University of Colorado, Aerospace Engineering Sciences Department, BioServe Space Technologies, Boulder, CO 80309-0429, USA
Correspondence
Michael Benoit
Michael.Benoit{at}Colorado.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is postulated that the primary aspect of space flight affecting microbial cell suspension growth characteristics is that of weightlessness, rather than radiation (Klaus, 2002). Furthermore, it has been hypothesized that space flight indirectly causes the observed differences in bacterial cell suspension cultures as a consequence of altering the distribution of cells in their fluid environment. This altered distribution is thought to affect the extracellular environment, hence triggering a physiological response (Mattoni, 1968
; Thévenet et al., 1996
; Kacena et al., 1997
, 1999a
; Klaus et al., 1997
).
Several factors caused by the weightless environment of space flight are likely to alter the fluid environment appreciably. For example, the lack of sedimentation of cells in suspension may contribute to the accessibility of nutrients simply due to better cell distribution (Klaus et al., 1997). In addition, a decrease in the density of the medium immediately surrounding a population of actively metabolizing bacterial cells has been observed to create buoyant plumes in a 1 g environment (Brown, 1999
). Albrecht-Buehler (1991)
pointed out that the lack of microconvective currents around a growing bacterial cell in microgravity might mean that the cell would resemble a growing protein crystal in microgravity. McPherson (1993)
suggested that the lack of mixing in weightlessness creates a quasistable depletion zone around the nucleus of a growing crystal. The result is that crystal surfaces interface with a microenvironment of lower protein concentration than the bulk solution, which is ideal for the formation of crystals of higher order and larger size. Klaus et al. (1997)
postulated that a similar phenomenon may occur around growing bacterial cells in a weightless environment with respect to nutrients and by-products, and that this depletion zone may be partially responsible for the observed differences in space flight experiments. The net effect is that an individual cell may become separated from its immediate surroundings at a higher rate when sedimentation is present (1 g) than in conditions of weightlessness.
Interestingly, higher final biomass yields are commonly achieved in bioreactor engineering by controlled addition of a reduced carbon source, such as glucose, in what is known as fed-batch culturing (Robbins & Taylor, 1989; Yee & Blanch, 1992
). This technique prevents excessive accumulation of byproducts such as acetate and allows for a more prolonged growth phase (Frude et al., 1994
). If, as previously discussed, a depletion zone is formed around growing bacterial cells in microgravity, the effect of reducing mass transport might essentially be the same as that of controlled addition of glucose to the cell, thus similarly contributing to higher final cell density (Klaus, 1998
, 2002
). Todd & Klaus (1996)
explored this notion with theoretical calculations and predicted higher final cell densities if the glucose level at the cell boundary is reduced from the bulk solution concentration.
One substantial obstacle in determining how space flight affects bacterial cell cultures is the relatively small number of samples obtained over a large array of experiments. Flight opportunities to conduct experiments are not only infrequent and expensive, they are also subject to a number of unique hardware and operational constraints, and opportunities to exactly repeat an experiment are rare. In addition, results are sometimes contradictory. For example, no significant difference between final cell population density of flight and ground samples was reported by Bouloc & D'Ari (1991), Gasset et al. (1994)
, Thévenet et al. (1996)
or Kacena & Todd (1997)
. In some cases this lack of a difference was attributed to a specific parameter, such as cell motility (Thévenet et al., 1996
) or growth on agar substrate (Kacena & Todd, 1997
). These latter two experiments actually corroborate the concept that a quiescent fluid environment and lack of cell sedimentation affect cell growth in microgravity. Specific test conditions, growth medium and assay methods may account for other contradicting results (Klaus et al., 1997
). In addition to studies conducted in space, various ground-based methods have been designed to mimic certain aspects of microgravity. Two devices, the clinostat and the rotating wall vessel bioreactor, are used extensively for experiments with bacteria and other cell cultures (Klaus, 2001
).
