Bacillus Endospores Research Articles

Efficiency of Bacillus pseudomycoides RAY21 and Bacillus subtilis CYA27 Endospore Formulation on Biochar and Oil Spill Dispersant

Bacillus sp. is well known for its functional capabilities such as solubilizing phosphorus (P) and potassium (K), and fixing nitrogen (N2). These bacteria can form endospores under stressed conditions, allowing long-term survival and application in biotechnological fields. This study aims to isolate Bacillus sp. capable of forming endospores and evaluates their viability on different carriers, specifically biochar and oil spill dispersant (OSD), to enhance biodegradation in contaminated environments. Soil samples from the bamboo rhizosphere were heat-shocked to isolate endospore-forming Bacillus strains, with the isolate identified as Bacillus pseudomycoides RAY21 through 16S rRNA sequencing. This strain exhibited Gram-positive characteristics, formed endospores, and demonstrated potential on various media such as Pikovskaya, Alexandrov, and N-Free Mannitol. The physiological characterization indicated optimal growth in a pH range of 6-8, salinity up to 3.5%, and thermophilic properties. Endospores from B. pseudomycoides RAY21 and B. subtilis CYA27 were tested on biochar and OSD as carriers. The results showed that endospores adhered better to biochar, but their viability was more stable in OSD over time. Notably, B. pseudomycoides RAY21 on OSD degraded 23.43% of total petroleum hydrocarbons (TPH), outperforming B. subtilis CYA27 (21.62%). In conclusion, the study demonstrates the potential of using Bacillus endospores on OSD as an effective carrier for bioremediation, particularly in degrading petroleum hydrocarbons. Future research should focus on optimizing carrier materials and exploring field-scale applications for enhanced environmental cleanup.

UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores

Bacillus endospores (spores) are generally resistant to environmental and food processing-related stress including thermal and non-thermal processing in the food industry, such as pasteurization, and UV-C inactivation. Bacillus thuringiensis insecticidal crystals and spores as the active substances in commercial biopesticides can also be introduced to vegetable foods and their food processing environment due to pre-harvest treatment of edible crops. The resistance of B. thuringiensis biopesticide spores in comparison to the genetically closely related foodborne B. cereus against heat and UV-C treatment is investigated in this study. The results show that B. thuringiensis biopesticide spores with the commercial granulated product formulation are better protected and as such more resistant to both wet heat (D values at 90 °C: 50.1–79.5 min) and UV-C treatment (D values at 0.6 mW/cm2: 7.5–8.9 min) than the pure spore suspension. The enhanced UV-C resistance properties of B. thuringiensis-formulated spores also indicate that the B. thuringiensis spores in powder or granule formulation applied in the field might not be effectively inactivated by solar radiation (UV-A and UV-B) in a short period. Furthermore, the spores of one emetic B. cereus toxin-producing strain (LFMFP 254; a Belgian outbreak strain) were found more resistant to the wet heat at 90 °C (D90-value = 71.2 min) than other tested pure spore suspensions, although the spores of B. cereus 254 did not show different behavior against UV-C treatment. This result suggests that UV-C treatment can be applied as an effective inactivation method against B. cereus 254 spores.

Moisture-Enabled Germination of Heat-Activated Bacillus Endospores for Rapid and Practical Bioelectricity Generation: Toward Portable, Storable Bacteria-Powered Biobatteries.

Small-scale battery-like microbial fuel cells (MFCs) are a promising alternative power source for future low-power electronics. Controllable microbial electrocatalytic activity in a miniaturized MFC with unlimited biodegradable energy resources would enable simple power generation in various environmental settings. However, the short shelf-life of living biocatalysts, few ways to activate the stored biocatalysts, and extremely low electrocatalytic capabilities render the miniature MFCs unsuitable for practical use. Here, heat-activated Bacillus subtilis spores are revolutionarily used as a dormant biocatalyst that can survive storage and rapidly germinate when exposed to special nutrients that are preloaded in the device. A microporous, graphene hydrogel allows the adsorption of moisture from the air, moves the nutrients to the spores, and triggers their germination for power generation. In particular, forming a CuO-hydrogel anode and an Ag2 O-hydrogel cathode promotes superior electrocatalytic activities leading to an exceptionally high electrical performance in the MFC. The battery-type MFC device is readily activated by moisture harvesting, producing a maximum power density of 0.4mW cm-2 and a maximum current density of 2.2mA cm-2 . The MFC configuration is readily stackable in series and a three-MFC pack produces enough power for several low-power applications, demonstrating its practical feasibility as a sole power source.

Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli.

Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B.cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

Incubation temperature and culture medium formulation impact the accuracy of pour-plate techniques for the enumeration of industrial Bacillus assemblages

Aerobic plate counting assays based on the pour-plate technique are frequently used to enumerate microbial products; however, colony swarming and merging at the agar surface can reduce the accuracy of these assays. Some plating methods mitigate this risk through the inclusion of strategies including agar overlays; however, these interventions may be inadequate to mitigate swarming and merging of certain Bacillus colonies. In the present study, we assessed the accuracy of several pour-plate techniques for the enumeration of a mixed-species Bacillus assemblage. Tested modifications included a customized culture medium formulation, agar overlays, decreased incubation times and increased incubation temperature. Methods which produced countable plates were assessed for agreement with a Bacillus-specific plate counting assay and with total cell counts rendered by flow cytometry. While all tested pour-plate methods underestimated Bacillus endospore concentrations relative to flow cytometry and customized spread-plating, our results suggest that increasing incubation temperature and the inclusion of bile salts into culture medium formulations can improve the accuracy of pour-plate techniques when used to enumerate Bacillus assemblages by decreasing the incidence of spreading colonies. As Bacillus endospore preparations become more ubiquitous in the market, familiar enumeration methods such as the pour-plate technique may require methodological modifications to ensure that the cGMP compliance of Bacillus-based microbial products is assessed accurately.

Nutrient germination improves DNA recovery from industrial Bacillus subtilis endospores during qPCR enumeration assays

Growth-independent microbial enumeration methods such as quantitative PCR require the efficient extraction of genomic DNA from targeted cells. Bacillus endospores are popular inclusions in commercial products due to their hardiness and metabolic dormancy; however, this hardiness is known to render Bacillus endospores resistant to traditional DNA isolation techniques. Metagenomic studies have sought to address this resistance through nutrient-based germination of bacterial endospores in environmental samples. In the present study, we sought to apply this technique to the enumeration of microbial products using an industrial strain of Bacillus subtilis as a model organism. Germination was induced through incubation of axenic spore suspensions in an AGFK-based rich medium. Total spore count, dipicolinic acid release and OD600 absorbance were monitored over time to track the progression of spore populations through the stages of germination and outgrowth. Aerobic plate counts and flow cytometry were used to monitor cell populations for proliferation during the incubation period. Finally, quantitative PCR with taxon-specific primers was used to examine DNA recovery as a function of time. Results show that customized germination protocols, once appropriately validated for the species and product matrix under consideration, can result in more efficient DNA extraction and thus lower limits of detection for qPCR assays targeting industrial Bacillus endospores in microbial products.

Inactivation efficiency of Bacillus endospores via modified flow-through PUV treatment with comparison to conventional LPUV treatment

Water is a fragile resource, consequently there is a pressing need to develop sustainable environmental-friendly disinfection technologies. This constitutes the first study to compare efficacy of continuous low-pressure UV (LPUV) to that of pulsed UV light (PUV) for UV disinfection performance in flow-through water systems. Bacillus endospores were used as these are frequently deployed as biodosimeters for drinking water UV reactor validation protocols. Results showed a PUV system output of 2,052 mJ/cm2 (energy below 300 nm) was required for a 2 log inactivation of B.pumilus endospores, whereas a lower LPUV system output of 12 mJ/cm2 produced a similar level of disinfection. Measurements of dose incident to the samples (which were 10.75 cm from the PUV light source) showed supplementation of cultivation media with manganese sulphate monohydrate (MnSO4.H2O) employed to enhance the rate of endospore formation for propagation was shown to increase the UV resistance of different Bacillus spp. to both irradiation approaches (P < 0.05). Conventional LPUV light that is commonly-used by the drinking water industry is a reliable and superior approach to PUV which requires substantial modification for effective disinfection of continuous flow-through water. Caution must also be exercised when manipulating media formulation to propagate endospores as this may lead to inconsistencies in reporting efficacy of intervention treatments with implications for validation and environmental evaluation.

