Search Results (100)

As a non-pathogenic, Gram-positive strain, Bacillus subtilis is well-known for its efficient protein secretion mechanism and versatile microbial cell factory. However, the present B. subtilis expression vectors have drawbacks that prevent their industrial use, such as poor stability, low copy number, and low expression efficiency. In recent years, systematic optimization of expression vectors and elements has emerged as a key strategy for enhancing protein production efficiency. Among these efforts, constructing high-copy, stable vector backbones serves as the foundation for improving heterologous protein expression. Further optimization of critical regulatory elements—including regulatory genes, promoters, ribosome binding sites, signal peptides, and terminators—can significantly increase protein yield and process controllability. This review summarizes recent advances in B. subtilis expression systems, focusing on vector design and coordinated optimization of regulatory elements. Additionally, it discusses strategies for constructing efficient and controllable expression vectors, offering theoretical insights and technical guidance for future industrial applications. Full article

The continuous advancement of the food industry is accompanied by increased generation of animal waste, including poultry feathers. Composting presents a sustainable alternative to disposal methods such as incineration by converting waste into valuable fertilizer products. This study aimed to evaluate the impact of inoculation with the keratinolytic strain Bacillus subtilis P22 on the quality and maturity of compost produced from feathers combined with organic additives (wood shavings and lignite). The experiment involved evaluation of the keratinolytic potential of the tested strain, and characterization of its proteolytic enzymes, solid-state cultures and composting conducted at semi-technical scale. The B. subtilis P22 strain demonstrated the ability to solubilize 78% of feather material within 7 days of cultivation. The keratinolytic enzyme complex was likely dominated by polycatalytic alkaline serine proteases, i.e., subtilisins. The effectiveness of the inoculum was confirmed in laboratory solid-state cultures, where the dry mass loss in inoculated samples was twice that of the control containing only endogenous microflora. At the semi-technical scale, inoculation with B. subtilis P22 significantly accelerated compost maturation and mineralization (C/N = 10.2; N-NH₄⁺/N-NO₃⁻ = 0.4; Cw/Corg = 0.9) compared to the control. The final compost’s mineral composition indicates its potential for use as an organic soil amendment. Full article

The application of microbial alkaline proteases holds significant potential for eco-sustainable industrial processes by reducing chemical usage and lowering the costs of effluent treatment. In the search for novel proteases with industrial relevance, several microbial strains were isolated from alkaline baths of the Portuguese tannery agroindustry. The most promising protease-producing strains were selected for identification and further study. Two isolates demonstrated the highest proteolytic activity, reaching 0.51 ± 0.01 U mL⁻¹ and 0.70 ± 0.01 U mL⁻¹ after 7.5 h of submerged cultivation in nutrient broth. Based on API biochemical tests, molecular biology techniques, and GC-FAME analysis of membrane lipids, the isolates were identified as Bacillus subtilis and incorporated into INIAV’s collection of industrial microbial cultures as B. subtilis CCMI 1253 (BMR2) and B. subtilis CCMI 1254 (BMR1). The most promising protease producer, B. subtilis CCMI 1253 (BMR2), exhibited a maximum specific growth rate of 0.88 ± 0.10 h⁻¹. The proteases produced exhibited good extracellular proteolytic activity, with adaptability to industrial conditions, indicating their suitability for agroindustry applications such as leather making, detergent formulations and the treatment of effluents and protein residues. The results support the potential of microbial proteases as valuable tools in the bioeconomy and green chemistry. Full article

Bacterial nanocellulose (BNC) has gained considerable interest over the last decade due to its unique properties and versatile applications. However, the low yield and the high production cost significantly limit its industrial scalability. The proposed study explores the isolation of new BNC producers from date palm sap and the use of date waste extract as a sustainable carbon source to improve BNC productivity. Results revealed three potential BNC producers identified as Komagataeibacter sp. IS20, Komagataeibacter sp. IS21, and Komagataeibacter sp. IS22 with production yield of 1.7 g/L, 0.8 g/L and 1.8 g/L, respectively, in Hestrin-Schramm (HS) medium. The biopolymer characterization indicated the presence of type I cellulose, a high thermal stability, and a highly dense network made of cellulose nanofibrils for all BNC samples. The isolate IS22, showing the highest productivity, was selected for an optimization procedure using a full factorial design with date waste extract as a carbon source. The BNC yield increased to 6.59 g/L using 4% date waste extract and 2% ethanol after 10 days of incubation compared to the standard media (1.8 g/L). Two probiotic strains, including Bacillus subtilis (BS), and Lactobacillus plantarum (LP) were successfully encapsulated into BNC matrix through a co-culture approach. The BNC-LP and BNC-BS composites showed antibacterial activity against Pseudomonas aeruginosa. BNC–probiotic composites have emerged as a promising strategy for the effective delivery of viable probiotics in a wide range of applications. Overall, this study supports the use of date waste extract as a sustainable carbon source to enhance BNC productivity and reduce the environmental footprint using a high-yielding producer (IS22). Furthermore, the produced BNC demonstrated promising potential as an efficient carrier matrix for probiotic delivery. Full article

