal control experiments were conducting in Chapter 3, following the approach taken by Chalmers et al (Chalmers et al., 2010). Specifically, polystyrene microsphere, PSM, of know diameter are mixed with the sample. The analysis of spores and PSM separately, as well as in a mixture provides a new way to increase the accuracy of the MM measurements. The data from Electronic Cell Tracking
Velocimetry showed consistent results.
In Chapter 4, through CTV, both magnetically induced velocity, referred to as magnetophoretic mobility, and settling velocity of Bacillus atrophaeus spores was
24 quantitatively measured. The calculations indicate that the spores are present in the form of clusters of approximately 3 to 6 microns. Treatment of these clusters with EDTA or pH of 2.0 or below resulted in not only the disruption of the spore clusters, but also a significant decrease in magnetic susceptibility dramatically. X-Ray Photoelectron
Spectroscopy (XPS) was used to determine the valances states of Mn in the spores. The total mass of Mn associated with each spore cluster was determined by ICP- MS, with a value of 3.28 x 10-11 grams of Mn per spore cluster, while the mathematical estimation from mobility and XPS analysis retuned a value of 1.16 x 10-11 g, which is a reasonable agreement.
In Chapter 5, factors that affect the magnetic susceptibility of the spores during the growth and sporulation were explored. To optimize the intrinsic magnetism, physical conditions were manipulated to control the magnetism of Bacillus spores. The concentration of Mn in the culture seemed to be the most important factor while the sporulation time and oxygen availability did not affect the magnetism significantly. Mn oxidation state is considered important to the intrinsic magnetism also. This hypothesis is supported by the report that some Bacillus species can oxidize Mn(II) to Mn3O4 and
MnO2 enzymatically. Different concentrations of ions and heat shock effect have been examined as well, with no significant effect on the magnetism.
Genetic studies will be discussed in Chapter 6.
Further application of deposition system was studied and discussed in Chapter 7.
Besides separation of spores from water, it can also be applied to separate spores from liquid food, like milk or chicken broth before or after sterilization. A large portion of
25 spores were killed in the autoclave process, but the remaining magnetism of the survived spores can be detected by CTV and deposited on the deposition system. Low pH/ High concentration of heavy metal ions decrease the magnetic properties, and low pH killed the spores at the same time. However, addition of Mn after treatment can recover most of magnetism of the spores and the spores with recovered magnetism can be separated from deposition system again. These studies indicate that the deposition system can be used in food industry, environmental pollution control as well as biological warfare agent detection.
Chapter 8 will discuss the idea of the recycling of the spores from polluted water by heavy metal ions, i.e. lead. After treatment of water with high level of Pb, the spores lost their magnetism. However, the acid treatment first and using base to neutralize the acid afterwards assists in the recovery of magnetism of the spores. Then the spores could be deposited on the slides using magnetic deposition system and be used again to adsorb heavy metal ions from contaminated water.
Chapter 9 summarizes the conclusions and presents future work suggestions.
26 Chapter 2 Quantification of magnetic susceptibility in several strains of
Bacillus spores
This chapter is from the paper “Quantification of Magnetic Susceptibility in
Several Strains of Bacillus Spores: Implications for Separation and Detection” in
Biotechnology and Bioengineering, 2007, Vol. 98, 186-192. List of authors is as follows:
Kristie Melnik, Jianxin Sun, Aaron Fleischman, Shuvo Roy, Maciej Zborowski, and
Jeffrey J. Chalmers. I use it as one of my chapters because I contributed all the EDS spectroscopy figures, almost all the magnetophoretic measurements for three strains from
CTV in the table.
Abstract: Three strains of Bacillus: Bacillus atrophaeus (formally Bacillus globigii),
Bacillus thuringiensis, and Bacillus cereus were tested for their intrinsic magnetic susceptibility. All three strains when sporulated demonstrated significant magnetic susceptibility using an instrument referred to as Cell Tracking Velocimetry. Energy
Dispersive Spectroscopy also confirmed the presence of paramagnetic elements, Fe and
Mn, in the spore form of the bacteria. It was demonstrated that this magnetic susceptibility is sufficient to separate and deposit these spores on glass slides in a
27 magnetic deposition system. These results indicate the potential to separate spores with intrinsic magnetic susceptibility directly out of water or air samples.
2.1 Introduction
The ability to detect and/or separate a biological entity (i.e.,cell, spore, protein, etc) is essential in modern laboratory processes in biology. A majority of the common, rapid detection and separation technologies are either based on an intrinsic property of the biological entity (size and/or density) or properties imparted to the entity through the binding of a high affinity molecule conjugated to a detection reporter. Examples of such reporters are fluorescent molecules, radioactive atoms, or magnetic micro- or nano- particles. Recent examples of the use of biological warfare (BW) agents have lead to increased research on the development of rapid and specific detection technology for BW agents such as Bacillus anthracis spores. Magnetic based separation and detection technology for biological applications has grown in use during the last couple of decades for a number of reasons, including the availability of magnetic micro and nanoparticles and the improvement in the power of permanent magnets (Zborowski and Chalmers,
2005). Since most biological entities are diamagnetic, the biological entities to be detected and/or separated must first be labeled with a magnetic tag. A few notable exceptions are magnetic bacteria and deoxygenated hemoglobin (Zborowski et al., 2003;
Hergt et al., 2005).
A measure of either the intrinsic or imparted magnetic susceptibility on a biological entity, such as a cell or spore, is the magnetophoretic mobility, m, of that entity. Magnetophoretic mobility, which has analogies to electrophoretic mobility, is a
28 measure of the transport of the magnetic entity as a result of an imposed magnetic field in a viscous medium. We have and are using magnetophoretic mobility as a measure of the effectiveness of antibody-magnetic nanoparticle conjugated to label cells of interest as well as to design, optimize and operate magnetic cell separation technology (Zborowski et al., 2002). To quantify the magnetophoretic mobility of cells and micro-particles, an instrument was developed, and is continuing to be improved, which is referred to as Cell
Tracking Velocity, CTV, which measures on a cell-by-cell basis, the magnetophoretic mobility of a cell (or particle) (Zhang et al., 2005). Through the use of a well- characterized magnetic energy gradient, Sm, a microscope, CCD camera, and a sophisticated computer algorithm, the magnetically induced velocity of cells or particles is determined. The CTV system is sufficiently automated so that several hundred cells or particles can be tracked at one time which gives rise to the potential to analyze a population greater than 1,000 in less than 15 min. Once the velocity is determined, the magnetophoretic mobility of the entity of interest is determined by dividing the measured velocity by Sm since it is nearly constant in the microscopic region of interest
(approximately 149 T-A/mm2) (Chalmers et al., 1999).
Mathematically, the magnetophoretic mobility can take a number of forms, depending on the characteristics of the cell (particle) being studied (Zborowski et al.,
2002). For the current application in this report, the mobility, m, is given by:
v v V m c c c (2.1) 2 Sm B 3Dc 2 0
29 where vc is the magnetically induced velocity, B is the magnetic field induction, (in
Tesla, T), µ=4π×10-7 T-M/A, is the magnetic permeability of free space, ∆χ is the difference in the magnetic susceptibility of the moving cell (entity) and the suspending fluid, Vc is the volume of the cell (spore), Dc is the diameter of the cell, and η is the viscosity of the suspending fluid. Equation (2.1) assumes creeping flow and the magnetic susceptibility of the solution and particles are constant:
χ=constant (2.2)
In the case that the paramagnetic material becomes saturated in the magnetic field,
Equation (2.1) becomes:
vc 0 M s V m f (2.3) S m B 3Dc
where Ms is the saturation magnetization of the material, and B is the magnetic field.
Bacillus globigii (Bg) is routinely used as a BW surrogate for the infectious organism Bacillus anthracis (Stratis-Cullum et al., 2003; Hindson et al., 2005). The initial purpose of this study was to quantify the magnetophoretic mobility of immunomagnetically labeled Bg spores for the potential use of antibody/immunomagnetic tagging in a BW detection system; however, it was observed that in the presence of a magnetic energy gradient no immunomagnetic label was required to impart mobility. Our findings of the significant level of magnetic susceptibility of Bg spores and other Bacillus spore formers is reported below.
30 2.2 Materials and methods
Bacillus globigii (also named Bacillus atrophaeus) was twice obtained from the
ATCC (#9372) as well as the Bacillus Genetic Stock Center in the Department of
Microbiology at the Ohio State University. Bacillus cereus (6A1), Bacillus thuringiensis
(4D1) and Bacillus subtilis (W23) were also obtained from the Bacillus Genetic Stock
Center at The Ohio State University. An inoculating loopful was taken from reconstituted culture in LB broth, and streaked onto trypticase soy agar (TSA) in a three phase streak method. The inoculated TSA plate was incubated at 30oC for approximately 20 h. In the case of Bg, cultures were incubated at 30oC for 20 h or after detection of orange pigment was quite obvious. An isolated colony was taken from each TSA plate and used to inoculate 20 ml of either nutrient broth or LB broth which was subsequently placed on a shaker at 30oC for 16 h at 150 RPMs. A 5 ml aliquot was taken from the NB or LB and was used to inoculate 50, 200, or 250 ml of either modified G (mod G) or SG sporulation media (See Table 2.1 for composition) (Hornstra et al., 2006). The inoculated mod G media was placed on a shaker at 30oC for 48–72 h at 200 RPMs. After the 48–72 h period, cells were heat shocked at 60oC for 1 h and shaken at the 30 min halfway mark.
Serial dilutions prior to and post heat shock were conducted and suspensions were plated on TSA to determine the percent of sporulation. After heat shock treatment, spores were washed twice with dH2O and resuspended in dH2O for CTV analysis which has been described previously.
The magnetophoretic mobility of the spores was measured using our in-house instrument, Cell Tracking Velocimetry, CTV (Chalmers et al., 1999; Moore et al., 2000;
31 Zhang et al., 2005). Figure 2.1A presents an overview of the CTV system, while Figure
2.1B presents an enlarged view of the analysis region where the value of B ranges from
1.5 to 1.875 T, and the gradient, dB/dy, decreases correspondingly to the increase in B such that the product, dB2/dy, varies from 0.36 to 0.37 and back down to 0.36 T2/mm
2 (between -7 and -9 mm, Figure 2.1B). When this dB /dy value is divided by 2µ0, a nearly
2 constant, average value of Sm equals 149 T-A/mm is obtained. The Energy Dispersive
Spectroscopy (EDS) analysis was performed on a FEI Quanta 200 scanning electron microscope and the actual EDS analysis was conducted using Edax Genesis software. A completely separate device from the CTV system, the magnetic deposition instrument was created by a fringing field at the interpolar gap, and when combined with a thin flow channel pressed against the interpolar gap, a very high magnetic force operating approximately perpendicular to the flow of cells (spore) allows capture from the suspension. (Figs. 2.2 and 2.3A) (Zimmerman et al., 2006). Typically, cell (spore) suspensions (500µl each) are delivered in a continuous manner into flow channel (s) by syringes connected to inlet tubing, and evacuated from flow channels by outlet tubing leading to waste containers. The flow channel cross-section was 6.4 mm × 0.25 mm, the volumetric flow rate was 0.7 ml/h, the resulting average linear velocity of fluid across the interpolar gap region was 1.2 mm/s, and the average fluid volume element residence time in the interpolar gap region (taken as twice the interpolar gap width, or 2.54 mm) was approximately 2 s.
32 Mod G SG Compound Mass Concentration Compound Mass Concentration (M) (g) (M) -2 -2 (NH4)2SO4 2.0 g 1.5 x 10 KCl 0.5 1.4 x 10 -4 -3 CaCl2·2H2O 0.025g 1.7 x 10 MgSO4·7H2O 0.5 2.6 x 10 -5 -3 CuSO4·5H2O 5.0 mg 2.0 x 10 Ca(NO3)2 1 x 10 -6 -4 FeSO4·7H2O, 0.5 mg 1.8 x 10 MnCl2·H2O 1 x 10 -3 -6 MgSO4·7H2O 0.2 g 8.1 x 10 FeSO4 1 x 10 -4 MnSO4·4H2O 0.05 g 2.2 x 10 Glucose 1 -- -5 ZnSO4·7H2O 5.0 mg 1.7 x 10 Difco Nutrient 16 -- Broth -3 K2HPO4 0.5 g 2.9 x 10 Yeast Extract 2.0 g
Table 2.1 Media Recipes of Sporulation of Bacillus spores
The magnitude of the magnetic energy gradient at the deposition surface, in the middle of the interpolar gap was 1,870 T-A/mm2. Subsequently, the flow channel is disassembled and the plastic sheet with the magnetic cell deposition is evaluated for the presence of cells. The deposition forms a well-defined band visible by an unaided eye, defined by the breadth of the flow channel and the width of the interpolar gap.
33 2.1A
2.1B
FigureFigure 22.1.1 AA diagramdiagram ofof CellCell TrackingTrackingVelocimetry Velocimetry(A). (A). A Aan anenlargement enlargement in thein microscopicanalysis regtheion microscopic (B). analysis region (B)
34 gasket with channel cutout
cell N sample flow path
S
cell deposition flow surface channel lumen Figure 2.2 Schematic diagram of the open gradient, magnetic deposition system
35 A HB = constant flow channel lumen N
S
cell deposition surface
B
Deposited
Spores
Figure 2.3 An enlargement of the thin channel near the open gradient, deposition region (A) and a photograph of the deposited, magnetic spores (B) The circled regions correspond to the characteristic deposition bands obtained with this instrument.
36 2.3 Results
As mentioned previously, the initial intent of this study was to determine the magnetophoretic mobility of immunomagnetically labeled Bg spores; however, while the antibody labeled spores demonstrated a significant magnetophoretic mobility (data not shown), the unlabeled controls had an equally high magnetophoretic mobility.
0.14
0.12
0.10
0.08
0.06
0.04 Fraction of population(-) of Fraction 0.02
0.00 1e-6 1e-5 1e-4 1e-3 1e-2
Magnetophoretic mobility (mm3/A.T.s)
0.01 0.1 1 velocity (mm/s) Figure 2.4 A histogram of the magnetophoretic mobility of Bacillus globigii spores. A second x-axis provides the actual velocity of the spores in the CTV instrument.
37 Figure 2.4 is a histogram generated by CTV measurements of the magnetophoretic mobility of unlabeled B. globigii spores (n=1,810). In addition to the magnetophoretic mobility, a second x-axis is added to indicate the actual velocity that was measured. For comparison Figure 2.5 is a histogram of immunomagnetically labeled human T-cells (anti-CD3-MACS) from Zhang et al. (Zhang et al., 2006). In Figure 2.5 the unlabeled cells are slightly negative (move in the opposite direction to the magnetic field gradient) which is typical for almost all cells due to the diamagnetic nature of most of the constituents of cells (Zborowski et al., 2003). Figure 2.4 clearly demonstrates that in the absence of an external immunomagnetic label, the Bg spores have a magnetophoretic mobility an order of magnitude greater than that of the immunomagnetically labeled cells as depicted in Figure 2.5. The high level of magnetic susceptibility demonstrated by the unlabeled Bg spores lead to the use of scanning electron microscopic analysis with an Energy Dispersive Spectroscopy (EDS) for elemental analysis. Figures 2.6A is an EDS analysis of Bg spores, cultured in NB media, and sporulated in mod G media. Of the major elements recorded, Manganese (Mn) is the only element present that has significant paramagnetic properties. As a point of vegetative form of Bg and several other strains, no significant or positive magnetophoretic mobility was detected with any of the bacterial suspensions. Table 2.2 lists the magnetic susceptibility (in a gram formula weight basis) of various elemental and oxidation states of Mn.
To date, three additional strains of Bacillus have been tested for intrinsic magnetic susceptibility: B. cereus, B. subtilis, and B. thuringiensis. Table 2.3 lists the number of
38 distinct times that a strain of Bacillus was sporulated, the average magnetophoretic mobility, and the highest and lowest mean value of mobility measured. Figures 2.7A
60 CD3 CD3 Positive Negative (80.3%) 50
40
30
Frequency(%) 20
10
0 0 2e-4 4e-4 6e-4 Magnetophoretic Mobility (mm3/T-A-s)
Figure 2.5 A histogram, on a linear scale, of immunomagnetically labeled (anti-CD3- MACS) human lymphocytes from Zhang et al.
39 Compound Susceptibility Compound Susceptibility (GFW) (GFW) Mn 529 MnO2 2,210 β Mn 483 Mn3O4 12,400 MnO 4,850 Fe2O3 3,586 Mn2O3 14,100 Table 2.2 Media recipes for the sporulation of Bacillus species
A)
Continued
Figure 2.6 Spectra of the Energy Dispersive Spectroscopy analysis of the Bacillus globigii spores (A), and the vegetative cells (B). Note the lack of Mn peaks in the vegetative cells
40 Figure 2.6: Continued
B)
41 A)
B)
Continued
Figure 2.7 EDS spectra of B. globiggi spores sporulated inC) SG media (A), B. cereus (B), and B. thuringiensis (C).
42 Figure 2.7: Continued
C)
43 Strain Number of Sporlation Number of Average, High mean/low mean Representative times media CTV of Mobilities EDS sporulated measurements the mean spectra a mobilitiesa B. globigii 7 Mod G 22 4.4 × 10-4 2.8 × 10-3/-1.9 × 10-5 Figure 2.6A 3 SG 6 7.26 ×10-5 2.0 × 10-4/ 3.4 × 10-5 Figure 2.7A B. subtilis Mod G 4 1.27 ×10-4 1.01 × 10-4/-3.4 × 10-5 SG 2 4.0 × 10-5 B. cereus 1 SG 2 5.5 × 10-5 7.7 × 10-5/-2.5 × 10-5 1 Mod G 1 2.47 ×10-5 Figure 2.7B B. 1 Mod G 4 0.99 x 10-5 Figure 2.7C thuringiensis
44 Table 2.3 Sporulated Bacillus strains and magnetophoretic mobility measurements
a The reported, average of the mean magnetophoretic mobility was calculated from multiple CTV measurements of the mean mobility of a specific experiment. The average number of events to calculate the mean mobility was 900. The unit is mm3/T•A•s
44 through C are the EDS spectra of the corresponding spores listed in Table 2.3. All strains show a Mn peak after sporulation, as well as the mean magnetophoretic mobility which is higher than that of vegetative non-immunomagnetically labeled cells.
To demonstrate the potential to use this high level of intrinsic magnetic susceptibility to separate spores, a set of experiments were conducted with the Bg spores using a magnetic deposition system. This system is based on an open-gradient magnetic field separator and a thin-film magnetophoresis process developed for cell analysis
(Zborowski et al., 1991; Fang et al., 1999). Figure 2.3B presents a photograph of the slide with the characteristic bands of deposition from the experiment using the unlabeled
Bg spores. The two, circled, parallel lines are deposited Bg spores.
2.4 Discussion
To the best of our knowledge, no one has ever reported that Bacillus spores can have a significant, intrinsic magnetic susceptibility and the most probable element responsible for this intrinsic magnetic susceptibility is Mn. Like elemental and oxidized iron, manganese has significant paramagnetic properties. It is also well established that
Mn is an important constituent in many microorganisms and specifically in Bacillus subtilis strain SG-1 (Tebo et al., 1997). While vegetative strains of Bacillus are not able to oxidize and reduce Mn oxides (Ghiorse and Ehrlich, 1974), a significant amount of evidence exists, and documented, for the active oxidation of Mn on the surface of free spores (Rosson and Nealson, 1982; Devrind et al., 1986; Hastings and Emerson, 1986). It has even been demonstrated that spores rendered non-germinating in the laboratory continue to oxidize Mn(II) (Rosson and Nealson, 1982). More recently, a significant
45 number of studies have focused on the actual mechanisms of the oxidation and the proteins associated with the process (Glasfeld et al., 2003; Golynskiy et al., 2005; Webb et al., 2005).
The EDS analysis demonstrating the lack of Mn in the vegetative cells, a lack of magnetophoretic mobility in the vegetative cells; the presence of Mn in all of the spores tested, and the significant magnetophoretic mobility in a majority of the spores is consistent with the conclusion that Mn is responsible for this intrinsic magnetic susceptibility. It is significant to note that in Bacillus sp. strain SG-1, van Waasbergen
(van Waasbergen et al., 1993; van Waasbergen et al., 1996) reports that the transcription of one of the known Mn oxidation operons is induced during mid-sporulation. It is also likely that the significant variability observed in the magnetophoretic mobility between experiments as well as between different strains is, at least partially, the result of the Mn being in various stages of oxidation. Note the significant range (a factor of 29) in magnetic susceptibility in the various oxidation forms of Mn listed in Table 2. Finally, it should be noted that while the same procedure was used to sporulate the various strains, in some cases different ratios of media to vessel size were used, as was shaker speed. The variability could contribute to different degrees of oxidation of the spores. Current work is focused on the use of a variety of methods to further determine and quantify both the amount of Mn present, the oxidation state of the Mn, and the factors associated with its accumulation in the spores.
The initial separation of the spores in the magnetic deposition system was a proof of concept test to demonstrate that the spores could be easily separated using their
46 intrinsic magnetic susceptibility (Fig. 2.3B). While this separation took place with the spores suspended in an aqueous media, the magnetophoretic mobility, mathematically represented by Equation 2.1, is equally applicable to spores suspended in air. Further,
Equation 2.1 indicates that the magnetophoretic mobility is inversely proportional to the viscosity of the suspending fluid. Since water is approximately 1,000 times more viscous than air, an airborne suspension of spores could theoretically be separated in a similar system in which the magnetic energy gradient, Sm, was 1,000 times smaller. Future work will investigate these, as well as other questions.
