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-μm (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.
Numerous studies have shown that bacteria, in their natural environments, grow on surfaces as slime-encased biofilm communities. In contrast to unattached, planktonic bacteria, biofilm bacteria are quite resistant to disinfectants often by several orders of magnitude . Biofouling by bacteria is a major industrial problem and has been associated with the failure of heat exchangers, reverse osmosis water purification systems, and microbial corrosion . It is reasonable to assume that biofouling of reverse osmosis-based water purification systems might occur during spaceflights. If this problem is not remedied, it may seriously jeopardize the ability of humans to survive interplanetary travel. As well, biofilms may be quite beneficial for certain aspects of wastewater treatment such as nitrification and organic carbon removal . In this context, biofilms may be desirable components of wastewater treatment for spaceflight. Although biofilm formation is common on Earth, it has not been documented during spaceflight under weightlessness (i.e. microgravity). Here, we present the first report of biofilm formation under microgravity conditions during a space shuttle flight.
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).
2Materials and methods
2.1Strains 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 −80°C as described .
2.2Sample handling for spaceflight
Because of the logistics involved in flying 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 18 h at 37°C, centrifuged at 5000×g, and resuspended in 100 ml phosphate-buffered saline (PBS)  to a final concentration of 2.6×107 CFU ml−1. The bacteria suspension, 100 ml sterile R2A broth, and 40 autoclaved black 0.2 μm 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 4°C until needed. Twenty-four hour prior to launch, the materials were loaded into 150-μl 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 flight 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 then days 2–9 as shown in Fig. 1B,C respectively. In total, four replicates were performed during this flight. All experiments aboard the spacecraft were conducted at 22°C.
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. Biofilm 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 filters were placed into 4% (v/v) paraformaldehyde. The plates and filters were then transported back to SWT by commercial airline. The petri plates were incubated at room temperature (22°C for 20 h) until their arrival at SWT. Afterwards, they were incubated for a further 24 h at 37°C. For scanning confocal laser microscopy (SCLM), biofilm-coated polycarbonate filters were stained with the BacLight Live/Dead™ fluorescent 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).
3Results and discussion
Exposure of the polycarbonate filters to P. aeruginosa during spaceflight induced the formation of biofilms (Fig. 2A and Table 1). The majority of the cells stained green (viable) . A few cells stained red (non-viable) however, several of these ‘non-viable’ cells were actively motile – a finding that suggests that estimations of cell viability based solely on fluorescent staining patterns should be interpreted with caution. No significant difference in cell concentration was noted between the 1-day and 8-day biofilms (Table 1). As observed in Fig. 2B, vigorous agitation (vortexing) did not remove all the bacteria from the biofilm on the polycarbonate membranes (compare with Fig. 2A), implying that the dilution plating technique underestimated the quantities of adherent bacteria. This observation also suggests that microgravity-grown biofilms can adhere tightly to surfaces.
Table 1. Bacterial concentrations (±S.E.M.) in planktonic populations (expressed as log10 CFU ml−1) and in biofilms (expressed as log10 CFU cm−12)
aDuring the flight of STS-95, the sterile R2A broth became contaminated. Identification of representative colonies using the Biolog system  identified these contaminants as P. aeruginosa.
Planktonic P. aeruginosa in PBS
P. aeruginosa in R2A brotha
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-μm filter (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 confirmed as P. aeruginosa by their identical metabolic profiles to the original strain, using a MicroLog™ 1 bacterial identification system (Biolog Inc., Hayward, CA, USA) . These 10 isolates from STS-95 have been saved as strains RM-1 through RM-10.
We saw no discernible differences in the morphology of the microgravity-grown biofilms as compared to biofilms 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 biofilm 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, possibly through its influence on fluid dynamics . In contrast, microgravity caused little inhibition of DNA repair mechanisms in E. coli and B. subtilis, but did not affect 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 . It is possible that bacterial growth and gene expression within biofilms and biofilm structure may also be influenced by microgravity. Our experimental protocol did not allow us to discern any influence of microgravity on biofilm structures or physiology in this study. Investigations of the kinetics of biofilm formation and morphology of biofilm growth in microgravity will require future investigations to be conducted in environments such as the Space Station Mir or the International Space Station.
In conclusion, we have shown that biofilm formation can occur during microgravity. It is an issue that must be addressed during the design of water recycling systems for long-term spaceflights.
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.