Adenosine holds significant application value in the fields of food additives and pharmaceutical intermediate synthesis. Engineering strains to enhance their efficiency in utilizing fermentation substrates is considered an effective strategy for improving production yield. However, modifications to adenosine-producing strains remain challenging due to the complex physiological and metabolic regulation governing adenosine biosynthesis. In this study, the molecular regulatory mechanisms of adenosine biosynthesis in a high-yielding Bacillus subtilis strain were analyzed through transcriptome sequencing. Under conditions in which an additional 10 g/L glutamine and 6 g/L hypoxanthine were supplemented at 48 h of cultivation to promote adenosine synthesis, a total of 105 significantly differentially expressed genes (69 downregulated and 36 upregulated) were identified, with key genes related to adenosine biosynthesis primarily concentrated in the downstream purine metabolic pathway. Notably, core biosynthetic genes including purD, guaC, purH, and purN showed significant downregulation in the high-yielding strain, suggesting that adenosine accumulation might inhibit related gene expression through negative feedback mechanisms. Fermentation kinetic analysis revealed that biomass reached its peak at 48 h (OD₆₀₀ = 0.82), with a glucose consumption rate of 73.28% at this stage. Gene expression pattern analysis demonstrated that purD, guaC, purH, and purN maintained relatively stable expression levels during fermentation. However, the exogenous supplementation of inosine (6 g/L) and glutamine (10 g/L) induced significant inhibition of their expression—a trend paralleling that observed with exogenous adenosine addition. This research elucidates key regulatory nodes in the adenosine biosynthesis of Bacillus subtilis and provides theoretical support and candidate targets for the targeted modification of industrial strains through metabolic engineering strategies. Full article

This study evaluates various strains of soil bacterial for use in the development of new biopreparations. Mesophilic spore-forming bacteria were isolated from cultivated soil and analysed for their enzymatic activity, ability to decompose crop residues, and antagonistic properties towards selected phytopathogens. Notably, this is the first cytotoxicity assessment of soil bacterial metabolites on Spodoptera frugiperda Sf-9 (fall armyworm). Bacillus subtilis, Bacillus licheniformis, Bacillus velezensis, Paenibacillus amylolyticus, and Prestia megaterium demonstrated the highest hydrolytic potential for the degradation of post-harvest residues from maize, winter barley, and triticale. They exhibited antimicrobial activity against at least three of the tested phytopathogens and demonstrated the ability to solubilize phosphorus. Metabolites of B. licheniformis (IC50 = 8.3 mg/mL) and B. subtilis (IC50 = 144.9 mg/mL) were the most cytotoxic against Sf-9. We recommend the use of the tested strains in industrial practice as biocontrol agents, plant growth biostimulants, crop residue decomposition stimulants, and bioinsecticides. Future studies should focus on assessing the efficacy of using these strains under conditions simulating the target use, such as plant microcosms and greenhouses and the impact of these strains on the abundance and biodiversity of native soil microbiota. This research can serve as a model procedure for screening other strains of bacteria for agricultural purposes. Full article

Surface display technology has revolutionized whole-cell biocatalysis by enabling efficient enzyme immobilization on microbial cell surfaces. Compared with traditional enzyme immobilization, this technology has the advantages of high enzyme activity, mild process, simple operation and low cost, which thus has been widely studied and applied in various fields. This review explores the principles, optimization strategies, applications in the food industry, and future prospects. We summarize the membrane and anchor protein structures of common host cells (Escherichia coli, Bacillus subtilis, and yeast) and discuss cutting-edge optimization approaches, including host strain genetic engineering, rational design of anchor proteins, innovative linker peptide engineering, and precise regulation of signal peptides and promoters, to maximize surface display efficiency. Additionally, we also explore its diverse applications in food processing and manufacturing, additive synthesis, food safety, and other food-related industries (such as animal feed and PET packaging degradation), demonstrating their potential to address key challenges in the food industry. This work bridges fundamental research and industrial applications, offering valuable insights for advancing agricultural and food chemistry. Full article

