Paul W.

Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

Paul W. Baker* and Laura Leff

The Effect of Simulated Microgravity on

Bacteria from the Mir Space Station

The effects of simulated microgravity on two bacterial isolates,

Sphingobacterium thalpophilium and Ralstonia pickettii (formerly Burkholderia pickettii), originally recovered from water
systems aboard the Mir space station were examined. These
bacteria were inoculated into water, high and low concentrations of nutrient broth and subjected to simulated microgravity
conditions. S. thalpophilium (which was motile and had flagella) showed no significant differences between simulated microgravity and the normal gravity control regardless of the method
of enumeration and medium. In contrast, for R. pickettii (that
was non-motile and lacked flagella), there were significantly
higher numbers in high nutrient broth under simulated microgravity compared to normal gravity. Conversely, when R. pikkettii was inoculated into water (i.e., starvation conditions) significantly lower numbers were found under simulated microgravity compared to normal gravity. Responses to microgravity
depended on the strain used (e.g., the motile strain exhibited no
response to microgravity, while the non-motile strain did), the
method of enumeration, and the nutrient concentration of the
medium. Under oligotrophic conditions, non-motile cells may
remain in geostationary orbit and deplete nutrients in their vicinity, while in high nutrient medium, resources surrounding the
cell may be sufficient so that high growth is observed until
nutrients becoming limiting.
1. Introduction
Several studies have investigated the response of bacteria to the
effects of microgravity in space [1, 2, 3, 4, 5, 6, 7]. Initial studies revealed that E. coli exhibited a slight increase in generation time under microgravity conditions compared to normal
Mail address:
*Paul W. Baker, Department of Biological Sciences, Kent State University,
Kent, OH 44242, pbaker3@kent.edu
Paper submitted: 22.08.2003
Submission of final revised version: 11.11.203
Paper accepted: 25.11.2003

Z-Tec Publishing, Bremen

gravity controls but that cell size remained unaffected [6].

Subsequent experiments on E. coli performed under microgravity in a specially designed compartmentalized syringe showed
that there was shortened lag phase and an increase in the final
cell density compared to the normal gravity controls [1, 2, 3, 4,
5, 7]. Bacillus subtilis showed similar characteristics to E. coli
under microgravity [2, 3, 5]. Differences in bacterial responses
between microgravity and normal gravity are attributed to factors such as sedimentation, hydrostatic pressure and evaporation
that do not occur in microgravity as they do under normal gravity conditions [8]. Also the open-state of porins may change
under microgravity conditions [7] but the bacterial cell may
respond to these changes to counteract these affects.
Conducting microgravity experiments in space is expensive,
cumbersome and time consuming. Consequently, a technique,
clino-rotation, has been devised to simulate microgravity in
ground-based experiments using a rotary cell culture system
(RCCS-Synthecon, Houston, TX). Previous studies on E. coli
and B. subtilis have shown that clino-rotation yields similar
results to actual microgravity conditions [5]. It is unlikely that
space radiation affected the growth of bacteria in space because
a previous study showed no detrimental effect on bacteria incubated aboard the Mir space station [9]. Under clino-rotation,
cells do not sediment because of the effect of gravity because
the bottom of the vessel is constantly moving in a circular
motion around the horizontal axis [10]. Consequently, the cells
remain in a relatively stationary position similar to the effect of
microgravity on suspended cells [11]. A number of studies have
investigated the effect of simulated microgravity on microorganisms and have revealed differences compared to normal gravity controls [12, 13, 14, 15, 16, 17]. Bacteria, such as E. coli and
B. subtilis used in the experiments described above [1, 2, 3, 4,
5, 7], represent widely-used laboratory strains rather than bacteria that thrive naturally in the microgravity environment.
Many bacterial isolates have been recovered from the water
systems of the Mir and International Space Stations [18]. These
bacteria are a potential source of contamination that may affect
water quality [19]. Because bacteria within water systems are
exposed to extremely oligotrophic conditions, it is important to

