Michele N. Birmele

Intercontinental Dispersal of Bacteria and Archaea by Transpacific Winds

ABSTRACT Microorganisms are abundant in the upper atmosphere, particularly downwind of arid regions, where winds can mobilize large amounts of topsoil and dust. However, the challenge of collecting samples from the upper atmosphere and reliance upon culture-based characterization methods have prevented a comprehensive understanding of globally dispersed airborne microbes. In spring 2011 at the Mt. Bachelor Observatory in North America (2.8 km above sea level), we captured enough microbial biomass in two transpacific air plumes to permit a microarray analysis using 16S rRNA genes. Thousands of distinct bacterial taxa spanning a wide range of phyla and surface environments were detected before, during, and after each Asian long-range transport event. Interestingly, the transpacific plumes delivered higher concentrations of taxa already in the background air (particularly Proteobacteria, Actinobacteria, and Firmicutes). While some bacterial families and a few marine archaea appeared for the first and only time during the plumes, the microbial community compositions were similar, despite the unique transport histories of the air masses. It seems plausible, when coupled with atmospheric modeling and chemical analysis, that microbial biogeography can be used to pinpoint the source of intercontinental dust plumes. Given the degree of richness measured in our study, the overall contribution of Asian aerosols to microbial species in North American air warrants additional investigation.

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

Aim:  To incorporate into the lambda phage genome, a luxI‐based acyl‐homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux‐based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens.

Characterization and validation of a bioluminescent bioreporter for the direct detection of Escherichia coli

A two-component bacteriophage-based bioluminescent reporter system was developed for the detection of Escherichia coli in environmental samples. The bioreporter system consists of a luxI integrated lambda bacteriophage and a lux-based bioluminescent reporter cell that responds to the infection event through acyl-homoserine lactone (AHL) mediated quorum sensing and bioluminescent signal stimulation. This work addresses the ability of the bioreporter system to detect and quantify the target pathogen in response to two analytical challenges: (1) detection of target cells in the presence of lactonase-producing non-target organisms that could interrupt AHL signal transduction, and (2) detection of sub-lethally injured or physiologically stressed target cells. The bioreporter system was able to autonomously respond to lambda phage infection events with a target host E. coli at 1x10(8) cfu/mL against a background of lactonase-producing Arthrobacter globiformis at cell densities ranging from 1 to 1x10(8) cfu/mL. E. coli target cells stressed by carbon-limitation for 2 weeks (i.e., starvation) or exposure to iodine for 1 week at 2 and 20 ppm (i.e., disinfection) yield a reduced, but detectable, biosensor response. Conversely, short-term iodine exposure produces a significant increase in bioreporter response within the first 24 h. The signal response and limit of detection for the two-component bioreporter system were affected by the physiology and environment of the target, but the bioreporter maintained target specificity demonstrating its potential application for remote sensing of pathogens.

Disinfection of Spacecraft Potable Water Systems by Passivation with Ionic Silver

Microbial growth is common on wetted surfaces in spacecraft environmental control and life support systems despite the use of chemical and physical disinfection methods. Advanced control technologies are needed to limit microorganisms and increase the reliability of life support systems required for long-duration human missions. Silver ions and compounds are widely used as antimicrobial agents for medical applications and continue to be used as a residual biocide in some spacecraft water systems. The National Aeronautics and Space Administration (NASA) has identified silver fluoride for use in the potable water system on the next generation spacecraft. Due to ionic interactions between silver fluoride in solution and wetted metallic surfaces, ionic silver is rapidly depleted from solution and loses its antimicrobial efficacy over time. This report describes research to prolong the antimicrobial efficacy of ionic silver by maintaining its solubility. Three types of metal coupons (lnconel 718, Stainless Steel 316, and Titanium 6AI-4V) used in spacecraft potable water systems were exposed to either a continuous flow of water amended with 0.4 mg/L ionic silver fluoride or to a static, pre-treatment passivation in 50 mg/L ionic silver fluoride with or without a surface oxidation pre-treatment. Coupons were then challenged in a high-shear, CDC bioreactor (BioSurface Technologies) by exposure to six bacteria previously isolated from spacecraft potable water systems. Continuous exposure to 0.4 mg/L ionic silver over the course of 24 hours during the flow phase resulted in a >7-log reduction. The residual effect of a 24-hour passivation treatment in 50 mg/L of ionic silver resulted in a >3-log reduction, whereas a two-week treatment resulted in a >4-log reduction. Results indicate that 0.4 mg/L ionic silver is an effective biocide against many bacteria and that a prepassivation of metal surfaces with silver can provide additional microbial control.

