Advances in Space Research 39 (2007) 1225–1232

www.elsevier.com/locate/asr

Microgravity, bacteria, and the influence of motility

Michael R. Benoit *,1, David M. Klaus
BioServe Space Technologies, Aerospace Engineering Sciences Department, University of Colorado, Boulder, CO 80309-0429, USA

Received 23 November 2005; received in revised form 3 October 2006; accepted 19 October 2006

Abstract

Space microbiology studies date back to the 1960s, with most investigations reporting that increased bacterial populations occur in
flight compared to ground controls. Several exceptions to these findings, however, have created controversy and complicated explana-
tions of how, or whether, microgravity affects microorganisms. Upon closer examination of the literature, we identified a trend relating
cell motility to experimental outcome. Related studies conducted in microgravity analog devices, such as the clinostat or rotating wall
vessel bioreactor, further corroborate this trend. We review the literature regarding bacterial growth experiments conducted in space (and
using microgravity analogs) and analyze the influence of bacterial motility.
Ó 2006 COSPAR. Published by Elsevier Ltd. All rights reserved.

Keywords: Microgravity; Bacteria; Influence of motility; Space life sciences; Clinostat

1. Introduction contrast, however, a few seemingly comparable studies

reported no differences in final cell populations between
Space flight has been reported to cause many changes to space flight cultures and ground controls (Bouloc and
bacterial growth and behavior such as reducing lag phase D’Ari, 1991; Gasset et al., 1994; Thévenet et al., 1996; Kac-
(Kacena and Todd, 1997; Kacena et al., 1999; Klaus ena and Todd, 1997). In order to understand these appar-
et al., 1997; Mennigmann and Lange, 1986; Thévenet ent discrepancies in the literature, it is necessary to consider
et al., 1996), increasing final cell population (Brown et al., how microgravity is thought to influence bacterial growth.
2002; Kacena et al., 1999; Klaus et al., 1994; Mattoni,
1968; Mennigmann and Heise, 1994; Mennigmann and
2. Theories for indirect mechanisms of space flight effects on
Lange, 1986; Thévenet et al., 1996), reducing sensitivity
bacterial growth
to antibiotics (Lapchine et al., 1988; Tixador et al., 1994,
1985), increasing productivity of secondary metabolites
Despite early predictions (Pollard, 1965, 1967) and indi-
(Lam et al., 1998, 2002), and increasing transfer of genetic
cations (Zhukov-Verezhnikov et al., 1962) that bacteria are
material through conjugation (Ciferri et al., 1986). Final
too small to be affected by gravity, a majority of studies
cell population values have been reported more frequently
have since found that bacterial cultures grown during space
than any other measured variable to compare space flight
flight tend to produce higher final cell populations com-
cultures to ground controls. In general, these studies found
pared to identical ground controls (Klaus, 2002). Although
that space flight increased final cell populations. In
different research teams spanning multiple decades have
independently reported similar results, the exact manner
*
through which space flight causes changes to bacteria still
Corresponding author.
E-mail address: benoitm@stanford.edu (M.R. Benoit).
remains largely undetermined. A variety of theories and
1
Present address: Microbiology and Immunology Department, models have been posed in an attempt to identify specific
Stanford University, Stanford, CA 94305-5124, USA. cause-and-effect mechanisms, but none have yet been fully

0273-1177/$30 Ó 2006 COSPAR. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.asr.2006.10.009
1226 M.R. Benoit, D.M. Klaus / Advances in Space Research 39 (2007) 1225–1232
established (Todd and Klaus, 1996). A leading explanation
suggests that bacteria are indirectly affected by space flight
as a result of the quiescent fluid environment surrounding
bacteria in a liquid suspension culture (Klaus et al.,
1997). This has been proposed to arise due to two gravi-
ty-dependent phenomena: (1) the settling of cells through
their liquid medium and (2) the potential for buoyant con-
vection of less dense fluid in the vicinity of the cell.
In the microgravity environment of space flight, both of
these phenomena are reduced by up to six orders of magni-
tude, creating cell cultures that remain evenly distributed
and mixed-density fluids that remain quiescent. In the
absence of convection, Brownian motion (diffusion)
becomes the dominant transport mechanism. Cultures
grown on solid agar medium, however, are not equally sub-
ject to these fluid phenomena. Consequently, cultures
grown on agar are not expected to show differences in
growth due to space flight (Kacena et al., 1997). Converse-
ly, motile cells in suspension cultures will actively agitate
the quiescent fluid environment of microgravity, also
reducing fluid environment differences with 1 g cultures.
