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RESEARCH ARTICLE
E¡ect of simulated microgravity on growth and production of
exopolymeric substances of Micrococcus luteus space and
earth isolates
Laurie Mauclaire1 & Marcel Egli2
IMMUNOLOGY & MEDICAL MICROBIOLOGY
1
Laboratory for Biomaterials, Empa, Swiss Federal Institute for Materials Testing and Research, St. Gallen, Switzerland; and 2Space Biology Group, ETH
Zürich, Swiss Federal Institute of Technology, Zurich, Switzerland
Correspondence: Laurie Mauclaire,
Laboratory for Biomaterials, Empa, Swiss
Federal Institute for Materials Testing and
Research, Lerchenfeldstrasse 5, CH-9014 St.
Gallen, Switzerland. Tel.: 041 71 274 77 94;
fax: 041 71 274 77 88;
e-mail: [email protected]
Received 25 November 2009; revised 23
February 2010; accepted 16 March 2010.
Final version published online 12 May 2010.
DOI:10.1111/j.1574-695X.2010.00683.x
Editor: GianFranco Donelli
Keywords
attachment; biofilm; Gram-negative bacteria;
biofouling; exopolymeric substances;
microgravity.
Abstract
Microorganisms tend to form biofilms on surfaces, thereby causing deterioration
of the underlaying material. In addition, biofilm is a potential health risk to
humans. Therefore, microorganism growth is not only an issue on Earth but also
in manned space habitats like the International Space Station (ISS). The aim of the
study was to identify physiological processes relevant for Micrococcus luteus
attachment under microgravity conditions. The results demonstrate that simulated microgravity influences physiological processes which trigger bacterial
attachment and biofilm formation. The ISS strains produced larger amounts of
exopolymeric substances (EPS) compared with a reference strain from Earth. In
contrast, M. luteus strains were growing faster, and Earth as well as ISS isolates
produced a higher yield of biomass under microgravity conditions than under
normal gravity. Furthermore, microgravity caused a reduction of the colloidal EPS
production of ISS isolates in comparison with normal gravity, which probably
influences biofilm thickness and stability as well.
Introduction
Microorganisms tend to aggregate on surfaces and form
biofilms which can have detrimental effects on materials (e.g.
biocorrosion; Gu, 2007) or even endanger human health (e.g.
medical implants; Padera, 2006). Material scientists have
developed numerous strategies to prevent biofilm formation
such as using antiadhesive materials and/or materials pretreated with organic or inorganic antimicrobial agents (Zhao
et al., 2009). However, these antifouling materials, if they are
not detrimental for nontarget cells, simply delayed the formation of biofilm. However, an efficient strategy to limit
biofouling is to regularly remove the formed biofilm using
high mechanical stress and/or strong biocides. Unfortunately,
this is not always possible. For example, mechanical cleaning
and sterilization procedures cannot be carried out on all
surfaces including those of implants as well as surfaces inside
the International Space Station (ISS). These two particular
environments, medical implants and ISS surfaces, also share
another characteristic: reduced gravity conditions.
2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
c
Microgravity has been shown to have various effects on
eukaryotic cell functions, but the effect of this environment on
microorganisms is not as well characterized (Lynch & Matin,
2005; Crawford-Young, 2006). Low shear force affects microbial gene expression, cell morphology, physiological processes
and pathogenesis (Nickerson et al., 2004). Experiments conducted with microbial liquid cultures under microgravity
conditions revealed a shorter lag phase in comparison with
standard conditions, as well as prolonged exponential phase
(Kacena et al., 1999), formation of cellular aggregates (Purevdorj-Gage et al., 2006), a modification of the production of
secondary metabolites (Fang et al., 1997), a decreased adhesion to target cells (Thomas et al., 2002), and a decreased
sensitivity to antibiotics (Lynch et al., 2006). The Mir space
station was heavily colonized by biofilm, which damaged
quartz windows, corroded various metals and caused polymer
deterioration, contributing to the shortened lifetime of that
station (Matin & Lynch, 2005; Gu, 2007). A space flight
experiment confirmed the increased chance of formation of
stable biofilm under microgravity conditions (McLean et al.,
FEMS Immunol Med Microbiol 59 (2010) 350–356
351
Effect of simulated microgravity on Micrococcus luteus
2001). A ground-based experiment under low-shear modelled
microgravity showed that Escherichia coli biofilms were thicker
than their normal-gravity counterparts and exhibited increased resistance to stresses such as salt, ethanol or antibiotics
(Lynch et al., 2006). However, the impact of microgravity
conditions on initial cell attachment has not as yet been
investigated. Under normal gravity conditions, microbial cells
interact with the surface via different mechanisms depending
on the distance between the cell and the surface. Apart from
sedimentation and Brownian motion, Van der Waals forces
mainly lead to attachment within a distance of 4 50 nm. At
shorter distances (10–20 nm), electrostatic interactions start to
play a role, and specific interactions become relevant for
distances below 15 nm.