47 Chapter 3 Improvement of Cell Tracking Velocimetry (CTV) accuracy
by the inclusion of internal control in spore samples
3.1. Motivation
As discussed in the Chapter 1, magnetic separation has been developed rapid over last couple decades. Liying Yang (Yang, 2008), in her dissertation, summarized a number of commonly used, commercial magnetic cell separation systems, such as MACS® family system from Miltenyi Biotec; Magnetic Particle Concentrator (MPC®) from
Dynal (Invitrogen); CaptivateTM system from Molecular Probes Inc and Immunicon Inc;
Easysep® system from Stemcell technology and BDTMImag cell separation systems. In addition to these simple batch magnetic columns commercially available (MACS®), more advanced high-throughput flow systems are available in a research basis (Moore et al., 1998; Sun et al., 1998). Quadrupole magnetic flow sorter (QMS) can sort intrinsic or labeled magnetic cells continuously from the nonmagnetic population. The composition of the sorted cell fractions can be well controlled by changing the resistance to cell transport along the magnetic field gradient (Zborowski et al., 1999) or transport laminar thickness (Schneider et al., 2010). However, the more specific the separation; the quantitative information with respect to the paramagnetism associated with cells is required (Nakamura et al., 2001). 48 Nuclear Magnetic Resonance (NMR) and Superconducting Quantum Interference
Device (SQUID) allow measuring magnetic susceptibility of matter. They are expensive, relatively hard to operate and require relatively large amount of material when particles on the micro or nanoscale are studied (Reddy et al., 1996). Moreover, large volume of particles will be required to have the population susceptibility, not for each individual particle. Another method to measure is called “isomagnetic”. It is destructive to the cells plus it is not easy to measure since particles have to be analyzed one by one and measuring media have to be changed by trial-and-error. Other methods for determining magnetic susceptibility were discussed by Reddy et al, in 1996 (Reddy et al., 1996). In those methods, neither magnetically induced velocity is accurately determined nor is the magnetic energy gradient well defined. To satisfy the demand for quantification of labeled or the intrinsic paramagnetism, Cell Tracking Velocimetry (CTV) was developed.
CTV is an analytical device to quantify magnetically induced velocity of intrinsic or imparted magnetic particles. CTV consists of several key components: 1) a well characterized magnetic field energy gradient 2) a microscopic image acquisition system
3) a computer algorithm which can determine the velocity from the location of each particle in the region of image analysis (Nakamura et al., 2001). The CTV system is sufficiently automated to track hundreds of cells or particles in less than 15 min, on a cell-by-cell basis.
The magnetically induced velocity from CTV divided by the magnetic energy gradient, defines ‘magnetophoretic mobility’ (Melnik et al., 2007). This term is analogous to “electrophoretic mobility”, which describes the behavior of a particle
49 moving within an external magnetic field. In other words, it is a measure of the magnetic susceptibility. It has been used for years as a measure of the effectiveness of the conjugation between cells and antibody-magnetic particles, and also a tool to design, optimize and operate immunomagnetic separation (Zborowski et al., 2002).
Magnetophoretic mobility can be obtained immediately after an experiment, from the software associated with CTV. As the technology has improved over the last decade,Due, more and more quantitative studies have been conducted. Combined with flow cytometry, the apparent dissociation constant and antibody-binding capacity was determined in 2006 (Zhang et al., 2006). Intracellular magnetic nano-particle uptake by live cells (Jing et al., 2008) and non-specific bound particles per cell were determined quantitatively (Chalmers et al., 2010). CTV was used to demonstrate that the magnetophoretic mobility varies from cell lines, and when primary and secondary magnetically conjugated antibodies are used (Chosy et al., 2003). In this work, we will demonstrate that the total manganese per spore cluster can also be obtained from CTV and X-ray Photoelectron Spectroscopy (XPS), which is consistent with the result of the total amount determined from Inductive Coupled Plasma-Mass Spectroscopy (next chapter). Magnetophoretic mobility was also quantified for different spores of different
Bacillus strains. However, variation of paramagnetism of the spores from batch to batch and different magnetic properties from strain to strain were observed. Duplicate or triplicate tests were performed for each sample to decrease the experimental error. Thus, the question arises: how accurate is the CTV measurement with respect to the small
(relative to previous cells studied) spores?
50 The previous study showed the inclusion of polystyrene beads of a significantly different size, allowed an internal control with respect to any fluid motion which might introduce dispersion into the movement of the cells. This specific internal control also demonstrated that mixing the cells with the beads did not introduce any unusual artifacts
(Chalmers et al., 2010). Therefore, internal controls will be discussed in this chapter for the accuracy of CTV to measure the spores. Given the potential to measure the magnetic susceptibility of the spores, and Mn contributions (next chapter) which impart this magnetic susceptibility, we wish to further quantify both the amount of manganese, and the oxidation state as described in previous chapter, through the use of complementary instrumentation and internal controls.
3.2. Theory
3.2.1 Fundamental forces, velocities of particles in CTV
Besides obtaining the magnetophoretic mobility through the experimental determination of magnetically induced velocities, another application of CTV is to calculate the hydrodynamic diameter of cells or particles through the settling velocities
(Nakamura et al., 2001). Figure 3.1 shows the fundamental forces on a paramagnetic particle suspended in a solution. Fm, Fb, Fg, Fd are forces of magnetism, buoyancy, gravity and drag (when the particle moves), respectively. It is assumed Fd can be characterized as
Stokes drag (Nakamura et al., 2001). The forces can be defined mathematically as follows:
51 Fb
Fd
Fm Fg
FigureFigure 3.13.1SchematicSchematic illustration illustration of forcesof forces on paramagnetic on paramagnetic particles particles in solution in solution in the in the magnetic field magnetic field
B2 Fm= p fV p (3.1) 20 3 ()p f D p g Fg- Fb= (3.2) 6
Fd=3πpD p (3.3)
where µ0 is magnetic permeability of free space, p is the volumetric magnetic
susceptibility of the particle while f , of the fluid; B is the magnitude of the magnetic
flux density, p and f stand for the density for the particle and fluid respectively, Dp is
the particle’s diameter, g is the gravity, p is the velocity (settling velocity vertically and
B2 magnetic velocity horizontally), and is the viscosity of the fluid. measures 20 the local magnetic energy density.
52 It is assumed that all forces balance (Reddy et al., 1996) (i.e. there is no acceleration). Consequently, the velocity due to the magnetic force and settling velocity can be obtained:
2 2 p f D p B u (3.4) 18 2 0
() D2 g p f p (3.5) 18 dividing 3.4 by 3.5, one can obtain:
u sphere f S m (3.6) vsphere f g
Further arrangement of Equation 3.6 gives
u g f (3.7) v Sm
It was recently experimentally demonstrated, by Jin et al. (2008) that Bacillus atrophaeus spores are not only paramagnetic, but unlike Fe oxides, the magnetization does not saturate in an applied field up to at least 1.2 T.
The magnetically induced velocity is “normalized” with respect to the magnetic energy gradient to obtain the magnetophoretic mobility:
u m (3.8) B2 20
53 3.2.2 Algorithm of CTV and classification by CTV associated software
The location and velocities of particles can be presented through a series of digital frames, by CTV algorithm process. The CTV algorithm defines a contiguous set of pixels that exceed a minimum level of contrast with the background image as an object. Each particle is identified and labeled by CTV algorithm based on its size, shape and location of its center. Basically, one set of velocities and locations can be obtained from tracking the particles in each five of successive frames (frames 1-5, 2-6… 16-20). All those sets of velocities (for 20 frames, there would be 16 sets of velocities) can be combined and averaged to have a mean velocity for the entire data set (Nakamura et al., 2001). CTV corresponds to the real size of particles to each pixel of 1.53 µm × 1.5 µm.
3.2.3 Classification by Support Vector Machines (SVMs)
Given a set of training examples, each marked as belonging to one of two categories, an SVM training algorithm builds a model that predicts whether a new example falls into one category or the other. The hyperplane to classify the mixture is defined by: w x b 0
b where vector w is perpendicular to the hyperplane while is the offset of the w hyperplane from the vector w (Figure 3.2).
3.3 Materials and methods
3.3.1 Experimental apparatus
The experimental instrument CTV has been reported and summarized previously
(Chalmers et al., 1999; Nakamura et al., 2001). A suspension of Bacillus spores, PSM or 54 their mixture to be analyzed was pumped in to a glass channel (0.1 mm × 1.0 mm), which was mounted within either a constant magnetic energy gradient of 142 T.A/mm2 for permanent magnets, or a well defined electromagnetic field (Table 3.1). An Olympus microscope using a 5 × objective and 10 × photo eyepiece was used to monitor the movements of the particles and spores. Light source was provided by a 150 W lamp
FigureFigure 3.23.. 2SMVSMV classification classification schematic schmatic picture.picture
connected via cable with the microscope. The schematic diagram of CTV was shown in the previous chapter. The electromagnetic CTV system looks similar except for the permanent magnets. Figure 3.3 is a schematic diagram of the electric coils and the channel for the electromagnetic CTV system. 1, 2 — pole pieces and flux return yolk made of 1018 low-carbon steel; 3 — copper wire coil wound with 1900 turns of 18
American Wire Gage (AWG). Each electromagnet consists of a coil surrounding a central
55 pole piece which replaces the permanent magnets (Jin et al., 2008). The magnetic energy gradient for electromagnetic CTV can range from zero to a maximum of 111 T-A/mm2.
The current to the coils is supplied by two switching programmable DC power supplies
(Model 1697, BK Precision). They are operated in a constant current mode and have a range of 0 to 5 A controlled by CTV associated software. The dependence of the magnetic energy density gradient, Sm and the electric current is listed in Table 3.1. Figure
3.4 is an example of the computer screen output from the CTV associated software indicating the settling trajectories (vertical traces) and magnetically induced trajectories
(horizontal traces) of particles in ROI tracked by the CTV system. Unlabelled and untracked white spots are PSM while the tracked particles are spores.
3.3.2 Spores and particles
15.3 µm polystyrene beads from Spherotech (Lake Forest, IL) were used in this study. Spores were prepared as the method described in Chapter 2. The collected spores were freeze-dried and stored as dry powder for analysis.
We have previously presented the observation that Bacillus atrophaeus spores, when created in mod G medium, exhibited high paramagnetic susceptibility (Melnik et al. 2007; Jin et al. 2008). In addition we have also observed variability in the magnetic susceptibility of the spores from batch to batch. Since the focus of this work is to further characterize and quantify this magnetic susceptibility with a number of different analytical instruments and conditions, 15.3 µm polystyrene beads were added to the spores for CTV analysis as internal controls.
56 57
Figure 3.3 Electric coils and channel for the electromagnetic CTV Figure 3.3 Electric coils and the channel for the electromagnetic CTV Table 3.1 Current and its corresponding magnetic energy gradient. The current and Tableits 3.1correspondingCurrent and itsmagnetic corresponding energy magnetic in red are energy used gradient in the this chapter.
57 Figure 3.4 An example of the computer screen output of the CTV software indicating the settling trajectories (vertical traces) and magnetically induced trajectories (horizontal traces) of particles in ROI tracked by the CTV system.
3.3.3 Computer imaging and CTV algorithms
As described above, the object is defined by the CTV algorithm when a
contiguous set of pixels exceed a level of contrast with the background. Minimum and
maximum numbers of pixels per object were user-defined to identify two populations, in
this study, spores and polystyrene microspheres (PSM) respectively. Minimum and
maximum pixels for spores were 2, 20; while PSM was defined with minimum pixels of
50 and maximum of 150 (Figure 3.5).
58 Figure 3.5 An example of digital image analysis by “image view” software. The set, frame and timing information defines the pictures taken in one set; image information defines the area of interest.
59 3.3.4 SMV classification
SMV classification was applied to the mixture of two populations using Software
R, based on their properties: while settling velocities correspond to gravity (density, volume); the magnetically induced velocities correspond to magnetic force. The settling velocities and magnetically induced velocities from the spore standard and PSM standard alone were used as training data. The model was built between the particle types
(dependent variable) and settling, horizontal velocities (independent variables).
3.3.5 Magnetophoretic mobility (MM) from Cell Tracking Velocimetry
The magnetically induced velocity of cells or particles can be determined by using the defined magnetic energy gradient, Sm, a microscope and a CCD camera, and the associated software. By dividing the measured velocity by Sm, the MM of the target entity, m, is obtained (Equation 3.8). Sm is independent of the particle portion
(approximately 142 T∙A/mm2 or 20.52 T∙A/mm2 for the current we chose for the electromagnetic CTV) (Chalmers et al., 1999) for the permanent magnet version; alternatively, the magnitude of Sm can be varied with the electromagnet version (Jin et al.,
2008).
3.4 Result
3.4.1 Classification by 2 methods
Particle types were obtained immediately through “image view” software associated with the CTV. Since spores tend to aggregate, SMV in R code was also used to group the mixtures. Through the comparison of 2 methods, not only the accuracy of
60 classification can be determined, but also the accuracy of measuring the magnetic properties of each group.
Radial Kernel SMV was used to model the particle type with both their settling velocity and magnetic induced velocity. C-classification was assigned as SMV- type. We combined the standard spores’ velocities with PSM velocities to make up two mixture standards. SMV method approved its accuracy by only misclassifying 0.98%.
Comparing SMV classification with CTV associated software; there is only 2.1% disagreement (Details about the result directly from R software is in the appendix A and
B). The aggregation of spores made it more difficult to identify the particles; however the huge size difference between PSM and spore clusters decreased the risk of overlap.
3.4.2 Magnetic susceptibility measurements of Polystyrene Microspheres (PSM)
Given the quantitative focus of this work, we choose to measure the magnetic susceptibility of the spores with and without an internal “control”; consequently, we followed experimental protocols used in Chalmers et al. (Chalmers et al., 2010).
Specifically, diamagnetic, 15.3 µm (as reported by the manufacturer) polystyrene microspheres (PSM) were analyzed independently and, added to a suspension of the spore suspension prior to CTV analysis.
Figure 3.6A is a representative “dot” plot of only the PSM analyzed in the CTV system. The mean, one and three standard deviations for the settling velocity and magnetically induced velocity are presented as dashed in lines in this figure. In addition to the dot plots, histograms of the two velocities are presented in 3.6B and 3.6C. The data for four independent analysis of these PSM is presented in Table 3.2.
61 Table 2. CTV analysis of PSM
Experiment Settling Mean (x+σ)×103 (x-σ) ×103 (x+3σ) ×103 (x-3σ) ×103 Mean Mean magnetic /Magnetic Velocity Diameter susceptibly×106 (µm/s) (µm) 1 Settling 4.55 5.07 4.02 6.12 2.97 14.6 2 Settling 4.59 5.05 4.13 5.98 3.20 14.7 3 Settling 5.39 6.37 4.41 8.33 2.46 15.9 4 Settling 5.34 6.27 4.41 8.14 2.55 15.9 Mean Settling 4.97 1 Magnetic -0.542 0.706 -1.79 3.20 -4.29 -9.36 2 Magnetic -0.465 0.383 -1.31 2.08 -3.01 -9.30 3 Magnetic -0.380 -0.0531 -0.707 0.601 -1.36 -9.23 4 Magnetic -0.334 0.196 -0.864 1.26 -1.92 -9.21 Mean Magnetic -0.430 -9.28 + 0.092 + 0.0686
62 TableTable3. 3.22 CTVCTV analysis analysis of of PSM PSM
62 A)
-3 - x + +3 0.007
0.006 +3
0.005 + x - 0.004
Settling Velocity Settling (mm/s) -3 0.003
0.002 -0.006 -0.004 -0.002 0.000 0.002 0.004 0.006 Magnetic Velocity (mm/s) Continued
Figure 3.6 Scatter plot (A), and histograms of settling velocity (B) and magnetic velocity (C), for the PSM. In addition, the mean, one and three standard deviations from the mean are presented as dashed lines for the settling and magnetic velocity
63 Figure 3.6: Continued B)
0.07
0.06
0.05
0.04
0.03
0.02 FrequencyPopulation of
0.01
0.00 -0.004 -0.002 0.000 0.002 0.004 Magnetic Velocity (mm/s)
C)
0.06
0.05
0.04
0.03
0.02 FrequencyPopulation of
0.01
0.00 0.0025 0.0030 0.0035 0.0040 0.0045 0.0050 0.0055 0.0060 0.0065 Settling Velocity (mm/s) 64 Using a diameter of 15.3 µm on average, CTV measured settling velocity of 4.97
× 10-3 mm/s, a value of 9.8 m/s2 for g, and a viscosity of 0.98 x 10-3 kg/m-s, Equation 3.5 can be rearranged to solve for :
18 3 particle f v 2 39.0kg / m gDsphere
With this value of , the value of the volumetric magnetic susceptibility () , for each
PSM (or spores) can be determined by using Equation 3.7.
2 8 3 Using a value of Sm of 142 T-A/mm (1.42 x 10 N/m ), a value of calculated from Equation 3.5 , and a value of the magnetic susceptibility of water of -9.05 x10-6, the average value of the magnetic susceptibility of PSM can be calculated and is presented in
Table 3.2. Four PSM samples and four mixtures of spores and PSM were prepared for
CTV. For each sample, around 1000 particles were tracked and the mean of magnetic velocity, settling velocity were calculated. We take the average of the mean velocities from four experiments in each situation and list the values in Table 3.2. Note, the reported values for the magnetic susceptibility of polystyrene range from -7.5 x 10-6 to -
8.2 x 10-6 (Weast CR et al., 1987; Watarai and Namba, 2001), and in a previous publication using CTV were between -7.7 to -8.0 x 10-6 (Zhang et al., 2005; Jin et al.,
2008).
3.4.3 Magnetic susceptibility of measurements and calculations of the spores
Figure 3.7A is a dot plot of a mix of the spores and the PSM previously discussed, and Figure 3.7B is an enlarged view, with dotted lines representing the mean of the
65 A)
0.03
0.02
0.01
0.00 Settling Velocity (mm/s) Settling Velocity
-0.01 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Magnetic Velocity (mm/s)
B)
-3 x +3 0.008 +3
0.006 x 0.004
-3 0.002
Settling Velocity (mm/s) Velocity Settling 0.000
-0.002
-0.02 0.00 0.02 0.04 0.06 0.08 0.10 Magnetic Velocity (mm/s) Figure 3.7 Dot plots of a full scale (A) CTV measurement of Bacillus spores mixed with 15.3 µm PSM (A) and an enlarged view (B)
66 settling and magnetic velocities and dashed lines representing three times the standard deviation from the mean, just as previous CTV studies of PSM (i.e. Figure 3.6).
Table 3.3 also presents the mean and standard deviations of the PSM and spores when analyzed as a mixture and subsequently partitioned into PSM and spore populations using the mean and standard deviation values for the PSM when evaluated independently as was presented in Figure 3.7B. Comparing the mean magnetically induced velocity of the PSM alone and in the mixture with the spores shows a slight difference; however the difference is well within the standard deviations of the mean suggesting that the difference is not significant. Correspondingly, it is suggested that the values of the magnetically induced velocity and corresponding calculated values of the spore magnetic susceptibility (Equation 3.7) are representative of the actual magnetic susceptibility of the spores. Consequently, further analysis of the spores did not include the PSM controls.
In addition to the studies of mixtures of PSM and spores, four independent CTV analyses were conducted with spores by themselves (no PSM mixed with them). Figure
3.8 is a dot plot, and distribution of the settling and magnetic velocity for one of these experiments. Unlike the PSM (Figure 3.6), there is clearly a wide, non-normal distribution in the magnetic velocity of the spores, and a much wider distribution in the settling velocity. Significant clumping is present which can contribute to both the wider distribution in the settling velocity and the non-normal distribution in magnetic velocity.
If one makes the assumption the densities of the spores are uniform, and using the previously presented value of the spore density (Carrera et al., 2008) the diameter of the spore clusters can be determined using Equation 3.5 and are presented in Table 3.4.
67 A)
Experiment Mean (x+σ)×103 (x-σ) ×103 (x+3σ) ×103 (x-3σ) ×103 Magnetic Velocity Susceptibility (µm/s) ×106 PSM 1 -0.542 2.63 -3.71 8.98 -10.1 -9.33 PSM 2 -0.529 2.42 -3.48 8.32 -9.37 -9.33 PSM 3 -0.319 0.871 -1.51 3.25 -3.89 -9.20 PSM 4 -0.296 0.811 -1.40 3.02 -3.62 -9.20 PSM Mean -0.421 -9.26 + 0.132 + 0.0746 Spores 1 26.0* * * * * 214
68 Spores 2 41.7* * * * * 224 Spores 3 40.2* * * * * 122 Spores 4 40.9* * * * * 165 Spores Mean 37.2* * * * * 186 + 7.49 +52 Continued
Table 3.3 Mean, and standard deviation, of settling (A) and magnetic velocity (B) of the PSM and spores when analyzed as a mixture in the CTV system
* Boundaries were not listed because of non-normal distribution of the spores’ magnetic velocity
68 Table 3.3: Continued
B)
Particles Experiment Mean (x+σ)×103 (x-σ) ×103 (x+3σ) ×103 (x-3σ) ×103 Mean Velocity (µm/s) Diameter (µm) PSM 1 4.99 6.22 3.76 8.69 1.29 PSM 2 4.88 5.79 3.97 7.62 2.14 PSM 3 5.44 6.54 4.34 8.75 2.13
PSM 4 5.14 6.11 4.18 8.04 2.25 69 PSM Mean 5.11 + 0.243
Spores 1 1.54 4.40 -1.31 10.1 -7.02 3.76 Spores 2 2.37 6.35 -1.60 14.3 -9.56 4.67 Spores 3 4.35 9.27 -0.564 19.1 -10.4 6.32 Spores 4 3.27 7.52 -0.982 16.0 -9.48 5.48 Spores Mean 2.88 5.06 + 1.21 + 1.1
69 Experiment Settling Mean (x+σ)×103 (x-σ) (x+3σ) (x-3σ) Mean Mean magnetic /Magnetic Velocity ×103 ×103 ×103 Diameter susceptibly×104 (µm/s) (µm) 1 Settling 1.58 5.04 -1.87 11.9 -8.78 3.81 2 Settling 3.91 8.25 -0.434 16.9 -9.12 5.99 3 Settling 4.84 10.6 -0.953 22.2 -12.5 6.67 4 Settling 4.93 10.9 -1.07 22.9 -13.1 6.73 5+ Settling 3.02 4.47 1.57 7.38 -1.34 6.52 6+ Settling 3.15 5.32 0.988 9.64 -3.34 5.37
Mean Settling 3.57 5.85 + 1.12 1 Magnetic 24.6 * * * * 2.06 2 Magnetic 39.3 * * * * 1.33
70 3 Magnetic 41.7 * * * * 1.14 4 Magnetic 42.1 * * * * 1.13 5 Magnetic 4.65# * * * * 1.48
6 Magnetic 3.17# * * * * 0.966 Mean Magnetic 1.43E-+/-0.382 Table 3.4 Spore settling and magnetic velocity when not mixed with PSM
*Boundaries were not listed because of non-normal distribution of the spores’ magnetic velocity # These measurements were made with the electromagnetic CTV with a Sm value of 20.5 in contrast to the value of 140 with the permanent magnet system. + From Electronic CTV
70 A)
-3 -2 x +2 +3 0.014
0.012 +3 0.010
0.008 +2
0.006
0.004 x 0.002
0.000 SettlingVelocity (mm/s) -2 -0.002
-0.004 -3
-0.006 -0.004 -0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Magnetic Velocity (mm/s)
Continued
Figure 3.8 Representative dot plot (A), histogram of the magnetic velocity (B), and histogram of the settling velocity of the spores not mixed with PSM (C).
