Magnetic space shuttle experiments

Journal of Magnetism and Magnetic Materials 194 (1999) 96—101

Magnetic space shuttle experiments

Paul Todd!,*, John F. Doyle!, Paul Carter!, Howard Wachtel!, Mark S. Deuser",John C. Vellinger", John M. Cassanto#, Ulises Alvarado#, Jurgen Sygusch$,

Thomas B. Kent%

!Bioserve Space Technologies Center, Campus Box 429, University of Colorado, Boulder, CO 80309-0429, USA"SHOT, Inc., 5605 Featherengill Road, Floyd Knobs, IN 47119, USA

#Instrumentation Technology Associates, Inc., 35 E. Uwchlan Avenue, Suite 300, Exton, PA 19341, USA$Department of Biochemistry, University of Montreal, CP 6128, Station Centre Ville, Montreal, PQ H3C3J7, Canada

%FeRx, Inc., 18200 West Highway 72, Arvada, CO 80007, USA

Abstract

Applications of magnetic fields in separation science, biotechnology and gravitational biology as studied in thelow-gravity environment of space flight are reviewed. Ferromagnetism, ferrimagnetism, paramagnetism and diamag-netism have been applied in studies of collagen gel formation, bacterial growth and mixing in the free-fall condition oforbital space flight. ( 1999 Elsevier Science B.V. All rights reserved.

Keywords: Magnetic particles; Magnetotactic bacteria; Space research; Collagen; Mixing; Microgravity

1. Introduction

Applications of magnetic fields in separationscience, biotechnology and gravitational biology asstudied in the low-gravity environment of spaceflight are reviewed. Studies have utilized ferromag-netism, ferrimagnetism, paramagnetism and dia-magnetism. Specific areas of study include collagenassembly, bacterial growth, low-volume mixing andplant gravitropism.

*Corresponding author. Fax: #1-303-492-4341; e-mail:[email protected].

2. Collagen assembly

The magnetotactic bacterium Magnetospirillummagnetotacticum has been used as a source ofmobile magnetic particles in the anisotropic assem-bly of collagen gels on the ground and in lowgravity. These bacteria contain chains of magnetiteparticles (‘magnetosomes’) which provide theequivalent of uniform ferrimagnetic microparticles[1]. The bacteria were chemically fixed withglutaraldehyde and suspended in phosphate buffer.This bacterial particle suspension was introducedinto a solution of Type I collagen in acetic acid inthe presence or absence of a magnetic field usingthe ‘Fluid Processing Apparatus’, an automatedtest-tube for space research developed by BioServe

0304-8853/99/$ — see front matter ( 1999 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 8 8 5 3 ( 9 8 ) 0 0 5 5 0 - 2

Fig. 1. Operation of a Fluid Processing Apparatus (BioServeSpace Technologies, Boulder, CO) for testing magnetic orienta-tion of assembling collagen gels in the laboratory and in reducedgravity. Initially chamber A contains an acid solution of col-lagen, and chamber B contains phosphate buffer and mag-netotactic bacteria fixed in glutaraldehyde and washed withphosphate buffer. Chamber C contains fixative that preservesthe collagen matrix. The two solutions are separated by movablerubber septa. Downward movement of the septa and fluidsallows mixing of the two fluids by transport around the septumat the bulge in the glass tube. Placing the magnet inside chamberA causes motion of the bacteria; placing the magnet outsidedoes not and serves as control. Magnetic field strength at thepole face of the permanent magnet was about 400 mT. FromCarter [2].

Space Technologies Center for Space Commercial-ization (Boulder, CO). The mechanics of the experi-ment are illustrated in Fig. 1, which details theoperation of the FPA. The motion of these particlesin a magnetic field sets up streamlines during theassembly of the collagen gel, giving strong parallelfibres. The stability of these streamlines appearedto be affected by gravity, as revealed in macro-scopic and microscopic inspection of gels formedon the ground and in low gravity. When assembledcollagen gels were removed from FPAs and fixedand sectioned for scanning electron microscopy,a clear difference between gels assembled in thepresence and absence of bacterial particles anda magnetic field was seen, as shown in Fig. 2 [2].

Fig. 2. Comparison of the microstructure of collagen gels for-med in control (top) and in magnetically oriented (bottom)collagen assembly systems. Reference bar is 1 lm long.

