Ultraviolet-C inactivation and hydrophobicity of Bacillus subtilis and Bacillus velezensis spores isolated from extended shelf-life milk.

Bacterial spores are important in food processing due to their ubiquity, resistance to high temperature and chemical inactivation. This work aims to study the effect of ultraviolet C (UVC) on the spores of Bacillus subtilis and Bacillus velezensis at a molecular and individual level to guide in deciding on the right parameters that must be applied during the processing of liquid foods. The spores were treated with UVC using phosphate buffer saline (PBS) as a suspension medium and their lethality rate was determined for each sample. Purified spore samples of B. velezensis and B. subtilis were treated under one pass in a UVC reactor to inactivate the spores. The resistance pattern of the spores to UVC treatment was determined using dipicolinic acid (Ca-DPA) band of spectral analysis obtained from Raman spectroscopy. Flow cytometry analysis was also done to determine the effect of the UVC treatment on the spore samples at the molecular level. Samples were processed for SEM and the percentage spore surface hydrophobicity was also determined using the Microbial Adhesion to Hydrocarbon (MATH) assay to predict the adhesion strength to a stainless-steel surface. The result shows the maximum lethality rate to be 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9% from the sample B. velezensis strain LPL-K103 (B44). The difference in surface hydrophobicity for all isolates was statistically significant (P < 0.05). Flow cytometry analysis of UVC treated spore suspensions clarifies them further into sub-populations unaccounted for by plate counting on growth media. The Raman spectroscopy identified B4002 as the isolate possessing the highest concentration of Ca-DPA. The study justifies the critical role of Ca-DPA in spore resistance and the possible sub-populations after UVC treatment that may affect product shelf-life and safety. UVC shows a promising application in the inactivation of resistant spores though there is a need to understand the effects at the molecular level to design the best parameters during processing.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus.

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.

DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation.

This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/ß-type small, acid-soluble spore proteins. The spore cores' large amount of Ca2+-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores' proteinaceous coat may have given some slight protection against UV222 Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254 The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.

Efficacy of UVC-LED in water disinfection on Bacillus species with consideration of antibiotic resistance issue.

Ultraviolet light emitting diode (UV-LED) has attracted extensive attention as a new technology to replace traditional mercury lamp for water disinfection. This study reported for the first time the application of UVC-LEDs in range of 200-280 nm for the treatment of two Gram-positive tetracycline resistant bacteria (TRB) from Bacillus species and their tetracycline resistant gene (TRG). The results showed that UVC-LEDs can inactivate TRB up to 5.7-log and inhibit TRG expression, especially at 268 nm. The required fluence was approximate to that of the referential non-resistant bacteria using the same UVC-LED, but far less than that of TRB using mercury lamp. After UVC-LED irradiation, photoreactivation was the dominant mechanism to repair TRB, just like non-resistant bacteria. But contrary to non-resistant bacteria, the regrowth ratio of TRB was remarkably high at 24 h since the end of the irradition, nevertheless the number of the regrown bacteria in the irradiated water was still less than that in the non-irradiated water. Whereas TRB restored resistance after repair even applying 268 nm at a fluence up to 46.08 mJ/cm2 (maximum in this study). This study highlights the merits of UVC-LED to effectively inactivate TRB in a prompt, energy-efficient and resistance-reducing way, while future study on TRB regrowth and resistance resilience is needed.

Radiation-inducible radioprotective exopolysaccharides of Bacillus siamensis CV5 from irradiated roots of Cistanche violacea to decrease free radical damage produced by ionizing radiation.