71 Figure 3.8: Continued
B)
0.08
0.06
0.04
Frequencey of Population of Frequencey 0.02
0.00 -0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Settling Velocity (mm/s) C)
0.05
0.04
0.03
0.02
Frequency of the population the of Frequency 0.01
0.00 -0.002 0.000 0.002 0.004 0.006 0.008 0.010 Magnetic Velocity (mm/s)
72 3.5 Conclusion
Even though Bacillus spores have been studied for immunomagnetic separation
(Zhang, 2004) in my lab before, it is the first time that we planed to quantify the paramagnetism of spores as well as the quantity of Mn amount and Mn valence. To build a relationship between Mn valance state, quantity and the paramagnetism of the spores, it is crucial that we have accurate and sensitive instruments to support this analysis. As discussed before, the sizes of the spores due to aggregation, the variation in the paramagnetism make it more difficult to quantify the intrinsic magnetism of the spores.
By adding an internal control, the accuracy and efficiency of CTV was established.
We measured an average magnetic susceptibility of 9.28×10-6, for the polystyrene beads which is consistent with the published data (Weast CR et al., 1987; Watarai and
Namba, 2001). The magnetic susceptibility of spores ranges from 1 to 1.97×10-4, with diameter of the cluster from 3-6 µm. The relationship between Mn and magnetism will be studied and discussed in the next chapter.
73 Chapter 4 Quantification of both the presence, and oxidation state, of
Mn in Bacillus atrophaeus spores and its imparting of magnetic
susceptibility to the spores
Most of the following content has been submitted to Bioengineering and Biotechnology.
List of authors: Jianxin Sun, Maciej Zborowski, and Jeffrey J. Chalmers.
Abstract: Bacillus atrophaeus spores were previously reported to have significant magnetic susceptibility in a magnetic field due to the presence of manganese (Mn).
However, relative little is known about the total amount and distribution of the oxidation state of Mn associated with the specific strains’ spores. Using the instrument Cell
Tracking Velocimetry (CTV), both magnetically induced velocity (referred to as magnetophoretic mobility) and settling velocity were quantitatively measured. Visual observations, and calculated diameter using previously reported density, indicate that the spores are present in the form of clusters of approximately 3 to 6 µm. Treatment of these clusters with EDTA or pH of 2.0 (or below) resulted in not only the disruption of the spore clusters, but also a significant decrease in magnetic susceptibility, in some cases by almost two orders of magnitude. Since the magnetic susceptibility of Mn varies significantly between the three typical valance states of Mn, Mn(II), Mn(III), and Mn(IV), 74 X-Ray Photoelectron Spectroscopy (XPS) was used to determine the valance states of Mn in the spores. This analysis returned the following ratio: 0.41, 0.38, and 0.21, corresponding to the following valance states: Mn(II), Mn(III), and Mn(IV) respectively.
The total mass of Mn associated with each spore cluster was determined by Inductive
Coupled Plasma-Mass Spectroscopy (ICP-MS). A second, completely independent estimate of Mn mass associated with each spore cluster was made, by mathematically solving for the amount of Mn per spore cluster using the experimentally measured magnetophoretic mobility and the magnetic susceptibility of each of three valence states from the XPS analysis. ICP-MS returned a value of 3.28 × 10-11 g of Mn per spore cluster while the mathematical estimation from mobility and XPS analysis retuned a value of
1.16 × 10-11 g, which is a reasonable agreement, given the complexity of the two techniques. Finally, a discussion of potential applications of the spores’ magnetic properties presented.
4.1 Introduction
Bacillus atrophaeus has been introduced in the first two chapters and apparently manganese plays a very important role in the sporulation. Like iron, manganese (Mn) has important functions in many biological systems, among which a common occurrence is the association of Mn atoms with proteins involved in electron transport or oxidation reduction reactions. This is not surprising, given the various valence states of Mn, analogous to Fe (II, III). It was claimed that at least three valence states of Mn and the two valence states of Fe were found in biological systems. With the help of Mn/Fe, electrons were transferred, from commonly occurring reductants (e.g. S, Organics, H2,
75 - H2S, etc) to oxidants (e.g. O2, NO3 , CO2, etc) (Nealson and Saffarini, 1994). Recently, significant interest has focused on the role of Mn superoxide dismutase (MnSOD, SOD2) in diseases. While a number of metal atoms can be presented in superoxide dismutase,
MnSOD has been shown to be an essential antioxidant enzyme in the mitochondrial matrix (Pardo-Andreu et al., 2006). In addition, over-expression of MnSOD protein inhibits growth in a wide variety of human cancer cells (Valdivia et al., 2009).
Figure 4.1 Possible mechanisms of Bacterial Mn (II) oxidation. The numbers 1, 2, and 3 in the lower panel indicate different possible pathways for Mn(II) oxidation (Tebo, 2004)
A number of bacteria can oxidize Mn, and at least one proposed mechanism is from the organism Bacillus sp.SG-1. This mechanism has been developed from the observation that different types of Mn oxides were found on the spore coat of B. subtilis.
For example, hausmannite (Mn3O4), deposited on spores of this marine strain, was
76 reported by Hastings and Emerson in 1986. Lattice imaging revealed that the amorphous deposits (Mn3O4) covered the spore coat in isolated domains (Mann et al., 1988).
Moreover, Mn3O4 was deduced to be the intermediate in MnO2 formation on the spores.
Besides Mn3O4, Mn(IV) solid phase products were claimed to be formed in SG-1 oxidation via X-ray diffraction studies (Mandernack et al., 1995). Inconsistent with the
Mn3O4 intermediate hypothesis, Hem, Lind (Hem and Lind, 1983) and Murray et al.
(Murray et al., 1985) observed that Mn(III) minerals only slowly aged to Mn(IV). A recent review article demonstrates (Spiro et al., 2010) the current understanding of the presence of Mn oxides in a number of bacteria as well as summarizes current thinking on the biological, manganese oxidation pathways. Two proposed mechanisms of Mn oxidation are given in Figure 4.1. In an abiotic oxidation process, the intermediate of Mn oxidation is Mn3O4 or MnOOH; while in biological system, Mn(III)-Enzyme complex may be the intermediate, and MnO2 could be produced from either Mn(IV)-Enzyme complex or Mn(III). It was also reported that Mn oxides of lower valence state would be precipitated at a higher Mn concentration and the higher valence state would be formed at a lower Mn concentration (Mandernack et al., 1995). Furthermore, Mn oxidation, proceeding at a very slow rate abiotically can be accelerated by microorganisms by five orders of magnitude (Nealson et al., 1988; Wehrli, 1990; Tebo, 1991).
A number of Bacillus strains have been reported to aggregate during sporulation.
It was previously shown that different spore species possess different hydrophobic properties (Rosenberg, 1984; Koshikawa et al., 1989; Doyle, 2000; Flint et al., 2000).
Increase of the hydrophobicity contributed to the formation of spore clumps (Furukawa et
77 al., 2005). Hydrophobicity and surface charge play a role in microbial surface adhesion; however, the magnitude of surface charge in influencing attachment of microbes to surfaces is not well understood (Flint et al., 2000).
4.2 Theory
4.2.1 Magnetic properties
Like Fe, Mn has paramagnetic properties, and these properties are a strong function of the oxidation state of the metal atom. Table 4.1 lists the compound, molecular weight, specific gravity, and magnetic susceptibility (in cgs units) of one gram formula weight for the common forms of Mn and water (Weast et al., 1987). In addition to presenting magnetic susceptibility in the form of one gram formula weight, molar it is also commonly presented as a volumetric magnetic susceptibility () where the two forms are related by:
m (4.1) M w with Mw representing the molecular weight of the compound. This form of susceptibility is also presented in Table 4.1, in the SI unit system.
4.2.2 Bulk magnetic susceptibility of a mixture
It has been experimentally demonstrated that several strains of Bacillus spores can contain various forms of Mn. As is quantified in Table 4.1 these various forms of Mn have significantly different values of magnetic susceptibility, one can apply the following relationship (Zborowski and Chalmers, 2007) to determine the weighted average value of the magnetic susceptibility of a Bacillus spore based on its composition:
78 n V V V V V V biomass biomass Mn Mn MnO MnO Mno2 MnO2 Mn2O3 Mn2O3 Mn3O4 Mn3O4 i i i 1 V
(4. 2)
where i is the volume fraction and i is the volumetric magnetic susceptibility of species ‘i’. It should be noted that while Energy Dispersive Spectroscopy (EDS) Spectra of several spores (Melnik et al., 2007) indicate the presence of a number of different types of atoms in addition to the basic components of biomass (i.e. Na, Ca, Mg, Al), the only atom present that could results in a positive, magnetic susceptibility was Mn (i.e. Fe level was below EDS detection limits).
4.2.3 Calculated magnetic force and magnetically induced velocity as a result of Mn composition
The magnetic force exerted on a volume of a substance (V), characterized by volume magnetic susceptibility () is given by:
dB F VH 0 (4.3) dx where H and B0 are magnitudes of the applied magnetic field vectors. One can define a magnetophoretic mobility, m, such that:
v v m mag mag (4.4) S dB m H 0 dx and in the case of a microsphere undergoing creeping flow,
v ( )V m mag sphere f (4.5) 2 3D B0 sphere
20
79 Compound Molecular Specific Molar magnetic Volume magnetic Heat of Formation Free energy of weight gravity susceptibility susceptibility (SI) (kcal/mol) formation 6 3 + m x 10 (cm /mol) (kcal/mol) Mn 54.938 7.2 529 8.72 x 10-4 0 0 β Mn 54.938 7.2 293 4.82 x 10-4 0 0 MnO (II) 70.94 5.43-5.46 4850 4.56 x 10-3 -92.04 -86.77 -3 MnO2 (IV) 86.94 5.026 2280 1.55 x 10 -124.58 -111.49 -3 Mn2O3 (III) 157.87 4.5 14100 5.1 x 10 -229.50 -209.90 80 -3 Mn3O4 (III, IV) 228.81 4.856 12400 3.3 x 10 -331.65 -306.22
MnCl2 (II) 161 14,350 -112.00 -102.20
MnSO4•4H2O 14,600 -6 H2O 18 1.0 -13.0 -9.04 x 10 Table 4.1 Spore settling and magnetic velocity when not mixed with PSM
+ in cgs units, for one gram formula weight
80 where vmag is the magnetically induced velocity, Sm is the magnetic energy gradient (T-
2 A/mm ), B0 is the magnetic field intensity (T), 0 is the permeability of free space (T-
m/A), η is the viscosity of the fluid, Dsphere is the diameter of spores, sphere is the
magnetic susceptibility of the particle, and f is the magnetic susceptibility of the suspending buffer. Implicit in Equation 4.5, it is assumed that the sphere is linear polarized (either diamagnetic or paramagnetic); consequently, the magnetization of the sphere is directly proportional to the applied field.
In an analogous manner to a magnetically induced velocity (vmag), one can speak of a sphere’s sedimentation velocity (vg), which is the result of gravity acting on the differences in densities between the sphere and the suspending fluid:
v V s g sphere f sphere (4.6) g 3Dsphere
Division of Equation 4.5 by 4.6 (Jin et al., 2008) results in:
v sphere f S mag m (4.7) vg sphere f g
It was recently experimentally demonstrated, by Jin et al. (Jin et al., 2008) that B. atrophaeus spores are not only paramagnetic, but unlike Fe oxides, the magnetization does not saturate in an applied field up to at least 1.2 T. Jin et al, demonstrated that the mean magnetophoretic mobility of the spores (n >1000) is constant over the full range of values of Sm tested (the value of T varied from 0.5 to 1.4 over this range). It was reported the “wet” and “dry” densities of a number of Bacillus spores (Carrera et al., 2008), including B. atrophaeus with a “wet” density of 1.201 g/cm3. Using this value of density,
81 a viscosity of 0.93 x 10-3 kg/m-s, the experimentally determined settling velocity, and a
1 rearrangement of Equation 4.6, (and the assumption of a perfect sphereV D 3 ): sphere 6
1/ 2 18 Dsphere vg g (4.8) the average diameter of the spores, or more likely, the clusters of spores were determined.
vm Equation 4.7 indicates linear correlation between and Sm , and the slope for vg
Sm is defined by /g. Equation 4.7 can be further rearranged to give
v g m f (4.9) vg Sm
v 1 m . W A clear linear relationship is obtained with a slope of Sm ith the paired settling vg velocity and magnetic velocity and corresponding magnetic energy gradient, the
vm 1 experimentally determined value for the defined slope is 0.104, and vg S m rearranging, one obtains:
0.104g f (4.10)
Solving for the current case, the magnetic susceptibility of the spores is 2.04 x 10-4 in the
SI unit system. As a point of comparison, Table 4.2 lists the previously published intrinsic magnetic susceptibility of normal, human lymphocytes, Peripheral blood lymphocytes
(PBL), oxygenated human red blood cells (RBC), the met-hemoglobin form (chemically deoxygenated form) of RBC, and 6.7 µm polystyrene particles. 82 Through the further analysis with complementary instrumentation and internal controls, further quantification of both the amount of manganese and the oxidation state are studied in this chapter, with combination of measuring the magnetic susceptibility of the spores, and Mn contributions (Equation 4.2) which imparts this magnetic susceptibility.
4.3 Materials and methods
4.3.1 Strains and sporulation
B. atrophaeus (ATCC #9372) and Bacillus sp. SG-1 from Bacillus Genetic Stock
Center at the Ohio State University were cultured in Nutrient Broth and sporulated using
Mod G media as described before (Melnik, 2007). To create a greater amount of spores for a number of different analytical studies, the process was scaled up, using a Biostat B bioreactor. The working volume was 4L, the aeration rate was approximately 2L/min at an agitation rate of 300 rpm. The pH was maintained at 7.45 during sporulation. The collected spores were freeze-dried and stored as dry powder for future analysis.
4.3.2 Mn valence state determination by X-Ray Photoelectron Spectroscopy
X-Ray Photoelectron Spectroscopy (XPS) was used to quantify the valence states of Mn on the spore surface. XPS involves irradiating the surface of sample in high vacuum, and subsequently measuring the kinetic energy of excited electrons from the atoms in the sample (Moulder, 1992). A limitation of this technique is that only the top 1 to 10 nanometers of the surface being interrogated is analyzed.
83 Particles Volume, magnetic Reference
susceptibility (SI units)
Peripheral blood -9.04 × 10-6 (Chalmers et al., 2010)
lymphocytes
Oxy red blood cells -9.19 × 10-6 (Chalmers et al., 2010)
Met Hb red blood cells -5.43 × 10-6 (Chalmers et al., 2010)
6.7 µm Polystyrene -8.0 × 10-6 (Jin et al., 2008)
microspheres
Table 4.2 Experimentally reported average values of magnetic susceptibility of selected cells and polystyrene microspheres.
The lyophilized magnetic bacteria on carbon tape were used directly for XPS. Al
K-alpha X-ray was used as the X-ray source. For most samples and standards, such as
Mn3O4, a charge of 2.3eV was used to obtain a good background. For MnO2, no neutralizer was applied. Since the spectrum created by this instrument results from X- rays that can penetrate the sample to a depth of 1-10 nm, it was assumed that only information on the surface of dried spores was obtained. This instrument can typically detect elements with atom concentration above 0.1%.
4.3.3 Mn and Fe concentration determination by Inductively Coupled Plasma-Mass
Spectroscopy (ICP-MS).
Ten mg of spores were previously prepared, and desiccated spores were suspended in deionized distilled water and separated into two aliquots. One sample was
84 disrupted by sonication with intermittent treatments for 1 hour until the sample appeared to be homogeneous; the other aliquot was maintained in an unagitated state. Both aliquots were subsequently dissolved using a modified Na-citrate-bicarbonate-dithionite (CBD) method (Neaman et al., 2004). These dissolved samples were then added to 20 ml of 0.3
M sodium citrate (Na3C6H5O7-2H2O), 2.5 ml of 1 M sodium bicarbonate (NaHCO3), 0.5
º g of sodium dithionite (Na2S2O4), and subsequently stirred for 1 h at 80 C (in a water bath). This spore lysate was then centrifuged and the supernatant portion was collected for ICP study. ICP measurements were made on a Quantitative measurements performed on a ThermoFinnigan Element2 Inductively Coupled Plasma Sector Field Mass
Spectrometer.
4.3.4 Magnetophoretic mobility (MM) of treated spores from Cell Tracking
Velocimetry
As reported previously, Cell Tracking Velocimetry (CTV) was developed to quantify the MM of cells and particles that have intrinsic magnetic susceptibility or that have been imparted this susceptibility through binding of magnetic particle conjugated antibody. CTV can measure the MM of hundreds to thousands of cells (or particles) simultaneously, allowing significant populations size to be characterized (Chosy et al.,
2003; Zhang et al., 2005). The magnetically induced velocity of cells or particles can be determined by using the defined magnetic energy gradient, a microscope and CCD camera, and the associated software. By dividing the measured velocity by Sm, the MM of the target entity is obtained (Equation 4.4). Sm is independent of microparticle portion
(approximately 142 T∙A/mm2) (Chalmers et al., 1999) for the permanent magnet version;
85 alternatively, the magnitude of Sm can be varied with the electromagnet version (Jin et al.,
2008).
4.4. Results
We have previously presented the observation that B. atrophaeus spores, when created in mod G medium, exhibited high paramagnetic susceptibility (Melnik et al. 2007;
Jin et al. 2008). In addition, we have also observed variability in the magnetic susceptibility of the spores from batch to batch. Since the focus of this work is to further characterize and quantify this magnetic susceptibility with a number of different analytical instruments and conditions, we developed a scaled up process to make a sufficient amount of spores in one run to conduct all of our analytical tests, including
XPS, ICP and CTV. One big challenge of using flasks to sporulate is that the amount of spores is not enough from one batch. Moreover, the magnetism of spores change from batch to batch, since it is hard to control the aeration, pH and other conditions. In this study, we used a 4L Biostat B bioreactor which allowed 55 mg dried magnetic spores to be produced. Other than the use of the 4L bioreactor, all other aspects of spore production were the same as previously reported.
4.4.1 Magnetophoretic mobilities of PSM and spores
The mobilities of PSM and spores were obtained as described in Chapter 3. The summary is in Table 4.3. The internal control confirmed the accuracy of magnetophoretic mobility from CTV.
86 4.4.2 Treatment of spores with pH and chelating agents
Given the visual, microscopic images, the large distribution in settling velocity, and the calculated, mean diameters which ranged from approximately 3 to 7 µm, we attempted to disaggregate the spores with various surfactants or chelating agents. In addition, we tested incubating the spores in solutions of pH ranging from 5.0 to 0.6.
These treatments had a variety of significant effects on both the aggregation and magnetic susceptibility of the spores.
Figure 4.2A and 4.2B are examples of a dot plot of the spores prior to, and after treatment with EDTA in distilled water and Figure 4.3B is a dot plot after overnight treatment in a solution of pH 1.2. Visual inspections and numbers provided in Table 4.3 indicate significant decrease in clumping and corresponding decrease in magnetic susceptibility for the EDTA and the low pH treated spores.
4.4.3 XPS analysis of the spores
Figure 4.4A is a representative plot of a XPS analysis of spores over the whole spectra, and Figure 4.4B is an enlargement over the binding energies associated with Mn oxides. To assist in the interpretation of the spectra, spectra for B. cereus, B. sp SG-1, and
pure samples of Mn2O3, Mn3O4, MnO, and MnO2 are included in addition to the specific spores in this study. Using the spectral deconvolution software, GRAM (Thermo
Scientific, MA), the fraction of each oxidation state of Mn to specific peaks was determined to be 0.41, 0.38, and 0.21 for Mn2+, Mn3+, and Mn 4+ respectively. Table 4.4 87 Particles Treatment Experiment Mean Settling Mean Magnetic Mean Mean magnetic Velocity (µm/s) Velocity (µm/s) Diameter susceptibly×106 (µm) PSM NA Mean 4.97+0.46 -0.430 15.3 -9.28 N=4 + 0.092 +0.72 + 0.0686 Mixed with Mean 5.11 -0.421 -9.26 spores N=4 + 0.243 + 0.132 + 0.0746 NA Mean 3.57+ 1.27 36.9+ 8.31* 5.85+ 143+ 38 N=6 1.12 Mixed with Mean -2.88+ 1.21 37.2+ 7.49 5.06+ 186+ 52 PSM N=4 1.10 Control 0.778 25.7 2.67 437 EDTA treatment 1.03 5.58 3.08 6.26 Detergent Triton treatment 0.552 7.70 2.25 175 Effect Tween 0.502 6.81 2.15 170 treatment Bacillus pH effect Control 3.91 39.3 6.0 133 88 atrophaeus 5.0 4.60 6.86 6.4 19.7 spores 4.0 4.51 6.73 6.4 19.7 3.0 3.15 27.3 5.4 115 2 1.85 0.715 4.1 5.11 1.2 1.34 1.07 3.96 5.44 0.6 2.64 3.94 4.26 6.35 Table 4.3 CTV analysis of PSM and spores
* Two CTV systems were used for the measurements. For permanent magnet system, the magnetic energy gradient value is 142 T∙A/mm2, in contrast to the value of 20.5 T∙A/mm2 with electromagnetic CTV. Four measurements from permanent magnet system were made and two measurements from electromagnetic CTV. Due to different magnetic system, the average of magnetic velocities were obtained only from permanent magnetic system (n=4).