Therefore, based on this argument, the use of non-motile
suspension cultures provides the most likely experimental
system expected to result in differences between space flight
and 1 g conditions. In this context, we propose that dispar-
ities in cell motility (discussed below) can be used to
explain the seemingly contradictory findings in the
literature.
3. Summary of space flight effects on final cell populations of
bacteria
At least 11 primary publications state results for bacte-
rial cell numbers from space flight experiments. The find-
ings, listed in Table 1, were reported for space flight
cultures grown in suspension relative to similarly treated
ground controls. Although most of these studies reported
Table 1
Summary of space flight induced changes in final cell number compared to motility status for bacterial suspension cultures
Cell number Motility Bacterial species
Increased Non-motile S. Typhimurium
E. coli
B. subtilis
Undetermined
No difference Motile E. coli
B. subtilis motility is dependent on the growth medium used and was undetermined in one case. The group of Gasset, Tixador et al. published one set of
data from Gasset et al. (1994) as the untreated control in an antibiotic effectiveness study (Tixador et al., 1994), but this result was not listed here as two
separate findings.
increased final cell populations due to space flight, a few
exceptions were also noted (Bouloc and D’Ari, 1991; Gas-
set et al., 1994; Thévenet et al., 1996; Kacena and Todd,
1997). Attempting to understand the cause of these excep-
tions led us to critically analyze the individual experimental
protocols used by each group.
Of the studies reporting no differences in cell population
between space flight and ground control cultures (Bouloc
and D’Ari, 1991; Gasset et al., 1994; Kacena and Todd,
1997; Thévenet et al., 1996), Thévenet et al. (1996) was
the first to explicitly show that bacterial motility might be
responsible for the lack of a difference in growth. Kacena
and Todd (1997) showed that the use of a solid agar sub-
strate could also lead to no difference in final cell popula-
tion due to space flight. Both of these conclusions are in
agreement with the theory that space flight affects bacteria
indirectly (Klaus et al., 1997) through altered extracellular
fluid properties. The studies that do not agree with this
explanation on initial examination, however, are those of
Bouloc and D’Ari (1991) and Gasset et al. (1994). These
two studies are frequently cited in the literature as unex-
plained exceptions to the more typical observation that
space flight causes increased bacterial populations. Closer
inspection of the bacterial strains used in both of these
studies, however, reveals that their findings can also be
explained if the effects of bacterial motility are taken into
account. Specifically, Bouloc and D’Ari (1991) used Esche-
richia coli strain GC2852, the same strain that was later
used to explicitly observe the effects of bacterial motility
by Thévenet et al. (1996). Gasset et al. (1994) grew E. coli
ATCCÒ 25922, which also has a high degree of motility
(Kunin et al., 1995; Libby, 1998). Because bacterial motil-
ity was not reported as a variable in either experiment
(Bouloc and D’Ari, 1991; Gasset et al., 1994), its effect
on the outcome was not evident. Understandably, both
studies were conducted before Thévenet et al. (1996) dem-
onstrated the influence of motility on bacterial space flight
cultures.
Bacterial strain Works cited
BS-5 (P-22)/P-22 Mattoni (1968)
C-600 (k)/k
K12 MC4100 Gasset et al. (1994)
PhB405 Thévenet et al. (1996)
ATCC 4157 Klaus et al. (1994)
ATCC 4157 Brown et al. (2002)
ATCC 4157 Kacena et al. (1999)
Not reported Mennigmann and Lange (1986)
Not reported Mennigmann and Heise (1994)
ATCC 6051 Kacena et al. (1999)
GC2852 Bouloc and D’Ari (1991)
ATCC 25922 Gasset et al. (1994)
GC2852 Thévenet et al. (1996)
 M.R. Benoit, D.M. Klaus / Advances in Space Research 39 (2007) 1225–1232
After determining that bacterial motility may have
affected the results of these two studies, we revisited the lit-
erature in an effort to establish the degree of motility for all
of the bacterial cultures used in each of the above noted
space flight experiments. The findings, related to experi-
mental outcome in Table 1, are summarized as follows:
(1) Mattoni (1968) described the sedimentation of bacterial
cells in 1 g standing liquid cultures, implying that the bac-
teria were non-motile. (2) Ciferri et al. (1986) mentioned
that they accounted for increased growth of E. coli due
to space flight when comparing the exchange rates of cer-
tain genetic markers; however, they did not report the sta-
tistical significance of cell population values and we were
unable to determine or infer the motility of the strains used.
