The need for space and planetary exploration has increased dramatically. The National Aeronautics and Space Administration (NASA) already designs future manned missions with the aim to shed light on important questions that concern humanity (Nat Microbiol, 2022). However, microbes are considered in most cases an enemy to this attempt. These tiny life forms can adhere either on biotic or abiotic surfaces (Noirot-Gros et al., 2019), proliferate, and generate structurally complex and dynamic communities named biofilms. Such a growth lifestyle is a widespread survival mode among microbes providing them protection from harsh conditions while reaping the benefits of co-living at the same time (Hall-Stoodley et al., 2004; Jefferson, 2004). To that goal, microorganisms that participate in the biofilm formation synthesize an extracellular matrix (EPS) consisting of bio-macromolecules such as extracellular DNA (eDNA), polysaccharides and proteins, as well as they notably reprogram their metabolic activities compared to their non-biofilm-counterparts (Penesyan et al., 2021; Malviya et al., 2023). The ability of microbial adhesion on a surface is a complex phenomenon that relies on a variety of factors, such as the physicochemical features of the substrate, e.g., chemistry, roughness, wettability, charge, stiffness, topography, surface orientation, the environmental conditions, e.g., pH, temperature, pressure, O₂ availability, microgravity, osmolarity, specific ion and nutrient presence, as well as the kind of microorganism itself (Goller and Romeo, 2008; Acres et al., 2021; Zheng et al., 2021; Jha et al., 2022).

Biofilms’ mode thrives in wet environments such as those provided by water systems addressing potentially detrimental effects on safety (Kretschmer et al., 2021). Nevertheless, this is not an issue associated only with Earth’s environment. Despite the applied purification procedures, several microorganisms have been isolated from International Space Station (ISS) potable water systems, thus raising concerns about the quality of water and efficiency of facilities (Bruce et al., 2005; O’Rourke et al., 2020; Yang et al., 2021). Taking also into account the exposure to microgravity, microbial biofilms can display unpredicted behavior related to increased virulence and antimicrobial resistance (Yang et al., 2021). It is perfectly understood that for any extended space mission, including the upcoming travel to the Moon and Mars (Vélez Justiniano et al., 2023), uncontaminated water is really essential. Therefore, it is pivotal to diminish the microbiological threats that could possibly risk the crew’s health and mission success. More specifically, biofilm generation constitutes a significant threat to the life of astronauts who depend on clean water for drinking, cooking and hygiene purposes, whereas its accumulation in space water systems can lead to clogging of pipes and filters and subsequent corrosion of equipment (Liu et al., 2016).

The careful selection of surface materials is of vital importance, as surfaces are the foundation upon which biofilms grow. Currently, the construction of space water distribution systems involves the use of various materials based on their unique properties and suitability to withstand the harsh space environment (Finckenor, 2018; Ghidini, 2018). Among them, metallic surfaces such as stainless steel (SS), either passivated or electropolished and titanium alloy (Ti-6Al-4V) are primarily used as construction material of pipes, junctions, etc (Petala et al., 2020). Regarding the preservation of water, NASA intends to utilize biocidal silver in order to ensure the quality of potable water and prevent the harmful effects of microorganisms (Petala et al., 2017). The antimicrobial potential of silver is already well-known including deactivation of macromolecules such as DNA, RNA and proteins, cell membrane disruption and reactive oxygen species (ROS) generation (Kędziora et al., 2021). However, according to ESA and NASA documents (Rebeyre, 2012; Li et al., 2018; Li et al., 2019; Muirhead et al., 2020) as well the studies of our research group (Petala et al., 2017; Mintsouli et al., 2018), silver is depleted from water when it is in contact with metallic surfaces due to deposition phenomena. Based on those findings, the aim of the present work is to evaluate the contribution of space-relevant parameters to the potential biofilm formation inside water distribution systems. More specifically, the main objective is the impact of different kind of surface materials, and of shear stress conditions (i.e., stagnant vs. shaking) on the initial stages of biofilm formation. Preventing the process at this stage can inhibit the evolvement into mature biofilms, thereby diminishing the biofilm-related issues that could threaten an extended space-mission (Fu et al., 2021). To this regard, various parameters were tested during the formation of biofilm utilizing a widely-used strain, prior to implementing more complex studies with multi-strain or mixed-species cultures. For our purpose, Pseudomonas fluorescens SBW25, a well-studied bacterium for its biofilm formation capacity (Koza et al., 2009; Jerdan et al., 2020), is used as a model microorganism to investigate bacteria adhesion, which reflects the effectiveness of initial stages of biofilm attachment and is considered as the first step in biofilm formation that governs the mature biofilm structure cultivated on top wetted surfaces and in contact to the cultivation medium (Flemming and Wingender, 2010; Huang et al., 2015). P. fluorescens belongs to Gram-negative bacteria, which constitute predominantly identified species in space water systems and it has been isolated from ISS potable sources and Mir station (Bruce et al., 2005; Song and Leff, 2005). To our knowledge, it is the first time that the ability of these bacteria to form biofilms is studied at the solid-liquid interfaces, which are space applications related materials.

