Heparosan biosynthesis in recombinant Bacillus megaterium: Influence of N-acetylglucosamine supplementation and kinetic modeling.

Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin-an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30 g L-1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N-acetylglucosamine (S2) resulted in a significant 1.6-fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi-substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method.

Development and performance evaluation of a microbiological method for screening and LC-MS/MS for conformation of sulfonamides in animal-derived foods.

This study developed a highly sensitive microbiological method utilizing a novel microtiter plate to screen 10 sulfonamides in chicken muscles, eggs, and prawns. This plate was fabricated from agar incorporating trimethoprim and spread with Bacillus megaterium. After residue detection by bioassay, the same test solutions were analyzed by LC-MS/MS for accurate identification and quantification. It also proved eco-friendly compared to using other quantitative methods. The residual drugs were extracted with McIlvaine buffer and purified using an Oasis® MCX cartridge. A triethylamine/methanol/water (0.5:75:24.5, v/v/v) mixture was used as the eluate. The obtained LOD values of the bioassay ranged from 5 to 25 µg kg-1 allowing the detection of the target drugs at the MRLs established in Japan. Adhering to ISO/IEC 17025 standards, the performance of the bioassay was evaluated. Based on the inhibition zone size in bioassay results, quality control yielded a Z score within ±2, indicating reasonable control over the screening process. Proficiency testing of a chicken muscle sample spiked with sulfadimidine demonstrated the inhibition zone detection of the bioassay and quantified value alignment of LC-MS/MS with reference values. In a surveillance study of 91 samples, sulfamethoxazole was detected in one prawn sample.

Comparative genomic analysis of strain Priestia megaterium B1 reveals conserved potential for adaptation to endophytism and plant growth promotion.

In our study, we aimed to explore the genomic and phenotypic traits of Priestia megaterium strain B1, which was isolated from root material of healthy apple plants, to adapt to the endophytic lifestyle and promote plant growth. We identified putative genes encoding proteins involved in chemotaxis, flagella biosynthesis, biofilm formation, secretory systems, detoxification, transporters, and transcription regulation. Furthermore, B1 exhibited both swarming and swimming motilities, along with biofilm formation. Both genomic and physiological analyses revealed the potential of B1 to promote plant growth through the production of indole-3-acetic acid and siderophores, as well as the solubilization of phosphate and zinc. To deduce potential genomic features associated with endophytism across members of P. megaterium strains, we conducted a comparative genomic analysis involving 27 and 31 genomes of strains recovered from plant and soil habitats, respectively, in addition to our strain B1. Our results indicated a closed pan genome and comparable genome size of strains from both habitats, suggesting a facultative host association and adaptive lifestyle to both habitats. Additionally, we performed a sparse Partial Least Squares Discriminant Analysis to infer the most discriminative functional features of the two habitats based on Pfam annotation. Despite the distinctive clustering of both groups, functional enrichment analysis revealed no significant enrichment of any Pfam domain in both habitats. Furthermore, when assessing genetic elements related to adaptation to endophytism in each individual strain, we observed their widespread presence among strains from both habitats. Moreover, all members displayed potential genetic elements for promoting plant growth.IMPORTANCEBoth genomic and phenotypic analyses yielded valuable insights into the capacity of P. megaterium B1 to adapt to the plant niche and enhance its growth. The comparative genomic analysis revealed that P. megaterium members, whether derived from soil or plant sources, possess the essential genetic machinery for interacting with plants and enhancing their growth. The conservation of these traits across various strains of this species extends its potential application as a bio-stimulant in diverse environments. This significance also applies to strain B1, particularly regarding its application to enhance the growth of plants facing apple replant disease conditions.

Efficient Immobilization of heavy metals using newly synthesized magnetic nanoparticles and some bacteria in a multi-metal contaminated soil.

