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Ethanol as a renewable biofule is an appropriate and viable alternative to the challenging fossil fuels. Bacillus subtilis, a gram positive bacterium, seems to be a promising choice since it has many useful features. For example B.subtilis ferments broad range of sugars derived from lignocellulosic hydrolysis. Transformation of this cellulytic bacterium to an ethanologenic one was accomplished via metabolic engineering techniques and Ethanol production operon of Z.mobilis was introduced to the B.subtilis. SR1 and SR21 strains expressed plasmid-borne ethanologenic genes of Z.mobilis but the genes had been integrated into the SR22 genomic DNA. Also lactate dehydrogenase gene had been knocked-out in SR21 and SR22 strains. Defect of cell growth in SR21 and SR22, suggests that NAD+ oxidation by lactate dehydrogenase is important for anaerobic growth. Considering the impact of Fe2+ ion on alcohol dehydrogenase II activity, in further experiments Fe2+ was added to the culture media and improvement in growth rates was seen. Final yield of ethanol production of SR1, SR21, and SR22 strains were 53.8%, 86.7%, and 83.9% respectively.

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Plant essential oils are increasingly used as natural food preservatives. Understanding their chemical composition and their antimicrobial activity in the context of food health and safety seems indispensible. In the present study the chemical composition of essential oils of Mentha longifolia L. and Cuminum cyminum L., which were obtained using a Clevenger apparatus, was analyzed by GC/MS. The anti-microbial effect on growth of Bacillus cereus and Bacillus subtilis in different concentrations during 15 days incubation at 8 and 25°C was done in commercial barley soup. The taste acceptability of these essential oils was also assessed. The result indicated pulegon with 31.54% and cuminaldehyde with 29.02% were the most compounds found in Mentha longifolia L. and Cuminum cyminum L., respectively. These essential oils at the 8°C showed significant reduction of bacterial count. The lowest concentration of the essential oils inhibited bacterial growth compared to the controls. Essential oil of Mentha longifolia L. showed more antibacterial activity compared to Cuminum cyminum L. essential oil in similar concentrations. On the other hand, Bacillus cereus was more sensitive to these essential oils than Bacillus subtilis. A concentration of 0.15 µg ml^-1 of these essential oils showed the maximum taste acceptability. This study clearly demonstrated that these essential oils could be used in some food systems without any undesirable palatability.

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Biosurfactants are produced by microorganisms. Surfactin is one of the main lipopeptide biosurfactants produced by different species of Bacillus subtilis. This study aims to analyze the effect of starch-coated Fe⁰and Fe³⁺nanoparticles on the biosurfactant production of Bacillus subtilis. Out of 70 soil samples, 20 Bacillus were isolated and genome sequenced by biochemical methods and 16S rRNA gene. Quantitative and qualitative screening methods were used to isolate and detect biosurfactant production. For the aim of this study, 61 and 63 (Bacillus subtilis subsp. Inaquosorum) were selected. Then, hemolytic activity, surfactant production and reduction of surface tension in Minimal Salt Medium containing Fe⁰ and Fe³⁺ nanoparticles were examined after 48h, 72h and 96h of culture. The binding of the nanoparticles to the surfactant was confirmed by SEM. Strain 61 was the best bacterium and Fe³⁺ was the best nanoparticle and it was cultured. The results were compared with the results of non-nanoparticle bioreactor. Surfactin from strain 61 culture in the Fe³⁺ nanoparticle bioreactor after 72 hours of growth showed higher production than the same strain culture after 72 hours without Fe³⁺, if continuing the research, this strain can be commercialized in the future.
 