88 A)
-3 x +3 0.020
0.015
0.010 +3
0.005 x 0.000
-0.005 Settling Velocity (mm/s) Velocity Settling -3
-0.010
-0.015 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 Magnetic Velocity (mm/s)
B)
-3 x +3 0.008
0.006 +3
0.004
0.002 x 0.000
-0.002
Settling Velocity (mm/s) Settling -0.004 -3
-0.006
-0.008 -0.02 0.00 0.02 0.04 0.06 0.08 Magnetic Velocity (mm/s) Figure 4.2 Settling and magnetic velocity of the spores only in distilled water (A) and in distilled water containing EDTA (B)
89 A)
-3 x + +3 0.03
0.02 +3
0.01 +
x
0.00 Settling Velocity (mm/s) Velocity Settling -0.01 -3
-0.02 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 Magnetic Velocity (mm/s)
Continued
Figure 4.3 Setting and magnetic velocity of the spores in distilled water and after being suspended in a solution of pH 1.0 for 14 hours (B). Control (A)
90 -3 - x + +3 Figure 4.3: Continued 0.010
B) 0.008
0.05 0.006
0.004 +3 0.04 0.002 + x 0.000 - 0.03 -0.002 -3
-0.004
0.02 SettlingVelocity (mm/s)
-0.006 91
0.01 -0.008
Settling Velocity (mm/s) Settling -0.010 -0.010 -0.005 0.000 0.005 0.010 0.00 Magnetic Velocity (mm/s)
-0.01 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 Magnetic Velocity (mm/s)
91 presents more information with respect to this analysis.
4.4.4 Quantitative estimates of the amount of Mn on the spores
Using the average values of magnetic susceptibility of the spore clusters, the volume of these clusters calculated from the effective diameters of the spores (Table 4.2), the volumetric magnetic susceptibility of the various oxidation states of Mn, Equation 4.2 can be rewritten to obtain:
V V V V V (4.11) spore spore cluster MnO MnO MnO2 MnO2 Mn2O3 Mn2O3 Mn3O4 Mn3O4
Note the term corresponding to the magnetic susceptibility of the biomass was omitted as was the magnetic susceptibility of elemental Mn since only MnSO4 was a constituent of the media. In these XPS studies we were only able to differentiate between Mn2+, Mn3+,
4+ and Mn ; we are not able to specifically quantify the presence of Mn3O4. Consequently,
Equation 4.11 was rewritten:
spore clsuterVspore cluster VMn 2 Mn 2 VMn3 Mn3 VMn 4 Mn 4 (4.12)
The values of for each oxidation state of manganese, Mnx+, are listed in Table 4.1, and
the values of the volumes of corresponding Mn oxidation state, VMn x , can be determined from:
3 MW Mnx O y 1 1m V x Mn mass in gram Mn MW Mn 100 cm MnxOy
(4.13)
Using the values from Table 4.1, the corresponding values of VMn2 , VMn3 , VMn3 , are
Mn Mass × 9.81 × 10-7, Mn Mass × 1.2 × 10-7 and Mn Mass × 6.51 × 10-8, respectively.
92 A)
50
40
30
)
3
- 93 CPS (×10 20
10
1400 1200 1000 800 600 400 200 Binding Energy (eV) Continued Figure 4.4 Full spectra from XPS analysis (A). The box framed spectra contains Mn peaks,B) which is enlarged in (B) 93 Figure 4.4 Continued : B. atrophaeus ______B) 14 B. cereus ______B.sp. SG-1 ______
Mn2O3 ______
12 Mn3O4 ______MnO ______
MnO2 ______
)
3 10
-
8
CPS (×10 94
6
4
2
660 650 640 Binding Energy (eV) 94 Mn2+ Center Height FWHM Area Proportion1 Proportion2 Peak #1: 640.179 300 0.9030155 288.3691 0.060191 Peak #3: 641.2 278.5569 1.6 474.4229 0.099026 Peak #6: 642 236.5623 2 503.6249 0.105122 Peak #8: 642.9 209.9633 0.9824735 219.5819 0.045833 Peak #14: 645 93.44832 2.75 273.5482 0.057098 0.413234 Peak #16: 647.5 104.6154 1.979269 220.2088 0.045964
Mn3+ Center Height FWHM Area Proportion Proportion Peak #2: 640.7 673.178 0.806196 644.5823 0.134544 Peak #4: 641.4 388.5492 1 460.7613 0.096175 Peak #7: 642.2 335.8108 0.7656146 307.357 0.064155 Peak #10: 643.2 146.7075 1.65 284.4088 0.059365 0.380165276 Peak #12: 644.6 31.37408 3.5 124.2172 0.025928
Mn4+ Center Height FWHM Area Proportion Proportion Peak #5: 641.9 185.4045 1.5 326.5487 0.06816 Peak #9: 642.9 135.1923 1.1 176.6936 0.036881 Peak #11: 643.8 117.0724 1.2 166.7105 0.034797 Peak #13: 644.8 75 1.497097 132.3673 0.027629 0.2066 Peak #15: 645.8 69.9997 2.319403 187.478 0.039132 Table 4.4 Quantification of Mn in different chemical state from XPS
1 Proportion of each peak in that state of Mn 2 Proportion of that state of Mn of the total Mn in the sample
2 3 Substituting these values forVMn , VMn , VMn4 , the corresponding values of , and the
-20 product of spore cluster V spore cluster of 1.35 x 10 , into Equation 4.13, the value of Mn
(weight) per spore cluster is determined to be 1.16 x 10-11 g.
95 4.4.5 Estimates of the amount of Mn per spore
As described in the materials and methods above, independent experimental measurements, with respect to CTV and XPS analysis, of the amount of elemental Mn per spore were made using an Inductively Coupled Plasma- Mass Spectroscopy (ICP- MS) instrument. Table 4.5 presents the results of the instrumental analysis as well as calculations with respect to the amount of Mn per spore cluster. While it not known why more Mn per spore cluster was observed in the non sonicated compared to the sonicated samples, 3.28 x 10-11 versus 1.96 x 10-11, the general agreement with the estimate from
CTV and XPS analysis, 1.16 x 10-11 g Mn per spore cluster is encouraging. Further, the
ICP-MS analysis with respect to Fe present further underscores the observation that the magnetic susceptibility is the result of Mn atoms.
4.4.6 Total Mn mass balance
For the batch of spores used in this study, a total of 55 mg of spores were produced in 4 L of mod G medium. From the ICP analysis (Table 4.5), there is 0.268 g of
Mn per gram of spores; therefore, there is a total of 1.34 mg of Mn associated with all of the spores produced in that specific batch. Since the concentration of Mn in the mod G medium is 50 mg of MnSO44H2O per liter, a total of 4.48 mg Mn is available. Given a total of 1.34 mg of Mn was quantified with the spores, the spores were able to collected
30% of the available Mn in solution.
4.5 Discussion
As in our previous report, spores of B. atrophaeus can have a significant, intrinsic, elevated paramagnetic susceptibility. However, in addition to merely equating the
96 magnetic susceptibility to the presence of Mn, in this study we were able to measure not only the amount of Mn per spore cluster, but also estimate the ratio of the different oxidation states of Mn. As noted above, some debate in the literature exists with respect to what oxidation states of Mn can be associated with the spores as well as the mechanism by which the Mn is oxidized. Cleary, the XPS analysis in this study demonstrates that the three typical oxidation states, Mn2+, Mn3+, and Mn4+ are present in
B. atrophaeus, but only Mn2+ was introduced in the sporulation media.
The general agreement between the two sets of independent experimental approaches (ICP-MS and CTV/XPS) to estimate the amount of Mn per spore cluster, suggests that biologically relevant amounts of Mn can be detected and measured in biological entities if the valance state of the Mn is known. Conversely, if the total amount of Mn is known, CTV measurements of the magnetically induced velocity can be used to make estimates of the valence state of atoms. For example, Zborowski et al. 2003
(Zborowski et al., 2003), were able to quantify the difference in the magnetophoretic mobility between oxygenated and deoxygenated red blood cells and to demonstrate that this difference is consistent with theoretical calculations in the difference between oxygenated and deoxygenated of hemoglobin, which corresponds to the valence state of the Fe atom contained within the hemoglobin molecule.
The amount of Mn per spore cluster in this study is actually relatively high compared to previous reports on the amount of Fe per cell that has been estimated using the CTV instrument. For example, the Fe content in a typical, single adult red blood cell is on the order of 1.3 × 10-13 g. More recently, Chalmers lab reported that the CTV
97 instrument can quantify the motion of the 1 µm, dextran Fe oxide particles from Stem
Cell Technology (Chalmers et al., 2010). The Fe content of these 1 µm particles is approximately 3.4 x 10-13 grams. In addition, the CTV instrument can detect on the order of 700, non-specifically bound MACS® particles (Miltenyi Biotech Inc., Auburn, CA) to a single cell. Using the magnetically induced velocity of the cell with these non- specifically bound MACS® beads, and the Fe in from of Fe3O4, the amount of Fe associated with each of these cells with non-specifically bound MACS® particles is on the order of 5 x 10-15 g.
The significant drop in magnetic susceptibility of the spore cluster with low pH or
EDTA, (Figure 4.2 and 4.3) and the corresponding decrease in cluster size are consistent with the general observation in the literature that the Mn oxides concentrate on the outside surfaces of Bacillus spores. Given the limitation of XPS spectroscopy of only providing information up to a penetration of only 10 nm, it is possible that deeper layers within the spore coating have different ratios of Mn oxides.
A number of potential applications can be envisioned with the use of these magnetic spore clusters, including a surrogate for food pathogens and a scavenger for contaminating heavy metals. The relative high intrinsic magnetic susceptibility of these spores introduces the concept that they can be used to determine if a sterilization process adequately destroys spores. Spiro et al. 2010, in addition to reviewing Mn oxidizing bacteria, summarized the ability of Mn(IV) to bind environmental contaminates such as
Pb(II) and Zn(II). The combination of the high magnetic susceptibility and the heavy metal adsorption capabilities of these spores, presents the possibility to use the spores and
98 a magnetic separation system, our previously reported continuous flow through system (Sun et al., 1998), to develop a
system to clean contaminated water.
Sample Mass Concen- Concen- Total Mn Total Fe Mn (g) g Mn per g Mn per of tration tration of recovered recovered per cm3 spore spore cluster (dry) of Mn Fe from (g) (g) gram spores from ICP-MS spore (mg) ICP-MS (µg/ml) (µg/ml) 99 Control 0.00 < 0.025 0.2 --- 4.1 x 10-6 ------Spores, no 5.0 40 1 8.04×10-4 2.01×10-5 0.268 0.322 3.28 x 10-11 sonication Spores, 5.0 60.5 1.5 1.34×10-3 3.32×10-5 0.160 0.192 1.96 x 10-11 sonication
Table 4.5 ICP-MS anlaysis of Mn content in spores
99 Chapter 5 Factors affecting the intrinsic magnetism of Bacillus spores
using spectra and fermentation studies
5.1 Motivation
As discussed in Chapter 1 and Chapter 2, Bacillus atrophaeus spores have been used as a surrogate for Bacillus anthracis. Since the paramagnetism of Bacillus spores makes it possible to separate the spores from air or the water without magnetic labeling
(Melnik et al., 2007), it is of interest to find the factors/variables effecting this magnetism, from elemental level, chemical analysis, genetic study as well as fermentation studies.
Chapter 4 establishes the relationship between Mn oxidation and spores’ magnetic susceptibility. According to the magnetic susceptibility of different forms of Mn oxides, as well as the composition of the Mn compounds found outside the spores, Mn (III) in
Mn2O3, Mn3O4, or oxyhydroxide plays an important role in the spores’ paramagnetism.
Mn oxidation, proceeds at a very slow rate abiotically, but can be accelerated by five orders of magnitude by microorganisms (Nealson et al., 1988; Wehrli, 1990; Tebo,
1991). Moreover, both Mn (III) and Mn(IV) will be preferably formed at high pH and in the presence of oxygen (Tebo et al., 2004).
Early research reported that hausmannite (Mn3O4) deposited on spores of the 100 Bacillus SG-1 (Hastings and Emerson, 1986), and amorphous deposits (Mn3O4) in isolated domains were confirmed by lattice imaging (Mann et al., 1988). However, the absence of Mn(III) intermediate (Mn3O4 or MnOOH) spectral features (Bargar et al.,
2000) challenges the Mn3O4 intermediate theory (Webb et al., 2005). In other words, the former theory supports that MnO2 can be slowly aged from Mn3O4, and the latter argues that Mn3O4 can be disproportionate from MnO2. The thermodynamic and kinetic studies
(Figure 5.1) showed that Mn3O4 is favored above pH 4 and at a higher concentration of
Mn.
Figure 5.1 Mn oxides equations under different combinations of pH and Mn concentration (Hastings and Emerson, 1986)
101 The importance of the potential to reliably produce these magnetic spores, the complex mechanism of the formation of Mn oxides, and the variety of magnetophoretic mobility (MM) from different batches encourage us to find a reliable way to produce spores with consistent magnetism. In addition, the understanding of fermentation conditions can also provide hints that can make other strains magnetic.
While Chapter 3 is focused more on the technical improvements and Chapter 4 demonstrates the relation between Mn oxidation state and the magnetic susceptibility, this chapter is divided into two parts: 1) Searched for other intrinsic magnetic Bacillus strains and following the chemical analysis used in Chapter 2, to examine whether there were similarities between intrinsic magnetic strains 2) Screened important factors for fermentation and optimized the process by using a statistical approach referred to as a response surface method.
5.2Materials and methods
5.2.1 Strains, growth curve and sporulation
Bacillus atrophaeus was obtained from the ATTC (#9372), and Bacillus sp SG-1,
B. cereus, B. magaterium, B. thuringiensis, B. sublilis were obtained from the Bacillus
Genetic Stock Center in the Department of Microbiology at the Ohio State University.
The growth curve was obtained first before the sporulation. An inoculating loopful was taken from reconstituted culture in LB broth, and streaked onto trypticase soy agar (TSA) in a three phase streak method. The inoculated TSA plate was incubated at 30°C for approximately 20 hours. The spores were made similarly as the sporulation method described before (Melnik et al., 2007).
102 To study the influence of sporulating condition on magnetism of spores, we controlled manganese concentration, oxygen availability and sporulation time. Mn concentration was varied, since Mn3O4 was found to form at pH 7.5 and in a high concentration of Mn2+ (>30μM) on Bacillus sp. SG-1 spore coat, otherwise γ-MnOOH or
MnO2 was formed (Mann et al., 1988; Green and Madgwick, 1991). Mn within 200- 600
μm was used in sporulation media. From previous study, no magnetism appeared within
60 hours, and the longest sporulation time reported for Mn3O4 production was 8 days
(Mann et al., 1988). Thus sporulation time was designed to vary from 3 days to 8 days.
Besides Mn concentration and sporulation time, oxygen availability may also affect oxidation of Mn. Oxygen availability was controlled by changing the size of flasks and working volume, so that air and culture interface area was varied. Model of MM versus
Mn concentration, flask volume, sporulating time was built and statistical modeling software JMP was used for optimum surface screening. Other ion (i.e. Fe, Cu, Zn) effects examined were insignificant to spores’ magnetism, and so was heat shock effect.
5.2.2 Sporulating using the Applikon bioreactor
After optimizing the conditions for stronger paramagnetism, the original flask
(250ml each) sporulating process was scaled up by using an Applikon bioreactor. The working volume was 2L, and an aeration rate of approximately 2L/min and an agitation rate of 300 rpm were used. The pH was maintained at 7.45 during sporulation. The temperature was held at 30o C through electric heating blanket. The collected spores were freeze-dried and stored as dry powder for future analysis.
103 5.2.3 X-Ray Photoelectron Spectroscopy
X-Ray Photoelectron Spectroscopy (XPS) was used to examine the elements on spores’ surface. The lyophilized magnetic bacteria on carbon tape were used directly for
XPS. All parameters were used the same as Chapter 4.
5.2.4 Cell Tracking Velocimetry
Spores were first freeze-dried at -10oC for 24 hours. The dried spores were suspended in deionized water at room temperature; with a final concentration of 107 spores/ml. Around 1 ml sample from each trial was pumped through a syringe to the channel in the Cell Tracking Velocimetry (CTV) instrument. CTV can measure the MM of hundreds to thousands of cells (or particles) at the same time (Chosy et al., 2003;
Zhang et al., 2005). The same parameters were chosen for B. atrophaeus spores, while other spores with less clumping issues had a smaller range of particle size as well as a smaller maximum for identification. The magnetically induced velocity of cells and MM were examined the same way as described before.
5.3 Results and discussion
5.3.1 Paramagnetism of Bacillus spores
A question that is commonly asked is: “Are there any other organisms with intrinsic magnetic?” While not necessarily the first,, the magnetotactic bacteria producing magnetosomes were reported 35 years ago (Blakemore, 1975). Moreover, the unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) make the erythrocytes paramagnetic compared to the diamagnetic character of oxyhemoglobin
(Zborowski et al., 2003). It was discussed before (Chapter 2, Chpater 4) that the
104 magnetism of spores could result from the oxidation of Mn on spores’ surface. Since there are some microorganisms that can also oxidize Mn, it is possible that those strains can be magnetic.
We first checked some common Bacillus strains (Table 5.1). Besides B. atrophaeus, SG-1 which can oxidize Mn, is also found to be magnetic. On the other hand,
B. cereus, B. mageterium, B. subtilis, B. thuringiensis (Francis and Tebo, 2002) which have been reported not to oxidize Mn, are found much less magnetic than SG-1 and B. atrophaeus. The rest of this part of the study will focus on the physiological condition for culturing/ sporulating B. atrophaeus.
Magnetic Magnetic Velocity Settling Velocity MM ×104 susceptibility (µm/s) (µm/s) 3 (mm /T•A•s) ×104 B.sp 19.9 1.77 1.40 2.00 SG-1 Bacillus atrophaeus 25.7 0.778 1.81 4.37 Bacillus -2 cereus 0.343 0.247 2.42×10 0.184 Bacillus -2 -2 magaterium 0.813 1.17 5.72×10 9.18×10 Bacillus subtilis 7.47 1.33 0.53 0.826 Bacillus -2 thuringiensis 1.36 0.825 9.59×10 0.186 Table 5.1 Comparison of magnetic velocity, settling velocity, MM and magnetic susceptibility of different strains of Bacillus
105 5.3.2 EDS spectra for Bacillus subtilis and Bacillus magaterium spores
EDS spectra for B. cereus, B. globigii, B. thuringiensis spores were published in
2007 (Melnik et al., 2007). The spectra for the other strains listed in Table 5.1 are shown
in Figure 5.2. Consistent with Chapter 2, the strains with less magnetism have only small
Mn peaks. However, it should be noted that peak height is only qualitative.
A)
C 4000
3000
2000
Intensity (arb.Intensityunits) O 1000
N Al P Ca Mn Mg S 0 0 1 2 3 4 5 6 7 8 9 10 Energy (keV)
Continued
Figure 5.2 EDS spectra for B. sp SG-1 (A), B. magaterium (B), B. subtilis (C)
106 Figure 5.2: Continued
B)
Continued
107 Figure 5.2: Continued
C)
5.3.3 Surface Scan for Bacillus strains using XPS
XPS is a quantitative surface technique, which can quantify the percentage of the
elements as well as their oxidation states. In this study, spores surfaces were scanned and
the elements on surface were compared between strains. It was noticed that only B.
atrophaeus and B. sp. SG-1 have Mn peaks in the spectra, potential Mn peaks for the
other strains are below the detection level for this technology. The peaks for Mn are
highlighted with dash lines in Figure 5.3 (D).
108 A)
Binding Energy (eV)
Continued
Figure 5.3 (A) Bacillus atrophaeus (B) B. SG-1 The peaks circled in (A) and (B) represent Mn. (C) B. cereus surface scan (D) the comparison of Mn peak area between B. atrophaeus (yellow), B. SG-1 (blue), B. cereus (red), B. subtilis (light blue) and B. megaterium (violet) (from top to bottom)
109 Figure 5.3: Continued
B)
Binding Energy (eV)
Continued
110 Figure 5.3: Continued
C)
Binding Energy (eV)
Continued
111 Figure 5.3 Continued
D)
Binding Energy (eV)
Besides Mn, there are some other elements shared between SG-1 and atrophaeus
(Figure 5.3): there are P, Zn, N, C on the surface of the spores, but no detectable Cl, S and maybe Ca, Mg under 1%. 112 5.3.4 Selection of culturing and sporulating media
The fact that more than one strain in Bacillus can be paramagnetic during culturing and sporulating raises further interest to explore the factors that can make these spores magnetic. Variability in spores’ MM (Melnik et al., 2007) also leads us to find out how the physiological condition affects spores’ magnetism and to try to maximize the magnetism. To use the intrinsic magnetic separation in Bacillus or more strains, it is crucial that the factors for the magnetism are elucidated and potentially controlled. There are several steps before final collection of the spores (Melnik et al., 2007): TSA plate culture, NB culturing media and Mod G sporulating media. TSA plate can make B. atrophaeus produce a characteristic pigment, which indicates the purity or contamination.
Thus, different culturing media and sporulating media were investigated.
LB and NB are two common culturing media, and at the same time SG is one of the most common liquid sporulating media. Even Mod G is not used as often as SG media; it was the sporulating media when we found the intrinsic magnetism of spores.
From Table 5.2, it is clear that culturing media doesn’t have much significant influence on the magnetophoretic mobility (MM) of the spores. However, on the other hand, at 5% significance level, spores from Mod G media showed stronger magnetism.
113 Culturing Media Sporulation media Mean MM Standard Deviation (×104 ) (mm3/T•A•s) (×105)
Nutrient Broth Mod G 1.65 4.34
Nutrient Broth SG 0.611 3.37
LB Mod G 1.18 1.77
LB SG 0.379 1.77
Table 5.2 The effect of culturing media (LB, NB) and sporulating media (SG, Mod G) to the MM of B. atrophaeus
5.3.5 Modifications of sporulation using Mod G media
Different forms of Mn oxides were reported to be found surrounding spores (Rosson and Nealson, 1982; DE Vrind et al., 1986; Mann et al., 1988). Among them, Mn3O4 is the second most magnetic form and it is the most stable among Mn oxides, according to magnetic susceptibility and thermodynamic parameters of Mn compounds in Table 5.3.
We believe that sporulation condition finally changes Mn states from originally Mn2+ to
Mn (oxyhydro)oxides and thus the magnetic susceptibility of spores. As discussed in the methods and materials earlier in this chapter, Mn concentration, oxygen availability and sporulation time were varied during spore production process to determine their influence on the magnetophoretic mobility of the spores.