3. Bacterial growth in low gravity

Having observed that magnetotactic bacteriacan be manipulated in low gravity with shapedmagnetic fields, we then asked if shaped magneticfields could be applied to magnetotactic bacterialcells to replace the gravity body force in orbitalflight and, conversely to suspend cells as if thegravitational body force were absent in the labora-tory. First, the effects of reduced gravity on bacter-ial growth must be considered. It has been repeat-edly found that bacteria in unagitated suspendedcultures grow to higher cell densities than in 1 g, asshown in Fig. 3 from the work of Kacena et al. [3],reproduced here by permission. The higher growth

P. Todd et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 96—101 97

Fig. 3. Growth curves of E. coli cultures at 37°C in unagitatedsuspensions at 1 g (lower curve) and in low gravity on the spaceshuttle (upper curve), showing enhanced total growth in lowgravity. From Kacena et al. [3].

is thought to be due to the lack of crowding whencells do not sediment together due to gravity. Theapplication of a different body force, such as mag-netic, could be used to test this hypothesis. M. mag-netotacticum cell suspensions were flown on a spaceshuttle and a Mir mission with permanent magnetshaving pole-piece fields of about 400 mT, and cellswere maintained at one end of their container. Toattempt to mimic low-gravity in the laboratory,a vertical cylindrical culture system was designed asshown in Fig. 4 to maintain M. magnetotacticum cellsin suspension without sedimentation. Since growthexperiments and sedimentation both require severaldays, it is necessary to create a stable suspension ofbacteria. To accomplish this, the procedure used increating magnetically stabilized fluidized beds wasfollowed [4]. The magnetic flux distribution ofa 1600-turn coil operated at 0.1 A in the configura-tion of Fig. 4 is shown in Fig. 5. The vertical fielddistribution shows a trapping region within whichthe magnetic force on the cells will exceed the gravi-tational force on any cells brought into that regionby sedimenting into it from the top or by beingmagnetically captured from a few mm below. Thehorizontal (radial) field distribution is, in general,very uniform; however, two small peaks indicate thepresence of a trapping ring about 4 mm in from theedge of the coil, near the walls of the inner culturecontainer. Experimental testing of these systems isperformed using iron microparticles suspended inethanol or polyethylene glycol obtained from FeRx,Inc. (Arvada, CO).

Fig. 4. Photograph of magnetic coil culture system for levitatingmagnetotactic bacteria in the laboratory and in space flight. Coilis wrapped around a Lexan sheath into which a culture tube isinserted.

This configuration is compatible with the flight-approved ‘Fluid Processing Apparatus’ developedby the BioServe Space Technologies Center forSpace Commercialization (Boulder, CO) and itsoperating container, ‘Group Activation Pack’. Theisothermal locker, ‘Isothermal Containment Mod-ule’, also designed and operated by BioServe iscapable of the heat rejection required by the mag-netic culture system. Preliminary experiments havetherefore been performed and most parametersidentified for the implementation of ‘magneto-pseudogravity’ and its counterpart ‘magneto-pseudomicrogravity’.

4. Mixing small fluid volumes in low gravity

Several pieces of experimental hardware exist forthe study of crystal growth from solution in lowgravity [5]. Biomacromolecule crystallization has

98 P. Todd et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 96—101

Fig. 5. Magnetic field map of an example of a bacterial suspen-sion system of the type shown in Fig. 4. In this example, 0.1A was applied to a coil with 1600 windings. The axial (vertical)field (top graph) shows a trapping region, while the radial field(bottom graph) is nearly uniform.

been emphasized in space-borne crystal growth ex-periments. Supersaturation conditions are typicallyachieved by the outward diffusion of water (toconcentrate precipitant and solute) or inward diffu-sion of precipitant. When both the precipitant,such as polyethylene glycol, and the solute, such asa protein have high molecular weights(10—100 kDa) diffusive transport is too slow(D"10~7—10~6 cm2/s) to allow supersaturationand equilibration during a short (6-day) spaceshuttle flight. Each reaction volume is just a fewmicroliters, and there could be hundreds of experi-ments on a single space shuttle mission, so conven-tional impeller technology is inappropriate asa means of increasing mass transfer. Fig. 6 indicatesone solution to this problem that has been imple-mented. In the ‘materials dispersion apparatus’(MDA) built and operated by InstrumentationTechnology Associates (Exton, PA), most experi-ments are conducted in free-boundary diffusion

