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Milk rice pudding (MRP) is a commercial and popular dairy dessert, but owing to its characteristics and valuable ingredients, it may be contaminated by many pathogenic and spoilage microorganisms. So, this study aimed to improve the quality and safety of MRP by using cinnamon and clove essential oils. Concerning the evaluation of the minimum inhibitory concentration (MIC) for both oils with 0.2, 0.5, and 1% concentrations, B. cereus and B. subtilis were sensitive (+) to cinnamon and clove 0.5%, with inhibition zones of 13.3 and 14 mm for cinnamon and 11.3 and 12 mm for clove EO, respectively. While both bacteria were very sensitive (++) to cinnamon 1% (18.8 and 19.5 mm) and clove 1% (17.3 and 18.7 mm), respectively. Therefore, MRP was prepared by adding cinnamon and clove EOs at 0.6%. Treatments containing EOs showed a significant reduction of tested microorganisms compared to controls. B. cereus wasn’t detected in clove and cinnamon EO treatments at day 21 of the storage period, while B. subtilis vanished on day 14 for the cinnamon treatment and on day 21 for clove MRP. Moreover, the results revealed the enhancement of sensory characteristics of MRP supplemented with EOs without any significant alteration in their pH values. This study recommends the addition of cinnamon and clove EOs (0.6%) to MRP, as it isn’t only an excellent substitution of chemical preservatives with powerful antibacterial efficiency but also improves the overall acceptance of the product.

The spread of grape leaf diseases has a negative impact on the sustainable development of agriculture. Diseases induced by Uncinula necator significantly affect the quality of grapes. Bacillus biocontrol agents have been proven effective in disease management. However, limited research has been conducted on the impact of biocontrol agents on the assembly and potential functions of plant phyllosphere microbial communities. This study used high-throughput sequencing combined with bioinformatics analysis and culture omics technology for analysis. The results showed that biocontrol bacteria B. subtilis utilized in this study can significantly reduce the disease index of powdery mildew (p

Background: Meju is a base material for making soy sauce, soybean paste, and red chili pepper paste, which are representative ingredients of Korean cuisine. Objectives: This study aimed to isolate a predominant bacterial strain of B. subtilis from meju and to observe its inhibitory effects on an aflatoxigenic mold. Methods: We used yellow soybeans (Glycine max (L.) Merr.) grown in South Korea, and meju was produced according to the recommended methods of the Korea Food Research Institute. The identification of the strain was conducted based on its morphological and biochemical characteristics and 16S rDNA sequence. Evaluation of the bacterial effect against A. parasiticus ATCC 15517 was done in yeast extract–sucrose broth at 28 °C. Its inhibitory effect was evaluated using two approaches: mycelial weight and aflatoxin production. Aflatoxins were determined using high-performance liquid chromatography. Results: In the meju samples fermented for three months, a B. subtilis strain, K-0924 was identified. At the end of the incubation period of A. parasiticus, dry mycelial weight was significantly reduced by more than 80% (p < 0.01) and total aflatoxin production was inhibited by more than 63% (p < 0.05) in the presence of B. subtilis. Conclusions: These results indicate that B. subtilis K-0924 inhibits the growth and aflatoxin production of toxigenic Aspergillus, which can be contaminated with meju. We could expect more inhibition by other bacteria related to fermentation of meju, and further examination is necessary on species other than B. subtilis.

Peptones are one of the priciest culture components utilized in the cultivation of microorganisms. The objective of this work was to prepare feather hydrolysate peptone from chicken feathers using Bacillus subtilis ES5 (GenBank: OQ034477). The cell growth performances of all investigated microorganisms were shown to exhibit better results with feather hydrolysate peptone (FHP) in comparison to commercial peptone. As per the current findings, microbial-based FHP preparation is significantly less expensive than commercial peptones, and the suggested process provides cost-effective and eco-friendly solutions. Furthermore, the present investigation demonstrated opening the door to the prospect of converting chicken feather waste to peptone.

Combining probiotic bacteria is a promising strategy to increase the effectiveness and avoid side effects of probiotic therapy. Bacteria that find themselves in a common environment are able to both change their properties and show new ones under the influence of each other. The change of colonization and antagonistic properties, which provide bacteria with competitive advantages in the development of new spaces, deserves special attention. The aim of this research was to study the features of the mutual influence of probiotic bacilli: B. clausii, B. coagulans and B. subtilis on growth, swimming and swarming motility when co-cultivated on agar media of different solidity. Materials and methods. The study used commercial strains of bacilli from three probiotic preparations: Enterogermina, Lactovit forte and Subalin. The ability of the studied species of bacilli to influence each other’s growth was investigated by the agar block method (using 1.5 % nutrient agar) and the spot-on-lawn assay (using 0.7 % nutrient agar). The study of the mutual influence on swimming and swarming activity was carried out when bacilli were cultivated on 0.25 % and 0.70 % tryptone agar, respectively. The diameters of swimming halos and migration swarms formed by monoand mixed cultures were measured, and the areas covered by them were compared. The phenotypes of the meeting of swarms formed by cultures spotted on the swarm plates at different locations were also investigated. Results. The studied probiotic species of the bacilli did not show a strong ability to inhibit each other’s growth. The B. clausii culture had no inhibitory effect, and the B. coagulans culture demonstrated a moderate inhibitory influence on the growth of the other two species of bacilli when using both diffusion methods. The B. subtilis culture showed moderate or weak inhibitory activity against the B. clausii culture and weak or no inhibitory activity against the B. coagulans culture using the agar block method or spot-on-lawn assay, respectively. The B. coagulans + B. subtilis, B. clausii + B. coagulans and B. clausii + B. subtilis mixed cultures showed moderate, weak and no inhibitory activity against third cultures, respectively. The studied species of bacilli showed different swimming ability and swarming potential as well as the ability to influence each other’s motility. Swimming halos formed by the B. clausii + B. subtilis and B. clausii + B. сoagulans + B. subtilis mixed cultures covered significantly larger plate areas than the swimming halos formed by each culture separately during the same cultivation time. The highest swarming potential was observed in B. coagulans culture and B. cla usii + B. coagulans + B. subtilis mixed culture. The studied bacilli did not show the ability to merge swarms, but, on the contrary, their swarms at the point of contact formed visible “boundary” or “intermediate” lines, demonstrating the ability to identify nonself. Conclusions. The obtained results indicate the ability of the probiotic species of bacilli: B. clausii, B. coagulans and B. subtilis to mutually influence growth, swimming and swarm motility. The highest indicators of swimming and swarming of the triple mixed culture indicate an increase in the colonization potential of the studied bacilli when they are combined. The hypothesis of mutual induction of inhibitory compounds production by bacilli, which may increase the overall antagonistic potential of the triple mixed culture, is subject to further confirmation.

One of the latest trends in sustainable agriculture is the use of beneficial microorganisms to stimulate plant growth and biologically control phytopathogens. Bacillus subtilis, a Gram-positive soil bacterium, is recognized for its valuable properties in various biotechnological and agricultural applications. This study presents, for the first time, the successful encapsulation of B. subtilis within electrospun poly(3-hydroxybutyrate) (PHB) fibers, which are dip-coated with cellulose derivatives. In that way, the obtained fibrous biohybrid materials actively ensure the viability of the encapsulated biocontrol agent during storage and promote its normal growth when exposed to moisture. Aqueous solutions of the cellulose derivatives—sodium carboxymethyl cellulose and 2-hydroxyethyl cellulose, were used to dip-coat the electrospun PHB fibers. The study examined the effects of the type and molecular weight of these cellulose derivatives on film formation, mechanical properties, bacterial encapsulation, and growth. Scanning electron microscopy (SEM) was utilized to observe the morphology of the biohybrid materials and the encapsulated B. subtilis. Additionally, ATR-FTIR spectroscopy confirmed the surface chemical composition of the biohybrid materials and verified the successful coating of PHB fibers. Mechanical testing revealed that the coating enhanced the mechanical properties of the fibrous materials and depends on the molecular weight of the used cellulose derivatives. Viability tests demonstrated that the encapsulated B. subtilis exhibited normal growth from the prepared materials. These findings suggest that the developed fibrous biohybrid materials hold significant promise as biocontrol formulations for plant protection and growth promotion in sustainable agriculture.