114 Compound Susceptibility Heat of formation Free energy of (kcal/mole) formation (kcal/mole)
α Mn 529 0 0 β Mn 483 0 0
MnO2 4,850 -124.58 -111.49
Mn2O3 14,100 -229.50 -209.90 MnO 2,210 -92.04 -86.77
Mn3O4 12,400 -331.65 -306.22
MnCl2 N/A -112.00 -102.20
Table 5.3 Magnetic susceptibility and thermodynamic parameters of Mn compounds
The magnetophoretic mobilities and their corresponding conditions were listed in
Table 5.4 for these varied conditions. In the Mn column, number 1 stand for the regular concentration of Mn we used in the past; and other numbers represent the factor by which the Mn concentration was increased; i.e. 1.5 or 2 times of regular concentration. The actual concentration of Mn is listed in column ‘Mn concentration’ with unit of M. The unit for volume column is ml. Column N stands for the number of spores detected through CTV. For a clear demonstration of analysis, the one way ANOVA result is given in Figure 5.4. Within each diamond in Figure 5.4, its center shows the mean of each sample. The tops and bottoms of the diamonds represent the 95% confidence intervals
(CI). P-value for Mn concentration, sporulating days and working volume in 250 ml flask is <0.0001, 0.84, 0.60 correspondingly. At a significance level of 5%, Mn concentration is the only significant factor among the three.
115 Mn concentration MM(×104) Mn Volume (ml) Days Sample 3 N ×104 (M) (mm /T•A•s) 1 50 2.20 5 1 0.747 795 1 50 2.20 5 1 0.690 742 1 50 2.20 5 2 0.622 770 1 50 2.20 5 2 0.532 873 2 50 4.40 5 1 2.87 1851 2 50 4.40 5 1 2.35 1072 2 50 4.40 5 2 2.50 1339 2 50 4.40 5 2 2.40 1251 1 50 2.20 3 1 0.140 143 1 50 2.20 3 1 0.569 1381 1 50 2.20 3 2 0.823 301 1 50 2.20 3 2 0.620 557 1 50 2.20 3 2 0.201 1169 2 50 4.40 3 1 2.21 1192 2 50 4.40 3 1 3.27 1107 2 50 4.40 3 2 3.47 481 1.5 100 3.30 4 1 1.43 2309 1.5 100 3.30 4 1 1.45 2237 1.5 100 3.30 4 2 1.29 1611 1.5 100 3.30 4 2 1.28 1708 1 150 2.20 3 1 1.13 421 1 150 2.20 3 1 0.848 961 1 150 2.20 3 2 0.832 1936 1 150 2.20 3 2 0.856 1185 2 150 4.40 3 1 4.63 376 2 150 4.40 3 1 4.64 898 2 150 4.40 3 2 0.832 1936 2 150 4.40 3 2 0.856 1185 1 150 2.20 5 1 0.800 6317 1 150 2.20 5 1 1.17 7765 2 150 4.40 5 1 2.05 1784 2 150 4.40 5 1 3.02 2135 2 150 4.40 5 2 2.52 5125 2 150 4.40 5 2 2.36 1497 Table 5.4 The effect of Mn concentration, sporulation time and working volume during the sporulation on MM of B. atrophaeus
116 A)
B)
Continued
Figure 5.4 One way analysis of MM by Mn concentration (A), Volume (B), Sporulating days (C)
117 Figure 5.4: Continued
C)
5.3.6 Ion effects during fermentation
After Mn concentration has been found as a factor to optimize magnetism, other ions, such as Zn, Cu, Fe were also tested in a Design of Experiment (DOE) approach. Fe was chosen, because it is another paramagnetic element. Since Mn oxidase is believed to be a multicopper oxidase, Cu was selected as another factor. In addition, small amounts of Cu2+ were found to increase the Mn(II)-oxidizing activity of wild-type cells by a factor of five (Francis and Tebo, 1999). Increase or decrease in Cu concentration may help the spores oxidize Mn to a more magnetic state. Zn is an important co-factor for some enzymes in the bacteria, and also can assist in germination (Johnstone et al., 1982).
Table 5.5 lists the magnetophoretic mobility of spores under different ion
118 concentrations. Ideally, a larger range should be tested to find the optimal combination.
Due to limited time and complexity of the DOE, we only tried a smaller range of the concentration: doubled or halved the ion concentration relative to the Mod G media.
Zn concentration Cu concentration Fe concentration Magnetophoretic Mobility (µM) (µM) (µM) (×104) p-vaule=0.38 p-vaule=0.08 p-vaule=0.22 (mm3/T•A•s) Run 1 Run 2 8.7 10 1.8 1.46 1.13 3.6 1.21 1.61 40 1.8 2.23 1.85 3.6 1.46 2.23 34.8 10 1.8 1.52 1.97 3.6 1.57 1.68 40 1.8 1.40 1.34 3.6 1.84 2.78 17.4 20 1.8 1.22 1.33 Table 5.5 The effect of ions
The same one way ANOVA analysis and t-test between each pair (only one ion considered as a dependent variable) demonstrate no significant effect from those ions in the concentration range tested. P-value for each ion effect is also listed in Table 5.5.
However, this only applies to the range studied in this chapter. Extremely high or low level concentration may influence the final magnetophoretic mobility. However, in that case, toxicity should be well considered.
5.3.7 Iron effects during fermentation
Since Fe is another paramagnetic element, higher concentrations and also extremely low concentrations were tested during sporulation. Zn and Cu concentration
119 was kept at normal concentrations, while Fe concentration was defined from 0 to 180 µM.
An important observation was that when no Fe was present in the sporulating media; the
MM of spores didn’t decrease (Table 5.6), relative to normal conditions. This result agrees to previous conclusion that the major cause for magnetism is Mn (oxyhydro-) oxides, while the effect from Fe is negligible. P-value for iron effect is 0.69, which means at a significance level of 0.05, the effect is not significant. When the iron concentration is increased to as high as 180 µM, there appeared iron precipitates. It is highly likely that these precipitates cannot be taken by the bacteria as effectively as free ion in the media.
Zn concentration Cu concentration Fe concentration Magnetophoretic Mobility (×104) (µM) (µM) (µM) (mm3/T•A•s) Run 1 Run 2 17.4 20 0 2.04 2.12 18 1.14 2.4 90 2.37 2.34 180 1.92 2.30 Table 5.6 The effect of iron
5.3.8 Heat shock after sporulation doesn’t increase the intrinsic magnetism
Heat treatment of spore forming bacteria is typically used for the dual purposes: to kill unsporulated organisms present and to activate (heat shock) the spore forming bacteria, such as Bacillus, rendering them more predisposed to germination (Turnbull et al., 2007). Optimal heat shock conditions may vary from species and media (Turnbull et
120 al., 2007). For example, the activation of B. anthracis is equivalent at 60 °C for 90 min or boiling for 1 min (Turnbull et al., 2007).
Heat Shock conditions were investigated in this study since it has been suggested that heat shock may have a different effect on Bacillus atrophaeus compared to B. anthracis and Bacillus spores have been reported to clump during sterilization (Furukawa et al., 2005).
After sporulating for at least 72 hours, B. atrophaeus spores were heat shocked for 0, 30, 60 min. One way ANOVA analysis and t-student between any two treatments shows no significant change in MM by changing the heat shock time (Table 5.7), at a significance level of 5%. The p-value is 0.13. This is consistent with the report that B. anthracis didn’t demonstrate any significant changes as a function of heat shock temperature. However, MM was not measured by Turnbull et al. Moreover, aggregation of spores was observed before heat shock, which can minimize the effect of heat, but other heat shock condition may increase the spore aggregation on a larger scale.
MM (×104) Heat Shock time (mm3/T•A•s) 0 1.43 0 1.61 0 1.40 30 1.76 30 2.09 30 1.76 60 1.79 60 1.87 60 1.39 Table 5.7 The effect of heat shock after sporulation on MM 121 5.3.9 Response surface analysis
The factorial designs we discussed in earlier sections are for factor screening and predictive models build-up. However, the predictive capability of the resulting models is limited by response surface contains highly localized features. Response surface methodology was used to optimize the sporulating process. Response surface analysis was done for section 5.3.5- 5.3.9. And it showed an optimal sporulating condition using
2.8×10-4 M.
5.3.10 Fermentation by bioreactors
To produce larger quantities, and have a sufficient number of spores from a single batch for a number of different analytical studies, I attempted to scale-up the process of producing the magnetic spores. Before using a bioreactor, the typical working volume for cultivating the spores was either 250ml in 1L flask or 100ml in 250ml flask. The
Applikon and Biostat B bioreactor increased the working volume to 2L or 4L, respectively. The reliability of this process is indicated in Table 5.8. Based on hundreds and thousands of spores per trial, all the mean magnetophoretic mobilities listed below were within a 95% confidence interval with the average “mean MM” of 2.36×10-4. While it is encouraging that we can produce reliable intrinsic magnetic spores in a large quantity, we still need to consider the following question: Can we apply the same condition to B. sp. SG-1 or other slightly magnetic strains? B. sp SG-1 spores provide to be nearly as magnetic, when compared to B. atrophaeus spores using the unmodified culturing conditions. However, different strains showed different preference for growth and sporulation, and the optimized media for other strains still need further study.
122 5.4 Conclusion
The two directions of study in this chapter both demonstrate the importance of
Mn in the intrinsic magnetism of spores. Again, EDS spectra gave comparatively larger peaks for magnetic strains. From the surface scan of XPS, Mn was only found in magnetic strains: B. atrophaeus and B. sp. SG-1. From factor screening experiments, increasing Mn concentration led to increase of spores’ MM or stronger magnetism; while the change of other factors didn’t show much affect. With the defined factors, response surface analysis suggests a concentration of 2.8×10-4 M Mn can give the best result.
Followed the modification of the sporulating media, reliable magnetic spores were obtained from bioreactors on a larger scale. Thus enhances the probability to separate spores from air or water without magnetic labeling.
MM ×(104) Number of spores detected by CTV 1 2.43 615 2 2.47 1485 3 2.38 2737 4 2.35 1977 5 2.18 851 Table 5.8 MM of spores from bioreactors
123 Chapter 6 Genetic studies of Bacillus atrophaeus
6.1 Introduction
The enzyme responsible for Mn oxidation in Bacillus SG-1 spores is probably
MnxG (Van Waasbergen et al., 1996). The mnxG gene encodes a protein about 138 kDa in size and 1218 amino acids in length (3,654 nucleotides). The sequence of this gene shares similarities with the multicopper oxidase family, a group of diverse proteins that use multiple copper ions as cofactors in the oxidation of a variety of substrates (Figure
6.1). The secondary structure of MnxG may be a β-strand structure around the copper binding sites by prediction from the sequence. The putative oxidase is located in the exosporium, a loosefitting, membranous layer composed of protein, lipid, carbohydrate and polysaccharide (Francis and Tebo, 2002).
5353 138138 255255 348348 486486 590590 692692 779 875875 976 11061106 12011201
Figure 6.1 Six homologous domains to other multicopper oxidases in MnxG in B. SG-1. The start and end of each domain are shown above each domain
124 A related cluster that is involved in Mn oxidation may be an operon containing genes mnxA~mnxG (Bacillus sp. strain SG-1 spore-associated Mn oxidation gene cluster).
Upstream mnxA is the putative -35 and -10 regions, GGCAGA and TATATATT (Van
Waasbergen et al., 1996). Although sequences of those genes are known, little information is available regarding their function. mnxA, encodes a protein about 103 amino acids in length. mnxB, is a 465bp stretch of nucleotides. mnxC consists of 588 bp, and possibly encodes a disulfide oxidoreductase. mnxD encodes a protein of 255 amino acids long. For mnxE, mnxF, the length for both genes is about 300bp. The deduced proteins both have 100 amino acids.
By manipulating mnxG, we might make other strains, such as E.coli, oxidize Mn to magnetic Mn3O4. Thus, other organisms would be magnetic and detected by Cell
Tracking Velocimetry and could potentially be separated in magnetic separation systems.
We have found that Mn oxidation process is important to intrinsic magnetism and accordingly Mn oxidase plays an indispensable role in the magnetism. MnxG has been found to be involved in Mn oxidation (Francis et al., 2002), thus it might be the main reason for spores’ magnetism. The corresponding gene has been sequenced (Van
Waasbergen et al., 1996) and also disruptions of the operon have been created in 1993
(Vanwaasbergen et al., 1993). If we can make similar knockouts for B. atrophaeus spores and compare the magnetism between disruptive and wild type and also compare those for
B. SG-1, we will see whether this operon is essential for the magnetism observed in B. atrophaeus spores.
125 6.2 Materials and methods
6.2.1 Strains
B. atrophaeus was obtained from ATCC and B. SG-1 was obtained from Bacillus
Genetic Stock Center in the Ohio State University. Both strains were cultured at 37oC at
200 rpm till log phase. Then the genome of both strains was extracted followed Qiagen
Genomic tip kit.
6.2.2 Validation of mnxG homolog using PCR and sequencing
PCR was used to check for the presence of gene homologous to mnxG in Bacillus atrophaeus. Primers were designed based on conservative region of mnxG in Bacillus
SG-1 : 5’ AcI cAY gTI TTY cAY TAY cAY gTN cA and 5' AA IAR RTg RTc RTg
RAA RAA cc. The primers previously published were also used for PCR. (Van
Waasbergen et al., 1996). Chromosome DNA was extracted using Qiagen Genomic-tip as described above. The PCR cycle was as follows: 30 cycle of 94oC for 30s, 45 oC for 30s,
60 oC for 1 min; then 72 oC for 15 min and 4 oC for ever The PCR product of mnxG in
Bacillus SG-1. The PCR products were examined by DNA gel, and the band around 900 bp was then cut out of the geand used as the templates for a new cycle of PCR. Then product was subsequently inserted into TOPO 2.1 vector for sequencing.
6.3 Result and discussion
Figure 6.2 shows the PCR result for the amplification of the conservative fragment in mnxG gene. Around 900 bp, which is the size of the conservative sequence, was successfully amplified for B. atrophaeus and B. SG-1. However there were multiple
126 bands for both of the PCR product. Further studies need focus on optimizing PCR conditions or using other primers.
mnxG- knockout strains were requested from Tebo’s lab. Those strains couldn’t oxidize Mn anymore. By comparing the magnetophoretic mobility of the wild type spores and the mutant spores, we can determine whether the gene is responsible for the magnetism. However, we haven’t received any mutants yet.
1 2 3 4
Figure 6.2 DNA gel for PCR product. Conservative sequence in mnxG was amplified for B. atrophaeus (lane 1) and B.SG-1 (lane 4). Lane 2 is a positive control for the PCR, approximate 850 bp. Lane 3 is the marker
127 Chapter 7 Spore separation from the liquid food via magnetic
deposition system
Part of the following work (sample preparation, sterilization, acid treatment and magnetic deposition separation) was done by Yuki, Sing, Daniel and Doug under my supervision in CHBE 762.
7.1 Motivation
Food borne illness is usually caused by infectious microbes or toxins in contaminated food. The statistical survey from “Harv Mens Health Watch” reported that roughly 76 million Americans get sick from something they ate, about 350,000 end up in the hospital, and some 5,000 die from their infection. In the last decade, more than 10 infectious bacteria have been newly found to be associated with food borne disease
(Tauxe, 1997). Most bacteria can be killed during sterilization process; however, spores, extremely resistant to heat and other preservation treatments in comparison to vegetative cells, require high temperatures and long heating times for inactivation which are costly and detrimental to the nutritional and organoleptic quality of most food products (Kort et al., 2005). Unfortunately, many food poisoning cases were caused by canned food, among which spore forming bacteria, i.e. Clostridium botulinum, commonly
128 contribute (Devers and Nine, 2010). Bacillus cereus, a spore-forming Gram-positive strain, is another major pathogenic bacterium that causes food poisoning and produces gastrointestinal diseases of two types: emetic and diarrheal. Seventy-three grains including rice of the staple food in Korea have been reported contaminated by B. cereus, and thirteen outbreaks of diarrhea have been reported during seven years since 2001.
However, it was claimed the disease associated with B. cereus has been underestimated since the symptom is similar to the food poisoning caused by Staphylococcus aureus
(Kim et al., 2010). Consequently, spore survival and inactivation mechanisms are very important to finding new processes that can extend shelf life and meet regulatory mandates for improved food safety (Fujii et al., 2002; Uemura and Isobe, 2003). These concerns, along with defense of the food supply present an urgent need for techniques that can accurately and efficiently predict the presence of viable spore cells at every step of the food production process (Armstrong et al., 2006).
Unique challenges for detecting pathogens in food result from wide variations in the physical characteristics, chemical composition of food and the extent of food processing, etc (Swaminathan and Feng, 1994). Tests for pathogens must be sensitive, since the some food would not usually go through a process that could be lethal to the bacteria after sampling and before food consumption. Moreover, microorganisms in food may exist in a low level after processing but multiply to hazardous levels during food distribution and storage (Swaminathan and Feng, 1994).
Magnetic separation has been used for a long time for cells, and the advantages comparing to other techniques are apparent (Chapter 1 and 2). It is also claimed to be
129 very promising (Swaminathan and Feng, 1994) in sequestering the pathogens, and developing the specificity of antigen-antibody binding as well as a wide range of other receptor-ligand interaction, all of which will suggest this technique grow significantly.
Another direction of magnetic separation is to develop label-less, magnetic separation/detection through the use of the intrinsic paramagnetism.
Bacillus spores were reported in Chapter 2 with as high magnetophoretic mobility as labeled cells and a slide with deposited spores using magnetic deposition system was presented as well. B. atrophaeus spores with comparative high intrinsic magnetism indicate a potential to be separated without labeling from liquid food, using the deposition system.
Even though spores have been successfully separated from PBS and water, whether spores can be separated from food has not been demonstrated. In this chapter, a magnetic deposition system along with CTV will be used to separate spores spiked into liquid food; the recovery of magnetism of spores from acid (simulate juices) will also be discussed.
Magnetophoretic mobility was examined, compared and discussed along with magnetic separation. It can reflect the magnetic susceptibility of spores. The following formula obtained from Chapter 4, Equation 4.8 was used to analyze the average diameter of the spores or spore clusters.
1/ 2 18 Dsphere vg g (7.1)
130 7.2 Experimental scope
The rationale of why we used B. atrophaeus spores to mimic the hard-to-kill bacteria in food is as follows: 1) B. atrophaeus has been used for sterilization control organism (ATS, Inc), and it is a surrogate for B. anthracis (Robinson et al., 2010; Talbot et al., 2010). Other Bacillus, like Bacillus cereus, can cause food poisoning. 2) It is intrinsic paramagnetic, and can be separated using a deposition system without labeling, which makes the separation fast and cheap. 3) There is a potential that we can either find, or genetically engineer other bacteria/spores to be magnetic.
In this study, the effectiveness of the autoclave process typically used to kill spores was evaluated through spore separation before and after sterilization using the deposition system. The effect of pH treatment for spores and Mn recovery after pH treatment was also investigated. Since this project is at the initial, investigative, proof of concept stage, instead of using 103-104 cfu in milk or other liquid food (a typical threshold for contamination), we used a much higher concentration of the spores, to determine whether the separation would work.
It was soon discovered that juices were challenging for the use of the magnetic spores, due to the low pH. To get a better idea of how the pH influences magnetic properties of spores and the spores’ ability to sporulate after a low pH treatment, solutions of pH 1.8, pH 2.7, pH 5 and water (control) were used to suspend the spores.
After overnight low pH treatment, CTV and the deposition system were applied. Mn (20
µl, final concentration of 2×10-4M) was added in to the sample to examine the potential
131 for recovery of magnetism. Successful recovery of magnetism implies the spores in juices can be made magnetic by adding more Mn.
The chapter has three parts: 1) Apply food samples (water, chicken broth) with
107cfu/ml spores to deposition system. The mixture was tested by pumping it through the deposition system, to examine whether the spores can be deposited on the slides, while the food goes through the system to get collected.
This part mainly determines whether this instrument could be used to separate spores in the liquid food based on the spores’ magnetism. For food selection, water is chosen as a control. Filtered chicken broth was selected because it can be autoclaved and has a rich, viscous look. Milk was not chosen for autoclave because the proteins in milk would denature during sterilization. According to preliminary result from the lab, spores in milk can be separated using deposition system too (Data not shown).
2) To examine whether the magnetic properties of spores in food changed after sterilization. The food was spiked using the same concentration of spores. The autoclave cycle was 30 min, 121oC. In this step, both CTV and magnetic deposition system were applied.
3) pH treatment of the spores overnight. Solutions of pH 1.8, pH 2.7, pH 5 and water (control) were used. The difference of the magnetism prior to, and after low pH treatments was examined by CTV. The treated samples were applied to the deposition system also. After the spores suspensions were returned to a neutral pH, Mn was added to determine if the magnetism was restored.
132 7.3 Materials and methods
7.3.1 Strains and treatment
B. atrophaeus from ATCC (#9732) was obtained using the method described in
Chapter 5. The freeze-dried powder of B. atrophaeus spores were resuspended either in water or chicken broth to make a final concentration of spores to 107, 106, 105 and finally
104 cfu/ml separately. Vitamin water and chicken broth were purchased from nearby grocery store. The clumps in chicken broth, which may possibly block the magnetic deposition system, were filtered out before suspending the spores. The spore suspensions were sterilized at 121o C for 30 min. HCl was used instead of juice for the spore separation from juice. Different concentrations of HCl gave final pH’s as follows: 1.8,
2.7 and 5. The spore suspensions were vortexed for each trial. After sterilization or HCl treatment overnight, CTV and magnetic deposition system were applied again for the spores.
7.3.2 Cell Tracking Velocimetry
As reported previously, Cell Tracking Velocimetry (CTV) was developed to quantify the MM of cells and particles; either intrinsic paramagnetic or imparted magnetic through binding of antibody magnetic particle conjugates. CTV can measure the MM of hundreds to thousands of cells (or particles) simultaneously, allowing significant populations sizes to be characterized (Chosy et al., 2003; Zhang et al., 2005).
The spore suspension either in vitamin water or chicken broth (before or after sterilization) will be centrifuged at 4000 ×g to discard the supernatant. The pellet was resuspended again in distilled water with the final concentration of 107 cfu. A sample of
133 around 1ml each trial was pumped through the syringe into the channel and analyzed as previously described.
7.3.3 Separation of spores
The magnetic deposition device is created by a fringing field at the interpolar gap, and combined with a thin flow channel pressed against the interpolar gap creates very high forces for the cell (spore) capture from the suspension. 500-600 µl spores were delivered in a continuous manner into the flow channel (s) by syringes connected to inlet tubing, and evacuated from flow channels by outlet tubing leading to waste containers.
The flow channel cross-section was 6.4 mm × 0.25 mm and the volumetric flow rate was
0.7 ml/hr. The flow channel was disassembled with caution to avoid air intrusion which could change the deposition lines. The plastic sheet with the magnetic cell deposition was washed and stored in 70% ethanol.