Microgravity sci. technol. XV/1 (2004)

35

Paul W. Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

experimentally examine bacterial growth and survival under

oligotrophic conditions in a microgravity environment. In this
study, using bacteria isolated from the water supply aboard the
Mir space station, simulated microgravity experiments were
conducted to investigate, using fluorescent staining techniques,
bacterial survival and growth under different nutrient concentrations.
2. Materials and Methods
2.1 Experimental Design
Bacteria used were: Sphingobacterium thalpophilium (isolated
from water condensate on the Mir space station that was collected, heated, filtered and passed through a heat exchanger to
cool) and Ralstonia pickettii (isolated from the main SVO water
storage tank on the Mir space station). These bacteria were identified by NASA using the Vitek system (Bio Merieux, Inc.,
Hazelwood, MO). For experiments in low nutrient medium or
water, the bacteria were acclimatized in low nutrient medium by
initially growing them in flasks containing 100 ml 0.2% (w/v)
nutrient broth (NB, Difco Laboratories, Detroit, MI, USA) for
24 h. Then cells were transferred into 0.02% (w/v) NB and growing for 24 h. Finally, cells were transferred into 0.002% (w/v)
NB and growing for 24 h. The bacteria were centrifuged at 6000
g for 20 min in a Beckman centrifuge to remove the medium.
The affects of centrifugation on bacteria are assumed to be only
temporary [7]. For the experiments in the high nutrient medium
(0.2% (w/v) NB), bacteria were grown in 0.2% (w/v) NB for 24
h and then centrifuged at 6000 g for 20 min prior to use in experiments.
Bacteria were then transferred into slow turning lateral vessels (STLV, Synthecon, Houston, TX, 240 ml volume, 9.2 cm
internal diameter) to a final abundance of approximately 5 x 105
cells ml-1 based on DAPI staining (see below). Three replicates
were used for each treatment. The STLV contained sterile deionized water, 0.002% (w/v) or 0.2% (w/v) nutrient broth. The
standard experimental set-up consisted of three STLV rotating
on a rotary cell culture system (RCCS, Synthecon) around the
horizontal axis (simulated microgravity). The STLVs were rotated at 40 rpm (40 mg) and during normal operation the bacteria
appeared equally distributed throughout the STLVs. Negative
controls were established by placing another RCCS so that the
three STLV rotated around the vertical axis (normal gravity).
Samples were recovered from the STLV containing water or
0.002% (w/v) NB on days 0, 1, 2, 3, 7 and 14 after the start of
the experiments. Growth during experiments using 0.2% (w/v)
NB was much more rapid and samples were collected in these
experiments on days 1 and 2 after the start of the experiments.
One portion of each sample was used directly for enumeration
of live and dead bacteria and plating. Another proportion of
each sample was preserved (PBS buffer: 8% paraformaldehyde)
and stored at 4C until analysis using DAPI and fluorescent in
situ hybridization (FISH) staining methods.
Different methods of enumeration were used to determine the
36