Fig. 6. A diffusion cell in a sliding-block system, the materialsdispersion apparatus — MDA (Instrumentation Technology As-sociates, Inc., Exton, PA). PTFE-coated ferrous magnetic par-ticles are stored in the upper cavity until activation (beforelaunch). They are then drawn into the lower cavity by a perma-nent magnet, drawing with them the upper fluid to be mixedwith the lower fluid.

cells, 3 mm in diameter, in multi-well sliding blocks[6,7]. PTFE-coated ferrous magnetic micropar-ticles, 1—3 lm in diameter were transferred betweenupper and lower cavities of the diffusion cells at thetime of activation by the sliding of the blocks thatcontain the cavities, as illustrated in the three stepsof Fig. 6. Initially, the particles are held in the ‘top’cavity in the precipitant solution. A permanentmagnet is placed below the bottom of the opposingcavity. Upon activation the two fluids in the cavi-ties come into full contact, and the magnet drawsthe magnetic particles from the upper to the lowercavity, and presumably they drag the solution con-taining precipitant along with them. Because thesolutions are miscible, the precipitant solutionshould still wet the microspheres after they havecrossed the interface into the target solution. Al-though it is difficult to calculate the volume ofprecipitant fluid transferred, it was determined thatthe mixing goal was achieved on the basis of crys-tallization that occurred when magnetic particleswere used and did not occur when magnetic par-ticles were not used. The particles were, indeedtransferred between cavities under the influence ofthe magnets.

On a somewhat larger scale (a few milliliters) it ispossible to implement more conventional impellertechnology. There is interest in studying the behav-ior of immiscible aqueous solutions when theydemix in low gravity [8]. In the laboratory suchstudies at bench scale are usually performed in testtubes or test-tube-based countercurrent extraction


Fig. 7. Thin-layer multicavity automated countercurrent distribution apparatus, ADSEP (Space Hardware Optimization Technology,Floyd Knobs, IN), showing two disks with 22 cavities. One disk rotates to bring demixed fluids into new contacts. Magnetic stirring witha tumbling magnet is used to re-mix fluids from top and bottom cavities.

equipment, and agitation by shaking is the mostcommon means of re-mixing the phases. Multistagepartitioning studies are sometimes performed usinga two-disk multicavity thin-layer countercurrentdistribution apparatus similar to the ADSEP,a product of Space Hardware Optimization Tech-nology, Inc. (SHOT, Inc.) of Floyd Knobs, IN,shown in Fig. 7. Bench-scale countercurrent distri-bution products usually re-mix phases by a manualor automatic shaking procedure, in which the en-tire pair of disks is shaken by periodic externalforce [9]. Such shaking during a space flight wouldtransmit undesired inertial vibrations to otherpayloads, so a magnetic mixing method was de-vised. Initially simple magnetic stir bars driven byan external rotating magnet were used in thefashion of chemical laboratory mixers. Evidencewas obtained indicating that, in low gravity, the stirbars, which were in one cavity, did not impartadequate momentum to the opposite cavity to ef-fect complete mixing. This led to a novel magneticmethod in which a stirring magnet with a triangu-

lar cross section is caused to tumble by an alternat-ing current electromagnet that is periodically ac-tivated and de-activated by a computer programthat specifies the rate of tumbling. This techniqueassures momentum transfer between the contactedcavities; however, at the end of a mixing episode, itleaves the stirring magnet in an unspecified posi-tion in low gravity, where, in the absence of gravity,it can stay — possibly at the interface where it caninterfere with the rotation of the disks. The ADSEPand its predecessor, ORSEP [10] have been flownon two space shuttle and one sub-orbital rocketflights, and the magnetic mixing technology hasbeen applied, not only to phase mixing but also tocell culture reagent mixing and batch crystalliza-tion procedures.