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 A consortium of biofertilizers (Azotobacter chroococcum and Bacillus subtilis) was applied in conventional as well as organic matrix entrapped granular forms as sole nutrient source in two different doses for cultivation of wheat (Triticum aestivum L. cv. PBW-343). A double dose of conventional biofertilizers increased the growth of wheat plants as measured on 30, 60, 90, and 120 Days After Sowing (DAS) in terms of root and shoot length, number of roots and leaves, as well as fresh and dry weight of roots and leaves over the recommended dose (0.6 kg ha^-1) of the same biofertilizers. The entrapment of biofertilizers in an organic matrix further increased the efficacy of these biofertilizers over the non-entrapped conventional forms. An increase in the plant growth of wheat by application of higher dose of biofertilizers and entrapped biofertilizers was correlated to the availability of NO₃^-, NO₂^- and NH₄⁺ in the plant’s rhizosphere (0-15 cm) and its transport from soil to the plant leaves as well as productivity and yield of wheat in these experimental fields. The increase of 63.47 and 32.17% in wheat yield was recorded in 120-days old plants by the application of organic matrix entrapped biofertilizers in double dose over no fertilizers and un-entrapped biofertilizers in single dose. The results indicate that efficacy of biofertilizers can be enhanced by increasing the dose of biofertilizers and by providing suitable carriers to replace chemical fertilizers load for wheat cultivation with eco-friendly and organic nutrient technologies.

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Objective: Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides and chemical nerve agents. These compounds are hazardous for humans and the environment. Organophosphate hydrolase (OPH) is a homodimeric protein initially isolated from Pseudomonas diminuta MG and Flavobacterium species. This enzyme is able to degrade a broad spectrum of toxic OPs compounds. Using immobilized OPH commonly presents a variety of advantages versus the free form of the enzyme. Advantages include an increase in stability, cost reduction by simple recovery and reutilization of the enzyme, quick and easy separation of the reactant and product in the reaction medium. Methods: Plasmid pET-26b (+) was used to generate the OPH protein under the control of the T7lac promoter. E. coli BL21 (DE3) pLysS was used as the host for expression of the OPH enzyme. Recombinant OPH was secreted into the extracellular medium and the purified enzyme was immobilized on the surface of Bacillus subtilis spores by the adsorption method, for the first time. Results: Approximately 42% to 45% enzymatic activity was determined to be associated with spores. Optimal pH and temperature of the enzyme were not altered by the presence of the spores. Thermo and pH stabilities of the immobilized enzyme was higher than the free form of the enzyme. Conclusion: Bacillus subtilis spores are safe for humans and the environment. Therefore this system can be considered an environmentally friendly biocatalyst for degradation of OPs. 

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Root-knot nematodes are the most economically important plant pathogens in pistachio. The ability of Pseudomonas fluorescens strains VUPF5, VUPF52, Bacillus cereus strain PRC95 and Bacillus subtilis strain PRC96 were tested as biocontrol agents for Meloidogyne incognita on the pistachio cultivars Sarakhs and Badami. The effect of these bacterial strains on defense-related enzymes activity in pistachio was also investigated. Pistachio seedlings of both cultivars were treated with bacterial strains and then were inoculated with 2000 second-stage juveniles of nematode after two days. Evaluations were made for changes of Peroxidase (POX), PolyPhenolOxidase (PPO), Phenylalanine Ammonia lyase (PAL) and Total Phenolic Content (TPC) determined at 2, 4, 7, and 10 Days After nematode Inoculation (DAI). Results showed improved activity of POX, PAL and PPO in both cultivars. The most significant result for POX activity in the treated seedlings belonged to Pseudomonas strain VUPF5 at 7 DAI for Sarakhs and 10 DAI for Badami. However, this strain displayed an increase in PAL activity at 2 and 4 DAI in Badami and Sarakhs, respectively. Seedlings treated by the Pseudomonas strain VUPF52 at 10 DAI had the highest PPO activity among cultivars. TPC concentration was slightly higher, by 8.4% at 4 DAI, in Sarakhs seedlings treated with VUPF5, but no significant increase could be seen in the Badami cultivar compared with the control. In another experiment, 4 months after nematode inoculation in seedlings of both cultivars treated by bacterial strains, numbers of galls, egg masses, and second juveniles decreased compared with the non-treated seedlings.