abundance of the bacteria. As described below, each method

relies on a different approach for enumeration and can reveal
differences in the physiological state of the cells.
2.2 Live-Dead BacLight kit
Fluorescent staining using the Live-Dead stain kit was performed to enumerate cells with an intact membrane (live) and cells
with a compromised membrane (dead) using a slight modification of the manufacturer's instructions (Molecular Probes,
Eugene, Oregon, USA). To the sample, 1.5 l of SYTO 9 and
1.5 l propidium iodide (PI) were added, and incubated in the
dark for 15 minutes as recommended in the manufacturers
instructions (Molecular Probes, Eugene, OR, USA). The sample
was dispensed onto a 0.2 m black polycarbonate filter
(Osmonics, Inc., Minnetonka, MN, USA) and filtered as previously described [20]. STYO 9 and PI stained cells were counted under an epifluorescent microscope using filter set 31011
and 41004 (Chroma Technology, Battleboro, VT, USA), respectively.
2.3 Colony Forming Units (CFU)
Samples recovered from each STLV were serially diluted in
0.85% (w/v) sodium chloride solution and then plated onto agar
plates containing 0.2% (w/v) nutrient broth (Difco Laboratories,
Detroit, MI) in triplicate. After plates were incubated at 25C
for 4 days, colonies were counted.
2.4 DAPI
The total number of cells was determined using DAPI (4',6-diamidino-2-phenylindole) staining [21]. The bacteria were stained
with 2 ml of DAPI solution (0.5 mg ml-1) for 3 minutes and the
cells were counted under an epifluorescent microscope.
2.5 Fluorescent in situ hybridization
Using FISH, cells are detected based on hybridization of a labeled probe to the rRNA inside the cell; only active cells with sufficient rRNA content are detected. FISH was performed using
the method developed by Lemke et al. [22]. Bacteria were concentrated onto a 0.2 m Anodisc filter (Whatman, Clifton, N.J.,
USA) overlaid onto a 0.45 m HA membrane filter. The
Anodisc was washed with 1 ml 0.2 m filtered sterile deionized
water, washed with 0.1% (w/v) Nonidet P40, and then removed
and placed into a petri-dish. To each Anodisc, 40 l hybridization solution (6x SSC, 0.02 M Tris [pH 7], 0.1% SDS and
0.01% Poly A) containing 5 ng l-1 Texas red-labeled probe
EUB338 [5'-JGCTGCCTCCCGTAGGAGT-3'] [23] (SigmaGenosys, The Woodlands, TX, USA) was added and were incubated for 4 hours at 48C in the dark. After the incubation, 2
volumes of 400 l washing buffer (0.9 M NaCl, 0.02M Tris [pH
7.2] and 0.1% (w/v) SDS) were filtered through the Anodisc. To
each Anodisc, 80 l washing buffer was added and they were
incubated at 48C for 10 minutes in the dark and rinsed with 2
volumes 400 l filtered sterilized distilled water. Bacterial cells
were counted under an epifluorescent microscope.
Microgravity sci. technol. XV/1 (2004)

Paul W. Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

2.6 Motility and detection of flagella

Bacterial colonies were grown on 0.2 % nutrient broth spread
plates for 18-24 h, gently transferred onto a glass slide and airdried. The flagella were stained for 4 min using Flagella Stain
Droppers [0.6% Crystal Violet in ethanol, 2.0% tannic acid,
2.5% phenol and 5.7% aluminum potassium sulfate] (Difco).
The slides were examined microscopically under oil immersion.
Motility tests were performed using Bacto motility test medium
(Difco) and were incubated for 24 h at 25C. Pseudomonas
aeruginosa was used as a positive control.

2.7 Statistical Analysis

The data were analyzed to determine significant differences
using One-way ANOVA in the standard version SPSS statistical
package for Windows (SPSS Inc., Chicago, USA). Post hoc
tests were performed using Tukey's test. Significant differences
were defined as cases where P<0.05.
3. Results
In the experiment where S. thalpophilium (which was motile
and flagellated) was inoculated into 0.2% (w/v) NB, there was
no significant differences between cells incubated under simulated microgravity and normal gravity (Figure 1). Likewise, in
the experiments where S. thalpophilium was inoculated into
0.002% (w/v) NB and water, there were no significant differences between numbers of cells under normal gravity and simulated microgravity regardless of the method of enumeration used
(Figures 2 and 3).
In the experiment where R. pickettii (which was non-motile and not flagellated) was inoculated into 0.2% (w/v) NB, numbers on day 2 were significantly higher under simulated microgravity compared to normal gravity (Figure 4). Differences

Fig 1a)

Fig 1b)

Fig. 1.: Growth of S. thalpophilium inoculated into 0.2% (w/v) NB

(a) sampled on day 1 (b) sampled on day 2 using the Live/Dead kit
(SYTO 9 + PI), DAPI and FISH. The bars in black represent simulated microgravity while the white bars represent the normal gravity
controls. Values are averages of 3 replicates and error bars represent the standard error. No differences between simulated microgravity and normal gravity controls were significant.