ABSTRACT: In the current study, the proteolytic enzyme (serratiopeptidase) was used to enhance the efficacy of Bacillus subtilis (B. subtilis) probiotic as a growth promotor in broiler chicken. The effects of serratiopeptidase on the efficacy of different levels of B. subtilis as a growth promotor in broiler chicks were evaluated regarding growth performance traits, villus histomorphometric characterization, and intestinal microbiota count. Day-old broiler chicks (n = 120) were allocated into 4 groups having 3 replicates/group. In the control group (C), the basal diet was kept without supplementation. In treatment groups (P100, P150, and P200), the basal diet was supplemented with 100, 150, and 200 mg probiotics, respectively besides 30 mg proteolytic enzyme in the 3 treated groups for 4 wk. The performance parameters were significantly affected by the supplementation of serratiopeptidase to the B. subtilis treatment groups. Feed intake (FI), body weight gain (WG), feed conversion ratio (FCR), and dressing percent were significantly improved in the treatment groups as compared to the control group. Significantly, the lowest feed intake was recorded for the P200 group. The highest body weight gain and dressing percentage were recorded for the P200 group. An improved FCR was recorded in the P200 group (1.7) as compared to the control group. The different levels of B. subtilis supplemented with serratiopeptidase revealed significant improvements (P

Potassium deficiency is one of the important factors restricting cucumber growth and development. This experiment mainly explored the effect of Bacillus subtilis (B. subtilis) on cucumber seedling growth and the photosynthetic system under different potassium levels, and the rhizosphere bacteria (PGPR) that promote plant growth were used to solubilize potassium in soil, providing theoretical support for a further investigation of the effect of biological bacteria fertilizer on cucumber growth and potassium absorption. “Xinjin No. 4” was used as the test material for the pot experiment, and a two-factor experiment was designed. The first factor was potassium application treatment, and the second factor was bacterial application treatment. The effects of different treatments on cucumber seedling growth, photosynthetic characteristics, root morphology, and chlorophyll fluorescence parameters were studied. The results showed that potassium and B. subtilis had obvious promotion effects on the cucumber seedling growth and the photosynthesis of leaves. Compared with the blank control, the B. subtilis treatment had obvious effects on the cucumber seedling height, stem diameter, leaf area, total root length, total root surface area, total root volume, branch number, crossing number, gs, WUE, Ci, and A; the dry weight of the shoot and root increased significantly (p ≤ 0.05). Potassium application could significantly promote cucumber growth, and the effect of B. subtilis and potassium application was greater than that of potassium application alone, and the best effect was when 0.2 g/pot and B. subtilis were applied. In conclusion, potassium combined with B. subtilis could enhance the photosynthesis of cucumber leaves and promote the growth of cucumber.

Abstract Over 380 host plant species have been known to develop leaf spots as a result of the fungus Alternaria alternata. It is an aspiring pathogen that affects a variety of hosts and causes rots, blights, and leaf spots on different plant sections. In this investigation, the lipopeptides from the B. subtilis strains T3, T4, T5, and T6 were evaluated for their antifungal activities. In the genomic DNA, iturin, surfactin, and fengycin genes were found recovered from B. subtilis bacterium by PCR amplification. From different B. subtilis strains, antifungal Lipopeptides were extracted, identified by HPLC, and quantified with values for T3 (24 g/ml), T4 (32 g/ml), T5 (28 g/ml), and T6 (18 g/ml). To test the antifungal activity, the isolated lipopeptides from the B. subtilis T3, T4, T5, and T6 strains were applied to Alternaria alternata at a concentration of 10 g/ml. Lipopeptides were found to suppress Alternaria alternata at rates of T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). The T6 strain outperformed the other three by having the highest antifungal activity against Alternaria alternata (85.88%).

ABSTRACT The effects of surface mechanical constraints that may promote or prevent bacterial expansion on semi-solid surfaces are largely unknown. In this work, we have manufactured agar surfaces with different viscoelasticity, topography, and roughness. To capture the essential biophysics of the bacterial expansion we have developed a continuum model that faithfully reproduces the main patterns of the short-range and long-range expansion with two critical parameters: local interfacial forces and colony viscosity. Cohesive energy of the bacterial colony that determines the extent of exploration was dependent on agar surface viscoelasticity. On soft surfaces, bacteria produce low viscoelastic colonies that allow guided population of bacteria to traverse distances that are six orders of magnitude larger than the size of the individual bacterium. Bacteria growing on stiff surfaces produce colonies with significantly increased viscoelasticity that prevent bacterial exploration of new territory and allow formation of a very steep cliff at the edge of the colony. Upon flooding of the rough surfaces, we have induced aquaplaning and spreading of bacteria. A layer of water between the bacterium and surface results in a loss of traction allowing bacteria to spread across the otherwise inhibitory rough surface. The results shed new light on the bacterial ability to rapidly colonize new territories. IMPORTANCE How bacterial cells colonize new territory is a problem of fundamental microbiological and biophysical interest and is key to the emergence of several phenomena of biological, ecological, and medical relevance. Here, we demonstrate how bacteria stuck in a colony of finite size can resume exploration of new territory by aquaplaning and how they fine tune biofilm viscoelasticity to surface material properties that allows them differential mobility. We show how changing local interfacial forces and colony viscosity results in a plethora of bacterial morphologies on surfaces with different physical and mechanical properties.

Microorganisms are integral components of ecosystems, exerting profound impacts on various facets of human life. The recent United Nations General Assembly (UNGA) Science Summit emphasized the critical importance of comprehending the microbial world to address global challenges, aligning with the United Nations Sustainable Development Goals (SDGs). In agriculture, microbes are pivotal contributors to food production, sustainable energy, and environmental bioremediation. However, decades of agricultural intensification have boosted crop yields at the expense of soil health and microbial diversity, jeopardizing global food security. To address this issue, a study in West Bengal, India, explored the potential of a novel multi-strain consortium of plant growth promoting (PGP) Bacillus spp. for soil bioaugmentation. These strains were sourced from the soil’s native microbial flora, offering a sustainable approach. In this work, a composite inoculum of Bacillus zhangzhouensis MMAM, Bacillus cereus MMAM3), and Bacillus subtilis MMAM2 were introduced into an over-exploited agricultural soil and implications on the improvement of vegetative growth and yield related traits of Gylcine max (L) Meril. plants were evaluated, growing them as model plant, in pot trial condition. The study’s findings demonstrated significant improvements in plant growth and soil microbial diversity when using the bacterial consortium in conjunction with vermicompost. Metagenomic analyses revealed increased abundance of many functional genera and metabolic pathways in consortium-inoculated soil, indicating enhanced soil biological health. This innovative bioaugmentation strategy to upgrade the over-used agricultural soil through introduction of residual PGP bacterial members as consortia, presents a promising path forward for sustainable agriculture. The rejuvenated patches of over-used land can be used by the small and marginal farmers for cultivation of resilient crops like soybean. Recognizing the significance of multi-strain PGP bacterial consortia as potential bioinoculants, such technology can bolster food security, enhance agricultural productivity, and mitigate the adverse effects of past agricultural activities.

