Subtilis Endospores Research Articles

Unlocking Wearable Microbial Fuel Cells for Advanced Wound Infection Treatment.

Better infection control will accelerate wound healing and alleviate associated healthcare burdens. Traditional antibacterial dressings often inadequately control infections, inadvertently promoting antibacterial resistance. Our research unveils a novel, dual-functional living dressing that autonomously generates antibacterial agents and delivers electrical stimulation, harnessing the power of spore-forming Bacillus subtilis. This dressing is built on an innovative wearable microbial fuel cell (MFC) framework, using B. subtilis endospores as a powerful, dormant biocatalyst. The endospores are resilient, reactivating in nutrient-rich wound exudate to produce electricity and antibacterial compounds. The combination allows B. subtilis to outcompete pathogens for food and other resources, thus fighting infections. The strategy is enhanced by the extracellular synthesis of tin oxide and copper oxide nanoparticles on the endospore surface, boosting antibacterial action, and electrical stimulation. Moreover, the MFC framework introduces a pioneering dressing design featuring a conductive hydrogel embedded within a paper-based substrate. The arrangement ensures cell stability and sustains a healing-friendly moist environment. Our approach has proven very effective against three key pathogens in biofilms: Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus demonstrating exceptional capabilities in both in vitro and ex vivo models. Our innovation marks a significant leap forward in wearable MFC-based wound care, offering a potent solution for treating infected wounds.

Germicidal efficacy of continuous and pulsed ultraviolet-C radiation on pathogen models and SARS-CoV-2

Ultraviolet radiation’s germicidal efficacy depends on several parameters, including wavelength, radiant exposure, microbial physiology, biological matrices, and surfaces. In this work, several ultraviolet radiation sources (a low-pressure mercury lamp, a KrCl excimer, and four UV LEDs) emitting continuous or pulsed irradiation were compared. The greatest log reductions in E. coli cells and B. subtilis endospores were 4.1 ± 0.2 (18 mJ cm−2) and 4.5 ± 0.1 (42 mJ cm−2) with continuous 222 nm, respectively. The highest MS2 log reduction observed was 2.7 ± 0.1 (277 nm at 3809 mJ cm−2). Log reductions of SARS-CoV-2 with continuous 222 nm and 277 nm were ≥ 3.4 ± 0.7, with 13.3 mJ cm−2 and 60 mJ cm−2, respectively. There was no statistical difference between continuous and pulsed irradiation (0.83–16.7% [222 nm and 277 nm] or 0.83–20% [280 nm] duty rates) on E. coli inactivation. Pulsed 260 nm radiation (0.5% duty rate) at 260 nm yielded significantly greater log reduction for both bacteria than continuous 260 nm radiation. There was no statistical difference in SARS-CoV-2 inactivation between continuous and pulsed 222 nm UV-C radiation and pulsed 277 nm radiation demonstrated greater germicidal efficacy than continuous 277 nm radiation. Greater radiant exposure for all radiation sources was required to inactivate MS2 bacteriophage. Findings demonstrate that pulsed irradiation could be more useful than continuous UV radiation in human-occupied spaces, but threshold limit values should be respected. Pathogen-specific sensitivities, experimental setup, and quantification methods for determining germicidal efficacy remain important factors when optimizing ultraviolet radiation for surface decontamination or other applications.Graphical abstract

High-Pressure-Based Strategies for the Inactivation of Bacillus subtilis Endospores in Honey

Honey is a value-added product rich in several types of phenolic compounds, enzymes, and sugars recently explored in biomedical and food applications. Nevertheless, even though it has a low water activity (aW ≈ 0.65) that hinders the development of pathogenic and spoilage microorganisms, it is still prone to contamination by pathogenic microorganisms (vegetative and spores) and may constitute harm to special groups, particularly by immunosuppressed people and pregnant women. Thus, an efficient processing methodology needs to be followed to ensure microbial safety while avoiding 5-hydroxymethylfurfural (HMF) formation and browning reactions, with a consequent loss of biological value. In this paper, both thermal (pressure-assisted thermal processing, PATP) and nonthermal high-pressure processing (HPP), and another pressure-based methodology (hyperbaric storage, HS) were used to ascertain their potential to inactivate Bacillus subtilis endospores in honey and to study the influence of aW on the inactivation on this endospore. The results showed that PATP at 600 MPa/15 min/75 °C of diluted honey (52.9 °Brix) with increased aW (0.85 compared to ≈0.55, the usual honey aW) allowed for inactivating of at least 4.0 log units of B. subtilis spores (to below detection limits), while HS and HPP caused neither the germination nor inactivated spores (i.e., there was neither a loss of endospore resistance after heat shock nor endospore inactivation as a consequence of the storage methodology). PATP of undiluted honey even at harsh processing conditions (600 MPa/15 min/85 °C) did not impact the spore load. The results for diluted honey open the possibility of its decontamination by spores’ inactivation for medical and pharmaceutical applications.