Microgravity sci. technol. XV/1 (2004)

Fig. 2.: Growth and survival of S. thalpophilium inoculated into

0.002% (w/v) NB and monitored over 14 days. (A) SYTO 9 stained
cells; (B) PI stained cells; (C) SYTO 9 + PI stained cells; (D) CFU
cell counts; (E) DAPI stained cell counts and (F) FISH cell counts.
The bars in black represent simulated microgravity while the white
bars represent the normal gravity controls. Values are averages of 3
replicates and error bars represent the standard error. No differences between simulated microgravity and normal gravity controls
were significant.

37

Paul W. Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

depended on the method of enumeration with the total counts

(based on DAPI and live+dead cells) showing a quite large difference between the two treatments. In contrast, in the experiment where R. pickettii was inoculated into 0.002% (w/v) NB,
there were no significant differences in cell number between
simulated microgravity and normal gravity. When the
Live/Dead stain was used on R. pickettii grown in 0.002% (w/v)
NB an unexpected result was found. These data are shown in
figure 5 but were omitted from statistical analysis because many
of the bacteria stained with SYTO 9 (i.e., Live) and also with PI
(i.e., Dead).
In the experiment where R. pickettii was inoculated into
water, there were significant differences, based on staining with
PI and DAPI, on days 7 and 14 between simulated microgravity and normal gravity conditions (Figure 6). In contrast to the
experiment with 0.2% (w/v) NB, in this case, the total number
of cells (and number of dead cells) was lower under simulated
microgravity than normal gravity. There were no statistically
significant differences for CFU counts and bacteria stained with
SYTO 9, SYTO 9 + PI, and FISH.

4. Discussion
Previous studies using E. coli demonstrated that population
sizes were significantly higher under simulated microgravity
than under normal gravity conditions [1, 5]. In this study, when
S. thalpophilium was inoculated into high/low nutrient medium
or water, there were no significant differences, regardless of the

4a)

4b)
Fig. 3.: Growth and survival of S. thalpophilium inoculated into
water and monitored over 14 days. (A) SYTO 9 stained cells; (B) PI
stained cells; (C) SYTO 9 + PI stained cells; (D) CFU cell counts;
(E) DAPI stained cell counts and (F) FISH cell counts. The bars in
black represent simulated microgravity while the white bars represent the normal gravity controls. Values are averages of 3 replicates and error bars represent the standard error. No differences between simulated microgravity and normal gravity controls were significant.

38

Fig. 4.: Growth of R. pickettii inoculated into 0.2% (w/v) NB (a)

sampled on day 1 (b) sampled on day 2 using the Live/Dead kit
(SYTO 9 + PI), DAPI and FISH. The bars in black represent simulated microgravity while the white bars represent the normal gravity
controls. Values are averages of 3 replicates and error bars represent the standard error. No symbols between microgravity and gravity represent no significant difference; * represents significant difference at 95. Live+Dead and DAPI on day 2 were significantly
higher under simulated microgravity compared to the gravity controls.