The results of this study were therefore excluded from our
analysis. (3) Mennigmann and Lange (1986) studied B. sub-
tilis growth and spore formation using a nutrient medium.
Although B. subtilis is normally motile, growth medium
can affect its motility. Specifically, motility gene expression
in B. subtilis depends upon the availability of the protein
rD (Mirel et al., 2000). In nutrient broth (NB) sporulation
medium, rD concentrations increase during exponential
growth and peak early in the transition to stationary phase,
whereas with minimal medium, rD levels remain constant
and high throughout all growth phases (Mirel et al.,
2000). Therefore, B. subtilis cultures grown on NB medium
are expected to lose their motility. Mennigmann and Lange
(1986) described cell settling for 1 g control cultures, thus
supporting the likelihood that the B. subtilis cultures
became non-motile during their experiment. (4) As previ-
ously described, Bouloc and D’Ari (1991) used a highly
motile strain of E. coli (GC2852). (5) Mennigmann and
Heise (1994) repeated the study of Mennigmann and Lange
(1986) with B. subtilis grown in NB medium and they also
noted cell sedimentation in unstirred (1 g) cultures, imply-
ing reduced cell motility. In addition, they mentioned that
increased space flight populations were observed only for
cultures grown in nutrient broth, but not for cultures
grown in minimal medium (from another, unpublished
study). This discrepancy is consistent with the dependence
of B. subtilis motility on medium type (described above),
combined with the influence of motility on space flight’s
effect on bacterial growth. (6) Klaus et al. (1994) grew
E. coli (ATCCÒ 4157) on a minimal medium supplemented
with D-glucose as the sole carbon source, which has been
shown to prevent the production of flagella due to catabo-
lite repression, thus rendering the E. coli non-motile (Adler
and Templeton, 1967). Also, the cells appeared non-motile
under microscopic observation and suspension cultures set-
tled to the bottom of standing liquid cultures in 1 g (unpub-
lished data). (7) Gasset et al. (1994) studied two E. coli
strains grown on different medium: the first was E. coli
ATCCÒ 25922 grown on a peptone medium and the second
was a derivative of E. coli K12 MC4100 grown on M9 min-
imal medium supplemented with glucose. As mentioned
previously, the first strain is highly motile, especially when
grown on a complex medium (Kunin et al., 1995; Libby,
1227
1998). However, E. coli K12 cells do not produce flagella
when grown on minimal medium with glucose (Adler and
Templeton, 1967), and therefore, the second strain was
likely non-motile. From the growth curve data provided
for the second strain, the spaceflight final cell populations
appear noticeably higher than those of the ground controls
(although the significance of this difference was not report-
ed). (8) Because motility was a primary focus of their study,
Thévenet et al. (1996) stated whether each E. coli strain was
motile or non-motile and reported the motility accordingly.
(9) Kacena and Todd (1997) used solid agar medium,
therefore the role of motility was eliminated and this study
was excluded from our trend analysis. (10) Kacena et al.
(1999) grew E. coli (ATCCÒ 4157), under the same (non-
motile) conditions as Klaus et al. (1994), and they also
studied cultures of B. subtilis grown on minimal medium,
but the degree of B. subtilis motility was not specifically
established. (11) Brown et al. (2002) used E. coli (ATCCÒ
4157) grown in a minimal medium supplemented with
glucose (non-motile).