Purpose: To assess exopolysaccharides (EPS) of Bacillus siamensis CV5, isolated from irradiated roots of Cistanche violacea, for their induction by ionizing radiation (IR) and their antioxidant and radioprotective power.Materials and methods: Isolated bacteria from the roots of C. violacea were screened for EPS production. The most EPS-producing bacterium was selected and the response surface methodology (RSM) was applied to elucidate the IR dose effects on EPS production. Gamma irradiation effects on the morphology and functional groups of EPS were studied using microscopy and Fourier transform infra-red (FT-IR). The radioprotective potential of EPS on the survival of B. siamensis CV5 following IR was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Free radicals scavenging potentialities (FRSP) of non-irradiated and irradiated EPS were evaluated through 2, 2--diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and ferric reducing ability of plasma (FRAP) assays.Results: Twenty strains, isolated from irradiated roots of C. violacea, were screened for their EPS production. IR-resistant B. siamensis CV5 was the most EPS-producing strain. Its purified EPS contained rhamnose, fructose, mannose and glucose. RSM indicated that EPS of CV5 (CV5-EPS) are radiation inducible. Micrographs of CV5-EPS suggested an increase in the total area and a decrease in the Feret's statistical diameter following exposure to IR. FT-IR spectra of these EPS revealed an increase of various functional groups. The MTT survival assay demonstrated a positive correlation between the added quantity of CV5-EPS and the viability of irradiated CV5 (p < .01). DPPH, ABTS and FRAP assays indicated that the antioxidant activities of CV5-EPS increased significantly with the irradiation dose (p < .01).Conclusions: CV5-EPS were demonstrated as radiation-inducible and radioprotective biomolecules. This radioprotective potential of CV5-EPS could be associated with their antioxidant activities. In the future, irradiated EPS could be tested as a gel in cancer radiotherapy for minimizing the damage caused by rays to surrounding healthy tissues.

Survival of Extremotolerant Bacteria from the Mukundpura Meteorite Impact Crater.

Carbonaceous meteorites provide clues with regard to prebiotic chemistry and the origin of life. Geological Survey of India recorded a carbonaceous chondrite meteorite fall in Mukundpura, India, on June 6, 2017. We conducted a study to investigate the microbial community that survived the meteorite impact. 16S rRNA metagenomic sequencing indicates the presence of Actinobacteria, Proteobacteria, and Acidobacteria in meteorite impact soil. Comparative phylogenetic analysis revealed an intriguing abundance of class Bacilli in the impact soil. Bacillus thermocopriae IR-1, a moderately thermotolerant organism, was isolated from a rock, impacted by the Mukundpura meteorite. We investigated the resilience of B. thermocopriae IR-1 to environmental stresses and impact shock in a Reddy shock tube. Bacillus thermocopriae IR-1 survived (28.82% survival) the effect of shock waves at a peak shock pressure of 300 kPa, temperature 400 K, and Mach number of 1.47. This investigation presents the first report on the effect of impact shock on B. thermocopriae IR-1. The study is also the first report on studying the microbial diversity and isolation of bacteria from impact crater soil immediately after meteorite impact event.

Overproduction of thermoalkalophilic lipase secreted by Bacillus atrophaeus FSHM2 using UV-induced mutagenesis and statistical optimization of medium components.

Microbial enzymes of extremophilic origin serve as a vital source of stable industrial enzymes. The present study focused on overproduction of a thermoalkalophilic lipase produced by Bacillus atrophaeus FSHM2 through UV-induced random mutagenesis (5-45 min exposure to UV light) and factorial experimental design augmented to response surface methodology. Firstly, a UV-induced mutant (designated as UV-45) was developed after the exposure of wild strain to UV irradiation for 45 min which was able to secrete 3484.8 U/L lipase. Afterward, Plackett-Burman experimental approach augmented to central composite design was employed to optimize medium components (olive oil, maltose, glucose, sucrose, yeast extract, tryptone, urea, (NH4)2SO4, NaCl, CaCl2, and ZnSO4) for lipase production by the UV-45 mutant strain. The maximum lipase production of 5505.3 U/L were predicted in medium containing 5% of olive oil, 0.69% of glucose, 0.69% of sucrose, 2.5% of maltose, yeast extract (0.7 g/L), urea (0.44 g/L), (NH4)2SO4 (2.44 g/L), tryptone (1.19 g/L), NaCl (1.61 g/L), CaCl2 (3.81 g/L), and ZnSO4 (1.42 g/L). A mean value of 5161.3 ± 83.3 U/L of lipolytic activity was acquired from real experiments. To sum up, the lipolytic activity of wild type strain (1720.4 U/L) increased by 3-fold after UV-induced mutagenesis and medium components optimization (5161.3 U/L).