The aim of this study was to evaluate the role of surface type and treatment, as well as shear stress met in water systems of space missions-related infrastructure on biofilm formation.

To this direction, the ability of P. fluorescens SBW25 to adhere on different metallic surfaces under different applied conditions was examined, while Ti, passivated SS and SSEP surfaces, which are used in several parts of space water distribution systems (tubes, junctions, etc.), were selected as substrates for investigating bacterial adhesion, which is critical for later biofilm structure cultivated under culture medium (Flemming and Wingender, 2010; Huang et al., 2015). The behavior of P. fluorescens was evaluated after 24 h exposure, which is considered a premature stage of biofilm formation. Biofilms at an early stage are more fragile exhibiting vigorous metabolic activities and higher susceptibility to disinfectants and antimicrobial agents, thus it is easier to remove them at that point (Fu et al., 2021). In addition, these early biofilm configurations strongly affect the regulation of more developed structures. Hence, it is important to understand what factors affect their evolvement. The results of this study should be used to further expand its scope, so as to include the understanding of the behavior of more complex systems (containing different bacterial strains and/or species) close to real space environments.

The wetted surfaces found in water systems serve as the basis upon which microbes grow and generate their biofilm communities, thus it is important to evaluate their features. Based on the confocal measurements, it is found that passivated SS was the roughest surface among the three substrates, while SSEP was the smoothest. Ti surface exhibited an intermediate roughness. In addition, wettability experiments provided interesting results concerning the material properties. More specifically, wetting behavior was not uniform in all directions for the rough surfaces SS and Ti, respectively. A high degree of anisotropy was noticed particularly for SS. This could be attributed to the grooves and crevices of these materials (Tardelli et al., 2023). If water enters the grooves, the energy cost is higher for the molecules to disperse perpendicular compared to flowing along them. This occurs due to the fact that groove walls function as pinning points, providing additional support with the surface tension. As a result, the contact angles were larger when observing the droplet from the direction of the grooves (Park et al., 2021). It should be mentioned here that not only the wettability of a surface could affect the biofilm formation, but also the biofilm formation could impact the wetting characteristics of a surface (Recupido et al., 2020; Castigliano et al., 2021; Kampouraki et al., 2022; Recupido et al., 2022; Recupido et al., 2023). Moreover, rough surfaces are colonized more easily providing an increased contact area for cell adhesion, as well as a protected environment for microorganisms against shear stress (Zheng et al., 2021). This is attributed to their features such as grooves and irregularities which favor microbial attachment (Tardelli et al., 2023). However, there are some studies pointing out contradictory outcomes regarding this parameter. Wu et al. reported that Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus colonized a stainless steel electropolished surface more efficiently compared to the untreated rougher surface (Wu et al., 2018). In another study, increased roughness did not affect the adherence of Gram-positive Staphylococcus epidermis on titanium and ceramic substrates, while it enhanced the adhesion of Streptococcus sanguinis, especially on ceramic surfaces (Wassmann et al., 2017). However, the above observed results may not be exclusively attributed to roughness, but other factors as well, e.g., surface chemistry.