Abstract Commercial carotenoid production is dominated by chemical synthesis and plant extraction, both of which are unsustainable and can be detrimental to the environment. A promising alternative for the mass production of carotenoids from both an ecological and commercial perspective is microbial synthesis. To date, C30 carotenoid production in Bacillus subtilis has been achieved using plasmid systems for the overexpression of biosynthetic enzymes. In the present study, we employed a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system to develop an efficient, safe, and stable C30 carotenoid-producing B. subtilis strain, devoid of plasmids and antibiotic selection markers. To this end, the expression levels of crtM (dehydrosqualene synthase) and crtN (dehydrosqualene desaturase) genes from Staphylococcus aureus were upregulated by the insertion of three gene copies into the chromosome of B. subtilis. Subsequently, the supply of the C30 carotenoid precursor farnesyl diphosphate (FPP), which is the substrate for CrtMN enzymes, was enhanced by expressing chromosomally integrated Bacillus megaterium-derived farnesyl diphosphate synthase (FPPS), a key enzyme in the FPP pathway, and abolishing the expression of farnesyl diphosphate phosphatase (YisP), an enzyme responsible for the undesired conversion of FPP to farnesol. The consecutive combination of these features resulted in a stepwise increased production of C30 carotenoids. For the first time, a B. subtilis strain that can endogenously produce C30 carotenoids has been constructed, which we anticipate will serve as a chassis for further metabolic engineering and fermentation optimization aimed at developing a commercial scale bioproduction process.

Treatment of wastewater has been a hotspot of research since ages. Emerging technologies and methodologies have been postulated to resolve the issue worldwide. Almost 97.2% of our earth is covered with water bodies, out of which 12,500 water bodies are situated in India. As per the statistics, nearly 70% water bodies are polluted in Southern Asia. Presence of emerging pollutants exacerbate the quality of flowing water. Amongst all possible ways, microbial bioremediation has been considered one of the most thriving methods to treat wastewater. This research will manifest about the isolation of B. subtilis from soil followed by its characterisation and action in treatment of wastewater which was collected from industry. From the study, it was concluded that B. subtilis holds the potential of degradation. Significant decrease in values of BOD and COD were achieved.

Introduction: The number of published biofilm studies and novel ways for studying them has risen dramatically in recent years, ow-ing to the broad application of biofilms in medicine. Some bacteria develop biofilms that are highly resistant to antimicrobial agents, resulting in persistent infections. This necessitates the development of alternative methods for combating biofilms. In this regard, the application of cationic polymers is a good candidate for realization of this strategy. Aim: The aim of our study was to investigate the potential of a newly synthesized covalently attached star copolymer of N,N’-dimeth-ylaminoethyl methacrylate and hydroxyl-bearing oligo(ethylene glycol) methacrylate [P(DMAEMA-co-HOEGMA)] to silica surfaces and its quaternized version [P(QDMAEMA-co-HOEGMA)] for destruction of biofilms formed by Bacillus subtilis or Pseudomonas aeruginosa. Materials and methods: Model strains representing different genera and taxonomic groups were selected for the study. The anti-biofilm activities of two different newly synthesized cationic polymers were investigated by observation (live/dead staining) of the viability of bacterial cells within the biofilm.Results: The results obtained by the live/dead labeling of bacterial biofilms show a substantial decrease in the viability of population in the presence of cationic polymers, better expressed at B. subtilis.Conclusions: The studied two immobilized on silica wafers newly synthesized star copolymers exhibited potential for anti-biofilm effects. The results demonstrated combined potential for reducing the viability of bacterial cells within the biofilms and probably for loosening the biofilm matrix. The effect was better expressed in B. subtilis.

Abstract Background Despite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming. Results In this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes. Conclusions The novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.

Microorganisms promoted corrosion has caused significant loss to marine engineering and the antibacterial coatings have served as a solution that has gained attention. In this study, the chemical vapour deposition technique has been employed to grow three different types of diamond coatings, namely, ultrananocrystalline diamond (UNCD), nanocrystalline diamond (NCD), and microcrystalline diamond (MCD) coatings. The evolution of associated surface morphology and the surface functional groups of the grown coatings have demonstrated antibacterial activity in seawater environments. It is found that different ratio of sp3/sp2 carbon bonds on the diamond coatings influences their surface property (hydrophobic/hydrophilic), which changes the anti-adhesion behaviour of diamond coatings against bacteria. This plays a critical role in determining the antibacterial property of the developed coatings. The results show that the diamond coatings arising from the deposition process kill the bacteria via a combination of the mechanical effects and the functional groups on the surface of UNCD, NCD, and MCD coatings, respectively. These antibacterial coatings are effective to both Gram-negative bacteria (E. coli) and Gram-positive bacteria (B. subtilis) for 1–6 h of incubation time. When the contact duration is prolonged to 6 h or over, the MCD coatings begin to reduce the bacteria colonies drastically and enhance the bacteriostatic rate for both E. coli and B. subtilis.

Despite the exploration towards the environmentally friendly manufacturing process, two main challenges remain for lactic acid synthesis by microbial fermentation: resource cost and production efficiency. To alleviate environmental issues, lower raw material costs, and increase lactic acid manufacturing efficiency, this work aimed to evaluate characteristics of jackfruit seed starch (JFSS) extracted from fruit waste and explore the efficiency of lactic acid generation by co-fermentation of L. plantarum and B. subtilis in JFSS. JFSS consisted of a high amount of resistant starch and amylose, which was thermostable and displayed type A x-ray crystallization. Co-fermentation using JFSS as a substrate stimulated the growth of L. plantarum and enhanced lactic acid production over monocultures of L. plantarum and B. subtilis with insignificant by-product output. This study was valuable for improving lactic acid production efficiency and exploring the interaction between L. plantarum and B. subtilis.

The development of efficient, eco-friendly antimicrobial agents for air purification and disinfection addresses public health issues connected to preventing airborne pathogens. Herein, the antimicrobial activity of a nanoemulsion (control, 5%, 10%, and 15%) containing neem and lavender oils with polycaprolactone (PCL) was investigated against airborne bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Various parameters such as the physicochemical properties of the nanoemulsion, pH, droplet size, the polydispersity index (PDI), the minimum inhibitory concentration (MIC), the minimum bacterial concentration (MBC), and the color measurement of the emulsion have been evaluated and optimized. Our results showed that the antimicrobial activity of PCL combined with neem and lavender oil was found to be the highest MIC and MBC against all tested bacteria. The droplet sizes for lavender oil are 21.86–115.15 nm, the droplet sizes for neem oil are 23.92–119.15 nm, and their combination is 25.97–50.22 nm. The range of pH and viscosity of nanoemulsions of various concentrations was found to be 5.8 to 6.6 pH and 0.372 to 2.101 cP. This study highlights the potential of nanotechnology in harnessing the antimicrobial properties of natural essential oils, paving the way for innovative and sustainable solutions in the fight against bacterial contamination.

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications.