Simultaneous application of modified Fe3O4 with biological treatments in remediating multi-metal polluted soils, has rarely been investigated. Thus, a pioneering approach towards sustainable environmental remediation strategies is crucial. In this study, we aimed to improve the efficiency of Fe3O4 as adsorbents for heavy metals (HMs) by applying protective coatings. We synthesized core-shell magnetite nanoparticles coated with modified nanocellulose, nanohydrochar, and nanobiochar, and investigated their effectiveness in conjunction with bacteria (Pseudomonas putida and Bacillus megaterium) for remediating a multi-metal contamination soil. The results showed that the coatings significantly enhanced the immobilization of heavy metals in the soil, even at low doses (0.5%). The coating of nanocellulose had the highest efficiency in stabilizing metals due to the greater variety of surface functional groups and higher specific surface area (63.86 m2 g-1) than the other two coatings. Interestingly, uncoated Fe3O4 had lower performance (113.6 m2 g-1) due to their susceptibility to deformation and oxidation. The use of bacteria as a biological treatment led to an increase in the stabilization of metals in soil. In fact, Pseudomonas putida and Bacillus megaterium increased immobilization of HMs in soil successfully because of extracellular polymeric substances and intensive negative charges. Analysis of metal concentrations in plants revealed that Ni and Zn accumulated in the roots, while Pb and Cd were transferred from the roots to the shoots. Treatment Fe3O4 coated with modified nanocellulose at rates of 0.5 and 1% along with Pseudomonas putida showed the highest effect in stabilizing metals. Application of coated Fe3O4 for in-situ immobilization of HMs in contamination soils is recommendable due to their high metal stabilization efficiency and suitability to apply in large quantities.

UV protection and insecticidal activity of microencapsulated Vip3Ag4 protein in Bacillus megaterium.

In this study, secretable Vip3Ag4 protein was encapsulated in Bacillus megaterium and used for quantitative bioassays, in order to determine the UV photoprotective capacity of the cell, for preventing inactivation of the insecticidal activity of the protein. The non-encapsulated and purified protein was exposed to the UV light showing a LC50 of 518 ng/cm2 against Spodoptera littoralis larvae, whereas the exposed encapsulated protein exhibited 479 ng/cm2. In addition to the capability to accumulate Vip3 proteins for the development of novel insecticidal formulates, the B. megaterium cell has demonstrated to provide moderate protection against the deleterious action of UV light.

Immobilization of Pb(II) by Bacillus megaterium-based microbial-induced phosphate precipitation (MIPP) considering bacterial phosphorolysis ability and Ca-mediated alleviation of lead toxicity.

Inappropriate handling of lead (Pb)-containing wastewater that is produced as a result of smelting activities threatens the surrounding environment and human health. The microbial-induced phosphate precipitation (MIPP) technology was applied to immobilize Pb2+ in an aqueous solution considering bacterial phosphorolysis ability and Ca-mediated alleviation of lead toxicity. Pb immobilization was accompanied by sample characterization in order to explore the inherent mechanism that affected the immobilization efficiency. Results showed that Ca2+ use elevated the immobilization efficiency through the prevention of bacterial physisorption and chemisorption, an enhancement to the phosphatase activity and the degree of SGP hydrolysis, and the provision of nucleation sites for Pb2+ to attach. The formation of the Pb-GP complex helped the bacteria to maintain its activity at the commencement of catalyzing SGP hydrolysis. The nucleated minerals that were precipitated in a columnar shape through a directional stacking manner under MIPP featured higher chemical stability compared to non-nucleated minerals. As a result, there were three pathways, namely, bacterial physisorption, bacterial chemisorption, and substrate chelation, applied for Pb immobilization. The immobilization efficiency of 99.6% is achieved by precipitating bioprecipitates including Pb5(PO4)3Cl, Pb10(PO4)6Cl2, and Ca2Pb3(PO4)3Cl. The findings accentuate the potential of applying the MIPP technology to Pb-containing wastewater remediation.

A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Synbiotic Bacillus megaterium DSM 32963 and n-3 PUFA Salt Composition Elevates Pro-Resolving Lipid Mediator Levels in Healthy Subjects: A Randomized Controlled Study.

Beneficial health effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) are partly attributed to specialized pro-resolving mediators (SPMs), which promote inflammation resolution. Strategies to improve n-3 PUFA conversion to SPMs may, therefore, be useful to treat or prevent chronic inflammatory disorders. Here, we explored a synbiotic strategy to increase circulating SPM precursor levels. Healthy participants (n = 72) received either SynΩ3 (250 mg eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) lysine salts; two billion CFU Bacillus megaterium; n = 23), placebo (n = 24), or fish oil (300 mg EPA plus DHA; N = 25) capsules daily for 28 days in a randomized, double-blind placebo-controlled parallel 3-group design. Biomarkers were assessed at baseline and after 2 and 28 days of intervention. The primary analysis involved the comparison between SynΩ3 and placebo. In addition, SynΩ3 was compared to fish oil. The synbiotic SynΩ3 comprising Bacillus megaterium DSM 32963 and n-3 PUFA salts significantly increased circulating SPM precursor levels, including 18-hydroxy-eicosapentaenoic acid (18-HEPE) plus 5-HEPE, which was not achieved to this extent by fish oil with a similar n-3 PUFA content. Omega-3 indices were increased slightly by both SynΩ3 and fish oil. These findings suggest reconsidering conventional n-3 PUFA supplementation and testing the effectiveness of SynΩ3 particularly in conditions related to inflammation.

