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During the Spacelab mission D-2, in the experiment RD-UVRAD, precalibrated biofilms consisting of dry monolayers of immobilised spores of Bacillus subtilis (strain Marburg) were exposed, for defined intervals, to extraterrestrial solar radiation filtered through an optical filtering system, to simulate different ozone column thicknesses. After the mission, the biofilms were processed and optical densities indicative of any biological activity were determined for each exposure condition by image analysis. For the different simulated ozone column thicknesses, biologically effective irradiances were experimentally determined from the biofilm data and compared with calculated data using a radiative transfer model and the known biofilm action spectrum. The data show a strong increase in biologically effective solar UV irradiance with decreasing (simulated) ozone concentrations. The full spectrum of extraterrestrial solar radiation leads to an increment of the biologically effective irradiance by nearly three orders of magnitude compared with the solar spectrum at the surface of the Earth for average total ozone columns.

In order to understand radiation mechanisms of heavy ions in detail, it is necessary to study effects of single ions on individual biological test objects. Spores of Bacillus subtilis have been used as a suitable small biological test system to measure the inactivation in dependence on the radial distance to the tracks of charged particles. Accelerator experiments have been performed using a modified Biostack technique--biological objects sandwiched between nuclear track detectors. Results of these experiments using ions differing in their energy and atomic number will be discussed under following aspects: (i) methodological differences between the experiments and their possible influences on the results, (ii) common features which are independent on the particle type and energy, (iii) theoretical expectations and problems to find solid theoretical concepts which explain the results.

For radiobiological experiments in space, designed to investigate biological effects of the heavy ions of the cosmic radiation field, a mandatory requirement is the possibility to spatially correlate the observed biological response of individual test organisms to the passage of single heavy ions. Among several undertakings towards this goal, the BIOSTACK experiments in the Apollo missions achieved the highest precision and therefore the most detailed information on this question. Spores of Bacillus subtilis as a highly radiation resistant and microscopically small test organism yielded these quantitative results. This paper will focus on experimental and procedural details, which must be included for an interpretation and a discussion of these findings in comparison to control experiments with accelerated heavy ions.

Using spores of two Bacillus subtilis strains differing in repair capacity, we have studied repair and mutation induction in the spores after irradiation with very heavy ions up to uranium with specific particle energies up to 18.6 MeV/u. The results indicate that repair and mutation induction after heavy ion irradiation are closely related to each other and that both phenomena strongly depend on the atomic number and specific energy of the ions. The effects are discussed in comparison with results obtained after X-irradiation.

The evaluation of the Bacillus subtilis experiment has been completed. The biological and the physical results for this part of the Apollo-Soyuz Test Project (ASTP) Biostack experiment are given. This comprises dosimetric data for the cosmic radiation at that orbit as well as biological findings from two types of plastic detectors. Further, the frequency distributions of the physical quantities atomic number, energy and energy loss of the heavy ions within the sample of spores hit are presented. The biological hazard presented by cosmic HZE-particles has been much underestimated.

Mutation induction (resistance to sodium azide) in spores of Bacillus subtilis was investigated after irradiation with heavy ions from Neon to Uranium with specific particle energies between 0.17 and 18.6 MeV/u. A strong dependence of the mutation induction cross section on particle charge and energy was observed. From the results it was concluded that mutation induction in bacterial spores by very heavy ions is mainly caused by secondary electrons.

The effectiveness of cosmic HZE-particles on unicellular procaryotic, organisms was studied on Bacillus subtilis spores, which were accommodated in the Biostack I and II experiments on board Apollo 16 and 17. Identification of the spores that were hit was achieved by using the Biostack sandwich construction and by precise microscopical measurements of tracks of particles. Germination, outgrowth and the rate of cellular elongation were investigated. A method was developed to determine the charge of each individual HZE particle that penetrated a spore and its energy loss in the region of hit. An attempt was made to establish a connection between these physical characteristics and the biological effects produced.

In order to check the results of earlier Biostack experiments, new experimental techniques were developed for the Biostack III experiment in the Apollo-Soyuz test project (ASTP). These techniques resulted in an increased accuracy of localization down to 0.2 micrometers for the determination of the impact parameter, accompanied by an increase in the sample size available for biological investigation. In addition, colony forming ability, metabolic mutations, and mutations affecting UV- and x-ray sensitivity were rendered observable by these methods. The biological and physical results obtained so far from the evaluation of the Bacillus subtilis experiment within Biostack III confirm and extend the findings of the previous Biostack experiments. They also add to the questions about the mechanisms of action of the radiation field under investigation, since the observed effects cannot be interpreted in terms of standard concepts.