Diatomite and Na-X zeolite as carriers for bacteria in self-healing cementitious mortars

The use of bacteria in the self-healing process of cementitious composites has been recently widely considered. In this study, diatomite and the Na-X zeolite were proposed as carrier materials impregnated by the Bacillus subtilis endospores. The effect of the impregnation method on a bacterial survival rate (BSR) and cement composites modification method on the strength parameters, was investigated. Additionally, the influence of modification and healing method on the flexural and compressive strength recovery and surface crack repair ratio, was assessed. Test results indicated higher BSR for a vacuum impregnated than immersed samples (up to 31% after 28 days of storage for the impregnated Na-X zeolite). The BSR in the self-healing mortar may be promoted by local pH reduction in the vicinity of carrier particles, especially diatomite (pHPZC = 4.6). Mortars containing medium dissolved in the mixing water showed an increase in the flexural and compressive strength (up to 10.4% and 18.0%, respectively). The addition of bacteria immobilized on the both carrier materials caused a decrease of strength properties (to 16.6%), while the series containing both bacteria and the medium attained the strength properties similar or better than the reference series and the series with carrier materials alone. Series containing bacteria immobilized on carrier material and with medium showed a self-healing ability, which was confirmed by the compressive strength recovery and surface repair ratio of 32.6% and 92.6%, respectively. The increased precipitation of CaCO3 was confirmed by the SEM and EDS analysis.

A microbiological perspective of raw milk preserved at room temperature using hyperbaric storage compared to refrigerated storage

The effects of hyperbaric storage (HS, 50–100 MPa) at room temperature (RT) on endogenous and inoculated pathogenic surrogate vegetative bacteria (Escherichia coli, Listeria innocua), pathogenic Salmonella enterica and bacterial spores (Bacillus subtilis) were assessed and compared with conventional refrigeration at atmospheric pressure for 60 days. Milk stored at atmospheric pressure and refrigeration quickly surpassed the acceptable microbiological limit within 7 days of storage, regarding endogenous microbiota, yet 50 MPa/RT slowed down microbial growth, resulting in raw milk spoilage after 28 days, while a significant microbial inactivation occurred under 75–100 MPa (around 4 log units), to counts below 1 log CFU/mL throughout storage, similar to what was observed for B. subtilis endospores. While inoculated microorganisms had a gradually counts reduction in all HS conditions. Results indicate that HS can not only result in the extension of milk shelf-life but is also able to enhance its safety and subsequent quality. Industrial relevanceThis new preservation methodology could be implemented in the dairy farm storage tanks, or during milk transportation for further processing, allowing a better microbial control, than refrigeration. This methodology is very promising, and can improve food products shelf-life with a considerable lower carbon foot-print than refrigeration.

Small-scale, storable paper biobatteries activated via human bodily fluids

Herein, a fully paper-based biobattery composed of four microbial fuel cell (MFC) units is evaluated, one that can be prepared in advance, stored, and quickly activated with virtually any available fluid. The biobattery uniquely utilizes Bacillus subtilis endospores as the storable anodic biocatalyst; the dormant, robust nature of B. subtilis endospores should allow for device preinoculation with spores followed by prolonged storage of the fully fabricated paper battery until needed. A germinant paper layer strategically fabricated above the spore-loaded anode layer contains all of the necessary chemical germinants and nutrient components required for the endospores to begin germination, exit dormancy, and return to fully metabolic vegetative bacterial cells that can generate electrical energy. This mechanism allows for the battery to be simply initiated via a wide range of available liquids. Bioelectricity generation of the battery is successfully demonstrated after introduction of a variety of artificial bodily fluids, including saliva, sweat, and urine, along with tap water. Since the biobattery has the capability of serially linking all 4 of its MFCs through simple dynamic folding, the device’s total power output can be greatly enhanced; a single biobattery is able to achieve 0.56 V and 2.4 μW, which is beyond the ratings required for their intended application in single-use, disposable sensors. Therefore, this concept of integrating 4 spore-based MFCs into a single biobattery device with a built-in germinant layer offers a potential solution for stable, long-term storable power sources, displaying feasibility for integration with low power, disposable sensor applications.