Biosynthesis Optimization of Antibacterial-Magnetic Iron Oxide Nanoparticles from Bacillus megaterium.

The occurrence of antibiotic resistance on common bacterial agents and the need to use new generations of antibiotics have led to the use of various strategies for production. Taking inspiration from nature, using bio-imitation patterns, in addition to the low cost of production, is advantageous and highly accurate. In this research, we were able to control the temperature, shake, and synthesis time of the synthesis conditions of Bacillus megaterium bacteria as a model for the synthesis of magnetic iron nanoparticles and optimize the ratio of reducing salt to bacterial regenerating agents as well as the concentration of salt to create iron oxide nanoparticles with more favorable properties and produced with more antibacterial properties. Bacterial growth was investigated by changing the incubation times of pre-culture and overnight culture in the range of the logarithmic phase. The synthesis time, salt ratio, and concentration were optimized to achieve the size, charge, colloidal stability, and magnetic and antibacterial properties of nanoparticles. The amount of the effective substance produced by the bacteria was selected by measuring the amount of the active substance synthesized using the free radical reduction (DPPH) method. With the help of DPPH, the duration of the synthesis was determined to be one week. Characterizations such as UV-vis spectroscopy, FTIR, FESEM, X-ray, and scattering optical dynamics were performed and showed that the nanoparticles synthesized with a salt concentration of 80 mM and a bacterial suspension to salt ratio of 2:1 are smaller in size and have a light scattering index, a PDI index close to 0.1, and a greater amount of reducing salt used in the reaction during one week compared to other samples. Moreover, they had more antibacterial properties than the concentration of 100 mM. As a result, better characteristics and more antibacterial properties than common antibiotics were created on E. coli and Bacillus cereus.

Exploring the untapped practices in bacterial-fungal mixed-based cultures for acidic treatment of metal-enriched printed circuit board waste.

This study explores the extraction of metals from spent mobile phone printed circuit boards (SMPhPCBs) to address environmental and resource depletion concerns. The challenges in metal recovery from SMPhPCBs arise due to their complex composition and high metal content. While previous research has primarily focused on using bio-cyanide, bio-sulfate, and bio-ferric compounds from acidophilic bacteria, the potential of bio-oxalic acid for SMPhPCBs treatment and the alteration of their complex structure has not yet been explored. Additionally, this study suggests evaluating the untapped potential of Aspergillus niger in oxalic acid production through mixed cultures with bacteria, marking a pioneering approach. A unique culture of Bacillus megaterium and A. niger was created, inducing bio-stress by bacterial metabolites, including gluconic acid (2683 mg/l) and live/dead bacterial cells in a medium with glucose deficiency. Results demonstrated reducing sugar consumption and oxalic acid over-production in mixed cultures compared to pure cultures, ranging from 1350 to 4951 mg/l at an initial glucose concentration (IGC) of 10 g/l and 4276 to 7460 mg/l at IGC 20 g/l. This over-production is attributed to proposed fungal signaling mechanisms to bacteria. Metal extraction using organic acids and siderophores at 10 g/l pulp density, 24 h, and 60 °C yielded Mn (100 %), Pt (100 %), Pd (70.7 %), Fe (50.8 %), Co (48.3 %), Al (21.8 %), among others. The final valuable residue containing copper, gold, and silver holds potential for future recycling. The study concludes with XRD and FTIR analyses to assess the bioleaching effect on the bio-leached powder.

Bacillus megaterium: Evaluation of Chemical Nature of Metabolites and Their Antioxidant and Agronomics Properties.

Bacillus megaterium is particularly known for its abundance in soils and its plant growth promotion. To characterize the metabolites excreted by this specie, we performed successive liquid/liquid extractions from bacteria culture medium with different polarity solvents (cyclohexane, dichloromethane, ethyl acetate and butanol) to separate the metabolites in different polarity groups. The extracts were characterized regarding their total phenolic content, the amount of reducing sugar, the concentration of primary amines and proteins, their chromatographic profile by HPLC-DAD-ELSD and their chemical identification by GC-MS. Among the 75 compounds which are produced by the bacteria, 19 identifications were for the first time found as metabolites of B. megaterium and 23 were described for the first time as metabolites in Bacillus genus. The different extracts containing B. megaterium metabolites showed interesting agronomic activity, with a global inhibition of seed germination rates of soya, sunflower, corn and ray grass, but not of corn, compared to culture medium alone. Our results suggest that B. megaterium can produce various metabolites, like butanediol, cyclic dipeptides, fatty acids, and hydrocarbons, with diverse effects and sometimes with opposite effects in order to modulate its response to plant growth and adapt to various environmental effects. These findings provide new insight into bioactive properties of this species for therapeutic uses on plants.