Because bacterial motility offsets cell settling and dis-
rupts the quiescent fluid environment surrounding the cell,
motile bacterial cultures are not expected to show differenc-
es compared to 1 g (unstirred) control cultures (Kacena
and Todd, 1997; Klaus et al., 1997). Therefore, by classify-
ing strain motility as an independent variable, the seeming-
ly conflicting findings of bacterial space flight experiments
no longer appear inconsistent. Instead, by taking motility
into account, the results are generally in agreement with
the indirect effect theory based on altered extracellular fluid
properties. Although this agreement is consistently true for
E. coli cultures, B. subtilis are more difficult to characterize
due to the relationships between motility and growth media
described above. A reduction in motility for cultures grown
on NB medium provides a reasonable explanation for why
increased space flight populations of B. subtilis were
observed (Mennigmann and Heise, 1994; Mennigmann
and Lange, 1986). However, it does not account for the
findings of Kacena et al. (1999), who used a minimal medi-
um to grow B. subtilis. Although the motility of these cul-
tures was not explicitly established in this study, we would
expect that bacterial motility was maintained throughout
the experiment based on the growth conditions, and there-
fore, we would not expect the increased growth that was
observed due to space flight. This finding remains the only
outcome that we cannot explain due to motility in our
assessment of the literature.
4. Summary of microgravity analog effects on final cell
populations of bacteria
In addition to research conducted onboard spacecraft,
various ground-based microgravity analogs are also used
to provide an alternate means of isolating the effect of grav-
ity on bacteria. These simulations are typically accom-
plished through continuous rotation of an isotropic fluid
to maintain the cells in a low shear, distributed state,
1228 M.R. Benoit, D.M. Klaus / Advances in Space Research 39 (2007) 1225–1232
similar in some respects to actual weightless conditions.
This method is generically referred to as clinorotation
(Dedolph and Dipert, 1971) and includes variations of a
specially designed Rotating Wall Vessel (RWV) bioreactor
(Nickerson et al., 2003). Clinostats and RWV’s do not
remove the force of gravity from the cells; rather, the grav-
ity vector is continually reoriented through rotation of the
fluid-cell system (Klaus, 2001; Klaus et al., 1998). In this
manner, the effect of gravity can be separated into two fun-
damental components – motion and weight – thereby pro-
viding additional insight into the underlying mechanisms
associated with weightlessness (Klaus, 2004; Klaus et al.,
2004). Rotating Wall Vessel (RWV) bioreactors were
designed to produce a low-shear environment conducive
to growing three-dimensional tissue cultures. Functionally
similar to a clinostat, RWVs also rotate about a horizontal
axis (orthogonal to the gravity vector) and produce solid
body rotation of the fluid medium (Schwarz et al., 1992).
This is referred to as the ‘‘simulated microgravity mode’’
of an RWV, and it results in a state of low-shear cell sus-
pension similar to conditions experienced in space flight.
Control conditions are produced by rotating the RWV
about its vertical axis (referred to as ‘‘normal gravity
mode’’). Due to various differences between typical clino-
stat and RWV studies, we reviewed them separately.
4.1. Clinostats
Of the 11 space flight studies reviewed above, five also
used clinostats to supplement the primary findings of their
experiment: (1) Mattoni (1968) reported that slowly rotated
cultures produced higher final cell populations (although
he did not specify for which bacterial species, E. coli or
S. typhimurium). (2) Mennigmann and Heise (1994) report-
ed an increase in the final yield of biomass for B. subtilis
cultures grown in a clinostat. (3) Kacena et al. (1997)
reported no significant difference in the final cell popula-
tions of clinorotated E. coli (ATCCÒ 4157) and B. subtilis
(ATCCÒ 6051) cultures grown on agar substrate. (4) Kac-
ena et al. (1999) discovered an increase in the final cell pop-
ulation of clinorotated E. coli (ATCCÒ 4157) cultures, but
reported no significant difference for B. subtilis (ATCCÒ
Table 2
Summary of changes in final cell number due to clinorotation compared to motility status for bacterial suspension cultures
Cell number Motility Bacterial species
Increased Non-motile Not reported
E. coli
B. subtilis
No difference Undetermined B. subtilis
Motile P. aeruginosa
B. subtilis motility is dependent on the growth medium used and was undetermined in one case.
6051). (5) Brown et al. (2002) likewise reported an increase
in final cell population for E. coli (ATCCÒ 4157) cultures.
In addition to these five studies, which corresponded
directly to flight experiments, Klaus et al. (1998) reported
increased final cell numbers of clinorotated E. coli (ATCCÒ
4157) cultures, Benoit and Klaus (2005) found increased
populations for a different strain of E. coli (DH5a), and
Guadarrama et al. (2005) reported no difference in the
growth of Pseudomonas aeruginosa.