Hyperbaric Storage of Vacuum-Packaged Fresh Atlantic Salmon (Salmo salar) Loins by Evaluation of Spoilage Microbiota and Inoculated Surrogate-Pathogenic Microorganisms

Hyperbaric storage at low and room temperature (HS/LT, 60 MPa/10°C; HS/RT, 75 MPa/25°C; 30 days) of vacuum-packaged Atlantic salmon (Salmo salar) was studied and compared with storage under atmospheric pressure (AP, at 5, 10, and 25°C). Spoilage and inoculated surrogate-pathogenic (Bacillus subtilis endospores, Escherichia coli, and Listeria innocua) microorganisms were monitored during storage. Under AP, the spoilage microorganisms increased during storage time, reaching total aerobic mesophiles values higher than the acceptable limit after 15 and 5 days at AP/5°C and AP/10–25°C, respectively. Contrarily, both HS conditions inhibited and inactivated the spoilage microorganisms, reaching total aerobic mesophiles to values below the detection limit after 30 days under HS/RT. Under AP, surrogate-pathogenic microorganism counts increased, while for both HS samples B. subtilis endospores reached counts below the detection limit after 30 days while E. coli and L. innocua counts were also reduced. In conclusion, HS decreased initial population of spoilage and inoculated surrogate-pathogenic microorganisms, showing that besides the shelf-life extension (due to microbial growth inhibition), it also increased microbial safety (by microbial inactivation) of vacuum-packaged Atlantic salmon.

Temporal and spatial regulation of protein cross-linking by the pre-assembled substrates of a Bacillus subtilis spore coat transglutaminase.

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a “spotwelding” activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

Enhanced control of Bacillus subtilis endospores development by hyperbaric storage at variable/uncontrolled room temperature compared to refrigeration

The effect of hyperbaric storage on Bacillus subtilis endospores, as a new food preservation methodology with potential to replace the conventional refrigeration processes, was assessed and compared to refrigeration. To do so, three different matrices (McIlvaine buffer, carrot juice and brain-heart infusion broth, BHI-broth) were inoculated with B. subtilis endospores and stored at 25, 50 and 100 MPa at variable/uncontrolled room temperature (18–23 °C), under refrigeration (4 °C), and room temperature at atmospheric pressure (0.1 MPa), up to 60 days. Two different quantification procedures were performed to assay both vegetative and endospores (unheated samples) and endospores (heated samples), to assess germination under pressure.The results showed that hyperbaric storage yielded pronounced endospore loads reductions in carrot juice and BHI-broth at 50 and 100 MPa, while in McIlvaine buffer, lower endospore loads reductions were observed. At 25 MPa, the endospores germinated and outgrew in carrot juice. Under refrigeration conditions, both carrot juice and BHI-broth underwent endospore germination and outgrowth after 60 and 9 days of storage, respectively, while in McIlvaine buffer there were no endospore outgrowth.These results suggest that hyperbaric storage at room temperature might not only be a feasible preservation procedure regarding endospores, but also that the food product (matrix characteristics) seems to influence the microbial inactivation that occurs during hyperbaric storage.

Cold atmospheric pressure ambient air plasma inhibition of pathogenic bacteria on the surface of black pepper

In present study, the inhibition effect of low temperature plasma on Bacillus subtilis, Escherichia coli, Salmonella Enteritidis and B. subtilis endospores inoculated on the surface of black peppercorns was studied. Plasma was generated by Diffuse Coplanar Surface Barrier Discharge (DCSBD) at atmospheric pressure in ambient air. Plasma treatment time of 300 s led to log10 CFU/g reduction of B. subtilis from 7.36 to 2.30 and B. subtilis endospores from 4.42 to 2.39. Plasma treatment reduced the number of E. coli and Salmonella Enteritidis to below detection level (1.0 log10 CFU/g) from initial populations of 7.45 log10 CFU/g and 7.60 log10 CFU/g, respectively. The inactivation kinetics was explained by Weibull model. Decimal reduction times (D-values) for B. subtilis, E. coli, Salmonella Enteritidis, and B. subtilis endospores were determined as 43 s, 47 s, 58 s, and 142 s, respectively. The surface morphology observed by Scanning Electron Microscopy showed no significant changes after the plasma treatment. The influence of plasma on chemical bonds on the surface and inside the peppercorns was studied by Attenuated Total Reflectance – Fourier Transform Infrared Spectroscopy.

Sporobeads: The Utilization of the Bacillus subtilis Endospore Crust as a Protein Display Platform.

