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At least 109 records · Page 6

Microbial Characterization of Free Floating Condensate Aboard the Mir Space Station

Three samples of humidity condensate that had accumulated behind panels aboard the Russian space station Mir were collected and returned to earth for analysis. As these floating masses of liquid come into contact with the astronauts and the engineering systems, they have the potential to affect both crew health and systems performance. Using a combination of culturing techniques, a wide variety of organisms were isolated included Escherichia coli, Serratia marcescens, and a presumed Legionella species. In addition, microscopic analysis indicated the presence of protozoa, dust mites, and spirochetes. These findings suggest the need for more comprehensive microbial analysis of the environment through the use of new methodologies to allow a more thorough risk assessment of spacecraft. Copyright 2004 Springer-Verlag.

Spacecraft↗

Characterization of exopolymers of aquatic bacteria by pyrolysis-mass spectrometry

Exopolymers from a diverse collection of marine and freshwater bacteria were characterized by pyrolysis-mass spectrometry (Py-MS). Py-MS provides spectra of pyrolysis fragments that are characteristic of the original material. Analysis of the spectra by multivariate statistical techniques (principal component and canonical variate analysis) separated these exopolymers into distinct groups. Py-MS clearly distinguished characteristic fragments, which may be derived from components responsible for functional differences between polymers. The importance of these distinctions and the relevance of pyrolysis information to exopolysaccharide function in aquatic bacteria is discussed.

NASA Program CELSS↗

Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components

Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydrofluoric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.

Ribitol/analogs & derivatives↗

Extreme Ionizing-Radiation-Resistant Bacterium

There is a growing concern that desiccation and extreme radiation-resistant, non-spore-forming microorganisms associated with spacecraft surfaces can withstand space environmental conditions and subsequent proliferation on another solar body. Such forward contamination would jeopardize future life detection or sample return technologies. The prime focus of NASA s planetary protection efforts is the development of strategies for inactivating resistance-bearing micro-organisms. Eradi cation techniques can be designed to target resistance-conferring microbial populations by first identifying and understanding their physiologic and biochemical capabilities that confers its elevated tolerance (as is being studied in Deinococcus phoenicis, as a result of this description). Furthermore, hospitals, food, and government agencies frequently use biological indicators to ensure the efficacy of a wide range of radiation-based sterilization processes. Due to their resistance to a variety of perturbations, the nonspore forming D. phoenicis may be a more appropriate biological indicator than those currently in use. The high flux of cosmic rays during space travel and onto the unshielded surface of Mars poses a significant hazard to the survival of microbial life. Thus, radiation-resistant microorganisms are of particular concern that can survive extreme radiation, desiccation, and low temperatures experienced during space travel. Spore-forming bacteria, a common inhabitant of spacecraft assembly facilities, are known to tolerate these extreme conditions. Since the Viking era, spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Members of the non-sporeforming bacterial community such as Deinococcus radiodurans can survive acute exposures to ionizing radiation (5 kGy), ultraviolet light (1 kJ/m2), and desiccation (years). These resistive phenotypes of Deinococcus enhance the potential for transfer, and subsequent proliferation, on another solar body such as Mars and Europa. These organisms are more likely to escape planetary protection assays, which only take into account presence of spores. Hence, presences of extreme radiation-resistant Deinococcus in the cleanroom facility where spacecraft are assembled pose a serious risk for integrity of life-detection missions. The microorganism described herein was isolated from the surfaces of the cleanroom facility in which the Phoenix Lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. This bacterium exhibits very low 16SrRNA similarity with any other environmental isolate reported to date. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Deinococcus and represents a novel species. The name Deinococcus phoenicis was proposed after the Phoenix spacecraft, which was undergoing assembly, testing, and launch operations in the spacecraft assembly facility at the time of isolation. D. phoenicis cells exhibited higher resistance to ionizing radiation (cobalt-60; 14 kGy) than the cells of the D. radiodurans (5 kGy). Thus, it is in the best interest of NASA to thoroughly characterize this organism, which will further assess in determining the potential for forward contamination. Upon the completion of genetic and physiological characteristics of D. phoenicis, it will be added to a planetary protection database to be able to further model and predict the probability of forward contamination.