As with the space flight experiments described above,
results of clinorotation also generally demonstrated higher
final cell populations compared to static 1 g controls with
three exceptions (Guadarrama et al., 2005; Kacena et al.,
1997, 1999). Again, bacterial motility can be related to
the experimental outcomes (Table 2). The results reported
by Kacena et al. (1997) were obtained on solid agar sub-
strate and, as discussed above, would not be expected to
show an effect from fluid transport. Because Pseudomonas
aeruginosa is typically considered to be motile (Montie,
1998), the ‘no effect’ results reported by Guadarrama
et al. (2005) are in agreement with the proposed theory.
The differing results for B. subtilis (Kacena et al., 1999;
Mennigmann and Heise, 1994) however, again require fur-
ther clarification. As described previously, the discrepancy
could be related to the dependence of motility on growth
medium. B. subtilis cultures grown on minimal medium
are expected to remain motile throughout an experiment,
while cultures grown on NB medium are expected to lose
their motility. Since Kacena et al. (1999) used a minimal
medium, we expect that cell motility caused the lack of a
difference observed for clinorotated B. subtilis cultures.
On the other hand, because Mennigmann and Heise
(1994) used NB medium, we expect motility to be reduced,
leading to increased final cell populations, as observed.
4.2. Rotating wall vessel bioreactors
At least 12 ground-based studies have also reported final
cell numbers for bacterial cultures grown in the RWV bio-
reactor. The following results, summarized here in chrono-
logical order, were reported for simulated microgravity
mode compared to normal gravity mode: (1) Fang et al.
(1997a,b,c) reported lower dry cell weight for Streptomyces
clavuligerus cultures; (2) Fang et al. (1997a,b,c) found no
Bacterial strain Works cited
Not reported Mattoni (1968)
ATCC 4157 Klaus et al. (1998)
Kacena et al. (1999)
Brown et al. (2002)
DH5a Benoit and Klaus (2005)
Not reported Mennigmann and Heise (1994)
ATCC 6051 Kacena et al. (1999)
ATCC 29260 Guadarrama et al. (2005)
 M.R. Benoit, D.M. Klaus / Advances in Space Research 39 (2007) 1225–1232
difference in dry cell weight for motile cultures of Bacillus
brevis; (3) Fang et al. (1997a,b,c) found higher dry cell
weight for E. coli; (4) Fang et al. (2000) found lower dry cell
weight for Streptomyces hygroscopicus cultures; (5) Huite-
ma et al. (2002) reported slightly higher final population
densities for cultures of E. coli; (6) Wilson et al. (2002)
showed higher final cell populations for two strains of Sal-
monella enterica serovar Typhimurium grown on minimal
medium, but no differences for the same two strains grown
on complex medium; (7) England et al. (2003) reported no
difference in final cell populations of Pseudomonas aerugin-
osa cultures; (8) Lynch et al. (2004) showed no differences
for three strains of E. coli; (9) Baker and Leff (2004) found
increased populations of Ralstonia pickettii, but no differ-
ence for Sphingobacterium thalpophilium; (10) Baker et al.
(2004) found increased populations of E. coli (ATCCÒ
26); (11) Baker and Leff (2005a) reported no difference or
slightly lower populations of Sphingomonas paucimobilis
(ATCCÒ 10829), depending on the method used (LIVE/
DEADÒ BacLightä kit or DAPI), but increased cell num-
bers for an ISS isolate of the same species, and they also
reported lower or comparable cell numbers for Acinetobac-
ter radioresistens (ATCCÒ 49000) on day 7 (LIVE/DEADÒ
BacLightä kit or DAPI, respectively), and higher cell num-
bers for an ISS isolate of the same species; and (12) Baker
and Leff (2005b) studied bacteria isolated from the ISS
including Pseudomonas fluorescens, which showed almost
identical growth curves for both modes of growth, and
two strains of Stenotrophomonas maltophilia, one showing
increased cell numbers and the other no difference on day 7.
Based on the observed trend with bacterial motility for
space flight and clinostat studies, we likewise attempted
to determine the motility of the bacterial strains used in
these RWV bioreactor experiments. The results are again
described in chronological order: (1) Fang et al.
(1997a,b,c) described the S. clavuligerus cultures as filamen-
tous. (2) Fang et al. (1997a,b,c) studied Bacillus brevis,
which is motile and spore producing. (3) Fang et al.