Upon starvation, the soil bacterium Bacillus subtilis forms an intracellular, metabolically inactive endospore. Its core contains the DNA, encased by three protein layers protecting it against a multitude of environmental threats. The outermost layer, the crust, harbors great potential as a protein-displaying platform: a gene of interest can be translationally fused to a crust protein gene, resulting in endospores displaying the desired protein on their surface. To unlock this potential in a standardized fashion, we designed a suite of 12 vectors (Sporovectors), based on the BioBrick cloning standard. With these vectors, proteins can easily be fused N- or C-terminally to the six crust proteins CotV, CotW, CotX, CotY, CotZ, and CgeA under the control of the strongest crust gene promoter PcotYZ. All Sporovectors were evaluated with GFP and two different laccases. On the basis of our data, CotY and CotZ represent the best anchor proteins. But there are significant differences in activity and functional stability between the two tested laccases. Our vector suite is a powerful tool to generate and evaluate a vast variety of functionalized endospores. It allows quickly identifying the best anchor and fusion site for the protein of interest. Our findings demonstrate that the crust of B. subtilis endospores is an inexpensive and easy platform for displaying different proteins of interest.

Thermal characterization of Bacillus subtilis endospores and a comparative study of their resistance to high temperature pulsed electric fields (HTPEF) and thermal-only treatments

Heat characterization of Bacillus subtilis DSM 618 endospores at temperatures ranging from 90 to 120 °C was performed in water and reconstituted skimmed milk. DT values showed that B. subtilis endospores were more heat resistant in skimmed milk than in water and the calculated z-values indicated the same thermo-dependency regardless of the treatment medium used. Moreover, the effect of High-Temperature Pulsed Electric Fields (HTPEF) treatments on the inactivation of B. subtilis endospores in water and its comparison to thermal-only treatments by means of equal FT values at different temperatures was studied. HTPEF treatment consisted in bipolar square-wave pulses (10 μs, 38 kV/cm, 466 Hz) at maximum processing temperatures up to c. 123 °C. Inactivation values greater than c. 4.5 log10 cycles were obtained for both HTPEF and heat treatments with no significant differences between them. However, further studies at higher electric field strengths and temperatures could be performed to establish if greater inactivation levels can be achieved and to observe whether PEF has an additional effect over heat.

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.

Impact of surface structure and feed gas composition on Bacillus subtilis endospore inactivation during direct plasma treatment.

This study investigated the inactivation efficiency of cold atmospheric pressure plasma treatment on Bacillus subtilis endospores dependent on the used feed gas composition and on the surface, the endospores were attached on. Glass petri-dishes, glass beads, and peppercorns were inoculated with the same endospore density and treated with a radio frequency plasma jet. Generated reactive species were detected using optical emission spectroscopy. A quantitative polymerase chain reaction (qPCR) based ratio detection system was established to monitor the DNA damage during the plasma treatment. Argon + 0.135% vol. oxygen + 0.2% vol. nitrogen as feed gas emitted the highest amounts of UV-C photons and considerable amount of reactive oxygen and nitrogen species. Plasma generated with argon + 0.135% vol. oxygen was characterized by the highest emission of reactive oxygen species (ROS), whereas the UV-C emission was negligible. The use of pure argon showed a negligible emission of UV photons and atomic oxygen, however, the emission of vacuum (V)UV photons was assumed. Similar maximum inactivation results were achieved for the three feed gas compositions. The surface structure had a significant impact on the inactivation efficiency of the plasma treatment. The maximum inactivation achieved was between 2.4 and 2.8 log10 on glass petri-dishes and 3.9 to 4.6 log10 on glass beads. The treatment of peppercorns resulted in an inactivation lower than 1.0 log10. qPCR results showed a significant DNA damage for all gas compositions. Pure argon showed the highest results for the DNA damage ratio values, followed by argon + 0.135% vol. oxygen + 0.2% vol. nitrogen. In case of argon + 0.135% vol. oxygen the inactivation seems to be dominated by the action of ROS. These findings indicate the significant role of VUV and UV photons in the inactivation process of B. subtilis endospores.

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain.

Predation by Myxococcus xanthus induces Bacillus subtilis to form spore-filled megastructures.

Biofilm formation is a common mechanism for surviving environmental stress and can be triggered by both intraspecies and interspecies interactions. Prolonged predator-prey interactions between the soil bacterium Myxococcus xanthus and Bacillus subtilis were found to induce the formation of a new type of B. subtilis biofilm, termed megastructures. Megastructures are tree-like brachiations that are as large as 500 μm in diameter, are raised above the surface between 150 and 200 μm, and are filled with viable endospores embedded within a dense matrix. Megastructure formation did not depend on TasA, EpsE, SinI, RemA, or surfactin production and thus is genetically distinguishable from colony biofilm formation on MSgg medium. As B. subtilis endospores are not susceptible to predation by M. xanthus, megastructures appear to provide an alternative mechanism for survival. In addition, M. xanthus fruiting bodies were found immediately adjacent to the megastructures in nearly all instances, suggesting that M. xanthus is unable to acquire sufficient nutrients from cells housed within the megastructures. Lastly, a B. subtilis mutant lacking the ability to defend itself via bacillaene production formed megastructures more rapidly than the parent. Together, the results indicate that production of the megastructure facilitates B. subtilis escape into dormancy via sporulation.