Thermal treatment of skim milk concentrates in a novel shear-heating device: Reduction of thermophilic spores and physical properties

Endospores of thermophilic bacilli are a major concern for producers of dairy powders. In this study, we heat treated 10 different spore suspensions at 110 °C in skim milk and skim milk concentrate (36% dry matter) of the species Geobacillus stearothermophilus (10 min) and Anoxybacillus flavithermus (5 min) in a new shear-heating device. The highest log reduction in skim milk concentrate was 3.5. The death behavior of the spores was strain dependent. Particle formation and Maillard reaction were observed. By increasing the shear-rate up to 1500 s−1 the particle size was reduced for both heating times (D90 reduction: 57.4 and 77.0%, respectively). The particle size was lessened by a reduction of dry matter of 27%, compared to 36%. This work emphasizes, that heat treatment of concentrated dairy products represents a technological option to reduce thermophilic spores in skim milk concentrate and powders produced thereof.

Biocrystalline structures in the nucleoids of the stationary and dormant prokaryotic cells

Compaction and biocrystallization of the nucleoid are presently considered as a necessary and important stage in the transformation of the cell ultrastructure during change of microbial cultures strategies from growth to survival. Nucleoid biocrystallization in the stationary phase cells is achieved due to structural regularity of the DNA complexes with the histone-like Dps protein. Our experiments with Escherichia coli mutants, overproducers of the Dps protein, confirmed nucleoid biocrystallization in the late stationary phase cells. Since nucleoid biocrystallization was revealed in E. сoli cells without Dps overproduction at late stages of starvation, it is constitutive in the cycle of development of microbial populations. The present work concentrated on detection of the nucleoid biocrystalline structure in (1) long-starved (21 day in the chemostat mode) bacterial cells (genera Arthrobacter and Pseudomonas), (2) dormant ametabolic (anabiotic) cells of such prokaryotes as archaea and non-spore-forming bacteria, (3) endospores of bacilli, (4) streptomycete exospores, and (5) in the cells surviving in permafrost for (2‒3 Ma). The topics discussed include nucleoid biocrystallization as a necessary stage of maturation of the dormant microbial cells providing for survival and preservation of the species, dynamics of nucleoid biocrystallization during maturation of the dormant cells, and its possible role for the preservation of genetic information in the case of autolysis of most of the cells in a developing culture.

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.

Optimization of sporicidal activity and environmental Bacillus endospores decontamination by biogenic silver nanoparticle.

The intent of this study is to decontaminate Bacillus endospores and to determine the D-values using silver nanoparticles (AgNPs) synthesized from Streptomyces sp. cell filtrate. AgNPs synthesis was performed extracellularly followed by characterization using spectrophotometer, High Resolution Transmission Electron Microscope and x-ray diffraction pattern analysis. Subsequently, the optimized conditions for the decontamination and D-value estimation of Bacillus endospores were determined using the response surface methodology. The environmental spore decontamination study was performed in mice model. AgNPs were visibly and spectroscopically identified which were spherical with the size range of less than 20 nm. The synthesized AgNPs destroyed 1log10 CFU Bacillus endospores at around 20 min. The adherence of AgNPs to the surface of spore coat, pit formation and its complete structural loss was detected under field emission scanning electron microscopy. All the mice exposed to AgNP-treated spores showed no sign of pathological lesions. The results of our study strongly suggest that the application of AgNPs as a sporicidal agent could be a new approach in consistently eliminating the hazardous Bacillus spores.