In the present work, passivated SS was more prone to bacterial adhesion under static conditions, followed by Ti and SSEP, respectively. Apparently, there is a positive correlation between adhesion of P. fluorescens and surface roughness. Furthermore, it was observed that after 24 h exposure, the rough surfaces promoted the formation of bigger clusters varying in size and shape, compared to SSEP where single cells were mainly noticed. The distribution of these clusters exhibits a spatial organization enabling bacteria to communicate with each other through molecular mechanisms, i.e., quorum sensing (van Hoogstraten et al., 2023). Microscopic images similar to those for SSEP were captured for smooth glass and polydimethylsiloxane surface (PDMS), when exposed to cultures of P. aeruginosa PAO1 and P. aeruginosa PA-14, respectively (Epstein et al., 2013; Shah et al., 2019).

Besides surface roughness, chemical interactions can play crucial role in biofilm development. In fact, electropolishing is a technique that not only provides smoothness on a surface, but it also alters the exterior layer of the given material. Analyzing the XPS graphs from our previous works (Petala et al., 2016; Petala et al., 2017) for passivated SS and SSEP, respectively, it is shown that SS surface has a high peak corresponding to iron, an element which is absent from SSEP surface. Iron is a crucial agent for cell viability and biofilm development. High iron concentration acts as a stimulus for P. aeruginosa to form microcolonies and subsequently a mature biofilm, whereas low concentration hinders the biofilm growth (Soldano et al., 2020). Furthermore, it is reported that bacteria utilize elements such as iron and magnesium from the metal surfaces on which they are attached, in order to exploit them as terminal electron acceptors in their vital respiration process (Tuson and Weibel, 2013). When oxygen is absent, iron can serve as the terminal electron acceptor in the deep layer of the biofilm (Charron-Lamoureux et al., 2023). In our case, during static culture conditions where the diffusion of oxygen is poor, it might be presumed that microenvironments of low oxygen or no oxygen are derived, especially at the boundary region separating the solid from the liquid phase. Such a condition could impede important biological activities of bacteria which will search eventually for alternative solutions. As a consequence, the chemistry of the surface in terms of elemental composition might also be a crucial factor that determines the extent of bacterial adhesion. Apart from iron, other elements might also act as signaling molecules regulating gene expression and communication or the secretion of EPS which is essential for biofilm formation and maintenance (Wu et al., 2022).

In general, concerning the material type, it is proved that electropolishing constitutes a particularly effective treatment preventing bacterial accumulation. Owing to its better and more uniform texture, without local wetting changes compared to SS appears as a surface that could withstand early biofilm formation. The higher bacterial coverage of SS, as well as the formation of several big clusters indicated by the graph-shift to higher size ranges, implies also a faster kinetic model of bacterial colonization and the stimulation of the P. fluorescens adhesion. On the other hand, Ti is a chemically different surface. Due to the combination of high strength and low weight, it is also considered a great candidate for future space missions. Although it seems to resist bacterial adhesion compared to SS, the detection of many particles on the surface, which could act as nuclei, might potentially lead to high levels of biofilm formation in the long term.