Enhanced Inactivation of Bacillus subtilis Spores during Solar Photolysis of Free Available Chlorine

Aqueous free available chlorine (FAC) can be photolyzed by sunlight and/or artificial UV light to generate various reactive oxygen species, including HO(•) and O((3)P). The influence of this chemistry on inactivation of chlorine-resistant microorganisms was investigated using Bacillus subtilis endospores as model microbial agents and simulated and natural solar radiation as light sources. Irradiation of FAC solutions markedly enhanced inactivation of B. subtilis spores in 10 mM phosphate buffer; increasing inactivation rate constants by as much as 600%, shortening inactivation curve lag phase by up to 73% and lowering CTs required for 2 log10 inactivation by as much as 71% at pH 8.0 and 10 °C. Similar results were observed at pH 7.4 and 10 °C in two drinking water samples with respective DOC concentrations and alkalinities of 0.6 and 1.2 mg C/L and 81.8 and 17.1 mg/L as CaCO3. Solar radiation alone did not inactivate B. subtilis spores under the conditions investigated. A variety of experimental data indicate that the observed enhancements in spore inactivation can be attributed to the concomitant attack of spores by HO(•) and O3, the latter of which was found to accumulate to micromolar concentrations during simulated solar irradiation of 10 mM phosphate buffer (pH 8, 10 °C) containing [FAC]0 = 8 mg/L as Cl2.

Complexation of neptunium(V) with Bacillus subtilis endospore surfaces and their exudates

The neptunyl ion is very toxic and has the potential to be highly mobile in the environment. In an effort to understand how its interactions with biological surfaces may affect its movement in the environment, we investigated neptunyl interactions with Bacillus subtilis endospores and their exudates. The exudates were dominated by dipicolinic acid. Spectrophotometric investigations of the chemical form of neptunyl in exudate solutions are consistent with the formation of 1:1 neptunyl–dipicolinate complexes. Using neptunyl–endospore adsorption data and spectrophotometric measurements of neptunyl–dipicolinate complexes, we determined thermodynamic stability constants for both species. Neptunyl adsorption onto the endospore surface decreased with an increasing pH, which corresponds to increasing aqueous complexation of neptunyl by dipicolinate. Adsorption was also highly ionic strength dependent with adsorption increasing as ionic strength decreased. With stability constants determined in this work, we compared controls on neptunyl partitioning in a simulated system with B. subtilis endospores, vegetative cells, and generic natural organic matter. Neptunyl complexation by B. subtilis endospore exudates exerted the greatest biological control in the simulated systems.

Electrical and Optical Characterization of Cold Atmospheric Pressure Plasma Jet and the Effects of N<sub>2 </sub>Gas on Argon Plasma Discharge

In this study, we conducted experiments to investigate the electrical and optical characteristics of a non-equilibrium Ar-N2 plasma discharge at atmospheric pressure. To obtain the plasma discharge we used our indigenously designed plasma generating device named CAPPLAT (Cold Atmospheric Pressure Plasma Torch) which was manufactured by Cresur Corporation. The plasma discharge obtained with only Ar gas was quite filamentary. So, to achieve a homogeneous discharge N2 gas was admixed. The effects of different volumes of admixed N2 gas were also studied. The optical emission spectroscopy was used to study the active charged species in the plasma discharges. The further increased volume of N2 gas further suppressed the emission intensity of Ar metastables but at the same time the emission intensity of the second positive system of nitrogen molecules (N2(C3Πu) enhanced significantly. It can be concluded that in Ar- plasma discharge, argon metastables are the main energy carriers but when N2 gas is added to the feeding gas (Ar) for plasma generation, the second positive system of nitrogen molecules (N2(C3Πu) become the main energy carriers. On the other hand the addition of the N2 gas doesn’t change the electrical characteristics of plasma discharge significantly. To identify the effectiveness of the CAPPLAT as a tool for sterilization, highly environmental stress resistant bacterial (Bacillus subtilis) endospores were treated for different durations. We could successfully deactivate the population of 1.0X107 to 4.0X107 Bacillus endospores/ml. The details of this experiment are discussed in our next paper.