Impact of sunlight exposure on the residual efficacy of biolarvicides Bacillus thuringiensis israelensis and Bacillus sphaericus against the main malaria vector, Anopheles gambiae.

BACKGROUND: Biotic and abiotic factors have been reported to affect the larvicidal efficacy of Bacillus thuringiensis israelensis (Bti) and Bacillus sphaericus (Bs), although the extent to which they are affected has been poorly documented. This paper studies the effect of sunlight exposure on the efficacy of a new larvicide formulation based on both Bti and Bs, herein after referred to as BTBSWAX, applied against two different larval stages. METHODS: The emergence of inhibition exhibited by BTBSWAX at three different dosages (1 g/m2, 1.5 g/m2, and 2 g/m2) was monitored under semi-field conditions using a total of 32 containers comprising 16 that were covered and 16 that were uncovered. Two experiments were conducted using first- and second-instar larvae of Anopheles gambiae, respectively. RESULTS: BTBSWAX at 2 g/m2 in covered containers exhibited high emergence inhibition (> 80%) when larvae were exposed from 1st instar on day-6 post-treatment, whereas the emergence inhibition was only 28% in uncovered containers. For larvae exposed from 1st instar on day-12 post-treatment, the emergence inhibition was moderate (70%) in covered containers but was low (< 20%) in uncovered containers. For larvae exposed from 2nd instar on day-10 post-treatment, the emergence inhibition was moderate (31%) in covered containers but was very low (< 10%) in uncovered containers. Moreover, the residual efficacy of BTBSWAX was markedly affected by environmental stresses, including sunlight exposure (Hazard ratio (HR) = 0.12, p < 0.001 and HR = 0.63, p = 0.033 for BTBSWAX at 2 g/m2 against 1st and 2nd instar larvae, respectively). CONCLUSION: These findings emphasize the impact of environmental variables (e.g., sunlight exposure) on the residual efficacy of Bti and Bs biolarvicides in the field. They hence highlight the need to take these factors into account for larvicide formulation development processes. Moreover, studies of the ecology of Anopheles larvae in targeted areas are also crucial for the integration of larval control strategies into malaria transmission plans devised by national malaria control programmes of endemic countries.

A Closer Look at Dark Toxicity of the Photosensitizer TMPyP in Bacteria.

Photodynamic inactivation of bacteria (PIB) is based on photosensitizers which absorb light and generate reactive oxygen species (ROS), killing cells via oxidation. PIB is evaluated by comparing viability with and without irradiation, where reduction of viability in the presence of the photosensitizer without irradiation is considered as dark toxicity. This effect is controversially discussed for photosensitizers like TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluensulfonate). TMPyP shows a high absorption coefficient for blue light and a high yield of ROS production, especially singlet oxygen. Escherichia coli and Bacillus atrophaeus were incubated with TMPyP and irradiated with different light sources at low radiant exposures (µW per cm²), reflecting laboratory conditions of dark toxicity evaluation. Inactivation of E. coli occurs for blue light, while no effect was detectable for wavelengths >450 nm. Being more susceptible toward PIB, growth of B. atrophaeus is even reduced for light with emission >450 nm. Decreasing the light intensities to nW per cm² for B. atrophaeus, application of TMPyP still caused bacterial killing. Toxic effects of TMPyP disappeared after addition of histidine, quenching residual ROS. Our experiments demonstrate that the evaluation of dark toxicity of a powerful photosensitizer like TMPyP requires low light intensities and if necessary additional application of substances quenching any residual ROS.

X-ray-induced mutation of Bacillus sp. MR10 for manno-oligosaccharides production from copra meal.