Microgravity sci. technol. XV/1 (2004)

Paul W. Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

method of enumeration, between simulated microgravity and

normal gravity. In a microgravity environment, bacteria generally remain in a geostationary position, whereas under normal
gravity the microorganisms are subject to the forces of sedimentation that can redistribute nutrients, waste products, airbubbles and cells [4]. Previous studies have suggested that
motility and the ability to form filaments may lead bacteria to
exhibit growth dynamics under microgravity that are similar to
normal gravity [3]. Indeed the cyanobacterium, Synechocystis
sp., a filamentous bacterium, exhibited growth characteristics
that were similar under simulated microgravity compared to
normal gravity [12]. In the present study, during examination of
S. thalpophilium via epifluorescent microscopy, it was found
that sometimes there were short filaments of cells despite vigorous mixing. It was also found that S. thalpophilium was flagellated during normal growth conditions. Therefore, it would
appear that filament formation and motility reduced the effect of
microgravity. Previous studies using B. subtilis, which is motile and forms filaments, revealed that under simulated microgravity growth was not significantly higher than under normal gravity but under actual microgravity conditions growth was signi-

Fig. 5.: Growth and survival of R. pickettii inoculated into 0.002%

(w/v) NB and monitored over 14 days. (A) SYTO 9 stained cells;
(B) PI stained cells; (C) SYTO 9 + PI stained cells; (D) CFU cell
counts; (E) DAPI stained cell counts and (F) FISH cell counts. The
bars in black represent simulated microgravity while the white bars
represent the normal gravity controls. Values are averages of 3
replicates and error bars represent the standard error. No differences between simulated microgravity and normal gravity controls
were significant.

Microgravity sci. technol. XV/1 (2004)

ficantly higher [5]. However, under actual microgravity conditions, the differences between microgravity and normal gravity
for B. subtilis were lower than for E. coli, a non-motile microorganism.
When R. pickettii was inoculated into high nutrient medium,
there were greater numbers of cells using some enumeration
methods (on day 2) under simulated microgravity compared to
normal gravity. Considering that this microorganism is not flagellated, these results correlate well with previous studies that
suggest that non-motile microorganisms under microgravity
will remain in a geostationally orbit and therefore show enhanced growth [4]. It has also been shown that under microgravity,
that the open-state of the porins in the cell membrane is reduced
[7]. This may lead to a reduction in metabolism and therefore
the cells may grow in the high nutrient broth as if they were in
a low nutrient environment and form smaller cells. This may
account for the higher number of cells observed during simula-

Fig. 6.: Growth and survival of R. pickettii inoculated into water

and monitored over 14 days. (A) SYTO 9 stained cells; (B) PI stained cells; (C) SYTO 9 + PI stained cells; (D) CFU cell counts; (E)
DAPI stained cell counts and (F) FISH cell counts. The bars in
black represent simulated microgravity while the white bars represent the normal gravity controls. Values are averages of 3 replicates and error bars represent the standard error. No symbols between microgravity and gravity represent no significant difference; *
represents significant difference at 95%; ** represents significant
difference at 99%. Dead and DAPI counts were significantly lower
on days 7 and 14 under simulated microgravity compared to the
gravity controls.