7.3.4 Counting of B. atrophaeus spores
B. atrophaeus spore suspensions before and after the separation were serially diluted with sterile distilled water, and 10 μl of 10-2, 10-3 and 10-4 dilutions were plated on
TSA plates. Caution was taken to spread the inoculums evenly but to the edge of agar plates. The plates were kept upside down in 30°C incubator overnight. The colonies formed on the plates were counted, and the concentration of spores in the suspension was calculated based on the colony number reading, which was usually between 20 and 200.
134 7.4 Result
7.4.1 Magnetic deposition before treatment
Spores of different concentrations were pumped into the magnetic deposition system. High concentration like 107 cfu/ml were applied first to prove the basic concept: whether the magnetism of spores is strong enough to be separated using the current deposition system (under the current magnetic field). After deposition line was acquired several times, we lowered the concentration to 106,105 and finally to 104 cfu/ml. We succeeded in getting spores deposited in the middle of the slides (Figure 7.1). The suspensions before and after the deposition system were shown in Figure 7.2. Since there is an obvious pigment of the spores, the suspension before treatment was pale orange; after separation became clear.
The efficiency of depletion of paramagnetic spores from water was calculated with the colony numbers on TSA plates before and after the separation (Table 7.1). For all the trials, the plated volume of spores was 10 µl. For the controls, the suspensions were diluted to 10-4, and for the suspension after separation, 10-2. The efficiency of depletion is 98-99%.
Trial Control After Deposition System 1 83 61 2 87 160 Table 7.1 The colony counts on TSA plates before and after the spores going through the magnetic deposition system (The suspensions were diluted to 10-4, and for the suspension after separation, 10-2)
135 Figure 7.1 The slide with spores deposited. The circled regions correspond to the characteristic deposition bands obtained with this instrument.
After Before
Figure 7.2 The suspension of spores before and after magnetic deposition system
136 7.4.2 Magnetophoretic mobility and magnetic deposition after sterilization treatment
Table 7.2 showed that the average magnetophoretic mobility didn’t change much after sterilization. Moreover, the distribution of both settling velocities and magnetic velocities didn’t change much according to Figure 7.3. We plated the spores before and after treatment and found the survival rate was 136:1; which means most of the spores were inactivated but the surviving spores managed to keep the magnetism. The inactivation of most spores is also revealed by Figure 7.3, since the density of the scatter plot is obviously less than the spores in water, or chicken broth after sterilization. All of the samples could deposit on the slide through magnetic deposition system in a similar way shown in Figure 7.1.
Before Chicken Broth DI water after Sterilization after sterilization Sterilization Run 1 2.23 2.49 1.94 Run 2 2.11 1.83 1.74 Table 7.2 Magnetophoretic mobility (×104) (mm3/T•A•s) of B. atrophaeus before and after sterilization
137 A)
-3 x +3 0.025
0.020
0.015 +3
0.010
0.005 x
0.000
Settling VelocitySettling (mm/s) -0.005 -3
-0.010
-0.015 -0.05 0.00 0.05 0.10 0.15 0.20 Magnetic Velocity (mm/s)
Continued
Figure 7.3 Scatterplots of spores settling velocity vs magnetic velocity in DI water before(A) and after sterilization (B). C showed the scatterplot of the spores in chicken broth after sterilization. Long dash lines stand for three standard deviation from the average velocity; while dotted lines refer to the mean.
138 Figure 7.3: Continued
B)
-3 x +3 0.025
0.020
0.015 +3
0.010
0.005 x 0.000
Settling VelocitySettling (mm/s) -0.005
-0.010 -3
-0.015 -0.10 -0.05 0.00 0.05 0.10 0.15 Magnetic Velocity (mm/s) C)
-3 x +3 0.025
0.020
0.015 +3 0.010
0.005 x 0.000
SettlingVelocity (mm/s) -0.005 -3 -0.010
-0.015 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 Magnetic Velocity (mm/s)
139 A)
-3 x +3 0.025
0.020
0.015 +3 0.010
0.005 x 0.000
SettlingVelocity (mm/s) -0.005 -3 -0.010
-0.015 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 Magnetic Velocity (mm/s) Continued
Figure 7.4 Scatter plot of magnetic velocity vs settling velocity of spores treated in pH 2.7 (B) overnight and control (A). After that, Mn was added into the same sample (C)
140 Figure 7.4: Continued
B)
-3 x +3 0.020
0.015
0.010 +3 0.005 x
0.000 SettlingVelocity (mm/s)
-0.005 -3
-0.010 -0.02 0.00 0.02 0.04 0.06 0.08 Magnetic Velocity (mm/s) C)
-3 x +3 0.020
0.015
0.010
+3 0.005
Settling Velocity Settling (mm/s) x 0.000 -3
-0.005 -0.01 0.00 0.01 0.02 0.03 0.04 0.05 Magnetic Velocity (mm/s) 141 7.4.3. Magnetophoretic mobility of spores after pH treatment
Table 7.3 showed that the average magnetophoretic mobility changed significantly in the low pH solution. Moreover the distribution of both settling velocities and magnetic velocities also changed significantly when a comparison is made between
Figure 7.4A and Figure 7.4B. Figure 7.4A implies a much smaller range for magnetic velocity. The survival rate again showed lots of spores were inactivated in low pH.
Spores in pH 5 could be deposited on the slide and spores from lower pH treatment didn’t form obvious bands on slides, which is also consistent with the loss of magnetism.
7.4.4 Magnetism recovery after adding Mn back in to the spores’ suspension
After pH treatment, we added extra Mn into the spore suspension, and magnetism was recovered the next day according to the analysis from CTV (Table 7.3). The recovery of the magnetism not only implies that the surviving spores in juice or other low acid food can be separated by adding more Mn in a neutral pH conditions; but also the recovery of magnetism also provides a potential to recycle spores that can be used in other areas, like decontamination.
The sizes of spore clumps were also analyzed using the method described in
Chapter 4. It has been shown that spores form clumps in suspensions and that these clumps decrease the spore inactivation rate during heat sterilization (Furukawa et al.,
2005; Mamane-Gravetz and Linden, 2005). The sizes listed in Table 7.4 show clumping after sterilization. Even comparing to the pre-sterilization samples, there is no significant change in the size. The acid treatment again shows the similar result as Chapter 4, which
142 is the decrease in the size. However, it seems the size of spore clumps increases a little after addition of Mn, along with the recovery of magnetism.
Regarding to the recovery of magnetism, we may question that whether the addition of Mn to the collected spores after fermentation can change the magnetophoretic mobility. The data are listed in Table 7.5 and at a significance level of 5%; there is no increase of the magnetophoretic mobility for both magnetic and nonmagnetic spores. This may result from the formation of Mn oxides during the sporulation (Tebo et al., 2004), and in this case have been indicated in a “saturated” state for Mn.
Treatment Control After Sterilization After pH Treatment After addition of Mn Run 1 2 1 2 pH=1.8 pH=2.7 pH=1.8 pH=2.7 Size (µM) 4.7 5.0 4.6 4.3 2.8 3.7 3.4 4.1 Table 7.3 Sizes of spore clumps before and after treatment
Control(×104) Add Mn(×104) 1 2 1 2 Paramangetic 2.26 2.59 1.68 2.73 spores Diamagnetic 6.31×10-2 5.53×10-2 0.105 0.117 spores Table 7.4 Magnetophoretic mobility (mm3/T•A•s) of spores (without any treatment) before and after adding extra Mn
143 7.5 Discussion and conclusion
The first part of experimentation tested the magnetic properties of Bacillus spores under sterilization conditions. The magnetophoretic mobility, a measure of the magnetic properties of an object, is an excellent indicator for the effects of sterilization on Bacillus spores. The magnetophoretic mobilities measured during this project indicate no significant differences in the magnetic properties of the spores pre- and post-sterilization for both the chicken broth and water samples. Note that all the measurements were made based on at least hundreds of spores. Even though mean magnetophoretic mobilities were listed in the table above, the measurement was actually on a spore by spore basis. The observation that spores formed a deposition band for each sample analyzed is also a strong indicator that the sterilization process does not significantly alter the inherent magnetic present in Bacillus spores.
The effectiveness of the autoclave process can be obtained through comparison of plated sample colony counts. The spore population of CTV images before and after sterilization, as well as the scatterplots shown in this chapter also supports the loss of spores. A dramatic decrease in colony counts after sterilization can be noted in Figure 7.3
(B) and (C). This figures coupled with the consistency in measured magnetophoretic mobility indicate that spores which manage to survive the sterilization process, though few they may be, will remain unaltered as far as their magnetic properties are concerned.
The large size compared to a single spore (Carrera et al., 2008) from the calculation agrees to the fact that large spore clusters are detected through CTV, and thus another indirect proof that spores tried to aggregate under heat treatment.
144 The second part of the experiments focused on determining the effects of pH on the magnetic properties of Bacillus spores. The low pH conditions were designed to simulate conditions found in many commonly processed drinks such as grape and apple juice. Table 7.3 implies that the magnetophoretic mobility of Bacillus spores decreases with decreasing pH. The more acidic, the more free protons there are in the solution, which can compete with the Mn on the spore surface.
Further tests revealed that the lost magnetophoretic mobility could be regained.
The introduction of highly concentrated Mn solution to spores which were exposed to low pH’s caused an increase in magnetophoretic mobility. This indicates the possibility of Mn recovery on the surface of Bacillus spores. Reintroduction of magnetic properties to the spores allows for separation of spores from juices as a possible application of this technology.
145 Chapter 8 Potential application of Bacillus atrophaeus for contaminated
water with heavy metal ion
8.1 Introduction
8.1.1 Heavy metal contamination source
Heavy metal ions can be toxic for animals and human beings when they are not metabolized by the body and accumulate in the tissues. However, unfortunately, they have been excessively released into the environment due to rapid industrialization and have created a widespread problem (Ngah and Hanafiah, 2008). Cadmium, zinc, copper, nickel, lead, mercury and chromium are often detected in industrial waste water (Ngah and Hanafiah, 2008). As a result of food processing equipment, the storage equipment, the delivery equipment, As, Cd, Hg, Pb are most frequently monitored/observed toxic elements in food, and to a lesser extent, Al and Sn (Capar et al., 2007). Moreover, over
50% of lead emissions to ambient air originated from petrol in the last century (Jarup,
2003). E-waste, referring to discarded electronic appliances (i.e. computers and mobile telephones) is another source of heavy metal contamination to the environment
(Robinson, 2009). Many contaminants are spread into the air via dust, exposed to humans through ingestion, inhalation and skin adsorption (Mielke and Reagan, 1998).
146 Residents in areas where heavy metal recycling is conducted, typically have a much higher Pb, Cr concentration in their blood, and exhibit chromosomal aberrations at a rate of around 20-fold higher than normal. It was claimed that co-exposure to Co and Pb or
Cd may cause genotoxic effect (Hengstler et al., 2003).
8.1.2 Lead effect
It is generally reported that lead has no positive biological function but is considered one of most toxic metals. Unfortunately, it is has been used more often than other metals in a wide range of industrial applications (source and emissions in Table
8.1). The symptoms of acute lead poisoning are headache (Silbergeld, 1992), irritability, sleeplessness, restlessness, abdominal pain and various symptoms related to the nervous system (Jarup, 2003). The health effect of childhood Pb exposure is a growing concern
(Shotyk and Le Roux, 2005). Even the blood Pb levels are well below generally accepted safe levels, deleterious effects have already been seen (Shotyk and Le Roux, 2005).
Source Emission Natural Ratio emissions Anthropogenic/ natural Vehicles 88,739 2600 34.1 Non-ferrous metallurgy 14,815 2600 5.7 Fossil fuel combustion, stationary 11,690 2600 4.5 sources Iron and steel production 2,926 2600 1.1 Waste incineration 821 2600 0.3 Cement production 268 2600 0.1 Total 119,259 2600 45.8 Table 8.1 Predominant anthropogenic ources of Pb to Atmosphere, tons per year (Shotyk and Le Roux, 2005)
147 8.1.3 Nowadays techniques for contamination
For removing toxic heavy metals such as Hg2+, Cd2+, and Pb2+ from solutions, adsorption by activated carbons and ion exchange resins are considered as effective techniques (Delgado et al., 1998; Rengaraj et al., 2001), except the fact that they are not quite efficient to eliminate the heavy metal ions at a low concentration. While some researchers work on modifying activated carbon (Kadirvelu et al., 2001; Monser and
Adhoum, 2002), others are trying to find biological or chemical sorption surfaces to increase the removal of toxic ion, like polyaniline (Gupta et al., 2004; Wang et al., 2009).
Polyaniline was synthesized and used as an adsorbent for removal of Pb2+ from aqueous solution based on its electrochemically controlled ion-exchange properties. At the same time, biogenic manganese oxide showed considerably higher Pb sorption capacity than synthetic manganese oxides (Nelson et al., 1999). Moreover, Pb(II) will only be attached above pH 6, due to the competition of the protons (Villalobos et al., 2005). Higher pH also results in a higher adsorption of Pb to the storage tubes (even though negligible compared to adsorption treatment), and reduced pH is likely to increase soluble Pb in natural aquatic systems (Nelson et al., 2002). From the study of the mechanism for the
Pb sorption (Villalobos et al., 2005), no Mn(IV) reduction and Pb(II) oxidation has been found.
8.1.4 Recyclable biogenic Mn oxides on Bacillus atrophaeus spores
Based on the combination of the following concepts, a hypothetical process to recycle spores, as well as Mn oxides on their surfaces, was designed (Figure 8.1):
148 1) Biogenic Mn oxides can absorb Pb efficiently, and this has been discussed for many years (Catts and Langmuir, 1983; Young and Harvey, 1992; Tebo et al., 2004).
2) Pb can replace Mn in the crystal structures (Tebo et al., 2004), and the preliminary experiment result showed the appearance of Mn in the Pb solution after addition of spores, while the control (water), no exchange was observed.
Use magnetic Spores recover Spores ready to use deposition system magnetism to collect spores
Treat Pb polluted Treat spores with Treat spores with + water with spores H OH- and Mn
Heavy metal ions can Protons are Magnetism recovery. exchange with the Mn on competitors for Discussed in spore surface heavy metal ions previous part
Figure 8.1 Schematic picture of the recyclable manganese oxides on spores
3) H+ is a very strong competitor for Pb ion, as described above
4) Magnetism can be recovered after addition of Mn (proved in previous chapter) or base
(will be discussed this chapter)
The process will have the following steps: 149 1) Using spores to treat the contaminated water
(Optional) 2) The magnetic spores will be deposited on the slides through a magnetic deposition system, if the concentration of Pb or other heavy metal ion in the contaminated water is less than 100 µM.
3) If no or only little deposition (which means the concentration of heavy metal ion is high), then treat the spores with HCl
4) Addition of Mn and base can help recovery of the spores’ magnetism
5) Using deposition system to collect the spores and the magnetic spores will be ready to treat polluted water in a new cycle.
The benefits of recyclable spores are obvious: cheap, fast and low maintenance since spores are easy to store.
8.2 Materials and methods
8.2.1 Strains and sporulation
Bacillus atrophaeus from Bacillus Genetic Stock Center at the Ohio State
University was cultured in NB medium and sporulated using Mod G media as described before (Melnik, 2007). Biostat B bioreactor was used for the fermentation. The working volume was 4L, an aeration rate at approximately 2L/min and an agitation rate of 300 rpm were used. The pH was maintained at 7.45 during sporulation. The collected spores were freeze-dried and stored as dry powder for future analysis.
8.2.2 Pb sorption experiments
The beakers were washed with 1% HNO3 before experiment. Spores were grown as described above. Tests were performed at room temperature. Pb concentration in the
150 spore suspension was between 0 and 500 µM. Spores’ suspension was left for more than
24 hours to reach the equilibrium. 3 ml solutions were sampled every three hours for the first four times. The samples were centrifuged and the pellet was put back into the spore suspension and the supernatant was saved for future analysis. After equilibrium, 10 ml samples were removed and centrifuged at 13400 ×g for 30 min. Supernatant was removed from each tube and stored with addition of concentrated HNO3, to give a 1% acid solution to prevent Pb adsorption to the storage tubes. ICP test will be performed for all the samples collected earlier, and Pb stock solution will also be checked as a positive control.
8.2.3 Mn recovery
Mn was added to the Pb treated spore suspension to make the final concentration of 200 µM. The re-sorption took a couple days to finish. The same centrifugation was performed as described above. The spore samples after (pellet) Mn recovery were checked the magnetophoretic mobility using CTV.
8.2.4 Cell Tracking Velocimetry
As reported previously, Cell Tracking Velocimetry (CTV) was developed to quantify the magnetophoretic mobility of cells and particles that have intrinsic magnetic susceptibility or that have been imparted to this susceptibility through binding of antibody magnetic particle conjugates. The spores pellet prepared in the last section was resuspended in sterile distilled water, and the concentration for spores was 107 cfu/ml.
About 1 ml sample per trial was used for CTV as previously described.
151 Control pH=1 pH=6.65 (×104) Add Mn (×104) (×104) 220µM (×104) 24 h 72 h 96 h 6D 8D 3.80 0.122 0.635 0.982 1.26 NA* NA* NA*
3.89 0.157 1.72 NA* 1.70 1.26** 0.119** 1.72 4.43 -0.110 0.799 1.09 4.45 -0.120 4.35 4.49 5.04 2.43& -0.130 0.945 1.91 2.49
2.27& -0.110 0.488 0.721 1.50 1.50 152
Table 8.2 Magnetophoretic mobility (mm3/T•A•s) of spores after acid treatment and after adding base followed the acid treatment. Mn was also added to examine effect on the magnetism recovery
* Samples are not enough for all the tests ** There is some decrease after 6 days & Samples from another batch, which is the same as the one in previous chapter
152 8.3 Result and discussion
8.3.1 Magnetism recovery by increasing the pH after acid treatment
It was found that addition of Mn can help recover the magnetism of acid treated spores. However, whether increasing pH by adding base, instead of adding Mn, can bring up the magnetophoretic mobility is unknown. The spores were put in a solution with pH 1
(HCl). After 8 hours, the magnetophoretic mobility of the spores decreased significantly.
NaOH was added to make the final pH close to 7. Extra Mn was added after 4 days, which allowed enough time to equilibrate. Table 8.2 shows the increase of magnetophoretic mobility of most trials, of which the last two samples were from the batch used in Chapter 7, and the rest four from another batch. This phenomenon may result from the decrease of protons in the solution, which drove Mn back onto the oxides.
8.3.2 Pb effect on magnetophoretic mobility of spores
Spores were suspended in solution of 0.2, 1, 20, 100, 500 µM Pb.
Magnetophoretic mobility of spores was examined after different days. Before adding Pb, the magnetophoretic mobility of spores was 4.33×10-4; 4.29×10-4 based on two runs.
Table 8.3 shows that when the Pb concentration in suspension is below 50 µM there is no significant decrease of magnetophoretic mobility. However, when the Pb concentration becomes as high as 500 µM, spores lose their magnetic properties. Pb in the supernatant was discarded after centrifuge, and a final concentration of 220 µM Mn was added to spore suspension again. While one of the trials didn’t get the magnetophoretic mobility recovered much after the addition of Mn, the other trials seemed to recover the magnetism. This experiment determined the effects of different concentration of Pb on
153 the spores’ magnetism, and higher concentration of Mn may increase the magnetophoretic mobility more for Pb treated spores.
Concentration(µM) 0.2 1 5 50 500 MM (×104) Days 1 3.51 2.73 2.26 2.95 3.51 2.40 3.93 2 3.50 3.93 3.91 4.23 3.20 3.52 3 2.50 3.03 2.96 2.79 3.08 3.03 2.95 4 2.79 2.29 3.51 4.44 3.18 1.87 6 2.65 -0.290 0.314 Add Mn 5.45 Table 8.3 The Magnetophoretic Mobility of spores after days of Pb treatment
8.3.3 Addition of Mn after Pb treatment
Spores were added in 0.2, 1, 20, 100, 500 µM Pb solution, magnetophoretic mobility was examined on day 2 and day 4. After day 4, all the samples were centrifuged and Pb was discarded with the supernatant. Then, a final concentration of 660 µM Mn was added into the spore suspension (treated with 500 µM Pb before), and 220 µM Mn was added into the rest suspensions of spores. However, after Pb treatment, it didn’t seem the increase of Mn in the treated spore suspension recovery the magnetism more (Table
8.4).
154 Pb concentration 2 days 4 days Add Mn (µM) 0.2 3.4 3.8 4.2 1 3.1 4.0 4.2 20 2.6 3.0 4.2 100 0.79 1.3 2.5 500 0.36 0.53 0.28 0.29 -0.044 0.37 Table 8.4 MM (×104) of spore streated with Pb after 2 days and 4 days. Mn was added after 4 days, and the last column is MM after the addition of Mn
8.3.4 Spore recycling process simulation
The principles and the process of recycling spores were discussed earlier in this chapter. Table 8.5 showed the magnetophoretic mobility after step 1, 3, 4 in the process, with the spores having a beginning magnetophoretic mobility of 3.74×10-4, 3.81×10-4.
After six trials with different pH treatments in Step 3 mentioned previously, only 1 trial with pH 1 treatment succeeded in the recovery of magnetism. The reason for this low success rate could be the pH. As the pH decreases, the magnetophoretic mobility increases after addition of Mn and/or base. The increase of Mn concentration or base should also be examined in future for improvement.
However, there is also another possibility that Pb doesn’t exchange Mn. Instead,
Pb can be adsorbed on the layers or edges of MnO6 octahedral complexes, or it can be absorbed into interlayer regions (Tebo et al., 2004).
155 500 µM Pb Acid Base and Mn 5.30 1.43 (pH3) 1.17 -0.44 -0.035 (pH3) 0.535 1.17 1.23 (pH2) 2.19 0.535 0.361 (pH2) 6.06 5.43 0.363 (pH1) 34.9* 8.84 0.265 (pH1) 3.49 Table 8.5 Magnetophoretic mobility ((mm3/T•A•s)×105) of spores followed the treatment mentioned in Figure 8.1
* a significant increase in the MM
156 Chapter 9 Conclusions and future work
9.1 Conclusions
The present work has performed an experimental investigation on the mechanisms of intrinsic magnetism of Bacillus spores from many aspects. Separation of Bacillus spores was discussed in Huading’s Ph.D. dissertation in 2004 (Zhang, 2004); however, stable and high separation efficiency was not achieved due to many complex reasons.
Moreover, the spores were labeled with nanobeads, which are different from the topic of intrinsic magnetism.