In this study, we characterized growth behaviour, cell wall
properties and secretion pattern of exopolymeric substances
(EPS) of Micrococcus luteus grown under normal as well as
simulated microgravity conditions. The aim was to correlate
these parameters with the ability of the bacteria to attach
and form biofilm.
Micrococcus luteus is a Gram-positive, nonmotile, spherical,
saprotrophic bacterium that belongs to the family of the
Micrococcaceae. The obligate aerobe M. luteus is found in soil,
dust, water and air, and as part of the normal flora of the
mammalian skin. The bacteria also colonize the human
mouth, mucosae, oropharynx and upper respiratory tract.
Although M. luteus is nonpathogenic (risk class 1 organism), it
should be considered a nosocomial pathogen in immunocompromised patients. Micrococcus luteus is resistant to
reduced water potential and can tolerate desiccation and high
salt concentrations. It has been shown to survive in oligotrophic environments for extended periods of time (Greenblatt et al., 2004). Not surprisingly, M. luteus was found on
board the Mir station and the ISS during numerous microbial
surveys (see review by Gu, 2007). The organisms were detected
in the air system, growing on surfaces, and were involved in
the biodegradation of polymeric materials.
This study presents a comparison of planktonic growth,
cell wall characteristics and production of EPS of three M.
luteus strains: the type reference strain (DSMZ20030) and
two isolates from the ISS (LT100 and LT110).
Materials and methods
Random positioning machine (RPM)
The RPM is a laboratory instrument for the simulation of
microgravity. Originally, the machine was developed by Hoson
(Hoson et al., 1992) and manufactured by the Dutch Space,
Leiden, Netherlands. Samples mounted on a platform randomly change the position in the three-dimensional (3D)
space on the machine controlled by dedicated software running on a personal computer. The movement of the experi-
FEMS Immunol Med Microbiol 59 (2010) 350–356
mental platform suspended in the centre of two perpendicular
cardanic frames is realized by two independent running
engines. These engines are controlled by feedback signals from
encoders, mounted on the motor axes, and by ‘null position’
sensors on the frames. Rotation rate (o) and geometrical
distance
from
the
centre
of
rotation
(R) yield ‘g-contours’ through the equation g = o2R/g0
(g0 = 9.81 m s2), which provides guidelines for the design and
layout of experimental packages and for the interpretation of
the results. The RPM, also called the 3D clinostat, has been a
standard apparatus to simulate microgravity for many years
and several reference experiments have been carried out
demonstrating that results from the RPM are comparable with
the results obtained in space (More & Cogoli, 1996). The RPM
was operated in a random walk (basic mode) with a rotation
speed of 601 s1 in a temperature-controlled room (25 1 1C).
