Studying biofilms in a microgravity environment currently relies on one of two scenarios, collecting planktonic aggregates in rotating wall vessels or performing experiments in the microgravity environment of space on the International Space Station. While informative techniques, both have their limitations when studying surfaceattached microbial communities. A simulated microgravity biofilm reactor (SMBR) was developed to study biofilms in microgravity, coupled with the integration of microfabricated sensors for internal system monitoring. The establishment of simulated microgravity was demonstrated through computational fluid dynamic modelling revealing low fluid shear stress conditions (<1 mPa) throughout the reactor and on the wall surface. Microfabricated resistance temperature devices integrated in the reactor walls confirmed the capability for continuous sensor measurements during operation with the ability to perform traditional microbiology analyses on the sensor surface following an experiment. Microbiological analyses established that there were no significant differences in biofilm growth between sensor and wall surfaces within the reactor. With the integration of defined sampling surfaces, the SMBR allows for in-depth biofilm analysis in a repeatable and accessible manner allowing for a greater understanding of the effects of microgravity on biofilm.

Biofilms are self-organized communities of microorganisms that are encased in an extracellular polymeric matrix and often found attached to surfaces. Biofilms are widely present on Earth, often found in diverse and sometimes extreme environments. These microbial communities have been described as recalcitrant or protective when facing adversity and environmental exposures. On the International Space Station, biofilms were found in human-inhabited environments on a multitude of hardware surfaces. Moreover, studies have identified phenotypic and genetic changes in the microorganisms under microgravity conditions including changes in microbe surface colonization and pathogenicity traits. Lack of consistent research in microgravity-grown biofilms can lead to deficient understanding of altered microbial behavior in space. This could subsequently create problems in engineered systems or negatively impact human health on crewed spaceflights. It is especially relevant to long-term and remote space missions that will lack resupply and service. Conversely, biofilms are also known to benefit plant growth and are essential for human health (i.e., gut microbiome). Eventually, biofilms may be used to supply metabolic pathways that produce organic and inorganic components useful to sustaining life on celestial bodies beyond Earth. This article will explore what is currently known about biofilms in space and will identify gaps in the aerospace industry's knowledge that should be filled in order to mitigate or to leverage biofilms to the advantage of spaceflight.

Microbial biofilm contamination is a widespread problem that requires precise and prompt detection techniques to effectively control its growth. Microfabricated electrochemical impedance spectroscopy (EIS) biosensors offer promise as a tool for early biofilm detection and monitoring of elimination. This study utilized a custom flow cell system with integrated sensors to make real-time impedance measurements of biofilm growth under flow conditions, which were correlated with confocal laser scanning microscopy (CLSM) imaging. Biofilm growth on EIS biosensors in basic aqueous growth media (tryptic soy broth, TSB) and an oil-water emulsion (metalworking fluid, MWF) attenuated in a sigmoidal decay pattern, which lead to an & SIM;22-25% decrease in impedance after 24 Hrs. Subsequent treatment of established biofilms increased the impedance by & SIM;14% and & SIM;41% in TSB and MWF, respectively. In the presence of furanone C-30, a quorum-sensing inhibitor (QSI), impedance remained unchanged from the initial time point for 18 Hrs in TSB and 72 Hrs in MWF. Biofilm changes enumerated from CLSM imaging corroborated impedance measurements, with treatment significantly reducing biofilm. Overall, these results support the application of microfabricated EIS biosensors for evaluating the growth and dispersal of biofilm in situ and demonstrate potential for use in industrial settings.One-Sentence Summary This study demonstrates the use of microfabricated electrochemical impedance spectroscopy (EIS) biosensors for real-time monitoring and treatment evaluation of biofilm growth, offering valuable insights for biofilm control in industrial settings. Graphical Abstract The accurate real-time detection of biofilm growth and removal is effectively demonstrated through the combination of impedance biosensors and high-resolution confocal laser scanning microscopy.