(1997a,b,c) grew E. coli (ZK650) cultures according to
Fang and Demain (1997) in minimal medium supplement-
ed with glucose, which likely rendered the cultures
non-motile (1967). (4) Fang et al. (2000) described the
Streptomyces hygroscopicus cultures as filamentous. (5)
Huitema et al. (2002) did not report the strain or the motil-
ity of the E. coli used. (6) Wilson et al. (2002) grew two
strains of Salmonella enterica serovar Typhimurium on
both complex (LB broth) and minimal (M9) medium. Both
strains are normally motile, but motility was not checked in
this study. (7) England et al. (2003) studied Pseudomonas
aeruginosa, which is highly motile (Montie, 1998). (8)
Lynch et al. (2004) grew three strains of E. coli that were
all motile (A.C. Matin, personal communication). (9) Bak-
er and Leff (2004) stated the motility level of the cultures
used. (10) Baker et al. (2004) stated that E. coli (ATCCÒ
26) was non-motile. (11) Baker and Leff (2005a) studied
S. paucimobilis (ATCCÒ 10829), which is normally motile
by a single polar flagellum (Yabuuchi et al., 1990), and
1229
A. radioresistens (ATCCÒ 49000), which is normally non-
motile (Berlau et al., 1999). They also studied two strains
of the same species isolated from the ISS, with presumably
the same motility. (12) Baker and Leff (2005b) studied a
strain of P. fluorescence (normally motile (Korber et al.,
1994)), an unidentified species of Chryseobacterium, and
two strains of S. maltophilia – one motile and one non-
motile, and they pointed out how motility might affect their
results.
Although at least two exceptions exist, we observed a
trend linking motility with the effect of RWV growth on
bacterial cell populations (Table 3). Namely, higher growth
was observed for non-motile bacteria (Baker and Leff,
2004; Baker et al., 2004; Fang et al., 1997a,b,c; Baker
and Leff, 2005a,b), while no difference in final cell number
was observed for motile bacterial cultures (Baker and Leff,
2004, 2005a,b; England et al., 2003; Fang et al., 1997a,b,c;
Guadarrama et al., 2005; Lynch et al., 2004; Wilson et al.,
2002). Additionally, lower growth was observed for fila-
mentous cultures in the RWV (Fang et al., 1997a,b,c,
2000). In a rotating environment, the additional size and
mass of the growing filament will result in an increased sed-
imentation velocity. This added motion of larger cells (or a
chain like series of cells) renders the simulation of micro-
gravity less suitable, and therefore we do not expect the
growth of filamentous cultures in RWVs to compare with
that of planktonic cultures.
Collectively, the reported findings for RWV bacterial
growth initially indicate seemingly random results, but as
Table 3 suggests, motility can again be related to the differ-
ing experimental outcomes in most cases. One case that
remains unclear is the finding of increased Salmonella ent-
erica serovar Typhimurium growth in simulated micro-
gravity mode of the RWV by Wilson et al. (2002) for
cultures grown on minimal medium. Although Salmonella
enterica serovar Typhimurium are normally motile, their
motility was not explicitly determined during this study,
and no evidence exists suggesting that Salmonella enterica
serovar Typhimurium is also catabolite repressed (like
E. coli) when grown on minimal medium with glucose
(Lai et al., 1997). Two other results that did not match
the trend were described by Baker and Leff (2005a). Name-
ly, the S. paucimobilis ISS isolate showed increased cell
numbers but was expected to be motile (although this
was not confirmed during the study), and the A. radioresi-
stens (ATCCÒ 49000) strain showed the same or lower final
cell numbers for simulated microgravity mode cultures
despite the fact that this strain is normally non-motile.
Despite these exceptions, the overall trend relating motility
to the effect of simulated microgravity is robust considering
the large number of different bacterial species observed,
and that the effects of simulated microgravity are expected
to be less pronounced than those of space flight.