Vaishampayan, Parag A.↗

Extreme Ionizing-Radiation-Resistant Bacterium

There is a growing concern that desiccation and extreme radiation-resistant, non-spore-forming microorganisms associated with spacecraft surfaces can withstand space environmental conditions and subsequent proliferation on another solar body. Such forward contamination would jeopardize future life detection or sample return technologies. The prime focus of NASA s planetary protection efforts is the development of strategies for inactivating resistance-bearing microorganisms. Eradification techniques can be designed to target resistance-conferring microbial populations by first identifying and understanding their physiologic and biochemical capabilities that confers its elevated tolerance (as is being studied in Deinococcus phoenicis, as a result of this description). Furthermore, hospitals, food, and government agencies frequently use biological indicators to ensure the efficacy of a wide range of radiation- based sterilization processes. Due to their resistance to a variety of perturbations, the non-spore forming D. phoenicis may be a more appropriate biological indicator than those currently in use. The high flux of cosmic rays during space travel and onto the unshielded surface of Mars poses a significant hazard to the survival of microbial life. Thus, radiation-resistant microorganisms are of particular concern that can survive extreme radiation, desiccation, and low temperatures experienced during space travel. Spore-forming bacteria, a common inhabitant of spacecraft assembly facilities, are known to tolerate these extreme conditions. Since the Viking era, spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Members of the non-spore-forming bacterial community such as Deinococcus radiodurans can survive acute exposures to ionizing radiation (5 kGy), ultraviolet light (1 kJ/sq m), and desiccation (years). These resistive phenotypes of Deinococcus enhance the potential for transfer, and subsequent proliferation, on another solar body such as Mars and Europa. These organisms are more likely to escape planetary protection assays, which only take into account presence of spores. Hence, presences of extreme radiation-resistant Deinococcus in the cleanroom facility where spacecraft are assembled pose a serious risk for integrity of life-detection missions. The microorganism described herein was isolated from the surfaces of the cleanroom facility in which the Phoenix Lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. This bacterium exhibits very low 16SrRNA similarity with any other environmental isolate reported to date. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Deinococcus and represents a novel species. The name Deinococcus phoenicis was proposed after the Phoenix spacecraft, which was undergoing assembly, testing, and launch operations in the spacecraft assembly facility at the time of isolation. D. phoenicis cells exhibited higher resistance to ionizing radiation (cobalt-60; 14 kGy) than the cells of the D. radiodurans (5 kGy). Thus, it is in the best interest of NASA to thoroughly characterize this organism, which will further assess in determining the potential for forward contamination. Upon the completion of genetic and physiological characteristics of D. phoenicis, it will be added to a planetary protection database to be able to further model and predict the probability of forward contamination.

Vaishampayan, Parag A.↗

Microbial biomass and productivity in seagrass beds

Different methods for measuring the rates of processes mediated by bacteria in sediments and the rates of bacterial cell production have been compared. In addition, net production of the seagrass Zostera capricorni and bacterial production have been compared and some interrelationships with the nitrogen cycle discussed. Seagrass productivity was estimated by measuring the plastochrone interval using a leaf stapling technique. The average productivity over four seasons was 1.28 +/- 0.28 g C m-2 day-1 (mean +/- standard deviation, n = 4). Bacterial productivity was measured five times throughout a year using the rate of tritiated thymidine incorporated into DNA. Average values were 33 +/- 12 mg C m-2 day-1 for sediment and 23 +/- 4 for water column (n = 5). Spatial variability between samples was greater than seasonal variation for both seagrass productivity and bacterial productivity. On one occasion, bacterial productivity was measured using the rate of 32P incorporated into phospholipid. The values were comparable to those obtained with tritiated thymidine. The rate of sulfate reduction was 10 mmol SO4(-2) m-2 day-1. The rate of methanogenesis was low, being 5.6 mg CH4 produced m-2 day-1. A comparison of C flux measured using rates of sulfate reduction and DNA synthesis indicated that anaerobic processes were predominant in these sediments. An analysis of microbial biomass and community structure, using techniques of phospholipid analysis, showed that bacteria were predominant members of the microbial biomass and that of these, strictly anaerobic bacteria were the main components. Ammonia concentration in interstitial water varied from 23 to 71 micromoles. Estimates of the amount of ammonia required by seagrass showed that the ammonia would turn over about once per day. Rapid recycling of nitrogen by bacteria and bacterial grazers is probably important.

NASA Program CELSS↗

Evidence that elevated CO2 levels can indirectly increase rhizosphere denitrifier activity

We examined the influence of elevated CO2 concentration on denitrifier enzyme activity in wheat rhizoplanes by using controlled environments and solution culture techniques. Potential denitrification activity was from 3 to 24 times higher on roots that were grown under an elevated CO2 concentration of 1,000 micromoles of CO2 mol-1 than on roots grown under ambient levels of CO2. Nitrogen loss, as determined by a nitrogen mass balance, increased with elevated CO2 levels in the shoot environment and with a high NO3- concentration in the rooting zone. These results indicated that aerial CO2 concentration can play a role in rhizosphere denitrifier activity.