Biofilm contamination is a widespread issue that can occur anywhere when organisms attach to surfaces in the presence of water. In industrial environments, formation of biofilms can lead to component failure, material degradation, and biofouling or spoilage, which collectively come with significant economic costs. Microfabricated electrochemical impedance spectroscopy (EIS) sensors have emerged as a promising tool for monitoring biofilm as EIS sensors capture information about biofilm growth autonomously in real-time; however, sensors suffer from drift, and the technique lacks temporal interpretation of dynamic biofilm processes. In this work, microfabricated sensors featuring gold micro-interdigitated electrodes (μIDEs) were modified with an electrically conductive polymer layer resulting in EIS measurement variability that was significantly reduced compared to unmodified sensors, and enabled highly stable, time-resolved EIS measurements. EIS characterization of Pseudomonas aeruginosa biofilm in parallel with high-resolution confocal laser scanning microscopy (CLSM) was performed using a novel 3D-printed flow cell system, resulting in distinct changes to EIS data corresponding with consistent biofilm growth. We have shown that EIS microsensors can detect four stages of biofilm: (i) initial biofilm attachment to the sensor substrate, (ii) early-stage irreversible biofilm proliferation characterized by sparse biofilm coverage, (iii) mature biofilm detection characterized by uniform biofilm coverage, and (iv) changes due to detachment and regrowth of biofilm.

During the growth of a polycrystalline ice lattice, microorganisms partition into veins, forming an ice vein network highly concentrated in salts and microbial cells. We used microfabricated electrochemical impedance spectroscopy (EIS) sensors to determine the effect of microorganisms on the electrochemical properties of ice. Solutions analyzed consisted of a 176 μ S cm −1 conductivity solution, fluorescent beads, and Escherichia coli HB101-GFP to model biotic organisms. Impedance spectroscopy data were collected at −10 °C, −20 °C, and −25 °C within either ice veins or ice grains (i.e., no veins) spanning the sensors. After freezing, the fluorescent beads and E. coli were partitioned into the ice veins. The corresponding impedance data were discernibly different in the presence of ice veins and microbial impurities. The presence of microbial cells in ice veins was evident by decreased electrical characteristics (electrode polarization between electrode and ice matrix) relative to solid ice grains. Further, this electrochemical behavior was reversed in all bead-doped solutions, indicating that microbial processes influence sensor response. Linear mixed-effects models empirically corroborated the differences in polarization associated with the presence and absence of microbial cells in ice. We show that EIS has the potential to detect microbes in ice and differentiate between veins and solid grains.

Electrochemical techniques such as impedance spectroscopy offer a non-invasive approach to monitor microorganisms in natural and engineered environments. Here, we present data on the use of microfabricated impedance spectroscopy sensors for the detection of microbes in icy environments. Under controlled laboratory settings, the effects of different cell concentrations of the Antarctic isolate Flavobacterium sp. ANT 11 on impedance spectroscopy was investigated at ambient temperature (22°C) and -10°C. Results show that varying cell concentrations of the bacterial isolate generate unique spectral responses that vary with temperature. Subsequent tests on the performance of microfabricated impedance sensors on natural microbial communities in icy environments in the Beartooth Mountains, WY, USA, confirmed laboratory findings. Different environmental samples at varying microbial concentrations in liquid, semi-frozen (slushy), or frozen states generated discrete impedance spectra. While caution is advised to generalize these results due to the potential contribution of unknown environmental variables, our data provide fertile territory for research in cryo-microbiology, as information on the applicability of impedance spectroscopy under frozen conditions is limited. [2020-0150]

Micro-fabricated sensors enable the study of chemical and physical dynamics in aqueous environments such as rivers, lakes or oceans at low cost. Sensors must work reliably in these environments, which include both biological and chemical challenges. However, sensor thin films have not been studied in detail for aqueous applications, and more specifically how biotic interactions may change sensor material properties. In this study, the long-term effects of biofilm formation on the properties of aluminum (electric conductor) and a-SixNy:H (insulating material) were investigated. Material degradation caused by Escherichia coli K12 biofilm growth was determined by electrical sheet resistance measurements (collinear four-point-probe) and Fourier-transform infrared spectroscopy (FTIR) absorption spectra over a time period of 7 weeks. Changes of the surface topography were tested using scanning electron microscopy (SEM) and white light interferometry. Aluminum was found to be heavily degraded at three weeks, whereas a-SixNy:H was inert during the entire investigation period. As differences between thin film sensor materials are evident, more detailed investigations including a broader range of materials should be explored.