This study presents a sustainable and scalable biosynthesis method for zinc oxide (ZnO) nanoparticles using Bacillus subtilis, focusing on their application in photocatalytic cyanide degradation in aqueous solutions. The bacterial strain was molecularly identified through 16S rRNA gene sequencing and phylogenetic analysis. The optimized biosynthesis process yielded crystalline ZnO nanoparticles in the zincite phase with an average size of 21.87 ± 5.84 nm and a specific surface area of 27.02 ± 0.13 m²/g. Comprehensive characterization confirmed the formation of high-purity hexagonal ZnO (space group P63mc) with a bandgap of 3.20 eV. Photocatalytic tests under UV irradiation demonstrated efficient concentration-dependent cyanide degradation, achieving 75.5% removal at 100 ppm and 65.8% at 500 ppm within 180 min using 1.0 g/L ZnO loading. The degradation kinetics followed a pseudo-first-order model with rate constants ranging from 6.64 × 10⁻³ to 3.98 × 10⁻³ min⁻¹. The enhanced photocatalytic performance is attributed to the optimal crystallite size, high surface area, and surface defects identified through a microscopic analysis. These results establish biosynthesized ZnO nanoparticles as promising eco-friendly photocatalysts for industrial wastewater treatment. Full article

Microbial-induced carbonate precipitation technology allows concrete to detect and diagnose cracks autonomously. However, the concrete’s compact structure and alkaline environment necessitate the adoption of a proper carrier material to safeguard microorganisms. In this study, various bacterial strains, including Bacillus subtilis, Bacillus sphaericus, and Bacillus megaterium, were immobilized in lightweight expanded clay aggregates (LECA) to investigate their effect on the self-healing performance, mechanical strength, and freeze–thaw durability. Self-healing concrete specimens were prepared using immobilized LECA, directly added bacterial spores, polyvinyl acetate (PVA) fibers, and air-entraining admixture (AEA). The pre-cracked prisms were monitored for 224 days to assess self-healing efficiency through ultrasonic pulse velocity (UPV) and surface crack analysis methods. A compressive strength restoration test was conducted by pre-loading the cube specimens with 60% of the failure load and re-testing them after 28 days for strength regain. Additionally, X-ray diffraction and scanning electron microscopy (SEM) were conducted to analyze the precipitate material. The findings revealed that self-healing efficiency improved with the biomineralization activity over the healing period demonstrated by the bacterial strains. Compression and flexural strengths decreased for the bacterial specimens attributed to porous LECA. However, restoration in compression strength and freeze–thaw durability significantly improved for the bacterial mixes compared to control and reference mixes. XRD and SEM analyses confirmed the formation of calcite as a self-healing precipitate. Overall, results indicated the superior performance of Bacillus megaterium followed by Bacillus sphaericus and Bacillus subtilis. The findings of the current study provide important insights for the construction industry, showcasing the potential of bacteria to mitigate the degradation of concrete structures and advocating for a sustainable solution that reduces reliance on manual repairs, especially in inaccessible areas of the structures. Full article

Biotechnological research and application of microbial enzyme production have consistently been focal points for scientific inquiry and industrial advancement. In this study, Bacillus subtilis Y4X3 was isolated from saline–alkaline soil in Xinjiang, China. Extracellular enzyme production analysis revealed that B. subtilis Y4X3 can secrete various enzymes, including cellulase, xylanase, protease, and amylase. Sequencing and assembly of the complete genome of this strain revealed a genome size of 4,215,636 bp with 43.51% C + G content, including 4438 coding genes. Genome annotation was performed with databases to predict gene functions in B. subtilis Y4X3, and a variety of genes related to carbohydrate metabolism were identified. A cellulase-encoding gene was subsequently cloned from the genome and heterologously expressed in Escherichia coli. The optimum pH and temperature for the purified cellulase Cel5A were 5.0 and 60 °C, respectively. Stability analysis revealed that Cel5A remained stable at pH 5.0–9.0 and 20–60 °C; after 1 h at pH 9.0, the relative enzyme activity still exceeded 60%. Additionally, Cel5A was positively affected by various metal ions and exhibited good tolerance to multiple chemical reagents. The results indicate that B. subtilis Y4X3 has the potential to produce a variety of industrial enzymes and could serve as a promising candidate for more efficient and cost-effective industrial applications; the characterized thermostable and alkali-resistant cellulase Cel5A also has potential applications in biotechnology and industry. Full article

Killing spores is an important challenge for the development of the food industry. After germination, the resistance of spores disappears and they are more easily killed, which is currently the main strategy for their destruction. Therefore, study of the mechanism of spore germination is of great significance for improving methods of spore inactivation. Previous studies have shown that the hydration of the spore core region, accompanied by the disappearance of bacterial spore resistance, is a key step in the germination pathway of bacterial spores. However, the specific mechanism of this process has been studied very little. In this study, Bacillus subtilis PY79 was used as a model strain, and its single water glycerol channel protein (GlpF) was regarded as a starting point to explore the mechanism of water transport during spore germination. First, we constructed glpF mutants and overexpression strains and discovered that the deletion of glpF did not affect the growth of bacterial vegetative cells and spores. Further germination experiments on the spores of the glpF-deficient strain through detecting calcium dipicolinate and absorbance of spores showed that the germination rate of the mutant strain spores increased, while increasing the water activity did not affect the results caused by glpF deletion. Meanwhile, overexpressed glpF affected the permeability of the spore coat. Finally, when treating spores with ultra-high pressure, the spores lacking glpF were more likely to be inactivated. The above results have suggested that the glpF gene plays an important role in spore germination. Full article

Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of B. licheniformis and B. subtilis in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of B. licheniformis and B. subtilis strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that B. licheniformis released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of B. licheniformis and B. subtilis survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of B. licheniformis and B. subtilis on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of B. licheniformis and B. subtilis, less CO₂ was produced while dry matter degradation and CH₄ production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants. Full article

Waste biomass deriving from agricultural activities has different destinations depending on the possibility of applying it to specific processes. As the waste biomass is abundant, cheap, and generally safe, it can be used for several applications, biogas production being the most relevant from the quantitative point of view. In this study, we have used a set of agricultural by-products (agro-waste) deriving from the post-harvest treatment of cereals and legumes as the growth substrate for selected biosurfactant-producing microbial strains. The agricultural by-products were easily metabolized and highly effective for the growth of microorganisms and the production of rhamnolipids and surfactin by Pseudomonas aeruginosa and Bacillus subtilis, respectively. In particular, the use of corn chaff (“bee-wings”) was suitable for the production of rhamnolipids. Indeed, in corn-chaff-based media, rhamnolipids yields ranged from 2 to 18 g/L of fermentation broth. This study demonstrated that the use of waste raw materials could be applied to reduce the carbon footprint of the production of biosurfactants without compromising the possibility of having a suitable fermentation medium for industrial production. Full article

Horizontal gene transfer (HGT) plays a pivotal role in bacterial evolution, shaping the genetic diversity of bacterial populations. It can occur through mechanisms such as conjugation, transduction, and natural transformation. Bacillus subtilis, a model Gram-positive bacterium, serves not only as a robust system for studying HGT but also as a versatile organism with established industrial applications, such as producing industrial enzymes, antibiotics, and essential metabolites. In this study, we characterize a novel method of plasmid transfer, termed Cell-to-Cell Natural Transformation for Plasmid Transfer (CTCNT-P), which efficiently facilitates plasmid transfer between naturally competent B. subtilis strains. This method involves co-culturing donor and recipient cells under antibiotic stress and achieves significantly higher efficiency compared to traditional methods such as Spizizen medium or electroporation-mediated transformation. Importantly, we demonstrate that CTCNT-P is applicable for plasmid transformation in wild B. subtilis isolates from natural environments and other Bacillus species, including Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis. The simplicity and efficiency of CTCNT-P highlight its strong potential for industrial applications, including genetic modification of wild Bacillus strains for synthetic biology and the development of biocontrol agents. Full article

Acid phosphatases (ACPase, EC 3.1.3.2) are hydrolytic enzymes widely distributed in both plant and animal tissues. Despite their ubiquitous presence, the production and specific activity of ACPase in these sources remain suboptimal. Consequently, the development of microbial cell factories for large-scale ACPase production has emerged as a significant research focus. In this study, we successfully expressed the phosphatase PAP2 family protein (acid phosphatase) from Acinetobacter nosocomialis 1905 in Bacillus subtilis 168. The specific activity of the crude enzyme solution was 59.60 U/mg. After purification, the enzyme activity increased to 86.62 U/mL, with a specific activity of 129.60 U/mg. Characterization of the enzyme revealed optimal activity at 45 °C and a pH of 6.0. The Km value was determined to be 0.25 mmol/L using p-nitrophenylphosphoric acid disodium salt as the substrate. Additionally, the enzyme activity was found to be enhanced by the presence of Ni²⁺. Dissolved oxygen and medium were subsequently optimized during fermentation on the basis of a commercially available 5 L bioreactor. The recombinant strain B. subtilis 168/pMA5-Acp achieved maximal volumetric enzyme activity of 136.9 U/mL after 12 h of fermentation under optimized conditions: an aeration rate of 1.142 VVM (4 lpm), an agitation speed of 350 rpm, and an optimal ratio of lactose to fish powder (7.5 g/L:12.5 g/L). These optimizations resulted in a 5.9-fold increase in volumetric enzyme activity, a 4.9-fold increase in enzyme synthesis per unit cell volume, and a 48.6% increase in biomass concentration. This study establishes a comprehensive technological framework for prokaryotic fermentation-based ACPase production, potentially addressing the bottleneck in industrial-scale applications. Full article