While Huading’s work used Bacillus cereus, which can cause food poisoning; this study mostly focused on Bacillus atrophaeus. Immunomagnetic separation and also determination of corresponding magnetophoretic mobility of labeled and unlabeled spores were designed for B. atrophaeus initially. However, the findings that the unlabeled spores were as magnetic as the labeled ones, which enabled the spores to be deposited through a magnetic deposition system, indicate the potential to separate spores directly out of water or air samples without labeling. Two more Bacillus strains were examined for their intrinsic magnetism (Chapter 2). It turned out that only Bacillus spores are paramagnetic, not any vegetative cells. Elemental factors were screened using Energy
Dispersive Spectroscopy (EDS). Compared to vegetative forms, there are Mn 157 peaks in the spectra of B. atrophaeus spores and B. sp. SG-1 spores. X-ray Photoelectron
Spectroscopy (XPS) also demonstrates the presence of Mn on the paramagnetic strains and the absence for the nonmagnetic.
Next, the study of the mechanism was divided into different directions: to quantify the state and amount of Mn per spore (cluster) and spores’ magnetic susceptibility and establish a relationship between them; to screen Bacillus for other paramagnetic strains and to optimize the sporulating condition to get stable and strong magnetism. Before the quantification, the accuracy to analyze Bacillus spores has to be proved. Internal control experiments were conducted in Chapter 3, according to the approach taken by Chalmers et al.(Chalmers et al., 2010). Specifically, spores and polystyrene microspheres (PSM’s) of a known diameter are mixed. The analysis of spores and PSM separately, as well as in a mixture demonstrates enough accuracy for spores’ magnetophoretic mobility.
X-Ray Photoelectron Spectroscopy (XPS) was used to determine the valance states of Mn in the spores, since the magnetic susceptibility of Mn varies significantly between different valance states. The total mass of Mn associated with each spore cluster was determined by ICP- MS. The consistency between CTV, XPS and ICP establishes a relationship between spores’ magnetism and the valence state, the quantity of Mn. This relationship demonstrates that Mn is the most important factor for the magnetism.
More Bacillus spores (on both liquid sporulating media or on solid plates) were examined, and three common strains sporulated in Mod G media were reported in
Chapter 5. Bacillus sp. SG-1 was also found paramagnetic. Both EDS and XPS spectra
158 showed the presence of Mn for the magnetic spores. At the same time, physical conditions were manipulated to control the magnetism of Bacillus spores. Different culturing media: LB and NB were applied, and no difference in the magnetophoretic mobility was noted. However, there was a huge increase in the magnetism when the Mod
G sporulating media was used as opposed to SG media. Modifications were made to Mod
G media for optimization. Different ion concentrations, working volume, sporulating time, heat shock time were changed to find the most important factor(s). The concentration of Mn in the culture seemed to be the most important factor while the sporulation time and oxygen availability did not affect the magnetism significantly. Mn oxidation state is considered important to the intrinsic magnetism as well. This hypothesis is supported by the report that some Bacillus species can oxidize Mn(II) to
Mn3O4 and MnO2 enzymatically. Different concentrations of ions have been examined, with no significant effect on the magnetism.
After the exploration of the mechanism for the intrinsic magnetism, further applications of the spores were also studied and discussed. Huading’s work tried to separate the spores from liquid food using IMS and MS separation column (Miltenyi
Biotec, Germany). The study in Chapter 7 tried to separate spores from water, milk or chicken broth before or after sterilization. A large portion of spores were inactivated in the autoclave process, but the remaining magnetism of the surviving spores can be detected by CTV and deposit spores on the slides. Low pH levels decreased the magnetic properties, and inactivated the spores at the same time. However, addition of Mn after treatment could recover most of magnetism of the spores and the spores with recovered
159 magnetism could be separated from deposition system again. These studies indicate that the deposition system can be used in food industry, environmental pollution control as well as biological warfare agent detection.
Another application was to use the spores to treat Pb polluted water. For a comparatively low concentration of Pb, the spores still had magnetism as strong as before the adsorption of lead. Thus, they can be separated directly via a magnetic deposition system. However, when Pb concentration becomes as high as 500 µM, the spores lose their magnetism, addition of acid (overnight) and base (and/or Mn) subsequently can bring back the magnetism of spores, which enables the collection of spores via the magnetic deposition system. The recycled spores can be used to treat other industrial waste water again.
In summary, intrinsic magnetism of spores is highly related to the Mn, either in the sporulating media or the amount and valance state of Mn on the spores’ surface. Cell
Tracking Velocimetry provided enough accuracy for the analysis of spores’ magnetophoretic mobility during the study. The intrinsic magnetism also enables the spores to be separated from air or liquid food. The effectiveness to adsorb heavy metal ions gives the spores another application in treating waste water. The recovery of magnetism after treatment by adding acid and base sequentially, makes the recycling of the spores for decontamination possible.
160 9.2 Future work
9.2.1 Other paramagnetic strains
In total, six strains were discussed in this dissertation; however, the screening populations should be broadened in future. As discussed before, many organisms can oxidize Mn (Tebo et al., 2004), including a diversity of bacteria, fungi, algae, and even eukaryotes (Nealson, 2006). Among the prokaryotes, the ability to oxidize Mn is also quite widespread, e.g. cyanobacteria, a diversity of heterotrophic rods and cocci, the sheathed (Leptothrix-like) and budding (Hyphomicrobium-like) bacteria and endospore forming bacteria (Nealson, 2006). Even within Bacillus, there are more than 10 strains found the Mn oxidase. Those strains, however, haven’t been applied to CTV yet. CTV,
EDS, and maybe ICP-MS should also be applied to the strains, to explore a general rule for all the paramagnetic strains.
9.2.2 Optimized media
Mn-oxidizing bacteria can be easily cultured in liquid or solid media, so long as
Mn(II) toxicity is avoided. Before culturing other potential paramagnetic strains using the optimized media in Chapter 5, a quick checklist needs to be made following the guideline for Mn oxidizers provided by Nealson (Nealson, 2006). There is not a requirement for carbon sources, since Mn oxidation in culture occurs only after growth has slowed or ceased. The oxidation of Mn is strongly pH dependent (Figure 5.1 and Figure 9.1), therefore, it is important to control the pH of the medium. By the addition of buffer neutrality, one can avoid indirect Mn oxidation due to pH changes. In the case of Mod G,
K2HPO4 is the buffer.
161 As discussed in Chapter 5, Mn source is the important factor for the magnetism.
Even though it was claimed 10 µM can be toxic to bacteria (Nealson, 2006). It seems not very destructive to B. atrophaeus spores. However, the concentration may need to be lowered for other non-spore-forming strains.
Figure 9.1 Thermodynamic stability of manganese oxides phases
The optimized media can be used to sporulate or culture other strains and after the bacteria get collected, CTV can be used to examine the magnetophoretic mobility. The paramagnetic strains can also be used in food industry or decontamination for the environment.
9.2.3 Continuous study for decontamination
Even though the results of using spores for Pb polluted water are exciting for the initial stage, there are still improvements need to make for reliable and efficient recycling.
162 For the reliability, there were only two successful trials, and it seems that more acidic solution followed by Mn (and/or base) can help recover the magnetism. For future study, a pH at maximum level of 1 should still be used. Meanwhile, ICP –MS will be used to check the exchange of Mn for Pb, when we put spores into the waste water. This can also demonstrate the reason for heavy metal adsorptions: either ion exchange or some destructive change to the biogenic oxides. Higher Mn concentration or lower pH, as well as longer equilibrium time will also be recommended in future work.
9.2.4 Mn oxidase expression level
After optimization of media and genetic study, we also would like to know “Does the physiological condition influence the magnetic susceptibility by changing expression level of MnxG?” Polyacrylamide Gel Electrophoresis and 2D-electrophoresis can be used to check the expression level of MnxG. The comparison of MnxG expression level between strong magnetic spores and weak magnetic spores, and the difference in magnetophoretic mobility will demonstrate whether the final Mn product is related to Mn oxidase expression.
Mn oxidase activity can be checked using leucoberbelin blue. Leucobase in the compound is the specific substrate for Mn(III) and Mn(IV), and leucobase can be oxidized in a few seconds to give a blue color. Mn (II) and other metals do not influence leucobase (Krumbein and Altmann, 1973).
163 References
Arakawa, E.T., Lavrik, N.V. and Datskos, P.G. (2003). Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores. Applied Optics 42(10): 1757-1762.
Armstrong, G.N., Watson, I.A. and Stewart-Tull, D.E. (2006). Inactivation of B-cereus spores on agar, stainless steel or in water with a combination of Nd : YAG laser and UV irradiation. Innovative Food Science & Emerging Technologies 7(1-2): 94-99.
Bargar, J.R., Tebo, B.M. and Villinski, J.E. (2000). In situ characterization of Mn(II) oxidation by spores of the marine Bacillus sp. strain SG-1. Geochim. Cosmochim. Acta 64: 2777-2780.
Bazylinski, D.A., Frankel, R.B., Heywood, B.R., Mann, S., King, J.W., Donaghay, P.L. and Hanson, A.K. (1995). Controlled Biomineralization of Magnetite (Fe3o4) and Greigite (Fe3s4) in a Magnetotactic Bacterium. Applied and Environmental Microbiology 61(9): 3232-3239.
Bernhardt, M., Pennell, D.R., Almer, L.S. and Schell, R.F. (1991). Detection of Bacteria in Blood by Centrifugation and Filtration. Journal of Clinical Microbiology 29(3): 422- 425.
Blakemore, R. (1975). Magnetotactic Bacteria. Science 190(4212): 377-379.
Bravo, A., Gill, S.S. and Soberon, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49(4): 423-435.
Burke, S.A., Wright, J.D., Robinson, M.K., Bronk, B.V. and Warren, R.L. (2004). Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis. Appl Environ Microbiol. 70: 2786–2790.
Buttner, M.P., Cruz, P., Stetzenbach, L.D., Klima-Comba, A.K., Stevens, V.L. and D., C.T. (2004). Determination of the efficacy of two building decontamination strategies by
164 surface sampling with culture and quantitative PCR analysis. Appl. Environ. Microbiol. 70: 4740-4747.
Capar, S.G., Mindak, W.R. and Cheng, J. (2007). Analysis of food for toxic elements. Analytical and Bioanalytical Chemistry 389(1): 159-169.
Carrera, M., Zandomeni, R.O. and Sagripanti, J.L. (2008). Wet and dry density of Bacillus anthracis and other Bacillus species. Journal of Applied Microbiology 105(1): 68-77.
Catts, J.G. and Langmuir, D. (1983). Adsorption of Cu, Pb, and Zn onto Birnessite (Delta-Mno2). Abstracts of Papers of the American Chemical Society 185(Mar): 43-Geoc.
Chalmers, J.J., Haam, S., Zhao, Y., McCloskey, K., Moore, L., Zborowski, M. and Williams, P.S. (1999). Quantification of cellular properties from external fields and resulting induced velocity: Cellular hydrodynamic diameter. Biotechnology and Bioengineering 64(5): 509-518.
Chalmers, J.J., Haam, S., Zhao, Y., McCloskey, K., Moore, L., Zborowski, M. and Williams, P.S. (1999). Quantification of cellular properties from external fields and resulting induced velocity: Magnetic susceptibility. Biotechnology and Bioengineering 64(5): 519-526.
Chalmers, J.J., Xiong, Y., Jin, X., Shao, M., Tong, X., Farag, S. and Zborowski, M. (2010). Quantification of Non-Specific Binding of Magnetic Micro- and Nanoparticles Using Cell Tracking Velocimetry: Implication for Magnetic Cell Separation and Detection. Biotechnology and Bioengineering 105(6): 1078-1093.
Chalmers, J.J., Zborowski, M., Sun, L.P. and Moore, L. (1998). Flow through, immunomagnetic cell separation. Biotechnology Progress 14(1): 141-148.
Chalmers, J.J., Zhao, Y., Nakamura, M., Melnik, K., Lasky, L., Moore, L. and Zborowski, M. (1999). An instrument to determine the magnetophoretic mobility of labeled, biological cells and paramagnetic particles. Journal of Magnetism and Magnetic Materials 194(1-3): 231-241.
Chalmers, J.J., Zhao, Y., Nakamura, M., Melnik, K., Lasky, L., Moore, L. and Zborowski, M. (1999). An Instrument to determine the magnetophoretic mobility of paramagnetic particles and labeled, biological cells. J. of Magnetism and Magnetic Materials 194: 231- 241
Chosy, E.J., Nakamura, M., Melnik, K., Comella, K., Lasky, L., Zborowski, M. and Chalmers, J.J. (2003). Characterization of antibody binding to three cancer-related
165 antigens using flow cytometry and cell tracking velocimetry Biotech and Bioeng 82: 340- 351.
Chosy, E.J., Nakamura, M., Melnik, K., Comella, K., Lasky, L.C., Zborowski, M. and Chalmers, J.J. (2003). Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry. Biotechnology and Bioengineering 82(3): 340-351.
Crowley, J.D., Traynor, D.A. and Weather, D.C. (2000). Enzyemes and proteins containing manganese: an overview. Met Ions Biol Syst. 37(209-278).
Czerwieniec, G.A., Russell, S.C., Tobias, H.J., Pitesky, M.E., Fergenson, D.P., Steele, P., Srivastava, A., Horn, J.M., Frank, M., Gard, E.E. and Lebrilla, C.E. (2005). Stable isotope labeling of entire Bacillus atrophaeus spores and vegetative cells using bioaerosol mass spectrometry. Anal. Chem. 77: 1081-1087.
Czerwieniec, G.A., Russell, S.C., Tobias, H.J., Pitesky, M.E., Fergenson, D.P., Steele, P., Srivastava, A., Horn, J.M., Frank, M., Gard, E.E. and Lebrilla, C.E. (2005). Stable isotope labeling of entire Bacillus atrophaeus spores and vegetative cells using bioaerosol mass spectrometry. . Anal. Chem. 77: 1081-1087.
DE Vrind, J.P.M., DE Vrind-DE Jong, E.W., DE Voogt, J.H., Westbroek, P., Boogerd, F.C. and Rosson, R.A. (1986). Manganese oxidation by spores and spore coats of a marine Bacillus Species. Appl Environ Microbiol. 52: 1096-1100.
Delgado, E., Garcia, M.A., Hernandez, E., Mansilla, N., Martinez-Cruz, L.A., Tornero, J. and Torres, R. (1998). Synthesis and characterization of oxo- and thiophosphorylcyclopentadienyl Ti(IV) thiolate complexes. Crystal structures of [(eta(5)- C5H4P(S)Ph-2)(eta(5)-C5H4SiMe3)TiCl2] and [(eta(5)-C5H4P(S)Ph- 2)(2)Ti(SPh)(2)]center dot C4H10O. Journal of Organometallic Chemistry 560(1-2): 27- 33.
Devers, K.G. and Nine, J.S. (2010). Autopsy Findings in Botulinum Toxin Poisoning. J Forensic Sci.
Devrind, J.P.M., Boogerd, F.C. and Devrinddejong, E.W. (1986). Manganese Reduction by a Marine Bacillus Species. Journal of Bacteriology 167(1): 30-34.
Doyle, R.J. (2000). Contribution of the hydrophobic effect to microbial infection. Microbes and Infection 2(4): 391-400.
Fang, B., Zborowski, M. and Moore, L.R. (1999). Detection of rare MCF-7 breast carcinoma cells from mixtures of human peripheral leukocytes by magnetic deposition analysis. Cytometry 36(4): 294-302. 166 Faye, R.S., Aamdal, S., Hoifodt, H.K., Jacobsen, E., Holstad, L., Skovlund, E. and Fodstad, O. (2004). Immunomagnetic detection and clinical significance of micrometastatic tumor cells in malignant melanoma patients. Clinical Cancer Research 10(12 Pt 1): 4134-4139.
Filion, G., Laflamme, C., Turgeon, N., Ho, J. and Duchaine, C. (2009). Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization. Journal of Microbiological Methods 77(1): 29-36.
Flint, S.H., Brooks, J.D. and Bremer, P.J. (2000). Properties of the stainless steel substrate, influencing the adhesion of thermo-resistant streptococci. Journal of Food Engineering 43(4): 235-242.
Francis, C.A., Casciotti, K.L. and Tebo, B.M. (2002). Localization of Mn(II)-oxidizing activity and the putative multicopper oxidase, MnxG, to the exosporium of the marine Bacillus sp. strain SG-1. Arch. Microbiol. 178: 450-456.
Francis, C.A. and Tebo, B.M. (1999). Marine Bacillus Spores as Catalysts for Oxidative Precipitation and Sorption of Metals. J. Molec. Microbiol. Biotechnol. 1(1): 71-78.
Francis, C.A. and Tebo, B.M. (2002). Enzymatic Manganese(II) oxidation by metabolically dormant spores of diverse Bacillus Species. Appl. Environ. Microbiol 68: 874-880.
Francis, C.A. and Tebo, B.M. (2002). Enzymatic Manganese(II) oxidation by metabolically dormant spores of diverse Bacillus species. Applied and Environmental Microbiology 68(2): 874-880.
Fritze, D. and Pukall, R. (2001). Reclassification of bioindicator strains Bacillus subtilis DSM 675 and Bacillus subtilis DSM 2277 as Bacillus atrophaeus. Int. J. Sys. Evol.Microbiol 51: 35-37.
Fujii, K., Ohtani, A., Watanabe, J., Ohgoshi, H., Fujii, T. and Honma, K. (2002). High- pressure inactivation of Bacillus cereus spores in the presence of argon. International Journal of Food Microbiology 72(3): 239-242.
Furukawa, S., Narisawa, N., Watanabe, T., Kawarai, T., Myozen, K., Okazaki, S., Ogihara, H. and Yamasaki, M. (2005). Formation of the spore clumps during heat treatment increases the heat resistance of bacterial spores. International Journal of Food Microbiology 102(1): 107-111.
Ghiorse, W.C. and Ehrlich, H.L. (1974). Effects of Seawater Cations and Temperature on Manganese Dioxide-Reductase Activity in a Marine Bacillus. Applied Microbiology 28(5): 785-792. 167 Glasfeld, A., Guedon, E., Helmann, J.D. and Brennan, R.G. (2003). Structure of the manganese-bound manganese transport regulator of Bacillus subtilis. Nature Structural Biology 10(8): 652-657.
Golynskiy, M.V., Davis, T.C., Helmann, J.D. and Cohen, S.M. (2005). Metal-induced structural organization and stabilization of the metalloregulatory protein MntR. Biochemistry 44(9): 3380-3389.
Gould, F., Blair, N., Reid, M., Rennie, T.L., Lopez, J. and Micinski, S. (2002). Bacillus thuringiensis-toxin resistance management: Stable isotope assessment of alternate host use by Helicoverpa zea. Proceedings of the National Academy of Sciences of the United States of America 99(26): 16581-16586.
Green, A.C. and Madgwick, J.C. (1991). Microbial formation of manganese oxides App. Eviron. Microbiol 57: 1114-1120.
Gupta, R.K., Singh, R.A. and Dubey, S.S. (2004). Removal of mercury ions from aqueous solutions by composite of polyaniline with polystyrene. Separation and Purification Technology 38(3): 225-232.
Hastings, D. and Emerson, S. (1986). Oxidation of Manganese by Spores of a Marine Bacillus - Kinetic and Thermodynamic Considerations. Geochimica Et Cosmochimica Acta 50(8): 1819-1824.
Hem, J.D. and Lind, C.J. (1983). Nonequilibrium models for predicting forms of precipitated manganese oxides. Geoch. et Cosmoch. Acta 47: 2037-2046.
Hengstler, J.G., Bolm-Audorff, U., Faldum, A., Janssen, K., Reifenrath, M., Gotte, W., Jung, D.L., Mayer-Popken, O., Fuchs, J., Gebhard, S., Bienfait, H.G., Schlink, K., Dietrich, C., Faust, D., Epe, B. and Oesch, F. (2003). Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co-exposures to cadmium, cobalt and lead as more dangerous than hitherto expected. Carcinogenesis 24(1): 63-73.
Hergt, R., Hiergeist, R., Zeisberger, M., Schuler, D., Heyen, U., Hilger, I. and Kaiser, W.A. (2005). Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools. Journal of Magnetism and Magnetic Materials 293(1): 80-86.
Hindson, B.J., McBride, M.T., Makarewicz, A.J., Henderer, B.D., Setlur, U.S., Smith, S.M., Gutierrez, D.M., Metz, T.R., Nasarabadi, S.L., Venkateswaran, K.S., Farrow, S.W., Colston, B.W. and Dzenitis, J.M. (2005). Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation. Analytical Chemistry 77(1): 284-289.
168 Hornstra, L.M., de Vries, Y.P., de Vos, W.M. and Abee, T. (2006). Influence of sporulation medium composition on transcription of ger operons and the germination response of spores of Bacillus cereus ATCC 14579. Appl Environ Microbiol 72(5): 3746- 3749.
Jakubovics, N.S. and Jenkinson, H.F. (2001). Out of the iron age: new insights into the critical role of manganese homeostasis in bacteria. Microbiol 147: 1709-1718.
Jarup, L. (2003). Hazards of heavy metal contamination. British Medical Bulletin 68: 167-182.
Jin, X., Zhao, Y., Richardson, A., Moore, L., Williams, P.S., Zborowski, M. and Chalmers, J.J. (2008). Differences in magnetically induced motion of diamagnetic, paramagnetic, and superparamagnetic microparticles detected by cell tracking velocimetry. Analyst 133(12): 1767-1775.
Jing, Y., Mal, N., Williams, P.S., Mayorga, M., Penn, M.S., Chalmers, J.J. and Zborowski, M. (2008). Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis. Faseb Journal 22(12): 4239-4247.
Johnson, Y.A., Nagpal, M., Krahmer, M.T., Fox, K.F. and Fox, A. (2000). Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus , closely related species of bacilli. J. Microbiol. Methods 40: 241-254.
Johnstone, K., Stewart, G.S.A.B., Scott, I.R. and Ellar, D.J. (1982). Zinc Release and the Sequence of Biochemical Events during Triggering of Bacillus-Megaterium Km Spore Germination. Biochemical Journal 208(2): 407-411.
Kadirvelu, K., Thamaraiselvi, K. and Namasivayam, C. (2001). Adsorption of nickel(II) from aqueous solution onto activated carbon prepared from coirpith. Separation and Purification Technology 24(3): 497-505.