The understanding of the radiobiological action of heavy ions requires the knowledge of the dependence of the inactivation probability on the distance between the particle's trajectory and the biological test organism (the impact parameter). Spores of Bacillus subtilis with a cytoplasmic core of about 0.22 micrometer cross section are suitable test objects for the study of this radial inactivation probability in its microscopic details. The spores are irradiated at low fluences of some 10(6) ions/cm2 with very heavy ions at different specific energies up to 10 MeV per atomic mass unit u while in fixed contact with visual nuclear track detectors. The methods are described by which the biological response of individual cells can be evaluated and the impact parameter be determined with an accuracy typically better than 0.2 micrometer. The results demonstrate that the common characteristics of inactivation, e.g., an effective range of inactivation extending to at least 3 micrometers, a nonmonotonic dependence of the inactivation probabilities on the radial distance, and the fact that the inactivation probability even for direct central hits on the cytoplasmic core is substantially below one, are nearly independent of the particle energy and type. The results are incompatible with the assumption that the radiobiological effectiveness can be attributed to the dose of secondary electrons as currently understood. They also demonstrate that the widely held notion of an "overkill" at low impact parameters does not apply for the spores even with the most densely ionizing ions.

Bacillus subtilis spores were flown in the Biostack experiment aboard the Apollo 16 command module. The spores embedded in plastic foils were stacked between physical track detectors. The energy loss spectrum of the heavy particles of cosmic radiation was determined. Biological studies were restricted to the high-energy loss component of these particles. Spores that had received single hits whose positions were determined with a typical accuracy of +/- 1 micrometers, were investigated for radiation effects on germination and outgrowth. It was found that germination was not influenced by a hit by an HZE particle, but outgrowth was reduced significantly.

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.

The object of the Biostack experiment is to study the biological effects of high ZE particles of cosmic radiation in order to obtain information on the mechanism of these particles in biological matter. For this purpose individual local evaluation methods have been developed which allow one to identify each biologically effective particle and to correlate the individual hitting particle with the biological effect produced. The Biostack experimental package contains a series of monolayers of selected biological objects (Bacillus subtilis spores, Arabidopsis thaliana seeds, Vicia faba radiculae, Artemia salina eggs) with each layer sandwiched between several different cosmic ion track detectors (nuclear emulsions, cellulose nitrate, polycarbonate). By this arrangement a variety of biological effects due to a single penetrating particle can be analysed. Influence on cellular and tissue development, nuclear damages, and mutation induction are the main investigated effects. These space flight findings will be completed by results of balloon flight and accelerator experiments.

The effect of vacuum on bacterial cells is related to water desorption. Below water vapour pressure the inactivation remains constant, independent of total pressure and exposure time. In subsequent growth, the lag-phase of the survivors is delayed. Combined treatment with vacuum and radiation (X-rays or uv of 254 nm wavelength) results in synergistic effects, whereas vacuum and heat can act antagonistically. The vacuum inactivated cells (indicated as loss of colony-forming ability) are completely damaged. They do not show cellular elongation, phage production or respiration. The cellular membrane becomes permeable by vacuum exposure: biomolecules are released from the cells when re-suspended after vacuum treatment.

To improve our understanding of Bacillus subtilis growth and biofilm formation under different environmental conditions, two versions of a microfluidic reactor with two channels separated by a polydimethylsiloxane (PDMS) membrane were developed. The gas phase was introduced into the channel above the membrane, and oxygen transfer from the gas phase through the membrane was assessed by measuring the dissolved oxygen concentration in the liquid phase using a miniaturized optical sensor and oxygen-sensitive nanoparticles. B. subtilis biofilm formation was monitored in the growth channels of the microbioreactors, which were designed in two shapes: one with circular extensions and one without. The volumes of these microbioreactors were (17 ± 4) μL for the reactors without extensions and (28 ± 4) μL for those with extensions. The effect of microbioreactor geometry and aeration on B. subtilis biofilm growth was evaluated by digital image analysis. In both microbioreactor geometries, stable B. subtilis biofilm formation was achieved after 72 h of incubation at a growth medium flow rate of 1 μL/min. The amount of oxygen significantly influenced biofilm formation. When the culture was cultivated with a continuous air supply, biofilm surface coverage and biomass concentration were higher than in cultivations without aeration or with a 100% oxygen supply. The channel geometry with circular extensions did not lead to a higher total biomass in the microbioreactor compared to the geometry without extensions. [ABSTRACT FROM AUTHOR]