The objective of the present study was to test the hypothesis of B. subtilis and B. licheniformis supplementation to a negative control diet in comparison to a standard control diet, had the potential to improve the performance and nutrient digestibility of growing–finishing pigs. For this purpose, 384 fattening pigs of 85 d of age were allotted to three treatments: a standard diet, a negative control (NC) diet (5% soybean meal replaced by 5% rapeseed meal), or a NC diet + probiotic. After reaching a body weight of approximately 110 kg, all animals going to the slaughterhouse (87% of total pigs) were selected to measure carcass quality. Moreover, the apparent total tract digestibility of protein was evaluated at the end of the grower period. The results of this study indicate that supplementation of the tested Bacillus-based probiotic significantly improved average daily gain (ADG, +14.6%) and Feed:gain ratio (F:G, −9.9%) during the grower phase compared to the NC diet. The improvement observed during the grower phase was maintained for the whole fattening period (ADG, +3.9%). Probiotic supplementation significantly improved the total apparent faecal digestibility of dry matter and crude protein in pigs at the end of the grower period. The improvements observed with the additive tested could indicate that supplementation of the Bacillus-based probiotic was able to counteract the lower level of crude protein and standardised ileal digestible amino acids in the NC diet by means of improved protein digestibility.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease that causes significant yield losses in wheat production worldwide, including in Egypt. The use of biocontrol agents is among the best eco-friendly management strategies to control this disease, as they are more sustainable and environmentally friendly than traditional chemical control methods. In a comparative analysis, antioxidant enzyme activity and various management approaches were compared with two bacterial biocontrol agents, Bacillus subtilis and Pseudomonas putida. This study showed the remarkable efficacy of endophytic bacteria, B. subtilis and P. putida, in mitigating wheat stripe rust infection across three wheat varieties, namely Misr1, Gimmeiza11, and Sids12. B. subtilis exhibited superior performance compared to P. putida, resulting in infection types of 1 and 2.66, respectively, following inoculation. The highest reduction rate was observed with Tilit fungicide (500 ppm), followed by B. subtilis and Salicylic acid (1000 ppm), respectively. Variations in wheat varieties’ response to Pst infection were observed, with Misr1 exhibiting the lowest infection and Sids12 showing high susceptibility. Among the tested inducers, Salicylic acid demonstrated the greatest reduction in disease infection, followed by Indole acetic acid, while Oxalic acid exhibited the lowest decrease. Additionally, the study evaluated the activities of five antioxidant enzymes, including Catalase, Ascorbate peroxidase (APX), glutathione reductase (GR), Superoxide dismutase (SOD), and peroxidase (POX), in the wheat-stripe rust interaction under different integrated management approaches. The wheat variety Misr1 treated with Tilit (500 ppm), B. subtilis, Salicylic acid, Montoro (500 ppm), and P. putida exhibited the highest increase in all enzymatic activities. These findings provide valuable insights into the effectiveness of B. subtilis and P. putida as biocontrol agents for wheat stripe rust control in Egypt, emphasizing their potential role in sustainable, integrated, and environmentally friendly management practices.

Abstract Contamination of water with bacteria is one of the main causes of waterborne diseases. The photocatalytic method on the basis of bacterial inactivation seems to be a suitable disinfectant due to the lack of by-products formation. Herein, g-C3N4/Fe3O4/Ag nanocomposite combined with UV-light irradiation was applied for the inactivation two well-known bacteria namely, E. coli and B. subtilis. The nanocomposite was prepared by a hydrothermal method, and subsequently it was characterized by XRD, FT-IR, SEM, EDX and PL analyses. The optimum conditions established for the inactivation of both bacteria were as follows: nanocomposite dosage 3 g/L and bacterial density of 103 CFU/mL. In the meantime, the efficient inactivation of E. coli and B. subtilis took 30 and 150 min, respectively. The results also revealed that inactivation rate dropped with an increase in the bacterial density. It is also pointed out that OH˚ was found out to be the main radical species involved in the inactivation process. Finally, the kinetic results indicated that the inactivation of E. coli and B. subtilis followed the Weibull model. It is concluded that C3N4/Fe3O4/Ag nanocomposite along with UV-light irradiation is highly effective in inactivating E. coli and B. subtilis bacteria in the aqueous solutions.

Date palm root rot disease is one of the most important diseases of date palms and offshoots. It is caused by many soil-borne pathogenic fungi. Pathogenicity assays of the isolated fungi showed that the major causative agents of root rot disease in date palm plantlets were Fusarium oxysporum Schlecht. emend. Snyder & Hansen, F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg S1, F. proliferatum S2, Gibberella fujikuroi (Sawada) Wollenw., and Rhizoctonia solani J.G. Kühn. The most virulent fungus was F. oxysporum with a severity index of 82.16 % of root rot, while R. solani was the least harmful with a disease severity rate of 12.42 %. In laboratory tests, Bacillus subtilis reduced the radial mycelial growth of F. oxysporum on PDA medium by 86.6 %. The application of B. subtilis in combination with F. oxysporum substantially inhibited the severity of root rot disease relative to plantlets treated with only F. oxysporum. In addition, B. subtilis application in the presence or absence of F. oxysporum improved the plant physiology of plantlets, including total chlorophyll, total carotenoid, antioxidant enzyme levels (catalase and peroxidase), and total proline content.

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening > 2,000 extracts from a diverse collection of actinobacteria, we identified a compound, mirubactin C, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed using synthetic mirubactin C. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin C rescuing activity is not due simply to iron sequestration. The results support an emerging view that the functions of bacterial siderophores extend well beyond simply iron binding and uptake.

Microorganisms pose a serious threat for us humans, which is exemplified by the recent emergence of pathogens such as SARS-CoV-2 or the increasing number of multi-resistant pathogens such as MRSA. To control surface microorganisms and viruses, we investigated the disinfection properties of an AI-controlled robot, HERO21, equipped with eight 130-W low pressure UV-C mercury vapor discharge lamps emitting at a wavelength of 254 nm, which is strongly absorbed by DNA and RNA, thus inactivating illuminated microorganisms. Emissivity and spatial irradiance distribution of a single UV-C lamp unit was determined using a calibrated spectrometer and numerical simulation, respectively. The disinfection efficiency of single lamps is determined by microbiological tests using B. subtilis spores, which are known to be UV-C resistant. The required time for D99 disinfection and the corresponding UV-C irradiance dose amount to 60 s and 37.3 mJ•cm−2 at a distance of 1 m to the Hg-lamp, respectively. Spatially resolved irradiance produced by a disinfection unit consisting of eight lamps is calculated using results of one UV-C lamp characterization. This calculation shows that the UV-C robot HERO21 equipped with the mentioned UV-C unit causes an irradiance at λ=254 nm of 2.67 mJ•cm−2•s−1 at 1 m and 0.29 mJ•cm−2•s−1 at 3 m distances. These values result in D99 disinfection times of 14 s and 129 s for B. subtilis spores, respectively. Similarly, human coronavirus 229E, structurally very similar to SARS-CoV-2, could be efficiently inactivated by 3–5 orders of magnitude within 10 - 30 s exposure time or doses of 2 - 6 mJ•cm−2, respectively. In conclusion, with the development of the HERO21 disinfection robot, we were able to determine the inactivation efficiency of bacteria and viruses on surfaces under laboratory conditions.

Bacillus subtilis is traditionally classified as a PGPR that colonizes plant roots through biofilm formation. The current study focused on investigating the influence of various factors on bacilli biofilm formation. In the course of the study, the levels of biofilm formation by the model strain B. subtilis WT 168 and on its basis created regulatory mutants, as well as strains of bacilli with deleted extracellular proteases under conditions of changes in temperature, pH, salt and oxidative stress and presence of divalent metals ions. B. subtilis 168 forms halotolerant and oxidative stress-resistant biofilms at a temperature range of 22 °C–45 °C and a pH range of 6–8.5. The presence of Ca2+, Mn2+ and Mg2+ upsurges the biofilm development while an inhibition with Zn2+. Biofilm formation level was higher in protease-deficient strains. Relative to the wild-type strain, degU mutants showed a decrease in biofilm formation, abrB mutants formed biofilms more efficiently. spo0A mutants showed a plummeted film formation for the first 36 h, followed by a surge after. The effect of metal ions and NaCl on the mutant biofilms formation is described. Confocal microscopy indicated that B. subtilis mutants and protease-deficient strains differ in matrix structure. The highest content of amyloid-like proteins in mutant biofilms was registered for degU-mutants and protease-deficient strains.