Τhe impact of applied shear stress was also investigated to provide insights on biofilm formation. Herein, the effect was intense for all the studied surfaces exhibiting a significantly lower bacterial attachment compared to stagnant conditions. Similar to static mode, SSEP proved to accommodate remarkably less adherent bacteria compared to SS and Ti. However, in contrast to static conditions, SS and Ti presented the same behavior without differences in bacterial adhesion. Taking also into account the observed orientation of bacteria along the grooves of these surfaces, might imply that such irregularities could indeed serve as protected environments against shear stress. Since SS and Ti are of different chemical composition but of comparable roughness, it is suggested that gravity and texture features of wetted surfaces are more crucial factors in cells’ attachment and biofilm formation compared to surface chemistry for the tested materials. On the whole, it is important to test the material type under different conditions for a better surface evaluation before its application. For longer periods of biofilm growth, the application of hydrodynamic stress on P. fluorescens B52 biofilms over borosilicate glass substrates was critical and led to intensification of biofilm growth, most probably due to higher production of EPS that demonstrated modified macromolecule composition of extracellular matrix compared to the static mode. The formation of thicker and denser biofilms under the tested conditions was attributed to the increased rate of nutrient diffusion and the agitation-assisted attachment of the free-living counterparts to the already generated biofilm colonies (Hou et al., 2018; Jara et al., 2021). Other studies underline the impact of shear stress on the biofilm growth as well, however they all refer to more mature and well-structured biofilms and not to first stages of biofilm formation, as in this study (Tsagkari and Sloan, 2018; Jara et al., 2021). Furthermore, the use of a specific culture medium could influence the interpretation of the results. Different culture media could affect the growth and metabolism of bacteria due to varying concentrations in carbon and nitrogen sources, minerals and other nutrients. The aforementioned components are utilized by microorganisms for cell growth, energy demands and synthesis of extracellular polymeric substances, therefore their availability, as well as their concentration in the environment could either promote or hinder biofilm formation.

The ability of microorganisms to attach to a surface derives from their motility and their passive movement governed by processes such as Brownian motion, gravitational sedimentation and convective mass transfer (Krsmanovic et al., 2021). However, without the proximity between cells and surface, the attachment will not occur (Marra et al., 2022). During shaking mode, it is expected that fewer cells will be near the solid-liquid interface available for attachment compared to static conditions. The final number of cells close to interface is dependent on flow characteristics (leading to passive approach to the surface) or active movement towards to the surface due to cell motility. Yet, cell attachment is related to its contact with the surface and subsequent successful anchoring, which is driven by different underlying mechanisms. In our study, it is justified that the surface type dominates the final bacterial attachment, since applied experimental conditions and shear stress are the same for all materials, while surface coverage is significantly lower for SSEP compared to Ti and SS (Figure 5; Figure 6). Bacterial settlement along the grooves of Ti and SS (materials of different chemical composition) imply that texture might be an important feature affecting the initial adhesion. Motility could be the tool that enables bacteria to navigate through the surface irregularities and detect appropriate sites for attachment.

It should be stressed that in all cases, the stained bacteria or clusters are considered strongly attached to wetted surfaces, due to handling practices chosen in this study, to avoid misinterpretations and overestimations. Sample rinsing is a critical step before visualization. In the study of Reddersen et al., it was shown that the P. fluorescens coverage was reduced about ten times after rinsing of the substrate (Reddersen et al., 2021). This finding raises concerns about the proper way of sample handling. It is known that the accumulation of microorganisms on surfaces normally results to biofilm formation (Wang et al., 2023). However, it must be taken into consideration that initial attachment does not lead inevitably to irreversible adhesion, as microorganisms might firstly sense the surface by means of appendage proteins that they possess (Kimkes and Heinemann, 2019). In fact, the cells that are attached irreversibly on the substrate will divide, generate microcolonies and subsequently produce the EPS that is characteristic of biofilms (Donlan, 2001). In addition, biofilm formation is mainly evaluated when surfaces are placed horizontally (Jha et al., 2022). However, due to gravitational sedimentation of bacteria, there is a high rate of cells that might be loosely attached. Actually, when the impact of a surface is studied under specific conditions, we should be interested exclusively in the forces between the substrate and the microorganism such as van der Waals forces, electrostatic forces and hydrophobic interactions (Zhao et al., 2023). Appropriate rinsing assures the presence only of strongly attached cells, which might subsequently be responsible for biofilm formation. The fact that there is no standard assay about this process could lead to misinterpretation of results. In particular, the volume of washing buffer and the way of rinsing, i.e., dripping rinsing of the surface with washing buffer (Kumar Jha et al., 2020) vs. immersion of the surface in a washing buffer bath (Niboucha et al., 2022) could have a different effect. It should be highlighted that biofilms are a scientific field with so many contradictory studies concerning the contribution of important physicochemical parameters, thus there is an imperative need for a standardized method. Such issues can be avoided by employing non-invasive methods such as Optical Coherence Tomography (Hou et al., 2019). However, due to the lack of sufficient thickness and a defined structure during the early stages of biofilms, confocal or fluorescence microscopy are mainly suggested for evaluation of phenomenon.