Microbial strains and culture conditions
Three M. luteus strains were studied. Dr P. Landini (University of Milan) provided the two ISS isolates, LT100 and
LT110. The type reference strain was provided by the
German Resource Centre for Biological Material (M. luteusT
DSMZ20030). Crystal violet test on microtiter plates was
used to assess the ability of the three strains to form biofilm
under normal gravity conditions. Results showed that,
under normal gravity conditions, the three strains formed a
similar amount of biofilm (OD605 nm: 0.024 0.006 for
M. luteusT, 0.006 0.012 for LT100 and 0.013 0.012 for
LT110; n = 7). Bacteria were cultivated in tryptic soy broth
(TSB) and frozen stocks were reconstituted. TSB 5 mL was
inoculated with frozen stock and bacteria were allowed to
grow overnight. The bacterial suspension was dispersed on
tryptic soy agar plates and incubated at 25 1C. Three
colonies were picked from one plate and grown separately
overnight in liquid medium. The medium was made of (per
liter) 10 g TSB, 3.5 g NaNH4HPO4, 3.7 g KH2PO4, 7.5 g
K2HPO4, 0.25 g MgSO4, 2.8 mg FeSO4, 1.47 mg CaCl2,
1.98 mg MnCl2, 2.38 mg CoCl2, 0.17 mg CuCl2 and 0.29 mg
ZnCl2. First, a solution of phosphate and sodium salts was
prepared, the pH was adjusted to 7.1, and the medium was
autoclaved. Filter-sterilized stock solutions of glucose,
MgSO4 and the remaining salts were added to the cold
medium. Fresh medium was inoculated with the overnight
culture (dilution 1 : 100) and transferred into silicon tubes
(diameter 6 mm, length 25 cm, thickness 1 mm). Control
experiments showed similar growth rates within the silicon
tubes compared with the baffled flasks, indicating that
oxygen diffusion through the fine silicone tube was not
growth-limiting. The silicon tubes were sealed with clamps.
Particular attention was paid to avoid the presence of gas
bubbles, which would create shear forces under the RPM.
Tubes in which gas bubbles were detected at the end of the
c 2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
352
L. Mauclaire & M. Egli
G
µg
G
Code:
Proteins were quantified using the microBCA kit (Thermo
Scientific) where bovine albumin served as standard. Polysaccharides were quantified according to the method of
Dubois et al. (1956) using glucose as standard. EPS abundance was standardized with OD600 nm, which measures the
amount of cells present in the culture.
G
µg
G
µg
GG
Gµg
µgG
µgµg
Fig. 1. Experimental design.
experiment were discarded. Tubes loaded with medium
without bacteria were used as negative controls. Silicon
tubes were placed at 25 1C either under normal gravity
conditions on a shaker (70 r.p.m.) or on the RPM. Bacteria
were allowed to grow until they reached the beginning of the
stationary phase (c. 10 h). At this point the tubes were
opened, the culture was transferred into fresh medium
(dilution 1 : 100; OD600 nm = 0.02) and grown under either
normal or microgravity conditions until the middle of the
exponential phase was reached (about 7 h). A summary of
the experimental design is presented in Fig. 1. The three M.
luteus strains were exposed to four types of culturing
procedures: (1) preculture under normal gravity and culture
under normal gravity (GG), (2) preculture under normal
gravity and culture under microgravity (Gmg), (3) preculture under microgravity and culture under normal gravity
(mgG) and (4) preculture under microgravity and culture
under microgravity (mgmg).
Growth rates and yield of biomass
Growth rates were determined by measuring OD600 nm of
samples taken at three or more time points during the
exponential growth phase, each point being from independent triplicate experiments. The total yield of biomass was
estimated by measuring the OD600 nm at the beginning of the
stationary phase.
Composition of EPS
The cell suspension was pelleted (8500 g, 15 min, Heraeus)
and the medium discarded. Colloidal and capsular EPS were
extracted according to the protocol of Hirst et al. (2003).
Briefly, the cell pellet was resuspended in distilled water to
extract the colloidal fraction. After 4 h the suspension was
centrifuged, and the supernatant containing the colloidal
EPS was frozen for later analysis. The cell pellet was
resuspended in 0.1 M EDTA to extract the capsular fraction.
After 12 h, the cells were centrifuged and the supernatant
containing the capsular fraction was frozen for later analysis.
EPS is composed mainly of polysaccharides and proteins.
2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
c
Cell wall characteristics
Because bacterial surface properties could not be investigated directly, the cell wall of M. luteus was characterized
using the microbial adhesion to solvent (MATS) method.