39

Paul W. Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

ted microgravity in comparison to the normal gravity controls

since more nutrients would be available for growth.
When R. pickettii was inoculated into low nutrient medium,
there were no significant differences between simulated microgravity and normal gravity. In this experiment, many of the bacteria stained simultaneously with SYTO 9 and PI when using
the Live/Dead kit; this phenomenon that was not observed in the
other experiments. In addition, during the first 24 h of the experiment, there was a significant increase in the number of cells
even though the majority stained with PI. This was concomitant
with a significant increase in numbers of CFU and suggests that
the bacteria stained with PI were still viable but that their cell
membranes may have been compromised allowing the PI stain
to enter the cells.
When R. pickettii was inoculated into water, there were significant differences between simulated microgravity and normal
gravity. Specifically, the total number of cells (based on DAPI
staining) was greater at the end of the experiment under normal
gravity conditions compared to microgravity. This is likely due
to nutrient depletion in the zone surrounding the cells under
microgravity conditions. In this experiment, there were also
greater numbers of dead cells under normal gravity compared to
microgravity. These results suggest that perhaps the rate of cell
break down and autolysis for R. pickettii under starvation conditions is higher under simulated microgravity compared to normal gravity. In a previous study using E. coli, microgravity had
no effect on the rate of autolysis (3) but that study used high
nutrient concentrations relative to our study. In an experiment
where killed R. picketti cells were incubated under simulated
microgravity and normal gravity, there were no significant differences in the loss of cells between the treatments (data not
shown) suggesting that apparent differences in the breakdown
of dead cells observed were not attributable solely to physical
differences between the two treatments. It is possible that the
differences in persistence of dead cells between normal gravity
and microgravity are related to differences in starvation response; prior studies have shown that microgravity affects stress
responses [17] and this may be related to the starvation response. Regardless, the interpretation of the observations about the
occurrence of dead cells is complicated by the results obtained
when R. pickettii was incubated in 0.002% (w/v) NB where
some cells stained as both live and dead. This suggests that
defining (and enumerating) dead cells based on currently available technology is imprecise under these circumstances but that
microgravity can impact bacterial survival and the distribution
of cells in different physiological states in a given population.
In conclusion, responses to microgravity vary among bacterial taxa; perhaps because of differences in motility and the
ability to form filaments. The motile bacterium that formed filaments, S. thalpophilium, was not affected by microgravity. In
contrast, for the non-motile strain, R. pickettii, effects of microgravity depended on nutrient concentration: under high nutrient
conditions there were significantly higher numbers under simulated microgravity compared to normal gravity while the oppo40

site was true under starvation conditions (in water). Differences

between the two gravity conditions not only depended on the
medium used; they were also dependent on the method of enumeration, reflecting differences in the response of cells in different physiological states. This difference may be due to higher
available nutrients near the geostationary cells grown in the
high nutrient medium that would enable more cell divisions to
occur whereas under starvation conditions depletion of nutrients
around the cell occurs under microgravity.
Acknowledgements
Thanks to Mark C. Ott and Duane Pierson for providing the
bacterial strains and to Michelle L. Meyer for technical assistance. This work was supported by grant NAG 2-1497 from
NASA.

References
[1] Brown, R.B., Klaus, D., Todd, P.: Effects of space flight, clinorotation, and
centrifugication on the substrate utilization efficiency of E. coli.
Microgravity Sci. Tech., vol. 13, p. 24 (2002).
[2] Kacena, M.A., Merrell, G.A., Manfredi, B., Smith, E.E., Klaus, D.M. and
Todd, P.: Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis. Appl. Environ.
Microbiol., vol. 51, p. 229 (1999).
[3] Kacena, M.A., Smith, E.E. and Todd, P.: Autolysis of Escherichia coli and
B. subtilis cells in low gravity. Appl. Microbiol. Biotechnol., vol.
52, p. 437 (1999).
[4] Klaus, D., Simske, S., Todd, P. and Stodieck, L.: Investigation of space
flight effects on Escherichia coli and a proposed model of underlying physical mechanisms. Microbiol., vol. 143, p. 449 (1997).
[5] Kacena, M.A., Manfredi, B. and Todd, P.: Effects of space flight and
mixing on bacterial growth in low volume cultures. Microgravity Sci.
Technol., vol. 12, p. 74 (1999).
[6] Gasset, G., Tixador, R., Eche, B., Lapchine, L., Moatti, N., Toorop, P.,
Woldringh, C.: Growth and division of Escherichia coli under microgravity conditions. Res. Microbiol., vol. 145, p. 111 (1994).
[7] Goldermann, M., Hanke, W.: Ion channel are sensitive to gravity changes.
Micrograv. Sci. Tech., vol. 13, p. 35 (2001).
[8] Kacena, M. and Todd, P.: Growth characteristics of E. coli and B.
subtilis cultured on an agar substrate in microgravity. Micrograv. Sci.
Technol., vol. 10, p. 58 (1997).
[9] Takahashi, A., Ohnishi, K., Yokota, A., Kumagai, T., Nakano, T., Ohnishi,
T.: Mutation frequency of plasmid DNA and Escherichia coli following
long-term space flight on Mir. J. Radiation Res. (suppl.), vol. 43, p. S137
(2002).
[10] Klaus, D.M.: Clinostats and bioreactors. Gravit. Space Biol. Bull., vol 14,
p. 55 (2001).
[11] Klaus, D.M., Todd, P., Schatz, A.: (1998) Functional weightlessness during
clinorotation of cell suspensions. Adv. Space Res., vol. 21, p. 1315 (1998).