The present study demonstrates the effectiveness of X-ray radiation in strain improvement for defective lipase production by Bacillus sp. MR10 for further application in the fermentative production of manno-oligosaccharides (MOS) from agricultural by-product, defatted copra meal (DCM). The mutants obtained were screened based on their defective lipase activity together with their ß-mannanase production performance. Among 10 selected mutants, the strain M7 was the highest promising mutant regarding the smallest lipase activity (0.05 U/ml) and the retained ß-mannanase activity similar to the parental strain (22 U/ml) were detected. The mutant M7 effectively hydrolyzed DCM to MOS with low-degree of polymerization (DP) oligomers including mannotriose (M3), mannotetraose (M4), and mannopentose (M5) as the main products. Although the pattern of DCM hydrolysis products of mutant M7 was distinctly different from wild type, the biochemical and catalytic properties of purified ß-mannanase of mutant were similar to those of wild type. Both purified ß-mannanases with apparent molecular mass of 38 kDa displayed optimal activity at pH 5-7 and 45-55°C. Co2+ and Hg2+ nearly completely inhibited activities of both enzymes, whereas Ba2+, Fe3+, and 2-mercaptoethanol obviously activated enzyme activities. Both enzymes showed high specificity for locust bean gum, konjac mannan, DCM, and guar gum. Thus, the mutant M7 has a potential for commercial production of high-quality MOS from low-cost DCM for further application in the feed industry.

Action of antimicrobial photodynamic therapy on heterotypic biofilm: Candida albicans and Bacillus atrophaeus.

The increase in survival and resistance of microorganisms organized in biofilms demonstrates the need for new studies to develop therapies able to break this barrier, such as photodynamic therapy, which is characterized as an alternative, effective, and non-invasive treatment. The objective was to evaluate in vitro the effect of antimicrobial photodynamic therapy on heterotypic biofilms of Candida albicans and Bacillus atrophaeus using rose bengal (12.5 µM) and light-emitting diode (LED) (532 nm and 16.2 J). We used standard strains of B. atrophaeus (ATCC 9372) and C. albicans (ATCC 18804). The biofilm was formed in the bottom of the plate for 48 h. For the photodynamic therapy (PDT) experimental groups, we added 100 µL of rose bengal with LED (P+L+), 100 µL of rose bengal without LED (P+L-), 100 µL of NaCl 0.9 % solution with LED (P-L+), and a control group without photosensitizer or LED (P-L-). The plates remained in agitation for 5 min (pre-irradiation) and were irradiated with LED for 3 min, and the biofilm was detached using an ultrasonic homogenizer for 30 s. Serial dilutions were plated in BHI agar and HiChrom agar and incubated at 37 °C/48 h. There was a reduction of 33.92 and 29.31 % of colony-forming units per milliliter (CFU/mL) for C. albicans and B. atrophaeus, respectively, from the control group to the group subjected to PDT. However, statistically significant differences were not observed among the P+L+, P+L-, P-L+, and P-L- groups. These results suggest that antimicrobial photodynamic therapy using rose bengal (12.5 µM) with a pre-irradiation period of 5 min and LED for 3 min was not enough to cause a significant reduction in the heterotypic biofilms of C. albicans and B. atrophaeus.

Applicability of UV resistant Bacillus pumilus endospores as a human adenovirus surrogate for evaluating the effectiveness of virus inactivation in low-pressure UV treatment systems.

Recent studies have demonstrated the potential to use Bacillus pumilus endospores as a surrogate for human adenovirus (HAdV) in UV disinfection studies. The use of endospores has been limited by observations of batch-to-batch variation in UV sensitivity. This study reports on a propagation method that utilizes a commercially available medium to produce UV tolerant B. pumilus endospores with a consistent UV sensitivity. It is further demonstrated that the endospores of B. pumilus strain (ATCC 27142), produced using this protocol (half strength Columbia broth, 5 days incubation, with 0.1mM MnSO4), display a UV dose-response that is similar to that of HAdV. Endospore stocks could be stored in ethanol for up to two months at 4 °C without a significant change in UV sensitivity. Synergistic endospore damage was observed by pre-heat treatment of water samples followed by UV irradiation. UV tolerant B. pumilus endospores are a potential surrogate of HAdV for UV treatment performance tests in water utilities which do not have in-house research virology laboratories.