According to the extended DLVO theory (named after
Derjaguin, Landau, Verwey and Overbeek), the free energy
of hydrophobic interaction (DGbsb) between two similar
cells (b) immersed in solvent (s) can be calculated as
qffiffiffiffiffiffiffiffiffiffiffi
qffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffi
2
þ gLW
DGbsb ¼ 2ð gLW
s Þ 4ð gb gb
b ð1Þ
q
ffiffiffiffiffiffiffiffiffiffiffi
q
ffiffiffiffiffiffiffiffiffiffiffi
pffiffiffiffiffiffiffiffiffiffiffi
þ gþ Þ
þ gþ
g
g
g
g
s s
b s
b s
qffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffi
gLW
where gLW
is the apolar adhesive (Lifshitz–van
s
b der Waals, LW)
interaction energy between the bacteria and
qffiffiffiffiffiffiffiffiffiffiffi
the solvent, gþ
b gb is the polar cohesive interaction energy
between the p
electron
ffiffiffiffiffiffiffiffiffiffiffi acceptors and the electron donors of
the bacteria, gþ
s gs is the polar cohesive interaction energy
between theq
electron
ffiffiffiffiffiffiffiffiffiffiffi acceptors and the electron donors of
the solvent, gþ
b gs is the polar adhesive interaction energy
between the electron acceptors of the
bacteria and the
pffiffiffiffiffiffiffiffiffiffiffi
þ
electron donors of the solvent and g
b gs is the polar
adhesive interaction energy between the electron donors of
the bacteria and the electron acceptors of the solvent (van
Oss, 1995). By comparing bacterial cell affinity to monopolar and apolar solvents with similar gLW it is possible to
quantify the electron donor/electron acceptor interactions
and the surface hydrophobicity of the bacterial cell surface.
Bellon-Fontaine et al. (1996) developed and named this
method ‘microbial adhesion to solvents’ (MATS). The
monopolar solvent can be acidic (electron-accepting) or
basic (electron-donating), but each pair of solvents has a
similar component of Lifshitz–van der Waals surface tension
(Table 1). For example, by comparing adhesion of bacterial
cells to chloroform and hexadecane it is possible to estimate
Table 1. Surface tension properties of MATS solvents (Bellon-Fontaine
et al., 1996)
Solvent
Formula
g1
g
gLW
2
2
(mJ m ) (mJ m ) (mJ m2) Type of solvent
Chloroform
Hexadecane
Diethyl ether
Hexane
CHCl3
C16H34
C4H10O2
C6H14
27.2
27.7
16.7
18.4
3.8
0
0
0
0
0
16.4
0
Electron acceptor
Apolar
Electron donor
Apolar
FEMS Immunol Med Microbiol 59 (2010) 350–356
353
Effect of simulated microgravity on Micrococcus luteus
Table 2. Maximal growth rates of Micrococcus luteus type strain (T) and space isolates (LT100 and LT110) placed under normal gravity (G) and
simulated microgravity (mg) conditions. Average SD (n = 3)
Gmg
mgmg
G
G
0.26 0.02
0.19 0.06
0.19 0.03
mg
G
0.21 0.04
0.30 0.03
0.27 0.06
G
mg
0.31 0.04
0.30 0.02
0.22 0.06
mg
mg
0.31 0.02
0.28 0.05
0.27 0.04
Results
Microgravity influences growth rate
Compared with normal gravity, simulated microgravity conditions increased the maximal growth rate of both M. luteusT and
the ISS isolates (Table 2). Simulated microgravity conditions
also influenced the yield of biomass. Under normal gravity
conditions, total biomass at the beginning of the stationary
phase was 1.7 and 1.8 OD for M. luteusT and LT100, respectively. Under microgravity conditions, total biomass reached
3.9 and 3.3 OD for M. luteusT and LT100, respectively. By
contrast, microgravity conditions did not affect the yield of the
ISS isolate LT110 (0.8 and 0.9 for microgravity and normal
gravity conditions, respectively).
Microgravity influences abundance and
composition of EPS
The ISS isolates LT110 formed significantly more colloidal
carbohydrates than the Earth reference M. luteusT strain
regardless of whether they were grown under simulated
microgravity or normal gravity conditions (Fig. 2). Under
normal gravity conditions, the ISS isolate LT100 also formed
more colloidal proteins than the M. luteusT strain (Fig. 3).
Both LT110 and LT100 produced more colloidal carbohydrates and proteins under normal gravity conditions compared with microgravity conditions (Figs 2 and 3). The
response of M. luteus to microgravity conditions was a
FEMS Immunol Med Microbiol 59 (2010) 350–356
25
20
15
10
5
0
Carbohydrates, capsular
fraction (µg OD–1)
gs, i.e. the electron-donor property of the bacteria. Similarly, comparison of the adhesion of bacterial cells to diethyl
ether and hexane gives a qualitative measurement of gs1, i.e.
the electron-acceptor property of the bacteria.