Increasing demand for carbon neutrality has led to the development of new techniques and modes of low carbon production. The utilization of microbiology to convert low-cost renewable resources into more valuable chemicals is particularly important. Here, we investigated the ability of a cellulolytic bacterium, Bacillus subtilis 1AJ3, in switchgrass lignocellulose degradation. After 5 days of culture with the strain under 37°C, cellulose, xylan, and acid-insoluble lignin degradation rates were 16.13, 14.24, and 13.91%, respectively. Gas chromatography–mass spectrometry (GC-MS) analysis and field emission scanning electron microscopy (FE-SEM) indicated that the lignin and surface of switchgrass were degraded after incubation with the bacterial strain. Strain 1AJ3 can grow well below 60°C, which satisfies the optimum temperature (50°C) condition of most cellulases; subsequent results emphasize that acid-heat incubation conditions increase the reducing sugar content in a wide range of cellulosic biomass degraded by B. subtilis 1AJ3. To obtain more reducing sugars, we focused on β-glycoside hydrolase, which plays an important role in last steps of cellulose degradation to oligosaccharides. A β-glycoside hydrolase (Bgl-16A) was characterized by cloning and expression in Escherichia coli BL21 and further determined to belong to glycoside hydrolase (GH) 16 family. The Bgl-16A had an enzymatic activity of 365.29 ± 10.43 U/mg, and the enzyme’s mode of action was explained by molecular docking. Moreover, the critical influence on temperature (50°C) of Bgl-16A also explained the high-efficiency degradation of biomass by strain under acid-heat conditions. In terms of potential applications, both the strain and the recombinant enzyme showed that coffee grounds would be a suitable and valuable substrate. This study provides a new understanding of cellulose degradation by B. subtilis 1AJ3 that both the enzyme action mode and optimum temperature limitation by cellulases could impact the degradation. It also gave new sight to unique advantage utilization in the industrial production of green manufacturing.

Although antibiotics are valuable drugs for treatment of bacterial and some parasitic infections, their presence in animal products have a potential public health hazard. This study investigated antibiotic residues in goat raw milk and thermal effect on residues. Samples were collected randomly from different farms and retail outlets in Erbil city from January 1st to June 30th 2019. The residues were detected by disc diffusion assay against Bacillus subtilis bacteria on Muller Hinton agar. The occurrence rates among milk samples was 14.9%, also these residues in the samples was 13.5% and 16.4% in farm samples and retail store samples, respectively. No significant differences were found between sampling sites (farms vs sale points). Regarding the seasonal variations, spring was found to be associated with gradual decrease in antibiotic residues frequency in milk. Boiling for 5 minutes was the most effective treatment (among pasteurization and microwave heating) that inactivated antibiotic residues in 57.7% of positive samples. Such occurrence rate of residues is alarming and require authorities to observe and validate the quality of raw milk introduced to markets for consumers. Further evaluation of antibiotic stability period in raw milk is highly recommended.

Probiotics, also referred to as “living microorganisms,” are mostly present in the genitals and the guts of animals. They can increase an animal’s immunity, aid in digestion and absorption, control gut microbiota, protect against sickness, and even fight cancer. However, the differences in the effects of different types of probiotics on host gut microbiota composition are still unclear. In this study, 21-day-old specific pathogen-free (SPF) mice were gavaged with Lactobacillus acidophilus (La), Lactiplantibacillus plantarum (Lp), Bacillus subtilis (Bs), Enterococcus faecalis (Ef), LB broth medium, and MRS broth medium. We sequenced 16S rRNA from fecal samples from each group 14 d after gavaging. According to the results, there were significant differences among the six groups of samples in Firmicutes, Bacteroidetes, Proteobacteria, Bacteroidetes, Actinobacteria, and Desferribacter (p < 0.01) at the phylum level. Lactobacillus, Erysipelaceae Clostridium, Bacteroides, Brautella, Trichospiraceae Clostridium, Verummicroaceae Ruminococcus, Ruminococcus, Prevotella, Shigella, and Clostridium Clostridium differed significantly at the genus level (p < 0.01). Four kinds of probiotic changes in the composition and structure of the gut microbiota in mice were observed, but they did not cause changes in the diversity of the gut microbiota. In conclusion, the use of different probiotics resulted in different changes in the gut microbiota of the mice, including genera that some probiotics decreased and genera that some pathogens increased. According to the results of this study, different probiotic strains have different effects on the gut microbiota of mice, which may provide new ideas for the mechanism of action and application of microecological agents.

Abstract Background Cellulases are well known for their various industrial applications. They are naturally produced by different species of bacteria and fungi. Fermentation process of cellulase producers has limitation due to the high substrate cost required for cellulase induction and challenges to maintain the suitable condition for the respective cellulase production. Recombinant cellulase production could be the potential solution to these problems. In the current study, we investigated recombinant cellulase expression in Escherichia coli using cellulase gene from Bacillus subtilis. Results Extracellular cellulase production from B. subtilis strain was first confirmed on CMC agar and then the cellulase gene (1500 bp) was amplified from this strain and was further cloned in pET21a expression vector. In initial experimental studies, recombinant cellulase expression was achieved in inclusion bodies through shake flask level fermentation of transformed E. coli expression host BL21DE3. Attempts were made to express this 55 KDa His tagged recombinant cellulase into soluble form by modifications in fermentation conditions. Partially purified recombinant cellulase was obtained using Ni-NTA affinity chromatography. The activity of the purified enzyme was confirmed by 3,5-dinitrosalicylic acid (DNS) qualitative assay. Conclusion Soluble expression of active recombinant cellulase can be achieved by subtle alteration in the upstream process.

Chromium of anthropogenic origin contaminates the environment worldwide. The toxicity of chromium, a group I human carcinogen, is greatest when it is in a hexavalent oxidation state, Cr(VI). Cr(VI) is actively transported into the cell, triggering oxidative damage intracellularly. Due to the abundance of unspecific intracellular reductants, any microbial species is capable of bio-transformation of toxic Cr(VI) to innocuous Cr(III), however, this process is often lethal. Only some bacterial species are capable of sustaining the vegetative growth in the presence of a high concentration of Cr(VI) and thus operate as self-sustainable bioremediation agents. One of the successful microbial Cr(VI) detoxification strategies is the activation of chromate efflux pumps. This work describes transplantation of the chromate efflux pump from the potentially pathogenic but highly Cr resistant Bacillus pseudomycoides environmental strain into non-pathogenic but only transiently Cr tolerant Bacillus subtilis strain. In our study, we compared the two Bacillus spp. strains harboring evolutionarily diverged chromate efflux proteins. We have found that individual cells of the Cr-resistant B. pseudomycoides environmental strain accumulate less Cr than the cells of B. subtilis strain. Further, we found that survival of the B. subtilis strain during the Cr stress can be increased by the introduction of the chromate transporter from the Cr resistant environmental strain into its genome. Additionally, the expression of B. pseudomycoides chromate transporter ChrA in B. subtilis seems to be activated by the presence of chromate, hinting at versatility of Cr-efflux proteins. This study outlines the future direction for increasing the Cr-tolerance of non-pathogenic species and safe bioremediation using soil bacteria.

