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Presentation reviews the status in reference to the Environmental, Health and Safety (EHS) of the water quality and microbiology for the International Space Station. It includes information about crew training, hardware delivery, and those items that will be returned for study.
Spacecraft and space habitats supporting human exploration contain a diverse population of microorganisms. Microorganisms may threaten human habitation in many ways that directly or indirectly impact the health, safety, or performance of astronauts. The ability to produce and maintain spacecraft and space stations with environments suitable for human habitation has been established over 40 years of human space flight. An extensive database of environmental microbiological parameters has been provided for short-term (< 20 days) space flight by more than 100 missions aboard the Space Shuttle. The NASA Mir Program provided similar data for long-duration missions. Interestingly, the major bacterial and fungal species found in the Space Shuttle are similar to those encountered in the nearly 15-year-old Mir. Lessons learned from both the US and Russian space programs have been incorporated into the habitability plan for the International Space Station. The focus is on preventive measures developed for spacecraft, cargo, and crews. On-orbit regular housekeeping practices complete with visual inspections are essential, along with microbiological monitoring. Risks associated with extended stays on the Moon or a Mars exploration mission will be much greater than previous experiences because of additional unknown variables. The current knowledge base is insufficient for exploration missions, and research is essential to understand the effects of space flight on biological functions and population dynamics of microorganisms in spacecraft. Equally important is a better understanding of the immune response and of human-microorganism-environment interactions during long-term space habitation.
Microbiology of water management subsystem for manned space flight, discussing sterilization by heat and tests inside Integrated Life Support System /ILSS/
Current microbiological parameters specified to verify microbiological quality of Space Station Freedom water quality include the enumeration of total bacteria, anaerobes, aerobes, yeasts and molds, enteric bacteria, gram positives, gram negatives, and E. coli. In addition, other parameters have been identified as necessary to support the Water Recovery Test activities to be conducted at the NASA/MSFC later this year. These other parameters include aerotolerant eutrophic mesophiles, legionellae, and an additional method for heterotrophic bacteria. If inter-laboratory data are to be compared to evaluate quality, analytical methods must be eliminated as a variable. Therefore, each participating laboratory must utilize the same analytical methods and procedures. Without this standardization, data can be neither compared nor validated between laboratories. Multiple laboratory participation represents a conservative approach to insure quality and completeness of data. Invariably, sample loss will occur in transport and analyses. Natural variance is a reality on any test of this magnitude and is further enhanced because biological entities, capable of growth and death, are specific parameters of interest. The large variation due to the participation of human test subjects has been noted with previous testing. The resultant data might be dismissed as 'out of control' unless intra-laboratory control is included as part of the method or if participating laboratories are not available for verification. The purpose of this document is to provide standardized laboratory procedures for the enumeration of certain microorganisms in water and wastewater specific to the water recovery systems test. The document consists of ten separate cultural methods and one direct count procedure. It is not intended nor is it implied to be a complete microbiological methods manual.
Chemical/physical and microbiological characteristics are studied of typical bath and laundry waters collected during a 12 day test in which the untreated waste waters were reused for toilet flush. Most significant changes were found for ammonia, color, methylene blue active substances, phosphates, sodium, sulfates, total organic carbon, total solids, and turbidity in comparison with tap water baseline. The mean total number of microorganisms detected in the waste waters ranged from 1 million to 10 to the 7th power cells/m1 and the mean number of possible coliforms ranged from 10 to the 5th power to 1 million. An accumulation of particulates and an objectible odor were detected in the tankage used during the 12 day reuse of the untreated waste waters. The combined bath and laundry waste waters from a family of four provided 91 percent of the toilet flush water for the same family.
The Apollo-Soyuz Test Project procedures for potable water system servicing and the results of preflight and postflight chemical and microbiological analyses of the water are discussed. Tables show results of the analyses. The effectiveness of the water system is evaluated.