Microbial Monitoring of Common Opportunistic Pathogens by Comparing Multiple Real-Time PCR Platforms for Potential Space Applications

Because the International Space Station is a closed environment with rotations of astronauts and equipment that each introduce their own microbial flora, it is necessary to monitor the air, surfaces, and water for microbial contamination. Current microbial monitoring includes labor- and time-intensive methods to enumerate total bacterial and fungal cells, with limited characterization, during in-flight testing. Although this culture-based method is sufficient for monitoring the International Space Station, on future long-duration missions more detailed characterization will need to be performed during flight, as sample return and ground characterization may not be available. At a workshop held in 2011 at NASAs Johnson Space Center to discuss alternative methodologies and technologies suitable for microbial monitoring for these long-term exploration missions, molecular-based methodologies such as polymerase chain reaction (PCR) were recommended. In response, a multi-center (Marshall Space Flight Center, Johnson Space Center, Jet Propulsion Laboratory, and Kennedy Space Center) collaborative research effort was initiated to explore novel commercial-off-the-shelf hardware options for space flight environmental monitoring. The goal was to evaluate quantitative or semi-quantitative PCR approaches for low-cost in-flight rapid identification of microorganisms that could affect crew safety. The initial phase of this project identified commercially available platforms that could be minimally modified to perform nominally in microgravity. This phase was followed by proof-of-concept testing of the highest qualifying candidates with a universally available challenge organism, Salmonella enterica. The analysis identified two technologies that were able to perform sample-to-answer testing with initial cell sample concentrations between 50 and 400 cells. In addition, the commercial systems were evaluated for initial flight safety and readiness.

Alternative Water Processor Test Development

The Next Generation Life Support Project is developing an Alternative Water Processor (AWP) as a candidate water recovery system for long duration exploration missions. The AWP consists of biological water processor (BWP) integrated with a forward osmosis secondary treatment system (FOST). The basis of the BWP is a membrane aerated biological reactor (MABR), developed in concert with Texas Tech University. Bacteria located within the MABR metabolize organic material in wastewater, converting approximately 90% of the total organic carbon to carbon dioxide. In addition, bacteria convert a portion of the ammonia-nitrogen present in the wastewater to nitrogen gas, through a combination of nitrification and denitrification. The effluent from the BWP system is low in organic contaminants, but high in total dissolved solids. The FOST system, integrated downstream of the BWP, removes dissolved solids through a combination of concentration-driven forward osmosis and pressure driven reverse osmosis. The integrated system is expected to produce water with a total organic carbon less than 50 mg/l and dissolved solids that meet potable water requirements for spaceflight. This paper describes the test definition, the design of the BWP and FOST subsystems, and plans for integrated testing.

Antimicrobial Resources for Disinfection of Potable Water Systems for Future Spacecraft

As human exploration adventures beyond low earth orbit, life support systems will require more innovation and research to become self-sustaining and durable. One major concern about future space travel is the ability to store and decontaminate water for consumption and hygiene. This project explores materials and technologies for possible use in future water systems without requiring point-of-use (POU) filtering or chemical additives such as iodine or silver that require multiple doses to remain effective. This experimentation tested the efficacy of a variety of antimicrobial materials against biofilm formation in a high shear CDC Biofilm Reactor (CBR) and some materials in a low shear Drip Flow Reactor (DFR) which(also utilizes ultra violet light emitting diodes (UVLEDs) as an antimicrobial resource. Most materials were tested in the CBR using the ASTM E 2562-07 1method involving the Pseudomonas aeruginosa and coupon samples that vary in their antimicrobial coatings and surface layer topographies. In a controlled environmental chamber (CEC), the CBR underwent a batch phase, continuous flow phase (CFP), and a harvest before analysis. The DFR portion of this experimentation was performed in order to assess the antimicrobial capabilities of ultraviolet-A LEDs (UV-A) in potable water systems. The ASTM E 2647-08 was modified in order to incorporate UV-A LEDs and to operate as a closed, re-circulating system. The modified DFR apparatus that was utilized contains 4 separate channels each of which contain 2 UV-A LEDs (1 chamber is masked off to serve as a control) and each channel is equipped with its own reservoir and peristaltic pump head. The 10 DFR runs discussed in this report include 4 initial experimental runs that contained blank microscope slides to test the UVA LEDs alone, 2 that incorporated solid silver coupons, 2 that utilized titanium dioxide (Ti02) coupons as a photocatalyst, and 2 runs that utilized silver coated acrylic slides. Both the CBR and DFR experiments were analyzed for microbial content via heterotrophic plate counts (HPC) and acridine orange direct counts (AODC). Ofthe materials used in the CBR, only two materials performed as anti~icrobials under high shear conditions (a reduction of 5 or more logs) showing a>7 log reduction in viable microbes.