The coconut is an important economic crop in the Philippines which currently ranks as the world's second largest producer. This study characterised and identified endophytes from coconut tissue culture in order to gain an initial understanding of their potential uses as sources of bioproducts. The isolates were evaluated using morphological, biochemical and molecular methods. Gram staining results revealed that four out of five bacteria isolated were Gram positive. Isolate CEB 1 fermented all three sugars in the Triple Sugar Iron Test while the other four did not. 16S rDNA gene fragments were amplified from genomic DNA using the universal primers 16F27 and 16R1542. The 16S rDNA sequence were found to be homologous to Bacillus subtilis and Pantoea dispersa. Phylogenetic analyses showed significant clustering of bacterial isolates together with archived DNA of B. subtilis and P. dispersa. All isolated bacteria matched the characteristics of their molecular homologies. Isolate CEB 5, identified as B. subtilis, produced red pigments which are possibly pulcherrimin. Literature reports that pulcherrimin possesses antimicrobial activity against yeast species, microscopic fungi, and postharvest pathogens. P. dispera, on the other hand, has been reported to convert insoluble phosphorus into soluble form to enable plants to take up more phosphorus. Determination of the bioactivities of endophytes reported in this study may enable the discovery of novel bioproducts. [ABSTRACT FROM AUTHOR]

In a Mars exploration scenario, knowing if and how highly resistant Bacillus subtilis spores would survive on the Martian surface is crucial to design planetary protection measures and avoid false positives in life-detection experiments. Therefore, in this study a systematic screening was performed to determine whether B. subtilis spores could survive an average day on Mars. For that, spores from two comprehensive sets of isogenic B. subtilis mutant strains, defective in DNA protection or repair genes, were exposed to 24 h of simulated Martian atmospheric environment with or without 8 h of Martian UV radiation [M(+)UV and M(-)UV, respectively]. When exposed to M(+)UV, spore survival was dependent on: (1) core dehydration maintenance, (2) protection of DNA by α/β-type small acid soluble proteins (SASP), and (3) removal and repair of the major UV photoproduct (SP) in spore DNA. In turn, when exposed to M(-)UV, spore survival was mainly dependent on protection by the multilayered spore coat, and DNA double-strand breaks represent the main lesion accumulated. Bacillus subtilis spores were able to survive for at least a limited time in a simulated Martian environment, both with or without solar UV radiation. Moreover, M(-)UV-treated spores exhibited survival rates significantly higher than the M(+)UV-treated spores. This suggests that on a real Martian surface, radiation shielding of spores (e.g., by dust, rocks, or spacecraft surface irregularities) might significantly extend survival rates. Mutagenesis were strongly dependent on the functionality of all structural components with small acid-soluble spore proteins, coat layers and dipicolinic acid as key protectants and efficiency DNA damage removal by AP endonucleases (ExoA and Nfo), non-homologous end joining (NHEJ), mismatch repair (MMR) and error-prone translesion synthesis (TLS). Thus, future efforts should focus on: (1) determining the DNA damage in wild-type spores exposed to M(+/-)UV and (2) assessing spore survival and viability with shielding of spores via Mars regolith and other relevant materials. [ABSTRACT FROM AUTHOR]

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The ability to form endospores allows certain Gram-positive bacteria (e.g. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Despite thorough study of spore resistance to external stresses, precisely how long B. subtilis spores can lie dormant while remaining viable, a period that potentially far exceeds the human lifespan; is not known although convincing examples of long term spore survival have been recorded. In this study, we report the first data from a 500-year microbial experiment, which started in 2014 and will finish in 2514. A set of vials containing a defined concentration of desiccated B. subtilis spores is opened and tested for viability every two years for the first 24 years and then every 25 years until experiment completion. Desiccated baseline spore samples were also exposed to environmental stresses, including X-rays, 254 nm UV-C, 10% H2O2, dry heat (120°C) and wet heat (100°C) to investigate how desiccated spores respond to harsh environmental conditions after long periods of storage. Data from the first 2 years of storage show no significant decrease in spore viability. Additionally, spores of B. subtilis were subjected to various short-term storage experiments, revealing that space-like vacuum and high NaCl concentration negatively affected spore viability. [ABSTRACT FROM AUTHOR]

Vegetative wild-type and DNA repair-deficient (homologous recombination, recA and nucleotide excision repair, uvrB) Bacillus subtilis cells were exposed to UV-C radiation. Colony formation, DNA bipyrimidine photoproducts and gene expression were measured during cell recovery. Gene expression was measured after 60 min cell recovery where 50% (wild-type), 30% ( recA) and 8% ( uvrB), respectively, of the UV-C induced DNA photoproducts were repaired. We examined changes in the gene expression following UV exposure in wild-type and both repair-deficient strains. A set of known and unknown genes were found to be significantly up-regulated in wild-type B. subtilis cells, whereas no or lower gene induction was determined for both mutant strains. In addition, the possible roles of newly identified UV-responsive genes are discussed with respect to cellular recovery following exposure to UV irradiation. [ABSTRACT FROM AUTHOR]