5. Plant root gravitropism

Studies of root gravitropism have been enhancedby Kuznetsov and Hasenstein [11], who used the

100 P. Todd et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 96—101

Fig. 8. Diagram illustrating the test of the statolith hypothesisusing the diamagnetic property of amyloplast starch in plantroot cap columella cells. The root cap is shown by a fine line; oneof several columella cells is represented by a rectangle; the starchstatoliths are the particles within each cell. Left: normal down-ward root growth. Center: gravistimulated downward rootgrowth from horizontal orientation. Right: diamagneticallystimulated amyloplast movement and resulting tropic rootgrowth. Despite being vertical, the root curvature follows thedirection of the amyloplasts away from the steep-gradient mag-netic field. Representation based on work of Kuznetsov andHasenstein [11].

diamagnetic properties of starch-containingamyloplasts to demonstrate the role of amyloplastsin sensing a body force inside the gravity-sensingcell. Plant roots normally grow downward duringseed germination in direct response to the gravi-tational force vector. The sensor of the gravityvector is thought to be the columella cells(‘statocytes’) in the root cap whose amyloplastssediment in response to gravity. To add to tests ofthis hypothesis Kuznetsov and Hasenstein sub-jected germinating roots to very high magnetic fieldgradients to cause the starch-filled amyloplasts tomove away from the field (starch is diamagneticwith a dimensionless susceptibility of about!1]10~6). It was demonstrated that the amylo-plasts did indeed move away from the field, and theroots grew in the direction predicted by thestatolith hypothesis, which states that amyloplastsfunction as sensors of the gravity vector. Althoughspace shuttle experiments were not reported, themagnetotropic response was shown to occur duringfunctional weightlessness, or simulated low gravity,on a horizontally rotating clinostat, and whole-plant experiments in high-gradient magnetic fields

have demonstrated the same principle on Salyutspacecraft [12]. A diagram summarizing the obser-vations is shown in Fig. 8.

Acknowledgements

These studies were supported by NASA, Cana-dian Space Agency, SHOT Inc., InstrumentationTechnology Associates, Inc., the Colorado Ad-vanced Technology Institute and FeRx. We thankDrs. James Urban and John Hobbs for technicalguidance with magnetotactic bacteria, SimranNanda and Ryan Cooper for laboratory assistance.

References

[1] R.P. Blakemore, Ann. Rev. Microbiol. 36 (1982) 217.[2] P. Carter, M.S. Thesis, University of Colorado, Boulder,

1994.[3] M.A. Kacena, B. Manfredi, D. Hanna, P. Todd, Appl.

Microbiotechnol., 1998, in press.[4] L. Nixon, C. Koval, L. Xu, R.D. Noble, G.S. Slaff, Bio-

separation 2 (1991) 217.[5] M.G. Sportiello, P. Todd, C.-Y. Lee, C.E. Kundrot, S.C.

Schultz, L.S. Stodieck, J.M. Cassanto, Space ‘96. 5th Int.Conf. on Engineering, Construction and Operations inSpace, Albuquerque, 2—6 June, Amer. Soc. of Civil Engi-neers, New York, 1996.

[6] J.M. Cassanto, W Holemans, T. Moller, P. Todd, R.M.Stewart, Z.R. Korszun, in: F. Shahrokhi, G. Hazelrigg, R.Bayuzick (Eds.), Progr. Astronaut. Aeronaut. 127: SpaceCommercialization: Platforms and Processing, AmericanInstitute of Aeronautics and Astronautics, Washington,1990, p. 199.

[7] J. Sygusch, R. Coulombe, J.M. Cassanto, M.G. Sportiello,P. Todd, J. Crystal Growth 162 (1996) 167.

[8] J. Van Alstine, L.J. Karr, J.M. Harris, R.S. Snyder, S.B.Bamberger, H.C. Matsos, P.A. Curreri, J. Boyce, D.E.Brooks, in: M.Z. Atassi (Ed.), Immunobiology of Proteinsand Peptides IV, Plenum, Springfield, OR, 1987, p. 305.

[9] T.E Treffry, P.T. Sharpe, H. Walter, D.E. Brooks, in: H.Walter, D.E. Brooks, D. Fisher (Eds.), Partitioning inAqueous Two-Phase Systems. Theory, Methods, Uses andApplications to Biotechnology, Academic Press, Orlando,FL, 1985, p. 131.

[10] M.S. Deuser, J.C. Vellinger, R.J. Naumann, M.R. Guinn,P.W. Todd, Terrestrial applications of biomedical researchin space, Book of Abstracts LS-42, American Institute ofAeronautics and Astronautics Life Sciences and Biomedi-cal Conf., Houston, TX, 1995, p. 65.

[11] O.A. Kuznetsov, K.H. Hasenstein, Planta 198 (1996) 87.[12] V. Tchikov, Ph. D. Thesis, Moscow State University, 1993.


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Magnetic space shuttle experiments