Post-Flight Microbial Analysis of Samples from the International Space Station Water Recovery System and Oxygen Generation System

The Regenerative, Environmental Control and Life Support System (ECLSS) on the International Space Station (ISS) includes the the Water Recovery System (WRS) and the Oxygen Generation System (OGS). The WRS consists of a Urine Processor Assembly (UPA) and Water Processor Assembly (WPA). This report describes microbial characterization of wastewater and surface samples collected from the WRS and OGS subsystems, returned to KSC, JSC, and MSFC on consecutive shuttle flights (STS-129 and STS-130) in 2009-10. STS-129 returned two filters that contained fluid samples from the WPA Waste Tank Orbital Recovery Unit (ORU), one from the waste tank and the other from the ISS humidity condensate. Direct count by microscopic enumeration revealed 8.38 x 104 cells per mL in the humidity condensate sample, but none of those cells were recoverable on solid agar media. In contrast, 3.32 x lOs cells per mL were measured from a surface swab of the WRS waste tank, including viable bacteria and fungi recovered after S12 days of incubation on solid agar media. Based on rDNA sequencing and phenotypic characterization, a fungus recovered from the filter was determined to be Lecythophora mutabilis. The bacterial isolate was identified by rDNA sequence data to be Methylobacterium radiotolerans. Additional UPA subsystem samples were returned on STS-130 for analysis. Both liquid and solid samples were collected from the Russian urine container (EDV), Distillation Assembly (DA) and Recycle Filter Tank Assembly (RFTA) for post-flight analysis. The bacterium Pseudomonas aeruginosa and fungus Chaetomium brasiliense were isolated from the EDV samples. No viable bacteria or fungi were recovered from RFTA brine samples (N= 6), but multiple samples (N = 11) from the DA and RFTA were found to contain fungal and bacterial cells. Many recovered cells have been identified to genus by rDNA sequencing and carbon source utilization profiling (BiOLOG Gen III). The presence of viable bacteria and fungi from WRS and OGS subsystems demonstrates the need for continued monitoring of ECLSS during future ISS operations and investigation of advanced antimicrobial controls

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 that possess the appropriate optical properties for the manufacture of LEDs capable of producing monochromatic light at germicidal wavelengths. This report describes the testing of a commercial-off-the-shelf, high power Nichia UV-A LED (250mW A365nnJ for the excitation of titanium dioxide as a point-of-use (POD) 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 actively-cooled 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

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

Evaluation of an ATP Assay to Quantify Bacterial Attachment to Surfaces in Reduced Gravity

Aim: To develop an assay to quantify the biomass of attached cells and biofilm formed on wetted surfaces in variable-gravity environments. Methods and Results: 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 1.0 x 10(exp 5) cells per milliliter. After the final parabola on each flight test day, half of the material coupon samples were treated with either 400 micro-g/L 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 Earth gravity

Inflight Microbial Monitoring- An Alternative Method to Culture Based Detection Currently Used on the International Space Station

Previous research has shown that potentially destructive microorganisms and human pathogens have been detected on the International Space Station (ISS). The likelihood of introducing new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of the total microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS microbes requires that samples be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction

Inflight Microbial Monitoring-An Alternative Method to Culture Based Detection Currently Used on International Space Station

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS). The potential to introduce new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Previous research has shown that microorganisms introduced to the ISS are readily transferred between crew and subsystems and back (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and a 48-hour incubation time. This increases the microbial load while detecting a limited number of microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification, To identify and enumerate ISS samples requires that samples to be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganism at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction

Inflight Microbial Monitoring - An Alternative Method to Culture Based Detection Currently Used on the International Space Station

Microorganisms including potential human pathogens have been detected on the International Space Station (ISS). The potential to introduce new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Current microbial monitoring methods require enrichment of microorganisms and a 48-hour incubation time resulting in an increase in microbial load, detecting a limited number of unidentified microorganisms. An expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted

Dormancy and Recovery Testing for Biological Wastewater Processors

Bioreactors, such as aerated membrane type bioreactors have been proposed and studied for a number of years as an alternate approach for treating wastewater streams for space exploration. Several challenges remain before these types of bioreactors can be used in space settings, including transporting the bioreactors with their microbial communities to space, whether that be the International Space Station or beyond, or procedures for safing the systems and placing them into dormant state for later start-up. Little information is available on such operations as it is not common practice for terrestrial systems. This study explored several dormancy processes for established bioreactors to determine optimal storage and recovery conditions. Procedures focused on complete isolation of the microbial communities from an operational standpoint and observing the effects of: 1) storage temperature, and 2) storage with or without the reactor bulk fluid. The first consideration was tested from a microbial integrity and power consumption standpoint; both room temperature (25 C) and cold (4 C) storage conditions were studied. The second consideration was explored; again, for microbial integrity as well as plausible real-world scenarios of how terrestrially established bioreactors would be transported to microgravity and stored for periods of time between operations. Biofilms were stored without the reactor bulk fluid to simulate transport of established biofilms into microgravity, while biofilms stored with the reactor bulk fluid simulated the most simplistic storage condition to implement operations for extended periods of nonuse. Dormancy condition did not have an influence on recovery in initial studies with immature biofilms (48 days old), however, a lengthy recovery time was required (20+ days). Bioreactors with fully established biofilms (13 months) were able to recover from a 7-month dormancy period to steady state operation within 4 days (approximately 1 residence cycle). Results indicate a need for future testing on biofilm age and health and further exploration of dormancy length

Fiber Attachment Module Experiment (FAME): Using a Multiplexed Miniature Hollow Fiber Membrane Bioreactor Solution for Rapid Process Testing

Bioreactor research is mostly limited to continuous stirred-tank reactors (CSTRs) which are not an option for microgravity (g) applications due to the lack of a gravity gradient to drive aeration as described by the Archimedes principle. Bioreactors and filtration systems for treating wastewater in g could avoid the need for harsh pretreatment chemicals and improve overall water recovery. Solution: Membrane Aerated Bioreactors (MABRs) for g applications, including possible use for wastewater treatment systems for the International Space Station (ISS)

Microorganism Utilization for Synthetic Milk Production

A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms were optimized in the laboratory and the desired end-products, sugars and lipids, were analyzed. Trichoderma reesei, a known cellulolytic fungus, was utilized to drive the production of glucose, with the intent that the produced glucose would serve as the carbon source for milk fat production and be a substitute for the milk sugar lactose. Lipid production would be carried out by Rhodosporidium toruloides, yeast known to accumulate those lipids that are typically found in milk fat. Results showed that glucose and total lipid content were below what was expected during this phase of experimentation. In addition, individual analysis of six fatty acids revealed that the percentage of each fatty acid was lower than naturally produced bovine milk. Overall, this research indicates that microorganisms could be utilized to breakdown inedible solid waste to produce useable products