Magnetite (Fe3O4) is an iron oxide of mixed oxidation state [Fe(II), Fe(III)] that contributes largely to geomagnetism and plays a significant role in diagenesis in marine and freshwater sediments. Magnetic data are the primary evidence for ocean floor spreading and accurate interpretation of the sedimentary magnetic record depends on an understanding of the conditions under which magnetite is stable. Though chemical reduction of magnetite by dissolved sulfide is well known, biological reduction has not been considered likely based upon thermodynamic considerations. This study shows that marine and freshwater strains of the bacterium Shewanella putrefaciens are capable of the rapid dissolution and reduction of magnetite, converting millimolar amounts to soluble Fe(II)in a few days at room temperature. Conditions under which magnetite reduction is optimal (pH 5-6, 22-37 degrees C) are consistent with an enzymatic process and not with simple chemical reduction. Magnetite reduction requires viable cells and cell contact, and it appears to be coupled to electron transport and growth. In a minimal medium with formate or lactate as the electron donor, more than 10 times the amount of magnetite was reduced over no carbon controls. These data suggest that magnetite reduction is coupled to carbon metabolism in S. putrefaciens. Bacterial reduction rates of magnetite are of the same order of magnitude as those estimated for reduction by sulfide. If such remobilization of magnetite occurs in nature, it could have a major impact on sediment magnetism and diagenesis.
The trophic importance of bacterioplankton as a source of carbon and sulfur nutrition for consumers in meromictic lakes was tested using stable carbon (delta 13C) and sulfur (delta 34S) isotopic measurements. Studies in three lakes near Syracuse, New York, showed that most consumers ultimately derive their C and S nutrition from a mixture of terrestrial detritus, phytoplankton, and littoral vegetation, rather than from bacterioplankton. Food webs in these meromictic lakes are thus similar to those in other lakes that lack dense populations of bacterioplankton.
A number of microbiological issues are of critical importance to crew health and system performance in spacecraft water systems. This presentation reviews an army of these concerns which include factors that influence water treatment and disinfection in spaceflight such as biofilm formation and the physiological responses of bacteria in clean water systems. Factors associated with spaceflight like aerosol formation under conditions of microgravity are also discussed within the context of airborne infections such as Legionellosis. Finally, a spectrum of analytical approaches is reviewed to provide an evaluation of methodological alternatives that have been suggested or used to detect microorganisms of interest in water systems. These range from classical approaches employing colony formation on specific microbiological growth media to direct (i.e. microscopic) and indirect (e.g. electrochemical) methods as well as the use of molecular approaches and gene probes. These techniques are critically evaluated for their potential utility in determining microbiological water quality through the detection of microorganisms under the influence of ambient environmental stress inherent in spaceflight water systems.
Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.
Qualitative and quantitative laboratory results are important to the decision-making process. In some cases, they may represent the only basis for deciding between two or more given options or processes. Therefore, it is essential that handling of laboratory samples and analytical operations employed are performed at a deliberate level of conscientious effort. Reporting erroneous results can lead to faulty interpretations and result in misinformed decisions. This document provides analytical control specifications which will govern future test procedures related to all Water Recovery Test (WRT) Phase 3 activities to be conducted at the National Aeronautics and Space Administration/Marshall Space Flight Center (NASA/MSFC). This document addresses the process which will be used to verify analytical data generated throughout the test period, and to identify responsibilities of key personnel and participating laboratories, the chains of communication to be followed, and ensure that approved methodology and procedures are used during WRT activities. This document does not outline specifics, but provides a minimum guideline by which sampling protocols, analysis methodologies, test site operations, and laboratory operations should be developed.
The growth of bacteria by dissimilatory elemental sulfur reduction is generally associated with obligate anaerobes and thermophiles in particular. Here we describe the sulfur-dependent growth of the facultatively anaerobic mesophile Shewanella putrefaciens. Six of nine representative S. putrefaciens isolates from a variety of environments proved able to grow by sulfur reduction, and strain MR-1 was chosen for further study. Growth was monitored in a minimal medium (usually with 0.05% Casamino Acids added as a growth stimulant) containing 30 mM lactate and limiting concentrations of elemental sulfur. When mechanisms were provided for the removal of the metabolic end product, H2S, measurable growth was obtained at sulfur concentrations of from 2 to 30 mM. Initial doubling times were ca. 1.5 h and substrate independent over the range of sulfur concentrations tested. In the cultures with the highest sulfur concentrations, cell numbers increased by greater than 400-fold after 48 h, reaching a maximum density of 6.8 x 10(8) cells ml-1. Yields were determined as total cell carbon and ranged from 1.7 to 5.9 g of C mol of S(0) consumed-1 in the presence of the amino acid supplement and from 0.9 to 3.4 g of C mol of S(0-1) in its absence. Several lines of evidence indicate that cell-to-sulfur contact is not required for growth. Approaches for the culture of sulfur-metabolizing bacteria and potential ecological implications of sulfur reduction in Shewanella-like heterotrophs are discussed.