A clinostat is a device that partially simulates the microgravity conditions of space flight for suspension cell cultures. It does so by rotating a cylinder completely filled with a liquid medium (i.e. no air bubbles) and cells at a constant velocity. After initial start-up of the clinostat, the rotational velocity of the cylinder wall is transferred radially inward as a function of the viscosity of the medium. This continues until solid body rotation of the fluid medium occurs and virtually no relative fluid motion exists (Klaus et al., 1998). The cells in this situation are still under the direct influence of gravity; however, the gravity vector is continually being reoriented as the cells and fluid rotate together. If the clinostat is operated at an appropriate rotation rate, the cells will neither sediment nor be appreciably centrifuged to the container wall. Therefore, the lack of net sedimentation of the cells and the reduction of relative fluid motion represent functional features of space flight experiments that are mimicked with the clinostat in 1 g. Bacteria grown under clinorotation typically exhibit trends similar to comparable space flight data (Mattoni, 1968
; Mennigmann & Lange, 1986
; Klaus et al., 1998
; Kacena et al., 1999a
; Brown et al., 2002
). Although bacteria do not sediment to the container bottom during proper clinorotation, they still move, thus making it difficult to quantify exactly how much fluid mixing occurs as a result.
Rotating wall vessel (RWV) bioreactors are also used to maintain low-shear suspensions of cell cultures in a similar fashion to clinorotation. A review of several RWV applications has recently been published (Nickerson et al., 2003). For bacterial RWV experiments, findings pertaining to final cell number or dry cell weight, when reported, show varying results (Fang et al., 1997a
, b
, 2000
; Huitema et al., 2002
; Wilson et al., 2002
; England et al., 2003
; Baker & Leff, 2004
). This unpredictability further emphasizes the assertion that specific experimental parameters must be considered before general conclusions can be drawn.
A novel method is proposed here for evaluating the effect of reduced cell sedimentation in a suspension culture (without imparting resultant motion from rotation) using Escherichia coli cultures that have been genetically modified to be neutrally buoyant. These bacteria produce gas vesicles, which are intracellular, protein-coated, hollow organelles that are normally found in cyanobacteria and halophilic archaea (Li & Cannon, 1998). The vesicles are permeable to ambient gases by diffusion and provide the carrier cell with buoyancy, allowing the cells to move upwards in liquid. Cultures of genetically modified E. coli that produce gas vesicles sediment at a considerably reduced velocity, thus approximating neutral buoyancy. The modified cells were investigated as an alternative experimental model for further characterizing how the lack of cell sedimentation and reduction of related fluid motion, common to space flight and clinorotation, affect bacterial growth. This experimental model does not, however, reduce convective fluid mixing, which is also an important feature of microgravity (Albrecht-Buehler, 1991
). The study described here directly compared the growth of matched buoyant or non-buoyant E. coli strains in static controls versus clinorotated suspension cultures of each strain.
![]() |
HYPOTHESIS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. Non-buoyant E. coli grown under clinorotation will achieve a higher population density at or near the onset of stationary phase as compared to matched (non-buoyant) static controls.
2. Induced buoyant E. coli grown under clinorotation will not experience an increased cell population density at or near the onset of stationary phase relative to matched (buoyant) static controls.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiment design.
The induced E. coli DH5(pNL29) was used as the experimental culture; for simplicity it will be referred to as the experimental strain. Growth of E. coli DH5
(pNL29) without induction by IPTG was also used as a control, but the DH5
host strain of E. coli does not have tight regulation of the plasmid genes; therefore, the non-induced E. coli DH5
(pNL29) still produces some gas vesicles, resulting in partial buoyancy. This strain will be referred to as control I. Finally, the E. coli with the pBluescriptIISK cloning vector, which produced no gas vesicles, will be referred to as control II. It was expected that the relative difference in culture density at 60 h between static and clinorotated cultures for the control I strain would fall between the experimental strain (smallest difference) and the control II strain (largest difference).
Media and growth conditions.