A pulsed light system for the disinfection of flow through water in the presence of inorganic contaminants.

The use of ultraviolet (UV) light for water disinfection has become increasingly popular due to on-going issues with drinking water and public health. Pulsed UV light has proved to be an effective form of inactivating a range of pathogens including parasite species. However, there are limited data available on the use of pulsed UV light for the disinfection of flowing water in the absence or presence of inorganic contaminants commonly found in water sources. Here, we report on the inactivation of test species including Bacillus endospores following pulsed UV treatment as a flow through system. Significant levels of inactivation were obtained for both retention times tested. The presence of inorganic contaminants iron and/or manganese did affect the rate of disinfection, predominantly resulting in an increase in the levels of inactivation at certain UV doses. The findings of this study suggest that pulsed UV light may provide a method of water disinfection as it successfully inactivated bacterial cells and bacterial endospores in the absence and presence of inorganic contaminants.

Life cycle and spore resistance of spore-forming Bacillus atrophaeus

Bacillus endospores have a wide variety of important medical and industrial applications. This is an overview of the fundamental aspects of the life cycle, spore structure and factors that influence the spore resistance of spore-forming Bacillus. Bacillus atrophaeus was used as reference microorganism for this review because their spores are widely used to study spore resistance and morphology. Understanding the mechanisms involved in the cell cycle and spore survival is important for developing strategies for spore killing; producing highly resistant spores for biodefense, food and pharmaceutical applications; and developing new bioactive molecules and methods for spore surface display.

Determination of Oxygen Respiration Rates in Wetted Developmentally Arrested Spores of Streptomyces Species

Streptomyces species produce spores, which, while not as robust as endospores of Bacillus or Clostridium species, are capable of surviving for months or even years (Hopwood, 2006). During this time these spores remain viable, surviving by slowly degrading internal stores of carbon compounds, such as the carbohydrate trehalose. To enable metabolism to continue they must have access to an electron acceptor that allows the removal of the reducing equivalents that accumulate through metabolic activity. The most commonly used acceptor is oxygen. We describe the quantitative measurement of oxygen respiration rates by developmentally arrested spores of the streptomycete Streptomyces coelicolor (Fischer et al., 2013).

Extraction of Bacillus endospores from water, apple juice concentrate, raw milk and lettuce rinse solutions using tangential flow filtration

As part of investigative sampling there is a need to rapidly concentrate the target from large sample volumes to enable downstream detection using suitable diagnostic platforms. To this end the following describes a method based on tangential flow filtration (0.2 μm pore size) for concentrating Bacillus endospores from sample matrices (water, raw whole milk, apple juice concentrate and lettuce rinse samples). Bacillus endospores were selected as a model system as a representative select agent of significance. From optimization studies it was found that the recovery of endospores was in the order of 2.5% and could not be enhanced through increasing the flow rate or transmembrane pressure (TMP). However, recoveries could be increased to >80% (concentration factor 6.74–8.86) by backwashing the system with 10% w/v Tween 80 solution to detach spores adsorbed onto the internal surface of the filtration unit. The recovery of endospores from raw milk and apple juice concentrate was problematic due to the presence of solids or high viscosity. However, by performing an initial dilution (1:10) prior to passing through the Tangential Flow Filtration (TFF) system it was possible to achieve over 80% endospore recovery with no significant decrease in filter performance (Concentration Factor of 6–10). The recovery of endospores from lettuce surfaces was facilitated by rinsing with 4% w/v Tween 80 solution. When used in conjunction with tangential flow filtration system it was possible to recover endospores derived from lettuce rinse solutions at levels in the order of 300 cfu/ml. The results of the study illustrate that TFF is a useful approach for concentrating microbial targets from a diverse range of food matrices.