The Pd (II) Reduction Mechanisms in Bacillus megaterium Y-4 Revealed by Proteomic Analysis.

Many studies have been conducted on the microbial reduction of Pd (II) to palladium nanoparticles (Pd-NPs) due to the environmental friendliness, low cost, and the decreased toxicity of Pd (II) ions. In this study, we investigate the reduction mechanism of Pd (II) by Bacillus megaterium Y-4 through proteomics. The data are available via ProteomeXchange with identifier PXD049711. Our results revealed that B. megaterium Y-4 may use the endogenous electron donor (NAD(P)H) generated by nirB, tdh, and fabG and reductase to reduce Pd (II) to Pd-NPs. The expression levels of fabG, tdh, gudB, and rocG that generate NAD(P)H were further increased, and the number of reduced Pd-NPs was further increased with the exogenous electron donor sodium formate. Endogenous electron mediators such as quinones and flavins in B. megaterium Y-4 can further enhance Pd (II) reduction. The findings provided invaluable information regarding the reduction mechanism of Pd (II) by B. megaterium Y-4 at the proteome level.

Mitigation of arsenic accumulation in crop plants using biofertilizer.

Elevated levels of arsenic in crop plants have been found in various regions worldwide, especially where agricultural soils have been affected by arsenic-enriched aquifers and human activities including mining, smelting, and pesticide application. Given the highly toxic nature of arsenic, remediation should be carried out immediately to reduce this potentially toxic element transport from soil to crop plants. This study focused on the utilization of biofertilizer which is a combination of arsenic-accumulating microorganisms and adsorbent (carrier) in order to achieve high efficiency of arsenic immobilization and ability to apply in the field. Thirty-two bacterial strains were isolated from 9 soil samples collected from the Dongjin and Duckum mining areas in Korea using a nutrient medium amended with 2 mM sodium arsenite. Among isolates, strain DE12 identified as Bacillus megaterium exhibited the greatest arsenic accumulation capacity (0.236 mg/g dry biomass) and ability to resist up to 18 mM arsenite. Among the three agricultural waste adsorbents studied, rice straw was proved to have a higher adsorption capacity (0.104 mg/g) than rice husk and corn husk. Therefore, rice straw was chosen to be the carrier to form biofertilizer together with strain DE12. Inoculation of biofertilizer in soil showed a reduction of arsenic content in the edible part of lettuce, water spinach, and sweet basil by 17.5%, 34.1%, and 34,1%, respectively compared to the control group. The use of biofertilizer may open up the potential application in the field for other food plants.

Interface Engineering of PdPt Ultrafine Ethanol Electro-Oxidation Nanocatalysts by Bacterial Soluble Extracellular Polymeric Substances (s-EPS) to Break through Sabatier Principle.

Unsatisfactory performance of ethanol oxidation reaction (EOR) catalysts hinders the application of direct ethanol fuel cells (DEFCs), while traditional alloy catalysts (like PdPt) is cursed by Sabatier principle due to countable active site types. However, bacterial soluble extracellular polymeric substances (s-EPS) owning abundent functional groups may help breacking through it by contrusting different active sites on PdPt and inducing them to play synergy effect, which is called interface engineering. Using s-EPS to engineer catalysts is more green and consumes lower energy compared to chemical reagents. Herein, PdPt alloy nanoparticles (≈2.1 nm) are successfully in situ synthesized by/on s-EPS of Bacillus megaterium, an ex-holotype. Tryptophan residuals are proved as the main reductant. In EOR, PdPt@s-EPS shows higher activity (3.89 mA cm-2) than Pd@s-EPS, Pt@s-EPS, Pt/C and most reported akin catalysts. Its stability and durability are excellent, too. DFT modelling further demonstrates that, interface engineering by s-EPS breaks through Sabatier principle, by the synergy of diverse sites owning different degrees of d-p orbital hybridization. This work not only makes DEFCs closer to practice, but provides a facile and green strategy to design more catalysts.