Practically, bacterial cells were harvested by centrifugation
(8500 g, 15 min, Heraeus), washed twice in 0.1 M potassium
phosphate buffer, pH 7, and resuspended in the buffer to
obtain a solution with an OD400 nm of approximately 0.7
(A0). Bacterial suspension (1.2 mL) was vortexed for 90 s
with 0.2 mL of appropriate solvents (chloroform, hexadecane, diethyl ether, hexane). The emulsion was allowed to
stand for 15 min and the OD of the aqueous phase (A) was
monitored. The percentage of adherence was calculated as:
% adherence = (1 A/A0) 100.
Carbohydrates, colloidal
fraction (µg OD–1)
mgG
GG
µgG
Gµg
µgµg
25
20
15
10
5
0
M. luteusT
LT100
LT110
Fig. 2. Concentration of carbohydrates in the colloidal and capsular
fractions of EPS of Micrococcus luteus type strain and space isolates
(LT100 and LT110). The abundance of carbohydrates was standardized
with OD600 nm, indicating the amount of cells present in the culture. Bars
represents average values 95% confidence level (n = 3).
200
Protein, colloidal
fraction (µg OD–1)
Preculture
Culture
Micrococcus luteusT
Micrococcus luteus LT100
Micrococcus luteus LT110
GG
GG
Gµg
µgG
µgµg
150
100
50
0
M. luteusT
LT100
LT110
Fig. 3. Concentration of proteins in the colloidal fraction of EPS of
Micrococcus luteus type strain and space isolates (LT100 and LT110). The
abundance of proteins was standardized with OD600 nm, indicating the
amount of cells present in the culture. Bars represents average
values 95% confidence level (n = 3).
c 2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
354
L. Mauclaire & M. Egli
decrease in production of colloidal EPS (Figs 2 and 3). The
EPS capsular fractions did not contain any detectable proteins.
The abundance of carbohydrates in the capsular fraction was
higher for M. luteusT under persistent microgravity conditions
(Fig. 2). Under simulated microgravity conditions, the ratio of
colloidal vs. capsular carbohydrates decreased significantly for
M. luteusT and LT100. This result showed that the carbohydrates were more tightly bound to cells growing under
microgravity conditions than normal gravity.
Microgravity influences cell wall characteristics
Electron acceptor characteristic of cell wall Electron donor characteristic of cell wall
(adherence to diethyl ether and hexane) (adherence to chloroform and hexadecane)
Due to large measurement uncertainties inherent in the use
of the MATS method with hydrophilic cells, we could not
detect significant differences between the M. luteus cell walls
grown under different gravity conditions (Fig. 4). The cell
walls of M. luteusT grown under simulated microgravity
seemed to harbour fewer electron acceptors and more
electron donors compared with cells grown under normal
gravity conditions, possibly indicating that their cell walls
became less hydrophilic when grown under microgravity. A
similar trend was observed for LT110 (Fig. 4).
2
GG
µgG
Gµg
1.5
µgµg
1
0.5
0
1.5
1
0.5
0
M.luteusT
LT100
LT110
Fig. 4. Cell wall characterization of Micrococcus luteus type strain and
space isolates (LT100 and LT110) according to the MATS method. Upper
graph: microbial adherence to chloroform (colour bars) and to hexadecane (white bars). Lower graph: microbial adherence to diethyl ether
(colour bars) and to hexane (white bars). Bars represents average
values 95% confidence level (n = 3).
2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
c
Discussion
Biofilm formation can be divided into two main stages: cell
adhesion and biofilm proliferation. Cell adhesion depends on
the probability of a cell coming into close vicinity of a surface.
On the one hand, microgravity conditions decrease the
probability of cell attachment by suppressing sedimentation.
On the other, the probability of attachment is directly proportional to the amount of planktonic cells. In our study, this was
estimated by the growth rate and the yield of biomass.
Micrococcus luteus strains grew faster under microgravity
conditions. Furthermore, M. luteusT and LT100 had higher
yield of biomass under microgravity than under normal
gravity. In general, increased growth kinetics and higher overall end point biomass under microgravity conditions represent
a greater health risk for astronauts and an amplified deterioration of the materials present in the ISS. Furthermore, the
higher density of cells that can be reached under microgravity
compared with normal gravity, increases the probability of the
organisms attaching to surfaces and biofilm formation.
The mechanism by which microgravity affects bacteria
growth remains largely unknown. The most accepted explanation for the sensitivity of bacteria to microgravity is that they
respond indirectly to the quiescent fluid environment which
surrounds the cell cultured in suspension (Klaus et al., 1997).
Such an undisturbed fluidic microenvironment is reached by
two gravity-dependent phenomena: (1) sedimentation of the
cells and (2) convection of less dense fluid in the vicinity of the
cells. Microgravity conditions reduced these two phenomena
by six orders of magnitude, creating evenly distributed cell
cultures and mixed-density fluids that remain separated. In
the absence of convection, the Brownian motion (diffusion)
becomes the dominant transport mechanism. Motile organisms have the possibility to counteract the action of microgravity by actively moving within the medium. Reviews in the
literature (Benoit & Klaus, 2007) show a strong correlation
between motility and the effect of microgravity. In general,
microgravity causes an increase in the amount of nonmotile
cell in liquid compared with normal gravity. Exceptions to this
finding were mainly found with motile bacteria (Salmonella,
E. coli, Bacillus, Pseudomonas) or when experiments were
conducted on solid agar. Our finding of increased growth rate
and yield of biomass of the nonmotile M. luteus in liquid
medium is in agreement with the data from the literature.
Initial cell attachment depends on the surface properties
of both substrates and cell. As for most Gram-positive
bacteria, the cell wall of M. luteus contains anionic polymers
connected by covalent bonds through a linking oligomer
with a peptidoglycan, the main structural polymer of cell
walls (Naumova & Shashkov, 1997). Anionic polymers
account for 10–60% of the weight of the cell wall and can
be divided into three groups: teichoic acids, teichuronic
acids and other carbohydrate-containing polymers. In this
FEMS Immunol Med Microbiol 59 (2010) 350–356
355
Effect of simulated microgravity on Micrococcus luteus
study, it was not possible to draw a clear conclusion about
the influence of gravity on the cell wall of M. luteus. The cell
walls grown under microgravity seemed to harbour fewer
electron acceptors and more electron donors than the cell
walls of bacteria grown under normal gravity. This observation could be due to a significant enrichment of anionic
polymers in the cell walls of M. luteusT grown under
microgravity conditions. A major feature of all anionic
polymers is that they give a negative charge to the bacterial
surface, which is important for physiological functions. The
negative charge allows the cells to spread actively across the
environment, take up cations and store them to maintain
their cationic homeostasis. Anionic polymers also regulate
the activity of autolytic enzymes, which are important for
growth and bacterial cell division. Secondary functions of
anionic polymers include their involvement in phage reception and immunogenicity (Naumova & Shashkov, 1997).
Under normal gravity conditions, the ISS isolate LT110
produced more colloidal carbohydrates compared with the
M. luteusT strain. Strains isolated from the ISS produced larger
amounts of EPS compared with the Earth reference strain.
EPS is known to act as a protective barrier for cells against
stress factors such as dehydration, UV and gamma radiation
(Elasri & Miller, 1999; Niemira & Solomon, 2005; Chang
et al., 2007). In this respect, the space environment is harsher
than Earth’s environment, which might explain why growth
onboard the ISS selected strains forming high amounts of
EPS. Various studies have shown an increased resistance of
bacteria grown under microgravity to stresses such as ethanol,
hyperosmosis, low pH or antibiotics compared with normal
gravity conditions (see review by Nickerson et al., 2004). For
E. coli, the microgravity effect is regulated by the ss transcription factor, which makes bacteria resistant to multiple stresses
(Lynch et al., 2004). Escherichia coli formed more copious
biofilms under microgravity compared with normal gravity
conditions, and these biofilms were more resistant to stresses
such as NaCl, ethanol, penicillin and chloramphenicol (Lynch
et al., 2006). The authors did not directly quantify the EPS,
but the observations suggest an increased EPS secretion
under microgravity conditions. This was not the case in our
experiment with M. luteus. Bacteria secrete colloidal EPS to
shape their microenvironment and degrade macromolecules.
A mathematical model has been developed to quantify the
influence of gravity-dependent physical factors on extracellular transport processes (Klaus, 1998). This model showed
a quasi-stable accumulation of byproducts around the cells
in free suspension. Under microgravity, the bacteria would
be able to create a favourable microenvironment more
rapidly, as the excreted cofactors and/or enzymes may reach
the necessary concentration sooner than under normal gravity. Therefore, it is not surprising that the bacteria produced
less colloidal EPS under microgravity because they also need
less to shape their microenvironment.
FEMS Immunol Med Microbiol 59 (2010) 350–356
Numerous studies have demonstrated that the production of EPS is largely influenced by the shear stress working
on the cells. Biofilm grown on high shear force tends to
produce less viable cells and larger amounts of EPS, which is
denser and less porous than biofilm produced at low shear
force (Liu & Tay, 2002; Ramasamy & Zhang, 2005). Shear
force also increases erosion and sloughing events. In contrast, absence of shear force may significantly affect the 3D
structure of the biofilm. In our study, the formation of
biofilm was not monitored directly, but our findings are in
agreement with what is known about the relation between
shear stress and EPS secretion. LT110 produced significantly
fewer colloidal carbohydrates and LT100 produced significantly fewer colloidal proteins under simulated microgravity
conditions, which represent a low shear stress environment.
This indicates a possible decrease of biofilm abundance and
stability under microgravity conditions. However, this hypothesis prediction should be viewed with caution because
M. luteusT did not decrease its colloidal EPS production
under microgravity. In stark contrast, Lynch et al. (2006)
observed increased biofilm formation by E. coli under microgravity conditions.
Conclusions and outlook
In the study, we investigated the fouling potential of
M. luteus under normal and microgravity conditions. Micrococcus luteus strains isolated from the ISS were able to
produce large amounts of EPS compared with the Earth
reference strain. We also observed an increase in the growth
rate and total biomass yield under microgravity conditions.
These physiological changes may increase the chance of
M. luteus attaching to surfaces. However, under microgravity, the LT110 produced fewer colloidal carbohydrates and
LT100 produced fewer colloidal proteins compared with
normal gravity conditions, which probably influences biofilm thickness and stability. Further experiments will focus
on direct microscopic observations of biofilm formation of
M. luteus under microgravity conditions.
Acknowledgements
Special thanks go to Aline Hunziker and Kathrin Grieder
(Empa) for technical assistance, Stéphane Richard as well as
Kriss Westphal (ETHZ) for their advice on the RPM and
Linda Thoeny (Empa) for the manuscript revision.
References
Bellon-Fontaine M-N, Rault J & van Oss CJ (1996) Microbial
adhesion to solvents: a novel method to determine the
electron-donor/electron-acceptor or Lewis acid–base
properties of microbial cells. Colloid Surface B 7: 45–53.
c 2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
356
Benoit MR & Klaus DM (2007) Microgravity, bacteria, and the
influence of mobility. Adv Space Res 39: 1225–1232.
Chang WS, de Mortel M, Nielsen L, de Guzman GN, Li XH &
Halverson LJ (2007) Alginate production by Pseudomonas
putida creates a hydrated microenvironment and contributes
to biofilm architecture and stress tolerance under waterlimiting conditions. J Bacteriol 189: 8290–8299.
Crawford-Young SJ (2006) Effects of microgravity on cell
cytoskeleton and embryogenesis. Int J Dev Biol 50: 183–191.
Dubois M, Gilles KA, Hamilton JK, Rebers PA & Smith F (1956)
Colorimetric method for determination of sugars and related
substances. Anal Chem 28: 350–356.
Elasri MO & Miller RV (1999) Study of the response of a biofilm
bacterial community to UV radiation. Appl Environ Microb 65:
2025–2031.
Fang A, Pierson DL, Koenig DW, Mishra SK & Demain AL (1997)
Effect of simulated microgravity and shear stress on Microcin
B17 production by Escherichia coli and its excretion into the
medium. Appl Environ Microb 63: 4090–4092.
Greenblatt CL, Baum J, Klein BY, Nachshon S, Koltunov V &
Cano RJ (2004) Micrococcus luteus – survival in amber. Microb
Ecol 48: 120–127.
Gu J-D (2007) Microbial colonization of polymeric materials for
space applications and mechanisms of biodegradation: a
review. Int Biodeter Biodegr 59: 170–179.
Hirst CN, Cyr H & Jordan IA (2003) Distribution of
exopolymeric substances in the littoral sediments of an
oligitrophic lake. Microb Ecol 46: 22–32.
Hoson T, Kamisaka S, Masuda Y & Yamashita M (1992) Changes
in plant-growth processes under microgravity conditions
simulated by a three-dimensional clinostat. Bot Mag Tokyo
105: 53–70.
Kacena MA, Merrell GA, Manfredi B, Smith EE, Klaus DM &
Todd P (1999) Bacterial growth in space flight: logistic growth
curve parameters for Escherichia coli and Bacillus subtilis. Appl
Microbiol Biot 51: 229–234.
Klaus DM (1998) Microgravity and its implications for
fermentation biotechnology. Trends biotechnol 16: 369–373.
Klaus D, Simske S, Todd P & Stodieck L (2007) Investigation of
space flight effects on Escherichia coli and proposed model of
underlaying physical mechanisms. Microbiology 143: 449–455.
Liu Y & Tay JH (2002) The essential role of hydrodynamic shear
force in the formation of biofilm and granular sludge. Water
Res 36: 1653–1665.
2010 Empa - Laboratory for Biomaterials
c 2010 Federation of European Microbiological Societies
Journal compilation Published by Blackwell Publishing Ltd.
c
L. Mauclaire & M. Egli
Lynch SV & Matin A (2005) Travails of microgravity: man and
microbes in space. Biologist 52: 80–87.
Lynch SV, Brodie EL & Matin A (2004) Role and regulation
of ss in general resistance conferred by low-shear
simulated microgravity in Escherichia coli. J Bacteriol 186:
8207–8212.
Lynch SV, Mukundakrishnan K, Benoit MR, Ayyaswamy PS &
Matin A (2006) Escherichia coli biofilms formed under lowshear modelled microgravity in ground-based system. Appl
Environ Microb 72: 7701–7710.
Matin A & Lynch SV (2005) Investigating the threat of bacteria
grown in space. ASM News 71: 235–240.
McLean RJC, Cassanto JM, Barnes MB & Koo JH (2001) Bacterial
biofilm formation under microgravity conditions. FEMS
Microbiol Lett 195: 115–119.
More D & Cogoli A (1996) Gravitational and space biology at the
cellular level. Biological and Medical Research in Space (Moore
D, Bie P & Oser H, eds), pp. 1–106. Springer, Berlin.
Naumova IB & Shashkov AS (1997) Anionic polymers in cell
walls of Gram-positive bacteria. Biochemistry 62: 809–840.
Nickerson CA, Ott CM, Wilson JW, Ramamurthy R & Pierson DL
(2004) Microbial responses to microgravity and other low
shear stress environments. Microbiol Mol Biol R 68:
345–361.
Niemira BA & Solomon EB (2005) Sensitivity of planktonic and
biofilm-associated Salmonella spp. to ionizing radiation. Appl
Environ Microb 71: 2732–2736.
Padera RF (2006) Infection in ventricular assist devices: the role
of biofilm. Cardiovasc Pathol 15: 264–270.
Purevdorj-Gage B, Sheehan KB & Hyman LE (2006) Effect of
low-shear modelled microgravity on cell function, gene
expression, and phenotype in Saccharomyces cerevisiae. Appl
Environ Microb 72: 4569–4575.
Ramasamy P & Zhang X (2005) Effects of shear stress on the
secretion of extracellular polymeric substances in biofilms.
Water Sci Technol 52: 217–223.
Thomas WE, Trintchina E, Forero M, Vogel V & Sokurenko EV
(2002) Bacterial adhesion to target cells enhanced by shear
force. Cell 109: 913–923.
Van Oss CJ (1995) Hydrophobicity of biosurfaces – origin,
quantitative determination and interaction energies. Colloid
Surface B 5: 91–110.
Zhao L, Chu PK, Zhang Y & Wu Z (2009) Review antibacterial
coatings on titanium implants. J Biomed Mater Res B 91:
470–480.
FEMS Immunol Med Microbiol 59 (2010) 350–356