Effect of sonic stimulation on Bacillus endospore germination.

This study investigates the effect of sonic stimulation on Bacillus endospore germination. Germinating endospores in a microtiter plate were exposed to audible sound wave generated by an array of piezoelectric transducers. In situ germination kinetics was measured by terbium-dipicolinate fluorescence assay, optical density measurement and phase contrast microscopy. Fluorescence results revealed that sonic stimulation (5 kHz at 90 dB) promoted the germination speed by 43.7% ± 11.3% and final germination level by 61.7% ± 11.9% of Bacillus atrophaeus. This acoustic energy absorbed by endospores is postulated to change membrane permeability and increase enzyme activities; thereby, expediting the germination process. This also raises the likelihood of dormant endospores undergoing germination because of a rapid release of unidentified chemical mediators for quorum sensing. On the other hand, acoustic effect was not observed in B. subtilis endospores. This may be attributed to the different spore aspect ratio, 1.43 ± 0.05 for B. atrophaeus and 2.02 ± 0.08 for B. subtilis, which results in a difference in specific absorption rates towards audible sound waves. Our results demonstrate the modulation of endospore germination by an external field to shed light on germination mechanism and cell-wave interaction.

Improvement of Bacillus strains by mutation for overproduction of exopolygalacturonases.

Pectinases, produced by microorganisms, have wide range application in food industry, textile processing, paper making, coffee and tea fermentation, etc. It accounts for 10% of the global industrial enzymes produced. The most important and widely used commercial pectinase polygalacturonase is produced by alkalophilic strains of Bacillus sp. and Streptomyces sp. Here, we explored 29 bacterial strains isolated from rotten mango samples for polygalacturonase production and selected 16 strains through preliminary screening by well-plate method for enzyme activity. The maximum zone of inhibition of pectin was observed up to 28 mm in diameter but one strain ZM11 was exhibiting no activity. Quantitative dinitrisalicylic acid (DNS) assay for polygalacturonase enzyme was also performed for the selected bacterial isolates. All the strains bestowed significant enzyme activity with the highest activity of 2.4 U/µL exhibited by strain ZM3 (P ≤0.05). Characterization of the isolates was performed using different biochemical tests which also confirmed the isolates as members of the genus Bacillus. Mutation was induced to the selected strains by UV light and acridine orange for different periods of time. Qualitative and quantitative assays of the mutant bacterial isolates showed that the enzyme activity increased to 4.62 U/µL which clearly indicated that induced mutation enhanced the ability of Bacillus strains to produce more polygalacturonase enzyme up to 3-fold as compared to the wild strains (P ≤0.05). Molecular characterization by 16S rRNA sequences further confirmed that the bacterial isolates belong to Bacillus subtilis and B. amyloliquefaciens.

Study of the radiation effect of 99Mo/99mTc generator on Bacillus subtilis and Bacillus pumilus species.

In this work, molybdenum-99 loaded columns were challenged with Bacillus subtilis vegetative cells and Bacillus pumilus spores inside and outside the alumina column, and microbial recovery and radiation effect were assessed. Alumina was a barrier for the passage of microorganisms regardless the species, whilst spores were more retained than vegetative cells with a lower microbial recovery, without significant differences between 9.25 and 74 GBq generators. Bacillus pumilus biological indicator showed lower recoveries, suggesting a radiation inactivating effect on microorganisms.

Sterilization Resistance of Bacterial Spores Explained with Water Chemistry.