PHB production by Bacillus megaterium strain MNSH1-9K-1 using low-cost media.

Plastics are widely used for diverse applications due to their versatility. However, their negative impact on ecosystems is undeniable due to their long-term degradation. Thus, there is a rising need for developing eco-friendlier alternatives to substitute fossil-based plastics, like biopolymers. PHA are synthesized intracellularly by microorganisms under stressful conditions of growth and have similar characteristics to conventional polymers, like their melting point, transition temperatures, crystallinity, and flexibility. Although it is feasible to use biopolymers for diverse industrial applications, their elevated production cost due to the supplies needed for microbiological procedures and the low productivity yields obtained have been the main limiting factors for their commercial success. The present study assessed the ability of Bacillus megaterium strain MNSH1-9K-1 to produce biopolymers using low-cost media from different kinds of fruit-peel residues. The results show that MNSH1-9K-1 can produce up to 58 g/L of PHB when grown in a medium prepared from orange-peel residues. The data obtained provide information to enhance the scalability of these kinds of biotechnological processes.

Plant growth promoting bacteria and citric acid promote growth and cadmium phytoremediation in ryegrass.

Based on the growth-promoting effect of plant growth promoting bacteria on plants and the mobilization of Cd by citric acid, an experiment was designed in which the combined treatment of Bacillus megaterium and citric acid promoted ryegrass to repair Cd-contaminated soil. This study aimed to evaluate the effects of different treatments on the antioxidant enzyme activity, photosynthesis intensity, Cd accumulation, and rhizosphere cadmium migration under cadmium contamination conditions. And the soil morphology and structure changes were studied by infrared spectroscopy FourierTransformInfrared(FT-IR) and scanning electron microscope Energy Dispersive Spectrometer(SEM-EDS) before and after different treatments. The results show that the combined treatment of Bacillus megaterium and citric acid significantly improved the oxidative stress defense and plant photosynthesis and increased of rye biomass. rye biomass 1.28 times higher than CK treatment. Joint treatment significantly increased the amount of shoot accumulation of Cd, 2.31 times higher than CK treatment, increased the migration and accumulation of cadmium. FTIR and SEM-EDS also showed that the organic constituents such as O-H, C-O and C-N in soils as a major mechanism for mobilization of the heavy metal Cd. Thus, the combined treatment of Bacillus megaterium and citric acid can promote plant growth, improve the damage to ryegrass caused by single organic acid addition, and improve the plant extraction efficiency, which is a feasible way to repair Cd-contaminated soil through activated extraction system.

The novelty of this study is the combined application of bacteria and chelating agents to ryegrass to improve phytoremediation efficiency. Bacillus giganosus has a good role in promoting the growth of ryegrass. As citrate, a small molecule chelate, can activate heavy metal cadmium and detoxify heavy metals, so it was selected. This study revealed in detail the response of ryegrass to the heavy metal Cd after exogenous addition of Bacillus gigansus and citrate, which is important for the application of cadmium removal by phytoremediation.

Mining Lactonase Gene from Aflatoxin B1-Degrading Strain Bacillus megaterium and Degrading Properties of the Recombinant Enzyme.

Mycotoxins are toxic secondary metabolites mainly produced by filamentous fungal species that commonly contaminate food and feed. Aflatoxin B1 (AFB1) is extremely toxic and seriously threatens the health of humans and animals. In this work, the Bacillus megaterium HNGD-A6 was obtained and showed a 94.66% removal ability of AFB1 by employing extracellular enzymes as the degrading active substance. The degradation products were P1 (AFD1, C16H14O5) and P2 (C14H16N2O2), and their toxicity was greatly reduced compared to that of AFB1. The AttM gene was mined by BlastP comparison and successfully expressed in Escherichia coli BL21. AttM could degrade 86.78% of AFB1 at pH 8.5 and 80 °C, as well as 81.32% of ochratoxin A and 67.82% of zearalenone. The ability of AttM to degrade a wide range of toxins and its resistance to high temperatures offer the possibility of its use in food or feed applications.

Bacillus megaterium RTS1 enhances resistance of Lycopersicon esculentum to salinity stress through the improvement of antioxidant defenses.