Four RWV studies also demonstrated that medium type
influences how the RWV affects bacterial growth (Baker
and Leff, 2004, 2005b; Baker et al., 2004; Wilson et al.,
2002). First, as mentioned above, Wilson et al. (2002)
1230 M.R. Benoit, D.M. Klaus / Advances in Space Research 39 (2007) 1225–1232

Table 3
Summary of changes in final cell number due to RWV bioreactor growth (simulated microgravity mode compared to normal microgravity mode) versus
motility status
Population Motility Bacterial species Bacterial strain Works cited
Increased Non-motile E. coli ZK650 Fang et al. (1997c)
ATCC 26 Baker et al. (2004)
R. pickettii ISS isolate Baker and Leff (2004)
S. maltophilia ISS isolate 2 Baker and Leff (2005b)
A. radioresistens ISS isolate Baker and Leff (2005a)
Motile S. paucimobilis
Not reported E. coli Not reported Huitema et al. (2002)
Motile? Salmonella Typhirium (minimal medium) Wild type v3339 Wilson et al. (2002)
RpoS mutant v4973
No difference Motile Salmonella Typhirium (complex medium) Wild type v3339 Wilson et al. (2002)
RpoS mutant v4973
Bacillus brevis Nagano Fang et al. (1997b)
P. aeruginosa UG2 England et al. (2003)
Sphingo. thalpophilium ISS isolate Baker and Leff (2004)
E. coli AMS6 Lynch et al. (2004)
AMS150
AMS171
S. maltophilia ISS isolate 1 Baker and Leff (2005b)
P. fluorescens ISS isolate
S. paucimobilis ATCC 10829 Baker and Leff (2005a)
Non-motile A. radioresistens ATCC 49000
Decreased Filamentous Strep. clavuligerus ATCC 27064 Fang et al. (1997a)
Strep. hygroscopicus ATCC 29253 Fang et al. (2000)

found different results for two strains of Salmonella enteri-
ca serovar Typhimurium grown using two different types of
medium. This may or may not be related to bacterial motil-
ity. Second, Baker and Leff (2004) reported higher final cell
populations for non-motile R. pickettii cultures grown with
nutrient-supplemented medium, but under starvation con-
ditions (in water), the simulated microgravity mode cul-
tures produced lower final cell populations (compared to
normal gravity mode controls). For the motile S. thalpophi-
lium cultures, however, they reported no differences regard-
less of medium type. Third, Baker et al. (2004) reported
that culture growth under simulated microgravity mode
caused only small increases in cell number for unsupple-
mented minimal medium, compared to substantially
increased numbers for cultures grown with NB medium.
Fourth, Baker and Leff (2005a) reported differing results
when diluted medium was used. These findings indicate
that medium type may play a secondary role in how the
RWV (as well as space flight and clinorotation) can affect
bacterial growth. For example, different medium types con-
tain different nutrients, leading to different metabolic
byproducts. If the nutrients and byproducts from each
media have different diffusion coefficients (dependent on
molecular weight), their rates of mass transport to and
from cells will differ. Therefore, any subtle differences
established in the fluid environment between 1 g controls
and cultures grown in space flight (e.g., lack of convection),
or cultures grown in microgravity analogs (e.g., reduced
cell settling), might differentially affect bacterial growth
depending on medium type. Detailed information is
required (from computational and/or empirical means)
about net mass transport in the different environments to
understand the overall effect.
5. Conclusions
Our review of experimental results found in the litera-
ture suggests a strong correlation between motility and
the effect of space flight (or microgravity analogs) on the
final cell number of bacterial suspension cultures. In gen-
eral, space flight and microgravity analog devices caused
increased final cell numbers compared to 1 g controls for
non-motile bacteria grown in liquid suspension cultures.
In most cases, the studies reporting exceptions to these
findings were found to have either used motile strains or
solid agar medium. This trend supports the hypothesis
that space flight and microgravity analogs indirectly affect
the growth and behavior of bacteria in suspension cul-
tures. The extent to which this gravity-dependent mass
transport phenomenon applies to more complex, eukary-
otic cells and other higher organisms should also be
considered.
Furthermore, this trend illustrates how confounding fac-
tors, such as cell motility and growth medium, can compli-
cate our understanding of the subtle mechanisms by which
reduced gravity profoundly affects biological systems. For
completeness, future space flight (and microgravity analog)
studies should characterize the level of bacterial motility of
the culture under investigation, and draw conclusions
about the results accordingly.
 M.R. Benoit, D.M. Klaus / Advances in Space Research 39 (2007) 1225–1232
Acknowledgments
Funding was provided by NASA Graduate Student
Researchers Program Fellowship (NGT3-52386) and Bio-
Serve Space Technologies under NASA Cooperative
Agreement NCC8-242.
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