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Amylase are the most important group of industrial enzymes that hydrolyze starch into amylopectin. The industrial demand of amylase predominantly from the microbial origin has been recently increased and enhances the research for amylase with high stability at extreme industrial conditions. Thus, this study is aimed to characterize amylase from Bacillus subtilis IMD34 obtained from previous using agro waste. Extracellular amylase production was carried out in basal medium by submerged fermentation technique. The production medium with 10% potato peels at pH 6.8 and 35 °C enhanced amylase activity (720 U/mL) than other wastes such as banana peels and yam peels. This optimized media increased the bacterial growth rate and activity of amylase compared to the non-supplemented basal medium. Further, amylase was purified and assessed their molecular weight (~39 k Da) in 12 % SDS -PAGE. The enzyme activity was observed at pH 6.8 and 35°C and stable over a wide range of pH (4-10), and temperatures (20-50°C). In addition, enzyme activity was stimulated in the presence of Mg²⁺, Ca²⁺ and Mn²⁺ and was unpretentious after treating with surfactants (SDS, Triton X-100, Tween 80), organic solvents (ethanol, methanol, chloroform and acetone) and amylase inhibitors (EDTA and β-mercaptoethanol). These enzymic characteristics makes the amylase obtained in this study a promising candidate for various industrial sector application.
 

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Today, the use of self-healing concretes has attracted the attention of various researchers. In this paper, the effect of Bacillus subtilis bacteria with different concentrations were used for evaluation on concrete containing zeolite.The mentioned bacteria were produced in the laboratory and added to the concrete mixing plan. In order to evaluate the effect of Bacillus subtilis bacteria on specimen with zeolite, resistance and durability tests including compressive strength, ultrasonic, water penetration test and water absorption tests were performed on the specimen at different ages. Also, using scanning electron microscope (SEM) and optical microscope, the self-healing of concrete was evaluated. The results of the compressive strength test showed that the specimen containing zeolite with a concentration of 2.8×10⁸ cells/ml increased the compressive strength by 16.76% and the specimen without zeolite increased the compressive strength by 13.51%. Also, the presence of Bacillus subtilis bacteria in the mixing design led to a 15% decrease in water absorption of specimens without zeolite and 30% of specimens with zeolite. Based on the results of the experiments, the most suitable concentration, for the simultaneous improvement of the resistance parameters and durability among the different concentrations of Bacillus subtilis bacteria, is suggested to be 2.8 x 10⁸.

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Fire blight, caused by Erwinia amylovora bacteria, is one of the well-known plant diseases in the world including major diseases of the fruit trees, especially apples and pears. In recent years, due to chemical nature of the pesticides damaging human health and environment, the importance of biological control is considered as alternative measure to manage plant diseases. To investigate the possibility of biological control of the pathogens, healthy foliar samples of apple, pear, and quince trees were collected from different regions of Kerman Province, Iran, and then, biocontrol activity of antagonist agents was evaluated under laboratory conditions. On the basis of the results, some of the antagonists could decrease the symptoms of the disease by 14.28-79.59%. Laboratory evaluation included investigating the disease severity in immature pears, biocontrol activity of antagonist agents in the plate assay, inhibitory siderophore production, biofilm formation capacity, drought stress tolerance, and silver nanoparticle synthesis capability, which showed that these antagonist agents could potentially control the disease. Among the 9 well-performed antagonistic isolates from apples and pears, Vr87 isolate was selected. The studies were confirmed by amplifying part of 16S rDNA region of the isolate, using specific primers. By comparing the results on the NCBI website, the selected isolate was identified as Enterobacter sp. genus. Among all selected isolates as successful factors in controlling fire blight pathogen, including isolated isolates and isolates in the collection of Vali-e-Asr University, Bacillus subtilis strain BsVRU1 in the Vali-e-Asr University collection, with 73.5% inhibition, had higher inhibitory power than the other antagonist isolates against the pathogen of fire blight disease.