During space missions, water systems undergo different operational conditions, i.e., flow, non-flow, sloshing. To this regard, alterations in surface characteristics may also occur. For example, as described earlier (Petala et al., 2017), silver ions of potable water bulk may be depleted and migrate to water tank or water pipe wall. In this case, disinfection capacity of silver ions is lost in the water bulk and silver is deposited in various forms depending on the type of water surface. This behavior was examined in this study with respect to potential biofilm formation in case of water contamination. It was observed that silver deposition did not alter the adhesion of P. fluorescens regardless of deposition mechanism. More specifically, in the case of SS and SSEP, silver deposits in its metallic form, while in the case of Ti it deposits as oxide (Petala et al., 2017; Mintsouli et al., 2018). It is known that the ionic form of silver presents antibacterial activity, while the non-ionic form is inert (Durmus and Molina Jordá, 2021). This could be an important reason for the absence of antimicrobial effect. Nevertheless, chemical composition of SS and Ti was not altered drastically after silver deposition, while essential elements, such as iron still exist as it is shown from XPS graphs in previous work (Petala et al., 2017; Mintsouli et al., 2018). Interestingly, silver deposition occurs to a considerably lesser extent on SSEP compared to SS and Ti, indicating that biocidal silver ions concentration in bulk is better preserved when SSEP is the wetted surface.

Overall, both the material type and the application of shear stress are crucial parameters that could affect the biofilm formation in space water systems. More specifically, SSEP rises as the best repellent substrate among the three studied surfaces concerning the bacterial accumulation and the potentially subsequent biofilm formation. This finding highlights also the importance of surface treatment. Although passivated SS was more prone to bacterial colonization, electropolished SS was proved to be resistant. Even so, aging of surfaces should be addressed to investigate long term impacts. As a consequence, different treatments might result to different outcomes, something that should be taken into consideration during the manufacturing process. Moreover, the efficiency of material type against bacterial colonization is enhanced under applied shear stress, underlying the impact of shear on cells' behavior. Still, material type was crucial to biofilm surface coverage. However, deposition of silver on metallic surfaces does not influence bacterial adhesion. The silver depletion from water is challenging, as the decrease of concentration renders water susceptible to microbial contamination, while its deposition on metallic surfaces causes loss of its biocidal effect due to deactivation. In this case, the application of SSEP could offer a dual advantage, presenting simultaneously a better microbial resistance and a resistance to silver depletion from water based on our previous findings.

As space missions extend their duration, the potential risks associated with microbial contamination increase. Such circumstances could compromise both the astronauts’ health and integrity of spacecraft’s facilities. The water distribution systems constitute an excellent candidate for bacterial growth, colonization and eventually biofilm formation. Therefore, it is essential to perform risk analysis studies and prepare risk management plans related to contamination issues. Towards this direction, the present study indicates that the careful selection of materials used in the construction of space water systems is of vital importance. Parameters such as texture, treatment and chemistry of a surface should be strictly taken into consideration. Controlling the platforms of microbial communities’ growth could eliminate the possible harmful effects. Moreover, the applied conditions to which these surfaces are exposed is another critical aspect. Understanding the dynamics of bacterial biofilm formation could lead to the development of new effective antimicrobial strategies or optimization of existing.