Abstract The bacteria residing in the gut of honey bees (HB) has demonstrated a significant role in protecting bees against various pathogens, production of honey and wax. However, no information exists about the antibacterial potential of bacterial isolates from gut of Asian HB, Apis cerana Indica F. (Hymenoptera: Apidae), against human pathogens. This study aims to investigate the antibacterial and multienzyme potential of aerobic bacteria from A. cerana gut using culture dependent approach. A total of 12 HB gut bacteria were characterized morphologically and biochemically. These strains were further screened for their antimicrobial activity against pathogenic human microorganisms Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus licheniformis and Bacillus subtilis using cross streak (primary screening) and agar well diffusion methods (secondary screening). Preliminary characterization of cell-free supernatant (CFS) of two promising isolates was performed by measuring lactic acid concentrations, enzymatic digestion of antimicrobial compounds, stability over a range of temperature, pH and amplification of spaS (subtilin) and spoA (subtilosin) genes. In primary screening, among 12 HB isolates, eight strains showed statistically significant highest zones of inhibition (p≤0.05) against E. coli, K. pneumoniae and P. aeruginosa. 16S rRNA sequencing revealed that these isolates belong to Bacillus genus, identified as B. tequilensis, B. pumilus, B. xiamenensis, B. subtilis, B. amyloliquefaciens, B. safensis, B. licheniformis, B. altitudinis (Accession numbers: MT186230-MT186237). Secondary screening revealed that among eight isolates, B. subtilis and B. amyloliquefaciens showed statistically significantly strong inhibition (p≤0.05) against all tested pathogens. Antibiotic susceptibility testing revealed that both isolates were resistant to antibiotics and possesses proteolytic, lipolytic and cellulolytic activities. The nature of the compound causing inhibitory activity was found to be proteinaceous and showed stability over a wide range of temperature as well as pH. PCR study confirmed the presence of bacteriocins by successful amplification of important antimicrobial peptide biosynthesis genes spaS and spoA. These results suggest that the HB gut is a home to bacteria that possess antimicrobial activity and important enzymes with antimicrobial potential. To our knowledge, this is the first report demonstrating the antimicrobial potential of bacteria isolated from gut of HB (A. cerana) against human pathogens.

In the present study, the effect of B. subtilis (107cfu/gm) supplemented to the basal diet of two groups of New Zealand rabbit of 28 days (n=15/group) on the count of commensal microflora ( Bifidobacteria and Lactobacilli spp.) and some opportunistic pathogens (E. coli and C. perfringens type A) in the rabbit caeci was investigated. Results indicated that the count of Bifidobacteria and Lactobacilli spp. are significantly increased at all intervals (7, 14, 28 and 40 days post supplementation) in the supplemented group compared with non-supplemented group with P< 0.001. On the other hand, there is a significant decrease in the count of E. coli and C. perfringens specially at 40 days post supplementation from (5.8 log 10 cfu ±0.06) to (3.1 log 10 cfu ±0.07) and from (4.8 log 10 cfu ±0.09) to (2.3 log 10 cfu ±0.09) respectively with P< 0.001. B. subtilis proved to be tolerant to the simulated gastric and intestinal juice for one hour (100% tolerance). However, after two hours, the tolerance to the gastric juice decreased to 70%, with no effect of the artificial intestinal juice on the viability of the organism. In vitro, the antimicrobial effect of B. subtilis on C. perfringens type A by well diffusion method showed an inhibition zone of 10 mm. Results of in vitro effect of B. subtilis on C. perfringens count showed that at 103 dilutions of C. perfringens, its count was reduced 2.1×105 to 3×104 after adding B. subtilis, while at dilution of 102, the count was reduced from 2.4×105 to 7×103cfu/ml after adding B. subtilis. It could be concluded that B. subtilis can be used as a probiotic in rabbits ration due to its ability to increase the commensal microflora count beside its antibacterial effect against some opportunistic pathogens.

El chile xcat´ik (Capsicum annuum L.) es el segundo más consumido en la península de Yucatán, después del habanero. Los biofertilizantes microbianos son una opción factible de integrarse a los sistemas de producción para disminuir el uso de fertilizantes químicos. El objetivo del trabajo se basó en la evaluación del efecto de la aplicación de Bacillus subtilis, Trichoderma harzanium y un consorcio microbiano en las características agronómicas de la planta y la calidad del fruto del chile xcat´ik. El experimento se realizó en condiciones de invernadero, a través de un diseño experimental de bloques completos al azar con cuatro repeticiones. De acuerdo a los resultados se encontró que la aplicación de B. subtilis incrementó significativamente la altura de plantas respecto al testigo, así mismo la aplicación de B. subtilis y T. harzanium incrementó el volumen de raíz. Se observó que existen diferencias estadísticas en los contenidos de lípidos y proteínas en el fruto de plantas biofertilizadas con T. harzanium, y que en los frutos de plantas tratadas particularmente con B. subtilis y T. harzanium se acumulan mayores cantidades de fósforo.

Agar diffusion and turbidimetry were commonly used to evaluation of antibacterial activity of honey. However, there is no report about which one of these two methods is better. This study attempts to validate the agar diffusion method and turbidimetry used for growth inhibitory assay of ceiba honey against Bacillus subtilis ATCC 6633 based on validation parameters including Limit of Detection (LOD), linearity, precision, and selectivity. The samples were ceiba honey aqueous solutions in concentration 20%-100%. It was found that the minimum inhibitory concentration (MIC) of agar diffusion and turbidimetric methods were 35% and 30% respectively, considered as LOD. In agar diffusion method, plot of inhibitory zone diameter against log of honey concentration yields linear regression equation with r = 0.9804 and Vx0=1.06%, while r and Vx0of linear plot from % transmittance against log concentration in the turbidimetric method were 0.9748 and 1.24% respectively. The coefficient of variation (CV) in agar diffusion method were 1.78% for repeatability and 3.13% for intermediate precision) whereas CV in turbidimetric were 3.64% and 4.05% respectively. Agar diffusion and turbidimetric methods were selective because different source of ceiba honey could give different response in term of MIC. It can be concluded that the agar diffusion and turbidimetric method were valid and suitable for growth inhibitory assay of ceiba honey against Bacillus subtilis ATCC 6633 and there was no significant differences between these methods. The turbidimetry was more sensitive than the agar diffusion method because of its lower LOD and it has more simple experimental technique.

Abstract Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 °C. Moreover, it was active over the broad temperature region of 30–60 °C, and stable within the pH range of 4.5–10.0. In addition, Co2+ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low- concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the β-1,3-1,4 glucan linkage from barley. The Michaelis–Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 μmol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.

The self-binding of bacterial cells, or autoaggregation, is, together with surface colonization, one of the first steps in the formation of a mature biofilm. In this work, the autoaggregation of B. subtilis in dilute bacterial suspensions was studied. The dynamics of cell lysis, eDNA release, and bacterial autoaggregate assembly were determined and related to the spatial autocorrelation of bacterial cells in dilute planktonic bacterial suspensions. The non-random distribution of cells was associated with an eDNA network, which stabilized the initial bacterial cell-cell aggregates. Upon the addition of DNase I, the aggregates were dispersed. The release of eDNA during cell lysis allows for the entrapment of bacterial drifters at a radius several times the size of the dying bacteria. The size of bacterial aggregates increased from 2 to about 100 μm in diameter in dilute bacterial suspensions. The results suggest that B. subtilis cells form previously unnoticed continuum of autoaggregate structures during planktonic growth.

Copper ore processing generates a large amount of copper slag, which has properties that are similar to fine aggregate. Copper slag has a promising future in the construction industry as an alternative to fine aggregate. Up to 50% of fine aggregate substitutions have been successful. The performance of copper slag concrete could be improved by microbiologically induced calcium carbonate precipitation. The impact of micro-organisms on the mechanical properties and flexural behaviour of copper slag concrete was investigated in this study. Five concrete mixtures were created by replacing varying amounts of fine aggregate with copper slag, ranging from 0% to 100%. M30 grade concrete was used, and 1% to 2% of the bacterium Bacillus subtilis by weight of cement was added during the concrete casting procedure. Specimens of different shapes, such as cubes, cylinders, and prisms, were cast and examined at 7, 14, and 28 days. When treated with micro-organisms, the test results revealed that replacing 50% to 75% of the sand with copper slag produced concrete with superior mechanical properties and a greater density. With the optimal ratio of copper slag to micro-organisms, a suitable RCC beam was formed. Load–deflection patterns of bacterial copper slag concrete were used to investigate beam flexural behaviour, and the results were compared using ABAQUS modelling. Microbiologically induced calcium carbonate precipitation can alter regular copper slag concrete, resulting in enhanced concrete performance.