NASA Discipline Life Support Systems↗

Clean is not Sterile: A Planetary Science and Planetary Protection Perspective on Cleanroom Microbiology at NASA

The Astromaterials Acquisition and Curation Office at NASA is responsible for the curation of extraterrestrial samples from NASA’s past and future sample return missions. Our office curates samples from the moon, meteorites, comets, asteroids, cosmic dust and solar wind particles. All these samples are kept in cleanrooms to limit particulate and trace metal contamination, but none of these cleanrooms are specifically designed to control microbial contamination. During the early Apollo missions NASA scientists were very concerned with protecting the Earth from potential microbial contamination from the moon and with protecting the lunar samples from terrestrial microbes. NASA developed specialized equipment and clean rooms to keep these collections pristine. However, as we learned more about the lunar environment our concerns about microbial contamination lessened. Today none of the existing collections have microbial contamination requirements because they are not considered susceptible to microbial alteration under curation conditions (e.g. solar wind samples, and lunar samples) or have already been contaminated by terrestrial biology (meteorites collected in Antarctica). However, NASA’s OSIRIS-REx mission will land in 2023 with samples from a carbon rich asteroid that will be susceptible to microbial alteration. The Perseverance rover on Mars will begin to collect and cache samples that will be returned to Earth as soon as 2031. Martian samples may contain signs of extraterrestrial life and will have to be treated like the early Apollo samples. Martian samples will be isolated to protect the Earth, and must also be protected from terrestrial contamination. I will present microbial monitoring data from existing NASA cleanrooms and discuss how NASA is planning to use techniques from the pharmaceutical industry and academia to design new laboratories and equipment that will protect astromaterials and the earth from unwanted microbial contamination. I will also discuss a project to sample the external microbiome of the International Space Station. Results from this research will be used to design facilities for use on Mars that limit the amount of contamination associated with crewed missions.

Aaron B Regberg↗

New method for estimating bacterial cell abundances in natural samples by use of sublimation

We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

Evaluation Studies↗

Molecular microbial diversity of a spacecraft assembly facility

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.

Industry↗

CDC Bioreactor Configuration Method for Volume Level Control with Controlled Inlet and Outlet Flow

Environmental Control Life Support Systems and other microbiological biofilm studies often utilize small scale bioreactors. Among these options are the popular BioSurface CDC bioreactors, currently being utilized by Marshall Space Flight Center groups researching the impact of biofilms on life support systems. After a recent experimental regime, it was determined additional equipment could be added to augment the capabilities of the bioreactors. Previous research configurations such as ASTM E3161-21 relied on an outlet stream gravity draining from a side drain port located above sampling coupons. This limited applications to experimental conditions with a controlled inlet and uncontrolled outlet flow. With the introduction of a small single pole single throw (SPST) reed float switch and a peristaltic pump connected to the outlet drain and a chassis controller, the bioreactor is able to maintain a set level. The modification allows additional variables to be tested, including highly adjustable fill and flush cycles, bioreactor volume, draining and filling control. Once the configuration modifications were implemented through the installation of the new equipment, data was collected to ensure the stability of the level measurements. As the level control switch is a float switch, consideration was taken into account for effects of internal stirring speed, along with effects of inlet and outlet flow rate. Data presented in this study will illustrate the stability and effectiveness of the configuration changes in equipment made to the bioreactor. These configuration changes have demonstrated control method to conduct biofilm mitigation techniques for ECLSS hardware research. The controlled level capabilities allow for a constant drip feed flow rate into the bioreactors, a key aspect of the biofilm mitigation testing.

CDC Bioreactor↗

CDC Bioreactor Configuration Method for Volume Level Control with Controlled Inlet and Outlet Flow

Environmental Control Life Support Systems and other microbiological biofilm studies often utilize small scale bioreactors. Among these options are the popular BioSurface CDC bioreactors, currently being utilized by Marshall Space Flight Center groups researching the impact of biofilms on life support systems. After a recent experimental regime, it was determined additional equipment could be added to augment the capabilities of the bioreactors. Previous research configurations such as ASTM E3161-21 relied on an outlet stream gravity draining from a side drain port located above sampling coupons. This limited applications to experimental conditions with a controlled inlet and uncontrolled outlet flow. With the introduction of a small single pole single throw (SPST) reed float switch and a peristaltic pump connected to the outlet drain and a chassis controller, the bioreactor is able to maintain a set level. The modification allows additional variables to be tested, including highly adjustable fill and flush cycles, bioreactor volume, draining and filling control. Once the configuration modifications were implemented through the installation of the new equipment, data was collected to ensure the stability of the level measurements. As the level control switch is a float switch, consideration was taken into account for effects of internal stirring speed, along with effects of inlet and outlet flow rate. Data presented in this study will illustrate the stability and effectiveness of the configuration changes in equipment made to the bioreactor. These configuration changes have demonstrated control method to conduct biofilm mitigation techniques for ECLSS hardware research. The controlled level capabilities allow for a constant drip feed flow rate into the bioreactors, a key aspect of the biofilm mitigation testing.