Cultures were inoculated from 15 % (v/v) glycerol freezer (80 °C) stock in 5 ml liquid LuriaBertani Miller (LBM) broth (Sigma L-3152) with 100 µg ampicillin ml1 (AMP100) (Fischer BP1760). The 5 ml cultures were grown for 12 h (to early exponential phase) in a 15 ml conical tube tilted at a 30° angle, incubated at 37 °C, and aerated by rotation at 225 r.p.m. (2 cm diameter). A volume of 1 ml of each culture was then transferred to 50 ml of a defined minimal growth medium (medium E; Vogel & Bonner, 1956) supplemented with 5 g glucose l1 (autoclaved separately) and AMP100. Each culture was grown in a 250 ml Erlenmeyer flask, aerated by rotation at 225 r.p.m. (2 cm diameter), and incubated at 37 °C for 12 h. OD600 readings were taken with a Genesys 10 Series Spectrophotometer (Spectronic Unicam) and 10 mm path-length cuvettes. Cells were counted on a Hausser Scientific Improved Neubauer Haemocytometer with a minimum of 200 cells counted in 16 squares. An appropriate volume of culture was diluted to 1x107 cells ml1 in fresh 1x medium E plus 5 g glucose l1 and AMP100. The E. coli experimental culture was induced to activate gas vesicle production by adding an appropriate volume of 1000x (1 M) stock of IPTG (Sigma I-5502) to obtain a final concentration of 1 mM IPTG. Experimental cultures were pipetted in 4 ml volumes into one end of a fluid processing apparatus (FPA) and 4 ml of either control culture (non-induced control I strain or control II strain) was added to the other end, such that each FPA contained an experimental and a control culture. The FPA is a glass barrel that has an inner diameter of 1·35 cm and is 11·5 cm long. It is a specially designed test tube for use with the clinostat described below and for use on space flights (Klaus et al., 1997
). Rubber septa were used to seal each fluid chamber of the FPA such that no air space existed; however, the cultures were not rendered anoxic prior to filling of the FPA. Initial dissolved oxygen levels in the freshly filled FPAs were calculated to be 0·23 mM. It was anticipated, based on previous unpublished data, that the dissolved oxygen in the FPAs would be depleted in approximately 24 h. Fourteen FPAs were loaded in this fashion for each experimental run. Seven were placed on the clinostat and seven were placed in a test tube rack that was laid horizontally next to the clinostat at room temperature. The temperature was recorded every 15 min and the overall mean was 20·67±0·85 °C. Due to the limited capacity of FPAs on the clinostat, comparison of culture growth was performed at only one time point in order to readily obtain a sufficient number of samples. The appropriate time for population comparison was determined from the preliminary growth curve data (with duplicate samples only) illustrated in Fig. 1
. It can be seen in Fig. 1(a)
that the control II cultures reached stationary phase on the clinostat at
60 h; however, the static cultures appeared to enter stationary phase sooner (
48 h). As such, 60 h was chosen as the time point for sampling the control II cultures. The experimental cultures showed nearly identical growth curves for both clinostat and static cultures, with stationary phase not completely reached until 72 h (Fig. 1b
). At 60 h, however, there appeared to be the greatest difference between the clinostat and static cultures. Therefore, by choosing the sample time point of 60 h, we were investigating a worst-case scenario, since we expected no difference in cell population density. Also, this allowed for all cultures to be harvested at the same time point.
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Certain factors impaired the ability to directly compare growth between the different E. coli strains. For example, one complication is the possibility of the production of gas vesicles causing an additional metabolic burden on the induced cells. Gas vesicle production presumably requires energy that otherwise could contribute to new cell growth. Therefore, comparison of growth between different strains could be affected by both the buoyant nature of the cells and the metabolic burden of producing the vesicles. Another reason for avoiding comparisons between strains was that for each strain of E. coli, a single common starting stock of culture was used for both the clinostat and static cultures. For different strains, however, separate starting cultures were necessarily used. Although precautions were taken to ensure that the starting conditions were the same, differences in initial cell number could complicate comparing growth between the different strains. For these reasons, comparison of growth on the clinostat to the controls was limited to the differences between each individual strain only.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The findings presented here indicate that preventing cell sedimentation through induced cell buoyancy mitigated the resultant higher final culture density of clinorotated E. coli cell suspension cultures relative to matched static controls. While the clinostat typically produces the same trend of increased growth as reported for comparable space flight experiments, the magnitude of the difference is usually less pronounced. This may be a function of the complete lack of buoyancy-induced convective flow that occurs in the extracellular fluid under true microgravity conditions, thus reducing metabolic byproduct transport in addition to causing a lack of cell sedimentation (Brown, 1999).
The mean 5·6 % difference of clinorotated and static control I E. coli cultures fell between the 10·5 % difference of the control II strain and the insignificant difference of the experimental strain. This was also expected since the control I strain showed a relatively high percentage of cells with gas vesicles, resulting in some buoyancy, presumably due to leaky operon control in the E. coli DH5 host cells as discussed.