Kim, J.B., Jeong, H.R., Park, Y.B., Kim, J.M. and Oh, D.H. (2010). Food Poisoning Associated with Emetic-Type of Bacillus cereus in Korea. Foodborne Pathogens and Disease 7(5): 555-563.
Kort, R., O'Brien, A.C., van Stokkum, I.H.M., Oomes, S.J.C.M., Crielaard, W., Hellingwerf, K.J. and Brul, S. (2005). Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release. Applied and Environmental Microbiology 71(7): 3556-3564.
169 Koshikawa, T., Yamazaki, M., Yoshimi, M., Ogawa, S., Yamada, A., Watabe, K. and Torii, M. (1989). Surface Hydrophobicity of Spores of Bacillus Spp. Journal of General Microbiology 135: 2717-2722.
Krumbein, W.E. and Altmann, H.J. (1973). A new method for the detection and enumeration of manganese oxidizing and reducing microorganisms. Helgolander wiss. Meeresunters 25: 347-356.
Laflamme, C., Lavigne, S., Ho, J. and Duchaine, C. (2004). Assessment of bacterial endospore viability with fluorescent dyes. J. Appl. Microbiol. 96: 684-692.
Lighthart, B., Prier, K. and Bromenshenk, J.J. (2004). Detection of aerosolized bacterial spores (Bacillus atrophaeus) using free-flying honey bees (Hymenoptera: Apidae) as collectors. Aerobiologia 20: 3-4.
Lim, D.V., Simpson, J.C., Kearns, E.A. and Kramer, M.F. ( 2005). Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clin. Microbiol. Rev 18: 583-607.
Mamane-Gravetz, H. and Linden, K.G. (2005). Relationship between physiochemical properties, aggregation and u.v. inactivation of isolated indigenous spores in water. Journal of Applied Microbiology 98(2): 351-363.
Mandernack, K.W., Post, J. and Tebo, B.M. (1995). Manganese mineral formation by bacterial spores of the marine Bacillus strain SG-1: Evidence for the direct oxidation of Mn(II) to Mn(IV). Geochim. Cosmochim. Acta 59: 4393-4408.
Mann, S., Sparks, N.H.C., Scott, G.H.E. and De-Vrind-De Jong, E.W. (1988). Oxidation of Manganese and Formation of Mn3O4 (Hausmannite) by Spore Coats of a Marine Bacillus sp. App.Environ. Microbiol. 54: 2140-2143.
Marshall, K.C. (1979). Biogeochemical cycling of mineral-forming elements., Elsevier, Amsterdam.
Mcclintock, J.T., Schaffer, C.R. and Sjoblad, R.D. (1995). A Comparative Review of the Mammalian Toxicity of Bacillus Thuringiensis-Based Pesticides. Pesticide Science 45(2): 95-105.
McCloskey, K.E., Comella, K., Chalmers, J.J., Margel, S. and Zborowski, M. (2001). Mobility measurements of immunomagnetically labeled cells allow quantitation of secondary antibody binding amplification. Biotechnology and Bioengineering 75(6): 642- 655.
170 Melnik, K., Sun, J., Fleischman, A., Roy, S., Zborowski, M. and Chalmers, J.J. (2007). Quantification of magnetic susceptibility in several strains of Bacillus spores: Implications for separation and detection. Biotechnology and Bioengineering 98(1): 186- 192.
Mielke, H.W. and Reagan, P.L. (1998). Soil is an important pathway of human lead exposure. Environmental Health Perspectives 106: 217-229.
Monser, L. and Adhoum, N. (2002). Modified activated carbon for the removal of copper, zinc, chromium and cyanide from wastewater. Separation and Purification Technology 26(2-3): 137-146.
Moore, L.R., Zborowski, M., Nakamura, M., McCloskey, K., Gura, S., Zuberi, M., Margel, S. and Chalmers, J.J. (2000). The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry. Journal of Biochemical and Biophysical Methods 44(1-2): 115-130.
Moore, L.R., Zborowski, M., Sun, L.P. and Chalmers, J.J. (1998). Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter. Journal of Biochemical and Biophysical Methods 37(1-2): 11-33.
Moulder, J.F. (1992). Handbook of X-ray Photoelectron Spectroscopy.
Murray, J.W., Dillard, J.G., Giovanoli, R., Moers, H. and Stumm, W. (1985). Oxidation of Mn(II): initial mineralogy, oxidation state and aging. Geochim Cosmochim Acta 49: 463-470.
Nakamura, L.K. (1989). Taxonomic Relationship of Black-Pigmented Bacillus subtilis Strains and a Proposal for Bacillus atrophaeus sp.nov. Int. J. Sys. Bacterial. 39: 295-300.
Nakamura, M., Decker, K., Chosy, J., Comella, K., Melnik, K., Moore, L., Lasky, L.C., Zborowski, M. and Chalmers, J.J. (2001). Separation of a breast cancer cell line from human blood using a quadrupole magnetic flow sorter. Biotechnology Progress 17(6): 1145-1155.
Nakamura, M., Zborowski, M., Lasky, L.C., Margel, S. and Chalmers, J.J. (2001). Theoretical and experimental analysis of the accuracy and reproducibility of cell tracking velocimetry. Experiments in Fluids 30(4): 371-380.
Nealson, K. (2006). The Manganese-Oxidizing Bacteria. Prokaryotes, DOI: 222-231.
Nealson, K.H. and Saffarini, D. (1994). Iron and Manganese in Anaerobic Respiration - Environmental Significance, Physiology, and Regulation. Annual Review of Microbiology 48: 311-343. 171 Nealson, K.H., Tebo, B.M. and Rosson, R.A. (1988). Occurrence and mechanisms of microbial oxidation of manganese. Adv Appl Microbiol 33: 299-318.
Neaman, A., Mouele, F., Trolard, F. and Bourrie, G. (2004). Improved methods for selective dissolution of Mn oxides: applications for studying trace element associations. Applied Geochemistry 19(6): 973-979.
Nelson, Y.M. and Lion, L.W. (2003). Geochemical and hydrological reactivity of heavy metals in soils, CRC press LLC.
Nelson, Y.M., Lion, L.W., Ghiorse, W.C. and Shuler, M.L. (1999). Production of biogenic Mn oxides by Leprothrix discophora SS-1 in a chemically defined growth medium and evaluation of their Pb adsorption characteristics. Applied and Environmental Microbiology 65(1): 175-180.
Nelson, Y.M., Lion, L.W., Shuler, M.L. and Ghiorse, W.C. (2002). Effect of oxide formation mechanisms on lead adsorption by biogenic manganese (hydr)oxides, iron (hydr)oxides, and their mixtures. Environmental Science & Technology 36(3): 421-425.
Ngah, W.S.W. and Hanafiah, M.A.K.M. (2008). Removal of heavy metal ions from wastewater by chemically modified plant wastes as adsorbents: A review. Bioresource Technology 99(10): 3935-3948.
Nicholson, W.L. and Setlow, P. (1990). Farmington, WWiley-Interscience.
Nou, X.W., Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Guerini, M.N., Kalchayanand, N. and Koohmaraie, M. (2006). Improvement of Immunomagnetic separation for Escherichia coli O157 : H7 detection by the PickPen magnetic particle separation device. Journal of Food Protection 69(12): 2870-2874.
O' Connel, K.P., Bucher, J.R., Anderson, P.E., Cao, C.J., Khan, A.S., Gostomski, M.V. and Valdes, J.J. (2006). Real-time fluorogenic transcription-PCR assays for detection of bacteriophage MS2. . Appl. Environ. Microbiol. 72: 478–483.
O’Connell, K.P., Bucher, J.R., Anderson, P.E., Cao, C.J., Khan, A.S., Gostomski, M.V. and Valdes, J.J. (2006). Real-time fluorogenic transcription-PCR assays for detection of bacteriophage MS2. . Appl. Environ. Microbiol. 72: 478–483.
Oyston, P.C.F., Sjosted, A. and R.W.2004., T. (2004). Tularemia: bioterrorism defense renews interest in Francisella tularensis. Nat. Rev 2(967-978).
Pardo-Andreu, G.L., Sanchez-Baldoquin, C., Avila-Gonzalez, R., Yamamoto, E.T.S., Revilla, A., Uyemura, S.A., Naal, Z., Delgado, R. and Curti, C. (2006). Interaction of
172 Vimang (Mangifera indica L. extract) with Fe(III) improves its antioxidant and cytoprotecting activity. Pharmacological Research 54(5): 389-395.
Plomp, M., Leighton, T.J., Wheeler, K.E., Pitesky, M.E. and Malkin, A.J. (2005). Bacillus atrophaeus outer spore coat assembly and ultrastructure. Langmuir 21: 10710- 10716.
Raber, E. and McGuire.R. (2002). Oxidative decontamination of chemical and biological warfare agents using L-Gel. . J. Hazard. Mater B93: 339-352.
Reddy, S., Moore, L.R., Sun, L.P., Zborowski, M. and Chalmers, J.J. (1996). Determination of the magnetic susceptibility of labeled particles by video imaging. Chemical Engineering Science 51(6): 947-956.
Rengaraj, S., Yeon, K.H. and Moon, S.H. (2001). Removal of chromium from water and wastewater by ion exchange resins. Journal of Hazardous Materials 87(1-3): 273-287.
Robinson, B.H. (2009). E-waste: An assessment of global production and environmental impacts. Science of the Total Environment 408(2): 183-191.
Robinson, G.M., Lee, S.W.-H., Greenman, J., Salisbury, V.C. and Reynolds, D.M. (2010). Evaluation of the efficacy of electrochemically activated solutions against nosocomial pathogens and bacterial endospores. Lett. Appl. Microbiol. 50: 289-294.
Rosenberg, M. (1984). Bacterial Adherence to Hydrocarbons - a Useful Technique for Studying Cell-Surface Hydrophobicity. Fems Microbiology Letters 22(3): 289-295.
Rosson, R.A. and Nealson, K.H. (1982). Manganese Binding and Oxidation by Spores of a Marine Bacillus. Journal of Bacteriology 151(2): 1027-1034.
Rosson, R.A. and Nealson, K.H. (1982). Manganese binding and oxidation by spores of a marine bacillus. . J. Bacteriol. 151 1027-1034.
Saenz, A.J., Petersen, C.E., Valentine, N.B., Gantt, S.L., Jarman, K.H., Kingsley, M.T. and Wahl, K.L. (1999). Time-of-flight Mass Spectrometry for Replicate Bacterial Culture Analysis Rapid Commun Mass Spectrom 13: 1580–1585.
Saikaly, P.E., Barlaz, M.A. and de los Reyes III, F.L. (2007). Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate. Appl. Environ. Microbiol. 73: 6557-6565.
173 Schneider, T., Karl, S., Moore, L.R., Chalmers, J.J., Williams, P.S. and Zborowski, M. (2010). Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter. Analyst 135(1): 62-70.
Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R. and Dean, D.H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62(3): 775-806.
Schuler, D. (1999). Formation of magnetosomes in magnetotactic bacteria. J Mol Microbiol Biotechnol 1(1): 79-86.
Shotyk, W. and Le Roux, G. (2005). Biogeochemistry and cycling of lead. Biogeochemical Cycles of Elements 43: 239-275.
Silbergeld, E.K. (1992). Neurological perspective on lead toxicity, CRC Press.
Soberon, M., Pardo-Lopez, L., Lopez, I., Gomez, I., Tabashnik, B.E. and Bravo, A. (2007). Engineering modified Bt toxins to counter insect resistance. Science 318(5856): 1640-1642.
Spiro, T.G., Bargar, J.R., Sposito, G. and Tebo, B.M. (2010). Bacteriogenic Manganese Oxides. Accounts of Chemical Research 43(1): 2-9.
Srivastava, A., Pitesky, M.E., Steele, P.T., Tobias, H.J., Fergenson, D.P., Horn, J.M., Russell, S.C., Czerwieniec, G.A., Lebrilla, C.S., Gard, E.E. and Frank, M. (2005). Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry. Analytical Chemistry 77(10): 3315-3323.
Stratis-Cullum, D.N., Griffin, G.D., Mobley, J., Vass, A.A. and Vo-Dinh, T. (2003). A miniature biochip system for detection of aerosolized Bacillus globigii spores. Analytical Chemistry 75(2): 275-280.
Stratis-Cullum, D.N., Griffin, G.D., Mobley, J., Vass, A.A. and Vo-Dinh.T (2003). A miniature biochip system for detection of aerosolized Bacillus globigii spores. Anal. Chem. 75(275-280).
Sun, L.P., Zborowski, M., Moore, L.R. and Chalmers, J.J. (1998). Continuous, flow- through immunomagnetic cell sorting in a quadrupole field. Cytometry 33(4): 469-475.
Swaminathan, B. and Feng, P. (1994). Rapid Detection of Food-Borne Pathogenic Bacteria. Annual Review of Microbiology 48: 401-426.
174 Szinicz, L. (2005). History of chemical and biological warfare agents. Toxicology 214: 161-181.
Talbot, S.T., Russmann, H., Köhne, S., Niederwöhrmeier , B., Grote , G. and Scheper, T. (2010). Effects of inactivation methods on the analysis of Bacillus atrophaeus endospores using real-time PCR and MALDI-TOF-MS. Eng. Life Sci. 10(109-120).
Tauxe, R.V. (1997). Emerging foodborne diseases: An evolving public health challenge. Emerging Infectious Diseases 3(4): 425-434.
Tebo, B.M. (1991). Manganese(II) oxidation in the suboxic zone of the Black Sea. Deep Sea Res 38: S883-S905.
Tebo, B.M., Bargar, J.R., Clement, B.G., Dick, G.J., Murray, K.J., Parker, D., Verity, R. and Webb, S.M. (2004). Biogenic manganese oxides: Properties and mechanisms of formation. Annual Review of Earth and Planetary Sciences 32: 287-328.
Tebo, B.M., Ghiorse, W.C., vanWaasbergen, L.G., Siering, P.L. and Caspi, R. (1997). Bacterially mediated mineral formation: Insights into manganese(II) oxidation from molecular genetic and biochemical studies. Geomicrobiology: Interactions between Microbes and Minerals 35: 225-266.
Tobias, H.J., Pitesky, M.E., Fergenson D.P., Steele, P.T., Horn J., Frank M and Gard, E.E. (2006). Following the biochemical and morphological changes of Bacillus atrophaeus cells during the sporulation process using Bioaerosol Mass Spectrometry. J. Microbiol. Methods 67: 56-63.
Turnbough, C.L. (2003). Discovery of phage display peptide ligands for species specific detection of Bacillus spores. J. Microbiol. Methods 53: 263-271.
Uemura, K. and Isobe, S. (2003). Developing a new apparatus for inactivating Bacillus subtilis spore in orange juice with a high electric field AC under pressurized conditions. Journal of Food Engineering 56(4): 325-329.
Valdivia, A., Perez-Alvarez, S., Aroca-Aguilar, J.D., Ikuta, I. and Jordan, J. (2009). Superoxide dismutases: a physiopharmacological update. Journal of Physiology and Biochemistry 65(2): 195-208.
Van Waasbergen, L.G., Hildebrand, M. and Tebo, B.M. (1996). Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp. strain SG-1. J. Bacteriol. 12: 3517–3530.
175 van Waasbergen, L.G., Hildebrand, M. and Tebo, B.M. (1996). Identification and characterization of a gene cluster involved in manganese oxidation by spores of the marine Bacillus sp. strain SG-1. Journal of Bacteriology 178(12): 3517-3530. van Waasbergen, L.G., Hoch, J.A. and Tebo, B.M. (1993). Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG-1: protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation. Journal of Bacteriology 175(23): 7594-7603.
Vanwaasbergen, L.G., Hoch, J.A. and Tebo, B.M. (1993). Genetic-Analysis of the Marine Manganese-Oxidizing Bacillus Sp Strain Sg-1 - Protoplast Transformation, Tn917 Mutagenesis, and Identification of Chromosomal Loci Involved in Manganese Oxidation. Journal of Bacteriology 175(23): 7594-7603.
Villalobos, M., Bargar, J. and Sposito, G. (2005). Mechanisms of Pb(II) sorption on a biogenic manganese oxide. Environmental Science & Technology 39(2): 569-576.
Wang, J., Deng, B.L., Chen, H., Wang, X.R. and Zheng, J.Z. (2009). Removal of Aqueous Hg(II) by Polyaniline: Sorption Characteristics and Mechanisms. Environmental Science & Technology 43(14): 5223-5228.
Watarai, H. and Namba, M. (2001). Magnetophoretic behavior of single polystyrene particles in aqueous manganese(II) chloride. Anal Sci 17(10): 1233-1236.
Weast CR, Astle MJ and WH., B. (1987). CRC Handbook of Chemistry and Physics.
Weast, C.R., Astle, M.J. and Beyer, W.H. (1987). CRC Handbook of Chemistry and Physics.
Webb, S.M., Dick, G.J., Bargar, J.R. and Tebo, B.M. (2005). Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II). Proceedings of the National Academy of Sciences of the United States of America 102(15): 5558-5563.
Wehrli, B. (1990). Aquatic Chemical Kinetics: Reaction Rates of Processes in Natural Waters.
Yang, L. (2008). APPLICATION OF IMMUNOMAGNETIC CELL SEPARATION IN CANCER CELL DETECTION:DEVELOPMENT AND OPTIMIZATION. Chemical Engineering. Columbus, The Ohio State University. phD.
Young, L.B. and Harvey, H.H. (1992). The Relative Importance of Manganese and Iron- Oxides and Organic-Matter in the Sorption of Trace-Metals by Surficial Lake-Sediments. Geochimica Et Cosmochimica Acta 56(3): 1175-1186.
176 Zborowski, M. (2008). Magnetic Cell Separation. M. Zborowski and J. Chalmers, Elsevier. 32: 1-28.
Zborowski, M. and Chalmers, J.J. (2005). Immunochemical protocols, methods in molecular biology. Totowa, Humana Press.
Zborowski, M. and Chalmers, J.J. (2007). Magnetic Cell Separation Elsevier Science.
Zborowski, M., Fuh, C.B., Green, R., Sun, L.P. and Chalmers, J.J. (1995). Analytical Magnetapheresis of Ferritin-Labeled Lymphocytes. Analytical Chemistry 67(20): 3702- 3712.
Zborowski, M., Malchesky, P.S., Savon, S.R., Green, R., Hall, G.S. and Nose, Y. (1991). Modification of Ferrography Method for Analysis of Lymphocytes and Bacteria. Wear 142(1): 135-149.
Zborowski, M., Moore, L.R., Williams, P.S. and Chalmers, J.J. (2002). Separations based on magnetophoretic mobility. Separation Science and Technology 37(16): 3611-3633.
Zborowski, M., Ostera, G.R., Moore, L.R., Milliron, S., Chalmers, J.J. and Schechter, A.N. (2003). Red blood cell magnetophoresis. Biophysical Journal 84(4): 2638-2645.
Zborowski, M., Sun, L.P., Moore, L.R., Williams, P.S. and Chalmers, J.J. (1999). Continuous cell separation using novel magnetic quadrupole flow sorter. Journal of Magnetism and Magnetic Materials 194(1-3): 224-230.
Zhang, H. (2004). Immunomagnetic cell separation continued development of fundamental model of magnetophoretic mobility and further applications.
Zhang, H., Zborowski, M., Williams, P.S. and Chalmers, J.J. (2005). Establishment and implications of a characterization method for magnetic nanoparticles using Cell Tracking Velocimetry and magnetic susceptibility modified solutions. Analyst 130: 514 – 527.
Zhang, H.D., Moore, L.R., Zborowski, M., Williams, P.S., Margel, S. and Chalmers, J.J. (2005). Establishment and implications of a characterization method for magnetic nanoparticle using cell tracking velocimetry and magnetic susceptibility modified solutions. Analyst 130(4): 514-527.
Zhang, H.D., Williams, P.S., Zborowski, M. and Chalmers, J.J. (2006). Binding affinities/avidities of antibody-antigen interactions: Quantification and scale-up implications. Biotechnology and Bioengineering 95(5): 812-829.
177 Zhang, M., Li, W.Q., Yang, Y.C., Chen, B.C. and Song, F.P. (2005). Effects of readily dispersible colloid on adsorption and transport of Zn, Cu, and Pb in soils. Environment International 31(6): 840-844.
Zimmerman, P.A., Thomson, J.M., Fujioka, H., Collins, W.E. and Zborowski, M. (2006). Diagnosis of malaria by magnetic deposition microscopy. American Journal of Tropical Medicine and Hygiene 74(4): 568-572.
178 Appendix A: R code and result for classification of spores and PSM
179 install.packages('e1071') library('e1071') library(MASS) setwd('D:/research/ctv/analyzeddata/Rcode')######may need to reset the dir#########
TR<-read.table('standard3.prn',header=T) attach(TR)
############SMV method############################# model.ma <- svm(Particles~.,data = TR) summary(model.ma) x<-TR pred.train <- predict(model.ma, x)
# check accuracy using the same data table(pred.train, Particles) detach(TR)
# check accuracy using the other data
TR3<-read.table('standard4.prn',header=T)
TR3sub<-subset(TR3,select=-Particles) pred2<-predict(model.ma,TR3sub) pred<- predict(model.ma, TR3) table(pred,TR3$Particles) table(pred2,TR3$Particles)
180 ########acurrate!###################################
######### now group our mix population############## mix1<-read.table('mix1.prn',header=T) pred_mix1<-predict(model.ma, mix1) mix1_final<-cbind(mix1, pred_mix1) table(pred_mix1,mix1$Particles)
mix2<-read.table('mix2.prn',header=T) pred_mix2<-predict(model.ma, mix2) mix2_final<-cbind(mix2, pred_mix2) table(pred_mix2,mix2$Particles)
write.table(TR3_final,quote=F,row.names=F,file='TR3_result.txt') write.table(mix2_final,quote=F,row.names=F,file='mix2_result.txt') write.table(mix1_final,quote=F,row.names=F,file='mix1_result.txt')
181 Appendix B: Result from the R code in appendix A
182 Call: svm(formula = Particles ~ ., data = TR2)
Parameters:
SVM-Type: C-classification
SVM-Kernel: radial
cost: 1
gamma: 0.5
Number of Support Vectors: 96
( 48 48 )
Number of Classes: 2
Levels:
PSM SPOpred2
PSM SPO
PSM 1476 3
SPO 1 1750
183 pred2 PSM SPO
PSM 1294 23
SPO 0 1049
pred_mix1 PSM SPO
PSM 555 1
SPO 23 566
pred_mix2 PSM SPO
PSM 1286 3
SPO 17 912
184