CDC Bioreactor↗

CDC Bioreactor Configuration Method for Volume Level Control with Controlled Inlet and Outlet Flow

Environmental Control Life Support Systems and other microbiological biofilm studies often utilize small scale bioreactors. Among these options are the popular BioSurface CDC bioreactors, currently being utilized by Marshall Space Flight Center groups researching the impact of biofilms on life support systems. After a recent experimental regime, it was determined additional equipment could be added to augment the capabilities of the bioreactors. Previous research configurations such as ASTM E3161-21 relied on an outlet stream gravity draining from a side drain port located above sampling coupons. This limited applications to experimental conditions with a controlled inlet and uncontrolled outlet flow. With the introduction of a small single pole single throw (SPST) reed float switch and a peristaltic pump connected to the outlet drain and a chassis controller, the bioreactor is able to maintain a set level. The modification allows additional variables to be tested, including highly adjustable fill and flush cycles, bioreactor volume, draining and filling control. Once the configuration modifications were implemented through the installation of the new equipment, data was collected to ensure the stability of the level measurements. As the level control switch is a float switch, consideration was taken into account for effects of internal stirring speed, along with effects of inlet and outlet flow rate. Data presented in this study will illustrate the stability and effectiveness of the configuration changes in equipment made to the bioreactor. These configuration changes have demonstrated control method to conduct biofilm mitigation techniques for ECLSS hardware research. The controlled level capabilities allow for a constant drip feed flow rate into the bioreactors, a key aspect of the biofilm mitigation testing.

CDC Bioreactor↗

CDC Bioreactor Configuration Method for Volume Level Control with Controlled Inlet and Outlet Flow

Environmental Control Life Support Systems and other microbiological biofilm studies often utilize small scale bioreactors. Among these options are the popular BioSurface CDC bioreactors, currently being utilized by Marshall Space Flight Center groups researching the impact of biofilms on life support systems. After a recent experimental regime, it was determined additional equipment could be added to augment the capabilities of the bioreactors. Previous research configurations such as ASTM E3161 – 18 relied on an outlet stream gravity draining from a side drain port located above sampling coupons. This limited applications to experimental conditions with a controlled inlet and uncontrolled outlet flow. With the introduction of a small single pole single throw (SPST) reed float switch, a peristaltic pump connected to the outlet drain and a chassis controller, the bioreactor is able to maintain a set level. The modification allows additional variables to be tested, including highly adjustable fill and flush cycles, bioreactor volume, draining and filling control. Once the configuration modifications were implemented through the installation of the new equipment, data was collected to ensure the stability of the level measurements. As the level control switch is a float switch, consideration was taken into account for effects of internal stirring speed, along with effects of inlet and outlet flow rate. Data presented in this study will illustrate the stability and effectiveness of the configuration changes in equipment made to the bioreactor. These configuration changes are proving an effective control method to conduct biofilm mitigation techniques for ECLSS hardware research. The controlled level capabilities allow for a constant drip feed flow rate into the bioreactors, a key aspect of the biofilm mitigation testing.

CDC Bioreactor↗

Elucidating Microbial Adaptation Dynamics via Autonomous Exposure and Sampling

The adaptation of micro-organisms to their environments is a complex process of interaction between the pressures of the environment and of competition. Reducing this multifactorial process to environmental exposure in the laboratory is a common tool for elucidating individual mechanisms of evolution, such as mutation rates. Although such studies inform fundamental questions about the way adaptation and even speciation occur, they are often limited by labor-intensive manual techniques. Current methods for controlled study of microbial adaptation limit the length of time, the depth of collected data, and the breadth of applied environmental conditions. Small idiosyncrasies in manual techniques can have large effects on outcomes; for example, there are significant variations in induced radiation resistances following similar repeated exposure protocols. We describe here a project under development to allow rapid cycling of multiple types of microbial environmental exposure. The system allows continuous autonomous monitoring and data collection of both single species and sampled communities, independently and concurrently providing multiple types of controlled environmental pressure (temperature, radiation, chemical presence or absence, and so on) to a microbial community in dynamic response to the ecosystem's current status. When combined with DNA sequencing and extraction, such a controlled environment can cast light on microbial functional development, population dynamics, inter- and intra-species competition, and microbe-environment interaction. The project's goal is to allow rapid, repeatable iteration of studies of both natural and artificial microbial adaptation. As an example, the same system can be used both to increase the pH of a wet soil aliquot over time while periodically sampling it for genetic activity analysis, or to repeatedly expose a culture of bacteria to the presence of a toxic metal, automatically adjusting the level of toxicity based on the number or growth rate of surviving cells. We are on our second prototype iteration, with demonstrated functions of microbial growth monitoring and dynamic exposure to UV-C radiation and temperature. We plan to add functionality for general chemical presence or absence by Nov. 2013. By making the project low-cost and open-source, we hope to encourage others to use it as a basis for future development of a common microbial environmental adaptation testbed.