The higher final cell population density achieved in the clinorotated samples compared to static cultures of the E. coli control II strain (no gas vesicles) compares favourably with differences observed in the literature for a variety of different bacteria (Mattoni, 1968; Mennigmann & Heise, 1994
; Klaus et al., 1998
; Kacena et al., 1999a
; Brown et al., 2002
).
Gas-vesicle-producing E. coli that achieve neutral buoyancy offer a novel ground-based method for isolating the role of reduced cell sedimentation on bacterial suspension cultures. Plans for future work in this area include developing an optical monitoring system to allow continuous recording of clinostat and static culture population densities through all phases of growth.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker, P. W. & Leff, L. (2004). The effect of simulated microgravity on bacteria from the Mir space station. Microgravity Sci Technol XV, 3541.
Bouloc, P. & D'Ari, R. (1991). Escherichia coli metabolism in space. J Gen Microbiol 137, 28392843.[Medline]
Brown, R. B. (1999). Effects of space flight, clinorotation, and centrifugation on the growth and metabolism of Escherichia coli. PhD thesis, University of Colorado, Boulder.
Brown, R. B., Klaus, D. & Todd, P. (2002). Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli. Microgravity Sci Technol XIII, 2429.
Ciferri, O., Tiboni, O., Di Pasquale, G., Orlandoni, A. M. & Marchesi, M. L. (1986). Effects of microgravity on genetic-recombination in Escherichia coli. Naturwissenschaften 73, 418421.[Medline]
England, L. S., Gorzelak, M. & Trevors, J. T. (2003). Growth and membrane polarization in Pseudomonas aeruginosa UG2 grown in randomized microgravity in a high aspect ratio vessel. Biochim Biophys Acta 1624, 7680.[Medline]
Fang, A., Pierson, D. L., Mishra, S. K., Koenig, D. W. & Demain, A. L. (1997a). Gramicidin S production by Bacillus brevis in simulated microgravity. Curr Microbiol 34, 119204.
Fang, A., Pierson, D. L., Mishra, S. K., Koenig, D. W. & Demain, A. L. (1997b). Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and its excretion into the medium. Appl Environ Microbiol 63, 40904092.[Abstract]
Fang, A., Pierson, D. L., Mishra, S. K. & Demain, A. L. (2000). Growth of Streptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production. Appl Microbiol Biotechnol 54, 3336.[CrossRef][Medline]
Frude, M. J., Read, A. & Kennedy, L. D. (1994). Scale-up production of a recombinant Mycobacterium leprae antigen. Ann N Y Acad Sci 721, 100104.[Medline]
Gasset, G., Tixador, R., Eche, B., Lapchine, L., Moatti, N., Toorop, P. & Woldringh, C. (1994). Growth and division of Escherichia coli under microgravity conditions. Res Microbiol 145, 111120.[CrossRef][Medline]
Huitema, C., Beaudette, L. A. & Trevors, J. T. (2002). Simulated microgravity (SMG) and bacteria. Rivista Di Biologia 95, 497503.[Medline]
Kacena, M. & Todd, P. (1997). Growth characteristics of E. coli and B. subtilis cultured on an agar substrate in microgravity. Microgravity Sci Technol X, 5862.
Kacena, M. A., Leonard, P. E., Todd, P. & Luttges, M. W. (1997). Low gravity and inertial effects on the growth of E. coli and B. subtilis in semi-solid media. Aviat Space Environ Med 68, 11041108.[Medline]
Kacena, M. A., Manfredi, B. & Todd, P. (1999a). Effects of space flight and mixing on bacterial growth in low volume cultures. Microgravity Sci Technol XII, 7477.
Kacena, M. A., Merrell, G. A., Manfredi, B., Smith, E. E., Klaus, D. M. & Todd, P. (1999b). Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis. Appl Microbiol Biotechnol 51, 229234.[CrossRef][Medline]
Klaus, D. (1998). Microgravity and its implications for fermentation biotechnology. Trends Biotechnol 16, 369373.[CrossRef][Medline]
Klaus, D. M. (2001). Clinostats and bioreactors. Gravit Space Biol Bull 14, 5564.[Medline]
Klaus, D. M. (2002). Space microbiology: microgravity and microorganisms. In Encyclopedia of Environmental Microbiology, pp. 29963004. Edited by G. Britton. New York: Wiley.