Background and Objectives: Plant growth-promoting bacteria (PGPB) may reduce the negative effects of salinity stress. The aim of this study was to optimize Bacillus megaterium RTS1 and characterize the effect of the PGPB on the physiological characteristics of tomato (Lycopersicon esculentum). Materials and Methods: The Central composite design (CCD) of response surface methodology (RSM) was used to optimize Bacillus megaterium RTS1 to produce maximum cell biomass and spores. Then the effect of the PGPB on the physiological characteristics of tomato (Lycopersicon esculentum), including membrane stability, leaf relative water content percentage, anthocyanin and carotenoids content, chlorophyll photosynthetic parameters, sugar and starch level, superoxide anion and antioxidant activity under salt stress conditions. The NFB medium was inoculated with 5% bacterial culture and the fermentation was carried out in a 10-lit fermenter. Results: After optimization, the amount of cell biomass by the model was 9.45 log10 CFUs/mL, which showed a 1.2-fold increase compared to the non-optimized medium. Usage of bacteria under the optimal conditions of the culture medium may increase the stability of the membrane and improve the relative water content. Bacteria were able to prevent the excessive increase of anthocyanins. Oxidative stress led to an increase in the content of chlorophyll a, while causing the degradation of chlorophyll b. Bacterial inoculation led to an increase in the level of sugar and starch compared to the control. PGPB showed an increasing effect on the amount of superoxide anion production and caused a significant increase in the antioxidant activity under salinity stress conditions. Conclusion: The PGPB can be a promising way to boost physiological characteristics of tomato plant under salinity stress. Also, sporulation capacity of Bacillus megaterium with high bacterial cell density in fermenter produce a sustainable product for tomato plants.

Application of Bacillus spp. Phosphate-Solubilizing Bacteria Improves Common Bean Production Compared to Conventional Fertilization.

The use of phosphate-solubilizing bacteria (PSB) can be a sustainable strategy to increase phosphorus availability and promote satisfactory crop yields. The objective of this study was to evaluate whether inoculation with PSB in common bean increases (i) growth, (ii) nutrition, (iii) yield, and (iv) grain quality, and (v) reduces the chemical phosphorus application dose to obtain maximum yields. The experiment was conducted in an Oxisol using a randomized block design in a 4 × 4 factorial scheme, with four replicates, using the cultivar IAC 2051. The first factor was four doses of P2O5 (0, 20, 40 and 60 kg ha-1), and the second factor was four doses of PSB (0, 100, 200 and 300 mL ha-1). For leaf area and leaf chlorophyll content, the association of PSB inoculation with a P2O5 dose of 40 kg ha-1 promoted the best conditions for the common bean. P2O5 application increased yield by 79 kg ha-1 for each 10 kg ha-1 added. PSB inoculation at a dose of 192 mL ha-1 promoted P export of 15.3 kg ha-1, and the PSB dose of 159 mL ha-1 increased yield by 389 kg ha-1 (12%) compared to the control. Grain quality remained within the standards required by the consumer market, being little affected by the treatments. Improvements in common bean growth and nutritional and physiological status promoted by P2O5 application and PSB were essential in increasing yield, so these are sustainable production strategies.

Characteristics and application in cheese making of newly isolated milk-clotting enzyme from Bacillus megaterium LY114.

Milk-clotting enzyme (MCE) is a crucial active agent in cheese making. It is necessary to find traditional MCE substitutes due to the limited production of traditional MCE (e.g., calf rennet) and increased cheese consumption. Bacillus megaterium strain LY114 with good milk-clotting activity (MCA) (448 SU/mL) and a high MCA/proteolytic activity (PA) ratio (6.0) was isolated and identified from agricultural soil in Laiyang (Shandong, China) through 16S rRNA sequencing of 45 strains. The Bacillus megaterium LY114 MCE had a remarkable specific activity (7532 SU/mg) and displayed a 4.83-fold purification yield with 34.17% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified LY114 MCE was a metalloprotease with a molecular weight of 30 kDa. LY114 MCE was stable at pH 5.0-7.0 and temperature <40 °C. The highest MCA appeared at a substrate pH of 5.5 with 30 mM CaCl2. The Michaelis constant (Km) and maximal velocity (Vm) for casein were 0.31 g/L and 14.16 µmol/min, respectively. LY114 MCE preferred to hydrolyze α-casein (α-CN) rather than ß-casein (ß-CN) and had unique α-CN, ß-CN and κ-casein (κ-CN) cleavage sites. LY114 MCE hydrolyzed casein to generate significantly different peptides compared with calf rennet and fungal MCE as determined by SDS-PAGE analysis. Chemical index analysis and sensory evaluation confirmed the usefulness of LY114 MCE in cheese making. LY114 MCE had the potential to be used in dairy processing and enriched traditional MCE substitutes.