Microbiology↗

Method for determining stable isotope ratios of dissolved organic carbon in interstitial and other natural marine waters

A procedure is described for the analysis of the stable carbon isotopic composition of dissolved organic carbon (DOC) in natural waters from marine and higher-salinity environments. Rapid (less than 5 min) and complete oxidation of DOC is achieved using a modification of previous photochemical oxidation techniques. The CO2 evolved from DOC oxidation can be collected in less than 10 min for isotopic analysis. The procedure is at present suitable for oxidation and collection of 1-5 micromoles of carbon and has an associated blank of 0.1-0.2 micromole of carbon. Complete photochemical oxidation of DOC standards was demonstrated by quantitative recovery of CO2 as measured manometrically. Isotopic analyses of standards by photochemical and high-temperature sealed-tube combustion methods agreed to within 0.3%. Photochemical oxidation of DOC in a representative sediment pore-water sample was also quantitative, as shown by the excellent agreement between the photochemical and sealed-tube methods. The delta 13C values obtained for pore-water DOC using the two methods of oxidation were identical, suggesting that the modified photochemical method is adequate for the isotopically non-fractionated oxidation of pore-water DOC. The procedure was evaluated through an analysis of DOC in pond and pore waters from a hypersaline microbial mat environment. Concentrations of DOC in the water column over the mat displayed a diel pattern, but the isotopic composition of this DOC remained relatively constant (average delta 13C = -12.4%). Pore-water DOC exhibited a distinct concentration maximum in the mat surface layer, and delta 13C of pore-water DOC was nearly 8% lighter at 1.5-2.0-cm depth than in the mat surface layer (0-0.5-cm depth). These results demonstrate the effectiveness of the method in elucidating differences in DOC concentration and delta 13C over biogeochemically relevant spatial and temporal scales. Carbon isotopic analysis of DOC in natural waters, especially pore waters, should be a useful probe of biogeochemical processes in recent environments.

NASA Discipline Exobiology↗

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

NASA Discipline Number 04-10↗

Highly ordered vertical structure of Synechococcus populations within the one-millimeter-thick photic zone of a hot spring cyanobacterial mat

A variety of contemporary techniques were used to investigate the vertical distribution of thermophilic unicellular cyanobacteria, Synechococcus spp., and their activity within the upper 1-mm-thick photic zone of the mat community found in an alkaline siliceous hot spring in Yellowstone National Park in Wyoming. Detailed measurements were made over a diel cycle at a 61 degrees C site. Net oxygenic photosynthesis measured with oxygen microelectrodes was highest within the uppermost 100- to 200-microm-thick layer until midmorning, but as the day progressed, the peak of net activity shifted to deeper layers, stabilizing at a depth of 300 microm from midday throughout the afternoon. Examination of vertical thin sections by bright-field and autofluorescence microscopy revealed the existence of different populations of Synechococcus which form discrete bands at different vertical positions. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments from horizontal cryosections obtained at 100-microm-thick vertical intervals also suggested vertical stratification of cyanobacterial, green sulfur bacterium-like, and green nonsulfur bacterium-like populations. There was no evidence of diel migration. However, image analysis of vertical thin sections revealed the presence of a narrow band of rod-shaped Synechococcus cells in which the cells assumed an upright position. These upright cells, located 400 to 800 microm below the surface, were observed only in mat samples obtained around noon. In mat samples obtained at other time points, the cells were randomly oriented throughout the mat. These combined observations reveal the existence of a highly ordered structure within the very thin photic zone of this hot spring microbial mat, consisting of morphologically similar Synechococcus populations that are likely to be differentially adapted, some co-occurring with green sulfur bacterium-like populations, and all overlying green nonsulfur bacterium-like populations.

Non-NASA Center↗