Who We Are: A staff of approximately 14 BS, MS, and PhD‐Level Engineers and Scientists with experience in Aerospace, Civil, Environmental, and Mechanical Engineering, Chemistry, Physical Science and Water Pollution Microbiology. Our Primary Objective: To develop the next generation water recovery system technologies that will support NASA's long duration missions beyond low‐earth orbit.
Crystalline salt is generally considered so hostile to most forms of life that it has been used for centuries as a preservative. Here, we present evidence that prokaryotes inhabiting a natural evaporite crust of halite and gypsum are metabolically active while inside the evaporite for at least 10 months. In situ measurements demonstrated that some of these "endoevaporitic" microorganisms (probably the cyanobacterium Synechococcus Nageli) fixed carbon and nitrogen. Denitrification was not observed. Our results quantified the slow microbial activity that can occur in salt crystals. Implications of this study include the possibility that microorganisms found in ancient evaporite deposits may have been part of an evaporite community.
The goal of this study was to characterize sessile and planktonic microbiota that developed during two successive hydroponic cultures of lettuce in a closed chamber system. Coupons of polyvinyl chloride (PVC) placed in the nutrient solution lines were removed periodically, as were samples of the nutrient solutions and condensate from the air-handling system. The bacteria and fungi present on the coupons and in fluid samples were enumerated by direct plate counts. Disinfecting the hydroponic system with 0.1% hypochlorite and 0.1 N nitric acid reduced the bacterial densities in biofilm samples from 1 x 10 7 CFU/10 cm 2 to 1 x 10 1 CFU/10 cm 2 and eliminated culturable fungi; Staphylococcus sp., Pseudomonas sp., and Micrococcus sp. survived this procedure. Bacterial and fungal concentrations in all samples returned to predisinfection levels after 2 days of plant growth. Pseudomonas and Acremonium predominated both before and after disinfection. Fungal concentrations never exceeded 7 x 10 2 CFU/10 cm 2 . The coupon microbiota differed from that of the rhizoplane at harvest. Overall, the greatest numbers of species were found on the rhizoplane samples collected during the second crop. The microbial community changed little during individual crops or between successive crops. Diversity indices remained relatively constant for all samples.
The complex polar lipids of the hot spring cyanobacterial mat in the 50 to 55 degrees C region of Octopus Spring, Yellowstone National Park, and of thermophilic bacteria cultivated from this or similar habitats, were compared in an attempt to understand the microbial sources of the major lipid biomarkers in this community. Intact complex lipids were analyzed directly by fast atom bombardment mass spectrometry (FAB-MS), two-dimensional thin-layer chromatography (TLC), and combined TLC-FAB-MS. FAB-MS and TLC gave qualitatively similar results, suggesting that the mat contains major lipids most like those of the cyanobacterial isolate we studied, Synechococcus sp. strain Y-7c-s. These include monoglycosyl, diglycosyl, and sulfoquinosovyl diglycerides (MG, DG, and SQ, respectively) and phosphatidyl glycerol (PG). Though Chloroflexus aurantiacus also contains MG, DG, and PG, the fatty acid chain lengths of mat MGs, DGs, and PGs resemble more those of cyanobacterial than green nonsulfur bacterial lipids. FAB-MS spectra of the lipids of nonphototrophic bacterial isolates were distinctively different from those of the mat and phototrophic isolates. The lipids of these nonphototrophic isolates were not detected in the mat, but most could be detected when added to mat samples. The mat also contains major glycolipids and aminophospholipids of unknown structure and origin. FAB-MS and TLC did not always give quantitatively similar results. In particular, PG and SQ may give disproportionately high FAB-MS responses.
This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.