Bacterial spores can survive for long periods without nutrients and in harsh environmental conditions. This survival is influenced by the structure of the spore, the presence of protective compounds, and water retention. These compounds, and the physical state of water in particular, allow some species of bacterial spores to survive sterilization schemes with hydrogen peroxide and UV light. The chemical nature of the spore core and its water has been a subject of some contention and the chemical environment of the water impacts resistance paradigms. Either the spore has a glassy core, where water is immobilized along with other core components, or the core is gel-like with mobile water diffusion. These properties affect the movement of peroxide and radical species, and hence resistance. Deuterium solid-state NMR experiments are useful for examining the nature of the water inside the spore. Previous work in our lab with spores of Bacillus subtilis indicate that, for spores, the core water is in a more immobilized state than expected for the gel-like core theory, suggesting a glassy core environment. Here, we report deuterium solid-state NMR observations of the water within UV- and peroxide-resistant spores from Bacillus pumilus SAFR-032. Variable-temperature NMR experiments indicate no change in the line shape after heating to 50 °C, but an overall decrease in signal after heating to 100 °C. These results show glass-like core dynamics within B. pumilus SAFR-032 that may be the potential source of its known UV-resistance properties. The observed NMR traits can be attributed to the presence of an exosporium containing additional labile deuterons that can aid in the deactivation of sterilizing agents.

Generator and Setup for Emulating Exposures of Biological Samples to Lightning Strokes.

GOAL: We aimed to develop a system for controlled exposure of biological samples to conditions they experience when lightning strikes their habitats. METHODS: We based the generator on a capacitor charged via a bridge rectifier and a dc-dc converter, and discharged via a relay, delivering arcs similar to natural lightning strokes in electric current waveform and similarly accompanied by acoustic shock waves. We coupled the generator to our exposure chamber described previously, measured electrical and acoustic properties of arc discharges delivered, and assessed their ability to inactivate bacterial spores. RESULTS: Submicrosecond discharges descended vertically from the conical emitting electrode across the air gap, entering the sample centrally and dissipating radially toward the ring-shaped receiving electrode. In contrast, longer discharges tended to short-circuit the electrodes. Recording at 341 000 FPS with Vision Research Phantom v2010 camera revealed that initial arc descent was still vertical, but became accompanied by arcs leaning increasingly sideways; after 8-12 µs, as the first of these arcs formed direct contact with the receiving electrode, it evolved into a channel of plasmified air and short-circuited the electrodes. We eliminated this artefact by incorporating an insulating cylinder concentrically between the electrodes, precluding short-circuiting between them. While bacterial spores are highly resistant to electric pulses delivered through direct contact, we showed that with arc discharges accompanied by an acoustic shock wave, spore inactivation is readily obtained. CONCLUSION: The presented system allows scientific investigation of effects of arc discharges on biological samples. SIGNIFICANCE: This system will allow realistic experimental studies of lightning-triggered horizontal gene transfer and assessment of its role in evolution.

Bioprocess of Kosa bioaerosols: effect of ultraviolet radiation on airborne bacteria within Kosa (Asian dust).

Kosa (Asian dust) is a well-known weather phenomenon in which aerosols are carried by the westerly winds from inland China to East Asia. Recently, the frequency of this phenomenon and the extent of damage caused have been increasing. The airborne bacteria within Kosa are called Kosa bioaerosols. Kosa bioaerosols have affected ecosystems, human health and agricultural productivity in downwind areas. In order to develop a new and useful bacterial source and to identify the source region of Kosa bioaerosols, sampling, isolation, identification, measurement of ultraviolet (UV) radiation tolerance and experimental simulation of UV radiation conditions were performed during Kosa bioaerosol transportation. We sampled these bioaerosols using a Cessna 404 airplane and a bioaerosol sampler at an altitude of approximately 2900 m over the Noto Peninsula on March 27, 2010. The bioaerosol particles were isolated and identified as Bacillus sp. BASZHR 1001. The results of the UV irradiation experiment showed that the UV radiation tolerance of Kosa bioaerosol bacteria was very high compared with that of a soil bacterium. Moreover, the UV radiation tolerance of Kosa bioaerosol spores was higher than that of soil bacterial spores. This suggested that Kosa bioaerosols are transported across the atmosphere as living spores. Similarly, by the experimental simulation of UV radiation conditions, the limited source region of this Kosa bioaerosol was found to be southern Russia and there was a possibility of transport from the Kosa source area.

Isolation and screening of thermophilic bacilli from compost for electrotransformation and fermentation: characterization of Bacillus smithii ET 138 as a new biocatalyst.

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C5 and C6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216(T) and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 10(3) colonies per µg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C5 and C6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.