In this study, we have isolated and characterized proteolytic soil bacteria and their alkaline protease. Based on 16S rRNA sequence analysis, 12 isolates with the highest protease activity were classified as B. subtilis and B. cereus groups. B. subtilis D9 isolate showing the highest protease activity was selected for in vitro and in silico analysis for its ِِAKD9 protease. The enzyme has a molecular mass of 48 kDa, exhibiting optimal activity at 50 °C pH 9.5, and showed high stability till 65 °C and pH 8–11 for 1 h. Fe3+‏ stimulated, but Zn2+ and Hg2+ strongly inhibited the protease activity. Also, the maximum inhibition with PMSF indicated serine protease-type of AKD9 protease. AkD9 alkaline serine protease gene showed high sequence similarity and close phylogenetic relationship with AprX serine protease of B. subtilis isolates. Functional prediction of AKD9 resulted in the detection of subtilase domain, peptidase_S8 family, and subtilase active sites. Moreover, prediction of physicochemical properties indicated that AKD9 serine protease is hydrophilic, thermostable, and alkali-halo stable. Secondary structure prediction revealed the dominance of the coils enhances AKD9 activity and stability under saline and alkaline conditions. Based on molecular docking, AKD9 showed very promising binding affinities towards casein substrate with expected intrinsic proteolytic activities matching our obtained in vitro results. In conclusion, AKD9 alkaline serine protease seems to be a significant candidate for industrial applications because of its stability, hydrophilicity, enhanced thermostability, and alkali-halo stability.

Food-borne diseases are widespread all over the world, and food safety has attracted much attention. This study is the first to use plasma to activate acidic electrolyzed water (AEW) to obtain a new disinfectant for food processing. The germicidal efficacy of plasma-activated acidic electrolyzed water (PA-AEW) on B. subtilis suspension and biofilm was investigated. Furthermore, the synergistic effect of different bactericidal factors was inferred by investigating the physicochemical parameters of PA-AEW and the influencing factors of bactericidal effect. The results demonstrate that PA-AEW is a highly effective and rapid disinfectant. The killing logarithm (KL) value of PA-AEW on B. subtilis suspension could reach 2.33 log10CFU/mL with a sterilization time of 10 s, which is significantly higher than that of AEW (KL = 0.58 log10CFU/mL) and plasma-activated water (PAW) (KL = 0.98 log10CFU/mL) (significant difference, p < 0.01). Moreover, the KL value of the B. subtilis biofilm of PA-AEW was 2.41 log10CFU/mL, better than that of PAW and AEW (significant difference, p < 0.01), indicating that PA-AEW has important application prospects in food processing. The synergistic effect should come from the interaction between reactive chlorine species (RCS) and reactive oxygen and nitrogen species (RONS) in PA-AEW.

The strategy of existence of microorganisms in the structure of biofilms determines their adaptation to specific environmental conditions. Bacteria of the genus Bacillus are actively used as models for studying the processes of regulation and formation of biofilms. This work aims to assess the effect of proteases secreted by bacilli on the formation of biofilms, as well as on the resistance of recombinant strains to environmental stress factors. We compared the dynamics of biofilm formation by the wild strain B. subtilis 168 and the following strains with altered expression of extracellular proteases: non-protease strains (B. subtilis BG2036, B. subtilis BRB8, and B. subtilis BRB14) and strains with increased protease secretion (B. subtilis (aprBp), B. subtilis (gseBp), and B. subtilis (mprBp)). We also investigated the effect of ethanol and sodium chloride on the ability of the B. subtilis strains to form biofilms in a liquid medium. The bacterial strains were cultured at pH 7.4 and temperature 37 ?C in a synthetic E-medium in U-shaped 96-well plates. Biofilm formation was identified by incubation with crystal violet (CV). We found that the level of biofilm formation is higher (by an average of 10%) in the protease-deficient strains of B. subtilis. The recombinant strains expressing genes of serine proteases form biofilms with a reduced level (by an average of 40%). A strain with the expression of metalloendopeptidase is characterized by an increased level of biofilm formation (up to 10%), which is possibly due to the functional in vivo role of this new enzyme. The wild and protease-deficient strains of B. subtilis respond similarly to ethanol stress: biofilm formation is reduced in both of them. The wild strain exhibits a greater sensitivity to osmotic stress than the protease-deficient one.

Актуальність. Інфікування Pseudomonas aeruginosa визначає клінічний прогноз упацієнтів з муковісцидозом. Мукоїдніморфотипи P. Aeruginosa здатні до формування біоплівок та росту в них. З огляду на множинну резистентність біоплівок обговорюється можливість використання Bacillusspp. зметою санітарної обробки поверхонь, на яких можуть зберігатися патогени. Мета– вивчити біоплівкоутворюючі властивості клінічних ізолятів P. aeruginosa, виділених від дітей з муковісцидозом, та вплив на них Bacillus subtilis in vitro. Методи. Зразки мокротиння та/або слизу з глибокого мазку з задньої стінки глотки взяті від дітей хворих на муковісцидоз. Використано бактеріологічний, мікроскопічний, біохімічний та статистичний методи дослідження. Антагоністичні властивості P. Aeruginosa та B. Subtilis вивчали методом відстроченого антагонізму з використанням методики перпендикулярних штрихів та при спільному культивуванні в рідкому поживному середовищі з наступним висівом серійних розведень суспензій.Здатність до утворення біоплівоквизначали експрес-методом з використанням 96-луночних планшетів. Результати. При бактеріологічному дослідженні встановили, що P. aeruginosa виділяється з 34,21% (95% ДІ 32,50-35,92) зразків. При вивченні відстроченого антагонізму між P. Aeruginosa і B. subtilis, а також при їх спільному культивуванні в рідкому поживному середовищі виявили, що B. Subtilis володіють прямим помірним антагонізмом по відношенню до P. aeruginosa. Після спільної культивації культура бацил давала рясний ріст в усіх дослідах при всіх повтореннях, обмежуючи ріст P. aeruginosa. Серед виділених ізолятів P. aeruginosa встановлено здатність до біоплівкоутворення в 76,92% (95% ДІ 73,02-80,77) випадків, серед зразків B. subtilis – в 100%. При спільному культивуванні B. subtilis утворювали біоплівку в присутностіP. aeruginosa в більшості випадків. Висновки. Клінічні ізоляти P. aeruginosa, виділені від дітей з муковісцидозом помірно чутливі до дії B. subtilisinvitro. Біоплівкоутворення P. Aeruginosa in vitro може бути пригнічене дією B. subtilis.

Chromium in wastewater is classified as one of dangerous contaminants that requires further treatment before it can be discharged to water body. Concentration of chromium in water body, especially river, has increased as many industries are utilizing chromium as raw materials and then discharging its wastewater directly without any treatment. Biosorption is one of methods that widely used to treat heavy metal containing wastewater. Bacteria is the most common microorganism to be used as heavy metal treatment agent. Azotobacter s8, Bacillus subtilis and Pseudomonas putida had been proven to have a heavy metal resistant capability. Screening test showed that Minimum Inhibitory Concentration value of chromium for all bacteria were ranged from 100 to 250 mg/L of CrCl3. Chromium biosorption test by bacteria showed that Azotobacter s8 was able to remove 10.53%, and Bacillus subtilis was able to remove 5.68% chromium from 50 mg/L initial concentration while Pseudomonas putida showed no chromium removal. The chromium biosorption capacity by Azotobacter s8 was 580.08 mg/g and 349.30 mg/g for Bacillus subtilis.