Disinfection of Spacecraft Potable Water Systems by Photocatalytic Oxidation Using UV-A Light Emitting Diodes

Ultraviolet (UV) light has long been used in terrestrial water treatment systems for photodisinfection and the removal of organic compounds by several processes including photoadsorption, photolysis, and photocatalytic oxidation/reduction. Despite its effectiveness for water treatment, UV has not been explored for spacecraft applications because of concerns about the safety and reliability of mercury-containing UV lamps. However, recent advances in ultraviolet light-emitting diodes (UV LEDs) have enabled the utilization of nanomaterials with appropriate optical properties for the manufacture of LEDs that produce monochromatic light at germicidal wavelengths. This report describes the testing of a commercial off-the-shelf, high power Nichia UV-A LED (250mW λ365nm) for the excitation of titanium dioxide as a point-of-use (POU) disinfection device in a potable water system. The combination of an immobilized, high surface area photocatalyst with a UV-A LED is promising for potable water system disinfection since toxic chemicals and resupply requirements are reduced. No additional consumables like chemical biocides, absorption columns, or filters are required to disinfect and/or remove potentially toxic disinfectants from the potable water prior to use. Experiments were conducted in a static test stand consisting of a polypropylene microtiter plate containing 3mm glass balls coated with titanium dioxide. Wells filled with water were exposed to ultraviolet light from an activelycooled UV-A LED positioned above each well and inoculated with six individual challenge microorganisms recovered from the International Space Station (ISS): Burkholderia cepacia, Cupriavidus metallidurans, Methylobacterium fujisawaense, Pseudomonas aeruginosa, Sphingomonas paucimobilis, and Wautersia basilensis. Exposure to the Nichia UV-A LED with photocatalytic oxidation resulted in a complete (>7-log) reduction of each challenge bacteria population in <180 minutes of contact time. With continued advances in the design and manufacture of UV-A LEDs and semi-conducting photocatalysts, LED activated photochemical process technology promises to extend its application to spacecraft environmental systems.

Evaluation of an ATP Assay to Quantify Bacterial Attachment to Wetted Surfaces in Variable Gravity Conditions

The goal of this experimentation was to develop an assay to quantify the biomass of attached cells and biofilm formed on wetted surfaces in variable gravity environments. Liquid cultures of Pseudomonas aeruginosa were exposed to 30–35 brief cycles of hypergravity (≤ 2 g) followed by free fall (i.e., reduced gravity) equivalent to either lunar g (i.e., 0.17 normal Earth gravity) or micro g (i.e., < 0.001 normal Earth gravity) in an aircraft flying a series of parabolas. Over the course of two days of parabolic flight testing, 504 polymer or metal coupons were exposed to a stationary-phase population of P. aeruginosa strain ERC1 at a concentration of 1 x 10 cells/milliliter. After the final parabola on each flight test day, half of the material coupon samples were treated with either 400 μg L-1 ionic silver fluoride (microgravity-exposed cultures) or 1% formalin (lunar gravity-exposed cultures). The remaining sample coupons from each flight test day were not treated with a fixative. All samples were returned to the laboratory for analysis within 2 hours of landing, and all biochemical assays were completed within 8 hours of exposure to variable gravity. The intracellular ATP luminescent assay accurately reflected cell physiology compared to both cultivation-based and direct-count microscopy analyses. Cells exposed to variable gravity had more than twice as much intracellular ATP as control cells exposed only to normal terrestrial gravity.