OPTIMIZATION OF THE CULTURE MEDIUM FOR ENTOMOPATHOGENIC BACTERIA OF STRAIN BACILLUS THURINGIENSIS ONU 15

With the use of experimental design methods there were optimized the culture medium for the cultivation of strain Bacillus thuringiensis ONU 15, having entomopathogenic properties against dipteran pests of edible fungi. Optimization of culture medium was performed using the central composite orthogonal method. The parameters of optimization were the total number of bacteria and the number of endospores of strain Bacillus thuringiensis ONU 15. As a result of the research it is proposed the culture medium of the following composition: peptone – 7.3 g/l , yeast extract – 3.0 g/l, glucose – 2.5 g/l, K2HPO4 – 5.0 g/l, KH2PO4 – 5.0 g/l, Na citrate – 3.0 g/l, Na2HPO4 – 1.5 g/l, MgSO4 – 0.05 g/l, MnSO4 – 0.03 g/l, CaCl2 – 0.05 g/l. At the optimized culture medium the total number of bacteria, compared with the original culture medium, increased by 7 times and has reached the indicator – 184.6± 6.9х1010 CFU/ml.

Efficacy of Using HarmlessBacillusEndospores to Estimate the Inactivation ofCryptosporidium parvumOocysts in Water

The need to use complex in vitro cell culture, expensive equipment, and highly-trained technicians that are available only to specialist laboratories has significantly limited studies assessing the potential of pulsed UV light (PUV) to inactivate the waterborne parasite Cryptosporidium parvum in drinking water. This constitutes the first study to report on the use of different non-pathogenic Bacillus endospores as potential surrogate organisms to indicate the PUV inactivation performance of a C. parvum oocyst suspended in water. Findings showed that PUV effectively inactivated approximately 5 log10 CFU/ml Bacillus megaterium and Bacillus pumilus endospores suspended in water at a UV dose of 9.72 μJ/cm(2) that also inactivated statistically similar levels of C. parvum oocysts (P < 0.05), as determined by combined in vitro HCT-8 cell culture and quantitative PCR. Specifically, this study demonstrated that B. megaterium exhibited greater or similar PUV-inactivation kinetic data compared to that of similarly treated C. parvum over the UV dose range 6.4 to 12.9 μJ/cm(2). Therefore, the former may be used as an indicator organism for safely investigating the PUV-inactivation performance of this chlorine-resistant, waterborne parasite at the waste-water treatment plant level. Findings presented will impact positively on future water quality studies and on public health.

Identification of Bacillus anthracis via Raman Spectroscopy and Chemometric Approaches

Raman micro-spectroscopy was applied to compile a large-scale database of Raman spectra of single Bacillus endospores and to calculate classification functions, which were trained to discriminate between endospores of 66 strains from 13 Bacillus and Bacillus-related species including B. anthracis. The developed two-stage classification system comprising two support vector machines and one linear discriminant analysis classifier was then challenged by a test set of 27 samples to simulate the case of a real-world-scenario, when "unknown samples" are to be identified. In the end, all 27 test set samples including six B. anthracis strains were identified correctly. The samples thereby covered a diverse selection of species within the phylogenetically broad Bacillus genus and also included strains, which were not incorporated in the database before. All of them were correctly identified on the species level with accuracies between 88 and 100%. The sample analysis itself requires no biomass enrichment step prior to the analysis and qualifies the presented Raman spectroscopic approach to be a rapid analysis system in term of Bacillus endospore typing.

Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

The electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates was measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here provide new insight for Bacillus anthracis surrogate selection and for attachment/detachment and transport studies.

Raman Spectroscopic Detection of Anthrax Endospores in Powder Samples

Polymerase chain reaction (PCR) is becoming the method of choice for detecting microorganisms concealed in complex matrices or “hoax materials” like household dry powders, food, and soil.[1] However, adding samples directly to a PCR reaction is in most cases not possible because of the presence of PCR inhibitors in the sample.[2] Thus, in order to reliably use PCR, one must either enrich the culture prior to the analysis, which is time-consuming for fastidious organisms, or extract the total DNA directly from the sample, which requires an extraction technique capable of processing different sample types. However, since most DNA extraction methods are often not generic in that sense, they lead to unreliable DNA recovery yields.[2, 3]