To start with I’m gonna answer the question:
Why is it important to study the effects of spaceflight on microbial communities?
Bacteria can be found everywhere. To date plenty of different species as well as their behavior in different conditions were described. The biofilms were found as well on the Mir space station and were responsible for corrosion and blocked water purification system. As we’ve been exploring the space we have been coming across many challenges like understanding the effects of spaceflight on biofilms. Several in-flight studies have reported that the microgravity environment came across during spaceflight can modify bacterial growth and physiology, including cell density, antibiotic resistance and virulence which can be harmful to the health of the crew during long – term space exploration. The reason of that can be as well the fact that space travelers tend to have decreased immune system during missions.
So far the spaceflight studies has used the suspension cultures but they do not usually reflect the real world situations as in nature bacteria tend to exist in surface- associated microbial communities known as biofilms that exhibit resistance to environmental stress, antibiotics and host defense system. An experiment using simulated microgravity shown that E.COLI grown in simulated microgravity forms thicker biofilms and exhibits increased resistance to stress compared to normal gravity controls.
During another experiment it was observed that P. aeruginosa is able to form the biofilms during spaceflight. Unfortunately the scientists were not able to indicate the quantitative comparison of biofilms formed during spaceflight and normal gravity. What is more salmonella cultured during spaceflight exhibited increases in cellular aggregation and clumping what is connected with the biofilm formation. Despite the evidence that biofilm formation can be changed in a low gravity environment, the studies comparing biofilm formation during spaceflight and normal gravity have not been reported.
P. aeruginosa is a model organism for biofilm studies. In this publication the first evidence that spaceflight affects biofilm formation by p. aeruginosa, with increased number of viable cells, biomass and thickness observed in spaceflight biofilms compared to normal gravity controls.
Previous studies has examinated how planctonic p. aeruginosa cells respond to simulated microgravity and the space environment. Plenty of genes and proteins were identified and were regulated in a different ways during spaceflight. It was shown that the global regulator Hfq is responsible for responding to microgravity by P. aeruginosa
Materials and Methods
Inocula preparation
Overnight shaking cultures of all strains were cultured at 37C in Nutrient broth (NB) . To prepare the inocula, cultures were washed and resuspended in PBS.
mAUM was used as a growth media.o
The concentration of calcium chloride digydrate was lowered so that the precipitation was minimized during storing.
Sodium nitrate was added as an electron acceptor in order to enable cell growth when the conditions became anaerobic in FPAs
Fluid Processing Apparatus was used to study the biofilm formation, designed for growing cells during spaceflights.
We can described the FPA as a glass barrel. It can be divided into compartments by rubber stoppers.
During spaceflight the components that were placed in different compartments can be mixed using the plunging motion.
1st compartment: a modified artificial urine media (mAUM) was loaded into the 1st compartment containing the mixed cellulose membrane disc used as a biofilm substrate. Solid or GE insert was used. The mAUM provides a physiologically revelant environment for the study of biofilms formed inside as well as outside the human body.
2nd compartment: was filled with inoculum with phosphate buffer saline (PBS)
3rd compartment: was filled with paraformaldehyde solution for microscopy samples.
All the FPAs were autoclaved before use. After all the components had been added , all the FPAs were placed in a single group activation pack (GAP) that enables to mix all the compartments in all 8 FPAs at the same time.
The biofilms were formed under the static conditions in FPAs at 37C for 3 days. Then they were stored at 8C to minimize the growth and the microscopy samples were fixed with paraformaldehyde.
Samples were obtained approx. 6h after mission.
Firstly the membranes were washed and sonicated. Then the samples were spot – plated on NB agar and incubated.
All the steps were done to enable the viable cell counting. Biofilms were stained with a solution containing propidium iodide. After the staining procedure the biofilm images were obtained using the confocal laser scanning microscope.
Results
a)Biofilm Formation
The comparison between the biofilm formed during spaceflight and biofilm formed in normal gravity.
The number of viable cells in p. aeruginosa biofilms formed in mAUM during spaceflight increased three-fold compared to those formed in normal gravity which shows FIGure A. It was proved as well that spaceflight promots biofilm production by P.a where both biomass and mean thickness increased significantly (B,C)
It was observed that spaceflight induced an increase in number of viable cells, biomass and mean thickness f biofilm regardless of phosphate concentration or carbon source
b) Biofilm architecture
Biofilms formed during spaceflight exhibited a column- and – canopy structure that has not been observed on Earth while ones cultured in normal gravity shown flat structures. Such a form has been not decribed previously.
It is easy to see the increased thickness of the biofilm formed during spaceflight from side view images.
On earth P.a forms mushroom-shaped structured biofilms only under hydrodynamic conditions when glucose and carbon are available and flat ones with citrate. Under static conditions structured biofilms are not observed because of limited nutrient availability and aeration availability.
As shown in figure 2b the unusual structure can be seen clearly when slices of the biofilm were approx. 5.8um thick. What is more P.a formed colun – and canopy structured biofilms in mAUM as well as mAUM – highPi and mAUMg – highPi. Observing those structures was especially suprising due to the static environment used togrow the biofilm.
c) Motility Affects Spaceflight Biofilm Formation (has an influence)
Flagella-driven motility has been shown to affect P.a biofilm development. To examine if motility plays a key role in the formation of the column-and-canopy-shaped biofilms during spaceflight CLSM images of wild-type, mutants deficient in flaggela-driven motility and type IV pili-driven motility were compared. As shows the figure 2B the structure of mutant structure biofilm cultured during spaceflight showed dence biofilm. The differences are not easy to see when compared to those cultured in normal gravity. Type IV behaved similary to wild type by forming the column-and canopy structures and those frmed in normal gravity shown dence biofilms. These comparisons prove that like mushroom-shape structured biofilms formed on earth, flagella driven motility plays a key role of a colum-and-canopy structured biofilms.
d) effects of oxygen Availability on Biofilm Formation
To show the effect of Oxygen availability on biofilm formation during spaceflight the solid insert was changed for gas exchange insert that enables movement of gases via membrane.
An increase in biofilm formation was observed with GE inserts compared to solid ones under both normal and microgravity conditions. (Figure 3) However it is possible that increased oxygen availability inhibits the formation of the column-and canopy structure.
Discussion
Figure 4 sumarizes how biofilms formed under the spaceflight culture conditions compare with those formed under two lab conditions : static and hydrodynamic.
The sciencist have demonstrated that spaceflight can start changes in P.a biofilm growth and architecture. These findings are the first evidence that spaceflight influences community – level behaviours of bacteria and indicated the importance of understanding how both harmful and beneficial human-microbe interactions may be modified during spaceflight.