Abstract This study aims to evaluate the influence of B. subtilis AP91 spores addition on the mechanical properties of mortars. B. subtilis strain AP91, isolated from rice leaves of the needle variety, which has an early cycle of production, was used at the concentration of 105 spores/mL in mortars with cement-to-sand ratio of 1:3 (by weight) and water-to-cement ratio (w/c) of 0.63. These spores were added in two different ways: in the mixing water and by immersion in a solution containing bacterial spores. Scanning Electron Microscope (SEM) analysis showed crystals with calcium peaks on the EDS, which possibly indicates the presence of bioprecipitated calcium carbonate. The results obtained in the mechanical analysis showed that the bioprecipitation of CaCO3 by B. subtilis strain AP91 was satisfactory, particularly when the spores were added in the mixing water, increasing the compressive strength up to 31%. Thus, it was concluded that the addition of B. subtilis AP91 spores in the mixing water of cement mortars induced biocementation, which increased the compressive strength. This bioprecipitation of calcium carbonate may very well have other advantageous consequences, such as the closure of pores and cracks in cementitious materials that could improve durability properties, although more research is still needed on this matter.

Abstract Background Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region. In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter. Results The YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain. PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an α-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis. Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions. Conclusions The new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.

With the rapid development of industrial society, a large amount of energy and resources are consumed, causing some mercury (Hg) pollution problems or potential Hg pollution problems. The Hg pollution in fish and water environment affects human health and becomes a global problem. Selenium (Se) is an essential element and plays an important role in many physiological and biochemical processes. In this study, 6 strains of Bacillus were screened for their potential probiotic capacity about Hg and Se tolerance and bioadsorption in vitro, and Bacillus subtilis (B. subtilis) was selected for animal experiments because of its excellent Hg and Se tolerance and bioadsorption. Both Se-only group and selenium-rich B. subtilis can reduce Hg accumulation in organs following Hg exposure in common carp. Selenium-rich B. subtilis is better than selenium-only group in reducing Hg accumulation in organs of fish. Our results suggested that administration of Selenium-rich B. subtilis has the potential to combat dietary Hg toxicity in common carp.

Grafting generally means stress to a plant and this triggers antioxidant defense systems. An imbalance in reactive oxygen species may negatively affect the grafting success. Several research projects have studied the association with plant growth-promoting rhizobacteria (PGPR) and it has been documented that they enhance nutrient acquisition, regulate hormone levels, and influence the antioxidant response in crops. However, little is known about the strategy of inoculating grafted herbaceous plants with PGPR and its effect on the antioxidant response. The effects of inoculating a strain of Bacillus subtilis on the antioxidant metabolism of grafted tomato were evaluated. In this study, two different rootstocks were used for tomato (Solanum lycopersicum L. var. Rio Grande (RG)): [S. lycopersicum L. var. cerasiforme (Ch)] and eggplant [(Solanum melanogena L. (Ber)] to establish a compatible graft (RGCh) and a semicompatible graft (RGBer). Enzyme activities involved in the antioxidant defense system: superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and total phenols were measured during 4 weeks after grafting. The results show that for RGCh, during the first two weeks after grafting, the tendency was a decrease of the enzyme activity for SOD, CAT, PAL when inoculated with B. subtilis; while in the semicompatible graft RGBer, PPO and PAL decreased their activity after inoculation. For both combinations, the quantity of total phenols varied depending on the day. In both graft combinations, applications of B. subtilis resulted in 86 and 80% callusing compared with the uninoculated control where the percentages were 74 and 70% for RGCh and RGBer, respectively. The highest significant graft success (95%) was recorded 28 days after grafting for inoculated RGBer. These findings imply that B. subtilis induced antioxidant mechanisms in grafted plants and suggest that inoculation with this growth-promoting bacterium can represent a biotechnological approach to improve success in tomato grafting.

Industrial wastes, for instance, tannery wastes are rich soups of resistant and bioremediation-potent bacteria. In the present work, Chromium (Cr) and tannic acid (TA) resistance bacterial strains were isolated from tannery effluent and identified as Bacillus subtilis (MCC 3275) and Bacillus safensis (MCC 3283) based on its 16S Ribosomal RNA homology. Hexavalent Cr is highly toxic and mutagenic due to its high mobility and reactivity. Whereas, TA is known to inhibit enzyme activity, substrate deprivation, and interaction with membranes and matrix-metal ions. The developed In vitro co-cultured microcosm of B. subtilis and B. safensis was able to remove Cr(VI) up to 95% and TA up to 23%. The bacteria cultures separately were able to degrade Cr(VI) to 88% by B. subtilis and 91% by B. safensis and TA up to 27%. Plackett Burman design (PBD) followed by Response surface methodology (RSM) was applied for the optimization of physio-chemical parameters. The optimized conditions for co-culture development were recorded as K2HPO4 = 0.2 g/L, MgSO4 = 0.2 g/L, NH4Cl = 0.5 g/L, glucose – 0.2 g/L, TA – 5%, Cr = 200 ppm, incubation period of 96 h, agitation speed of 110 rpm, pH = 5.0, temperature= 30 °C and inoculum size = 3%. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) revealed the thorough mechanism of cellular uptake followed by degradation of Cr(VI) and TA. The efficiency of co-culture for other heavy metals was observed as follows: Zn 65%, Pb 63%, Cd 65%, and Ni 65%. Bioremediation using bacteria is an economical and environmentally better alternative to conventional remediation methods. The isolated bacteria are useful in the effluent treatment of tannery or related industries and in metal recovery in mining processes.

Photodynamic sterilization technology (PDT) is widely used in disease therapy, but its application in the food industry is still at the research stage because of the limitations of food-grade photosensitizers. Curcumin exhibits photosensitivity and is widely used as a food additive for its natural color. This study aimed to determine the effect of curcumin-mediated photodynamic technology (Cur-PDT) on Bacillus subtilis and to elucidate the anti-bacterial mechanism involved. First, the effects of curcumin concentration, duration of light irradiation, light intensity, and incubation time on the inactivation of B. subtilis were analyzed. It was found that Cur-PDT inactivated 100% planktonic cells with 50 μmol/L curcumin in 15 min (120 W). Then, the cell morphology, oxidation state and the expression of membrane structure- and DNA damage-related genes of B. subtilis vegetative cells were investigated under different treatment conditions. The membrane permeability of cells was enhanced and the cell membrane structure was damaged upon treatment with Cur-PDT, which were exacerbated with increases of treatment time and curcumin concentration. Meanwhile, the production of reactive oxygen species increased and the activities of the antioxidant enzymes SOD, GPX, and CAT decreased inside the cells. Furthermore, the Cur-PDT treatment significantly downregulated the mRNA of the membrane protein TasA and upregulated the DNA damage recognition protein UvrA and repair protein RecA of B. subtilis. These results suggested that curcumin-mediated PDT could effectively inactivate B. subtilis by inducing cell redox state imbalance, damaging DNA, and disrupting membrane structures.

Treatment options for infections caused by antimicrobial-resistant bacteria are rendered ineffective, and drug alternatives are needed—either from new chemical classes or drugs with new modes of action. Historically, natural products have been important contributors to drug discovery. In a recent study, the dimeric naphthopyrone lulworthinone produced by an obligate marine fungus in the family Lulworthiaceae was discovered. The observed potent antibacterial activity against Gram-positive bacteria, including several clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, prompted this follow-up mode of action investigation. This paper aimed to characterize the antibacterial mode of action (MOA) of lulworthinone by combining in vitro assays, NMR experiments and microscopy. The results point to a MOA targeting the bacterial membrane, leading to improper cell division. Treatment with lulworthinone induced an upregulation of genes responding to cell envelope stress in Bacillus subtilis. Analysis of the membrane integrity and membrane potential indicated that lulworthinone targets the bacterial membrane without destroying it. This was supported by NMR experiments using artificial lipid bilayers. Fluorescence microscopy revealed that lulworthinone affects cell morphology and impedes the localization of the cell division protein FtsZ. Surface plasmon resonance and dynamic light scattering assays showed that this activity is linked with the compound‘s ability to form colloidal aggregates. Antibacterial agents acting at cell membranes are of special interest, as the development of bacterial resistance to such compounds is deemed more difficult to occur.