Klaus, D. M., Lutteges, M. W. & Stodieck, L. S. (1994). Investigation of space flight effects on Escherichia coli growth. In SAE Technical Paper Series 941260, pp. 19. Warrendale, PA: SAE Publications.
Klaus, D., Simske, S., Todd, P. & Stodieck, L. (1997). Investigation of space flight effects on Escherichia coli and a proposed model of underlying physical mechanisms. Microbiology 143, 449455.[Medline]
Klaus, D., Todd, P. & Schatz, A. (1998). Functional weightlessness during clinorotation of cell suspensions. Adv Space Res 21, 13151318.[CrossRef][Medline]
Lam, K. S., Mamber, S. W., Pack, E., Forenza, S., Fernandes, P. & Klaus, D. (1998). The effects of space flight on the production of monorden by Humicola fuscoatra WC5157 in solid state fermentation. Appl Microbiol Biotechnol 49, 579583.[CrossRef][Medline]
Lam, K. S., Gustavson, D. R., Pirnik, D., Pack, E., Bulanhagui, C., Mamber, S. W., Forenza, S., Stodieck, L. S. & Klaus, D. M. (2002). The effect of space flight on the production of actinomycin D by Streptomyces plicatus. J Ind Microbiol Biotechnol 29, 299302.[CrossRef][Medline]
Li, N. & Cannon, M. C. (1998). Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli. J Bacteriol 180, 24502458.
Mattoni, R. H. T. (1968). Space-flight effects and gamma radiation interaction on growth and induction of lysogenic bacteria. BioScience 18, 602608.
McPherson, A. (1993). Effects of a microgravity environment on the crystallization of biological macromolecules. Microgravity Sci Technol VI, 101109.
Mennigmann, H. D. & Heise, M. (1994). Response of growing bacteria to reduction in gravity. In Fifth European Symposium on Life Science Research in Space SP-366, pp. 8387. Paris: European Space Agency.
Mennigmann, H. D. & Lange, M. (1986). Growth and differentiation of Bacillus subtilis under microgravity. Naturwissenschaften 73, 415417.[CrossRef][Medline]
Moatti, N., Lapchine, L., Gasset, G., Richoilley, G., Templier, J. & Tixador, R. (1986). Preliminary results of the antibio experiment. Naturwissenschaften 73, 413414.[CrossRef][Medline]
Nickerson, C. A., Ott, C. M., Wilson, J. W., Ramamurthy, R., LeBlanc, C. L., Honer zu Bentrup, K., Hammond, T. & Pierson, D. L. (2003). Low-shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis. J Microbiol Methods 54, 111.[CrossRef][Medline]
Robbins, J. W. & Taylor, K. B. (1989). Optimization of Escherichia coli growth by controlled addition of glucose. Biotechnol Bioeng 34, 12891294.[CrossRef]
Thévenet, D., D'Ari, R. & Bouloc, P. (1996). The SIGNAL experiment in BIORACK: Escherichia coli in microgravity. J Biotechnol 47, 8997.[CrossRef][Medline]
Tixador, R., Richoilley, G., Gasset, G., Templier, J., Bes, J. C., Moatti, N. & Lapchine, I. (1985). Study of minimal inhibitory concentration of antibiotics on bacteria cultivated in vitro in space (Cytos 2 Experiment). Aviat Space Environ Med 56, 748751.[Medline]
Todd, P. & Klaus, D. M. (1996). Theories and models on the biology of cells in space. Adv Space Res 17, 310.[Medline]
Vogel, H. J. & Bonner, D. M. (1956). Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218, 97106.
Wilson, J. W., Ott, C. M., Ramamurthy, R., Porwollik, S., McClelland, M., Pierson, D. L. & Nickerson, C. A. (2002). Low-shear modeled microgravity alters the Salmonella enterica serovar Typhimurium stress response in an RpoS-independent manner. Appl Environ Microbiol 68, 54085416.
Yee, L. & Blanch, H. W. (1992). Recombinant protein expression in high cell-density fed-batch cultures of Escherichia coli. Biotechnology 10, 15501556.[CrossRef][Medline]
Received 27 January 2004;
revised 30 July 2004;
accepted 7 October 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |