FEMS Microbiology Letters 195 (2001) 115^119
www.fems-microbiology.org
Bacterial bio¢lm formation under microgravity conditions
Robert J.C. McLean
a;
*, John M. Cassanto b , Mary B. Barnes a , Joseph H. Koo
c
a
c
Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666, USA
b
Instrumentation Technology Associates, Inc., 15 E. Uwchlan Ave, Suite 406, Exton, PA 19341, USA
Institute for Environmental and Industrial Science, Southwest Texas State University, 601 University Drive, San Marcos, TX 78666, USA
Received 30 June 2000 ; received in revised form 1 November 2000; accepted 8 December 2000
Abstract
Although biofilm formation is widely documented on Earth, it has not been demonstrated in the absence of gravity. To explore this
possibility, Pseudomonas aeruginosa, suspended in sterile buffer, was flown in a commercial payload on space shuttle flight STS-95. During
earth orbit, biofilm formation was induced by exposing the bacteria to sterile media through a 0.2-Wm (pore size) polycarbonate membrane.
Examination of these membranes by confocal microscopy revealed biofilms to be present and that these biofilms could persist in spite of
vigorous agitation. These results represent the first report of biofilm formation under microgravity conditions. ß 2001 Federation of
European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Bio¢lm; Microgravity; Space£ight; Bacterial adhesion; Pseudomonas aeruginosa
1. Introduction
Numerous studies have shown that bacteria, in their
natural environments, grow on surfaces as slime-encased
bio¢lm communities. In contrast to unattached, planktonic bacteria, bio¢lm bacteria are quite resistant to disinfectants often by several orders of magnitude [1]. Biofouling
by bacteria is a major industrial problem and has been
associated with the failure of heat exchangers, reverse osmosis water puri¢cation systems, and microbial corrosion
[2]. It is reasonable to assume that biofouling of reverse
osmosis-based water puri¢cation systems might occur during space£ights. If this problem is not remedied, it may
seriously jeopardize the ability of humans to survive interplanetary travel. As well, bio¢lms may be quite bene¢cial
for certain aspects of wastewater treatment such as nitri¢cation and organic carbon removal [3]. In this context,
bio¢lms may be desirable components of wastewater treatment for space£ight. Although bio¢lm formation is common on Earth, it has not been documented during space£ight under weightlessness (i.e. microgravity). Here, we
present the ¢rst report of bio¢lm formation under microgravity conditions during a space shuttle £ight.
* Corresponding author. Tel. : +1 (512) 245-3365;
Fax: +1 (512) 245-8713; E-mail: [email protected]
This investigation involved the active participation of
eight students ranging in ages from 6 to 13 year. The
educational aspects of this project have been described in
a separate publication (Krause et al., submitted for publication).
2. Materials and methods
2.1. Strains and growth conditions
Pseudomonas aeruginosa PAO-1, used in this study, was
a gift from V. Deretic, formerly located in the Department
of Microbiology, University of Texas Health Sciences
Center at San Antonio, TX, USA. This organism was
grown on LB broth and stored frozen at 380³C as described [4].
2.2. Sample handling for space£ight
Because of the logistics involved in £ying experiments
on the space shuttle, all materials had to be prepared at
Southwest Texas State University (SWT) over 1 week
ahead of time. Eleven days prior to the shuttle launch,
P. aeruginosa PAO-1 was streaked onto R2A agar (Difco)
from frozen stock, and checked for purity. The organisms
were then subcultured in 500 ml R2A broth and grown for
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 5 4 9 - 8
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then days 2^9 as shown in Fig. 1B,C respectively. In total,
four replicates were performed during this £ight. All
experiments aboard the spacecraft were conducted at
22³C.
2.3. Sample processing
Fig. 1. Schematic of osmotic dewatering device used to grow bio¢lms
under microgravity conditions with the locations of P aeruginosa bacterial suspension in PBS and R2A broth indicated. The polycarbonate
membrane (arrow) that covered the R2A broth was used to grow the
bio¢lms. The position of the 150-Wl vials during launch (A) and landing
(D) is shown. During the 9-day orbit (microgravity), the vials were
aligned for day 1 (B) and days 2^9 (C) as shown. This alignment enabled P. aeruginosa to be exposed to the media that di¡used through
the 0.2-Wm pores in the membrane. In this manner, bio¢lm formation
was induced on the membranes.
18 h at 37³C, centrifuged at 5000Ug, and resuspended in
100 ml phosphate-bu¡ered saline (PBS) [5] to a ¢nal concentration of 2.6U107 CFU ml31 . The bacteria suspension, 100 ml sterile R2A broth, and 40 autoclaved black
0.2 Wm pore size polycarbonate membranes (Osmonics
Inc, Livermore, CA, USA) were then shipped by overnight
courier to Instrumentation Technology Associates Incorporated (ITA), Exton, PA, USA where they were stored at
4³C until needed. Twenty-four hour prior to launch, the
materials were loaded into 150-Wl vials within ITA's type
III osmotic dewatering device (as shown in Fig. 1). This
device was then placed in the space shuttle, Discovery for
£ight STS-95 (Space Transport System-95), which lasted
from October 29 to November 7, 1998. The vials containing the bacteria and sterile broth were not aligned during
launch (Fig. 1A) and landing (Fig. 1D). During the 9-day
orbit (microgravity) the vials were aligned for day 1 and
After landing, the commercial payload was removed
and the samples removed from the osmotic dewatering
device. Due to logistical reasons (NASA safety inspection
of the shuttle, removal of over 80 other experiments, and
availability of lab space and personnel), sample processing
at the Kennedy Space Center did not occur until 28 h after
shuttle landing. Bio¢lm enumeration (performed by
R.J.C.M. at the Kennedy Space Center) consisted of placing duplicate membranes from each time exposure (1 day
and 8 days) into 10 ml PBS, and vortexing vigorously for
5 min. Bacteria in this suspension and other liquids were
then enumerated by dilution plating onto R2A agar (Difco
Laboratories, Detroit, MI, USA). The remaining polycarbonate ¢lters were placed into 4% (v/v) paraformaldehyde.
The plates and ¢lters were then transported back to SWT
by commercial airline. The petri plates were incubated at
room temperature (22³C for 20 h) until their arrival at
SWT. Afterwards, they were incubated for a further 24
h at 37³C. For scanning confocal laser microscopy
(SCLM), bio¢lm-coated polycarbonate ¢lters were stained
with the BacLight Live/Dead1 £uorescent stain (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. Filters were then placed on glass
slides, covered with a cover slip and examined with an
Olympus IX-70 inverted SCLM (Olympus America Inc.,
Melville, NY, USA) coupled with a Bio-Rad 1024 SCLM
System (Bio-Rad Laboratories, Hercules, CA, USA).
3. Results and discussion
Exposure of the polycarbonate ¢lters to P. aeruginosa
during space£ight induced the formation of bio¢lms (Fig.
2A and Table 1). The majority of the cells stained green
(viable) [6]. A few cells stained red (non-viable) however,
several of these `non-viable' cells were actively motile ^ a
¢nding that suggests that estimations of cell viability based
solely on £uorescent staining patterns should be interpreted with caution. No signi¢cant di¡erence in cell concentration was noted between the 1-day and 8-day bio¢lms
(Table 1). As observed in Fig. 2B, vigorous agitation (vortexing) did not remove all the bacteria from the bio¢lm on
the polycarbonate membranes (compare with Fig. 2A),
implying that the dilution plating technique underesti-
C
Fig. 2. Scanning confocal micrograph of 8d bio¢lms, stained with a LiveDead1 viability stain (A). Viable cells stain green, whereas non-viable cells
stain red [6]. Vigorous agitation for 5 min (B) failed to remove all the adherent bacteria. Scale bar is in Wm.
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Table 1
Bacterial concentrations ( þ S.E.M.) in planktonic populations (expressed
as log10 CFU ml31 ) and in bio¢lms (expressed as log10 CFU cm312 )
Sample
Log10 CFU
1-day bio¢lms
8-day bio¢lms
Planktonic P. aeruginosa in PBS
P. aeruginosa in R2A brotha
5.63 þ 0.10
5.68 þ 1.10
9.66 þ 0.26
9.63
Acknowledgements
a
During the £ight of STS-95, the sterile R2A broth became contaminated. Identi¢cation of representative colonies using the Biolog system
[7] identi¢ed these contaminants as P. aeruginosa.
mated the quantities of adherent bacteria. This observation also suggests that microgravity-grown bio¢lms can
adhere tightly to surfaces.
Contamination was a real possibility during this experiment due to the extensive transport and handling of the
materials. While the R2A broth in the osmotic dewatering
device (Fig. 1) did acquire bacteria in spite of the 0.2-Wm
¢lter (Table 1), all colonies observed had an identical morphology and resembled the original P. aeruginosa PAO-1
inoculum. To verify this, ten colonies were selected at
random and con¢rmed as P. aeruginosa by their identical
metabolic pro¢les to the original strain, using a MicroLog1 1 bacterial identi¢cation system (Biolog Inc., Hayward, CA, USA) [7]. These 10 isolates from STS-95 have
been saved as strains RM-1 through RM-10.
We saw no discernible di¡erences in the morphology of
the microgravity-grown bio¢lms as compared to bio¢lms
formed under conditions of full gravity. As stated earlier,
due to logistical reasons we were not able to gain access to
our experiment until 28 h after the shuttle had landed.
Owing to the unavoidable 28 h delay in processing the
samples, it is conceivable, though unlikely, that some bio¢lm growth could have occurred after the shuttle had
landed.
Microgravity has been shown to enhance the growth of
planktonic cultures of Escherichia coli and Bacillus subtilis
[8], possibly through its in£uence on £uid dynamics [9]. In
contrast, microgravity caused little inhibition of DNA repair mechanisms in E. coli and B. subtilis, but did not
a¡ect DNA repair mechanisms in Deinococcus radiodurans
[10,11]. Based on experiments performed using a positive
cDNA selection method, there is evidence of altered gene
expression in Salmonella typhimurium cultures grown
under simulated microgravity [12]. It is possible that bacterial growth and gene expression within bio¢lms and bio¢lm structure may also be in£uenced by microgravity. Our
experimental protocol did not allow us to discern any
in£uence of microgravity on bio¢lm structures or physiology in this study. Investigations of the kinetics of bio¢lm
formation and morphology of bio¢lm growth in microgravity will require future investigations to be conducted
in environments such as the Space Station Mir or the
International Space Station.
FEMSLE 9763 7-2-01
In conclusion, we have shown that bio¢lm formation
can occur during microgravity. It is an issue that must
be addressed during the design of water recycling systems
for long-term space£ights.
This project would not have been possible without the
generous support of the Institute for Environmental and
Industrial Science at Southwest Texas State University, the
personnel at ITA, NASA personnel including the crew of
the space shuttle Discovery (STS-95), and the active participation of eight students (Brent Krause, Justin Franke,
Thomas Sanchez, Keri Mounger and Kristen Scott from
Port Lavaca, TX; and Keller Tiemann, Malcolm McLean,
and Alistair McLean from Wimberley, TX). We thank
Cheryl Nickerson, Tulane University, for sharing her unpublished data and ideas with us. R.J.C.M. would like to
dedicate this paper to John H. Glenn Jr. and his fellow
astronauts in recognition of their service to humanity.
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