Microgravity sci. technol. XV/1 (2004)

Paul W. Baker and Laura Leff: The Effect of Simulated Microgravity on Bacteria from the Mir Space Station

[12] Erdmann, N., Effmert, U., Fulda, S., Oheim, S.: Effect of gravity changes
on the cyanobacterium Synechocystis sp. PCC 6803. Curr. Microbiol., vol.
35, p. 348 (1997).
[13] Fang, A., Pierson, D.L., Koenig, D.W., Mishra, S.K., Demain, A.L.: Effect
of simulated microgravity and shear stress on microcin B17 production by
Escherichia coli and on its excretion into the medium. Appl. Environ.
Microbiol., vol. 63, p. 4090 (1997).
[14] Hder, D.-P., Rosum A., Schfer, J., Hemmersbach, R.: Graviperception in
the flagellate Euglena gracilis during a shuttle space flight. J. Biotechnol.,
vol. 47, p. 261 (1996).
[15] Nickerson, C.A., Ott, C.M. Wilson, J.W., Ramamurthy, R., LeBlanc, C.L.,
Hner zu Bentrup, K., Hammond, T., Pierson, D.L.: Low-shear modeled
microgravity: a global environmental regulatory signal affecting bacterial
gene expression, physiology, and pathogenesis. J. Microbiol. Methods,
vol. 54, p. 1 (2003).
[16] Thiruvenkatam, R., Scholz, C.: Synthesis of poly (-hydroxybutyrate) in
simulated microgravity: an investigation of aeration profiles in shake flask
and bioreactor. J. Polym. Environ., vol. 8, p. 155 (2000).
[17] Wilson, J.W., Ott, C.M., Ramamurthy, R., Porwollik, S., McClelland, M.,
Pierson, D.L. and Nickerson, C.A.: Low-shear modeled microgravity
alters the Salmonella enterica sevovar typhimurium stress response in an
Rpo-S independent manner. Appl. Environ. Microbiol., vol. 68, p. 5408
(2002).
[18] Gu, J.-D., Roman, M., Esselman, T., Mitchell, R.: The role of microbial
biofilms in deterioration of space station candidate materials. Int. Biodeter.
Biodegrad., vol. 41, p. 25 (1998).
[19] Pierson, D.L.: Microbial contamination of spacecraft. Gravit. Space Biol.
Bull., vol. 14, p. 1 (2001).
[20] Boulos, L., M. Prvost, B. Barbeau, J. Coallier, R. Desjardins.:
LIVE/DEAD BacLight: application of a new rapid staining method for
direct enumeration of viable and total bacteria in drinking water. J
Microbiol. Methods vol. 37, p. 77 (1999).
[21] Porter, K. G., Y.S. Feig, Y.S.: The use of DAPI for identification and counting of aquatic microflora. Limnol. Oceanogr., vol. 25, p. 943 (1980).
[22] Lemke, M.J., McNamara, C.J., Leff, L.G.: Comparison of methods for concentration of bacterioplankton for in situ hybridization. J. Microbiol.
Methods, vol. 29, p. 23 (1997).
[23] Amann, R.I., Ludwig, W., Schleifer, K.-H..: Phylogenetic identification and
in situ detection of individual microbial cells without cultivation.
Microbiol. Rev., vol. 59, p. 143 (1995).

Microgravity sci. technol. XV/1 (2004)

41