Assessing the quality and eco-beneficial microbes in the use of silkworm excrement compost.

Sericulture has become widespread globally, and the utilization of artificial diets produces a substantial quantity of silkworm excrement. Although silkworm excrement can be composted for environmentally friendly disposal, the potential utility of the resulting compost remains underexplored. The aim of this study was to assess the quality of this unique compost and screen for eco-beneficial microbes, providing a new perspective on microbial research in waste management, especially in sustainable agriculture. The low-concentration compost application exhibited a greater plant growth-promoting effect, which was attributed to an appropriate nutritional value (N, P, K, and dissolved organic matter) and the presence of plant growth-promoting bacteria (PGPB) within the compost. Encouraged by the "One Health" concept, the eco-benefits of potent PGPB, namely, Klebsiella pneumoniae and Bacillus licheniformis, in sericulture were further evaluated. For plants, K. pneumoniae and B. licheniformis increased plant weight by 152.44 % and 130.91 %, respectively. We also found that even a simple synthetic community composed of the two bacteria performed better than any single bacterium. For animals, K. pneumoniae significantly increased the silkworm (Qiufeng × Baiyu strain) cocoon shell weight by 111.94 %, which could increase sericulture profitability. We also elucidated the mechanism by which K. pneumoniae assisted silkworms in degrading tannic acid, a common plant-derived antifeedant, thereby increasing silkworm feed efficiency. Overall, these findings provide the first data revealing multiple beneficial interactions among silkworm excrement-derived microbes, plants, and animals, highlighting the importance of focusing on microbes in sustainable agriculture.

Enhanced biodegradation of phenol under Cr(VI) stress by microbial collaboration and potential application of machine learning for phenol biodegradation.

Cr(VI) and phenol commonly coexist in wastewater, posing a great threat to the environment and human health. However, it is still a challenge for microorganisms to degrade phenol under high Cr(VI) stress. In this study, the phenol-degrading strain Bacillus cereus ZWB3 was co-cultured with the Cr(VI)-reducing strain Bacillus licheniformis MZ-1 to enhance phenol biodegradation under Cr(Ⅵ) stress. Compared with phenol-degrading strain ZWB3, which has weak tolerance to Cr(Ⅵ), and Cr(Ⅵ)-reducing strain MZ-1, which has no phenol-degrading ability, the co-culture of two strains could significantly increase the degraded rate and capacity of phenol. In addition, the co-cultured strains exhibited phenol degradation ability over a wide pH range (7-10). The reduced content of intracellular proteins and polysaccharides produced by the co-cultured strains contributed to the enhancement of phenol degradation and Cr(Ⅵ) tolerance. The determination coefficients R2, RMSE, and MAPE showed that the BP-ANN model could predict the degradation of phenol under various conditions, which saved time and economic cost. The metabolic pathway of microbial degradation of phenol was deduced by metabolic analysis. This study provides a valuable strategy for wastewater treatment containing Cr(Ⅵ) and phenol.

Enhancing the Antibacterial Impact of Lipopeptide Extracted from Bacillus licheniformis as a Probiotic against MDR Acinetobacter baumannii.

BACKGROUND: The antibiotic resistance of microorganisms is escalating rapidly. Infections caused by opportunistic pathogens in immunocompromised individuals have prompted researchers to seek for potent and safe antibacterial agents. The purpose of this investigation was to explore the suppression of virulence gene expression, specifically the pga operon genes responsible in biofilm formation in Acinetobacter baumannii, through the utilization of metabolites obtained from probiotic bacteria. METHODS: To assess the antimicrobial properties, standard strains of five probiotic bacteria were tested against a standard strain of multidrug-resistant (MDR) A. baumannii employing the agar gel diffusion technique. Following the identification of the most potent probiotic strain (Bacillus licheniformis), the existence of its LanA and LanM genes was confirmed using the polymerase chain reaction (PCR) test. High-performance liquid chromatography (HPLC) and fourier-transform infrared spectroscopy (FTIR) techniques were employed to identify the intended metabolite, which was found to be a lipopeptide nature. The minimum inhibitory concentration (MIC) values and anti-biofilm activity of the targeted metabolite were determined using a dilution method in 96-well microplates and field emission scanning electron microscopy (FE-SEM). Real-time PCR (qPCR) was utilized for comparing the expression of pga operon genes, including pgaABCD, in A. baumannii pre- and post-exposure to the derived lipopeptide. RESULTS: The MIC results indicated that the probiotic product inhibited the growth of A. baumannii at concentrations lower than those needed for conventional antibiotics. Furthermore, it was observed that the desired genes' expression decreased due to the effect of this substance. CONCLUSIONS: This research concludes that the B. licheniformis probiotic product could be a viable alternative for combating drug resistance in A. baumannii.

Enhanced organic degradation and microbial community cooperation by inoculating Bacillus licheniformis in low temperature composting.

Microbial activity and interaction are the important driving factors in the start-up phase of food waste composting at low temperature. The aim of this study was to explore the effect of inoculating Bacillus licheniformis on the degradation of organic components and the potential microbe-driven mechanism from the aspects of organic matter degradation, enzyme activity, microbial community interaction, and microbial metabolic function. The results showed that after inoculating B. licheniformis, temperature increased to 47.8°C on day 2, and the degradation of readily degraded carbohydrates (RDC) increased by 31.2%, and the bioheat production increased by 16.5%. There was an obvious enhancement of extracellular enzymes activities after inoculation, especially amylase activity, which increased by 7.68 times on day 4. The inoculated B. licheniformis colonized in composting as key genus in the start-up phase. Modular network analysis and Mantel test indicated that inoculation drove the cooperation between microbial network modules who were responsible for various organic components (RDC, lipid, protein, and lignocellulose) degradation in the start-up phase. Metabolic function prediction suggested that carbohydrate metabolisms including starch and sucrose metabolism, glycolysis / gluconeogenesis, pyruvate metabolism, etc., were improved by increasing the abundance of related functional genes after inoculation. In conclusion, inoculating B. licheniformis accelerated organic degradation by driving the cooperation between microbial network modules and enhancing microbial metabolism in the start-up phase of composting.

Detection of risk areas in dairy powder processes: The development of thermophilic spore forming bacteria taking into account their growth limits.

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.

Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Effect of Ba2+ on the biomineralization of Ca2+ and Mg2+ ions induced by Bacillus licheniformis.

The effect of barium ions on the biomineralization of calcium and magnesium ions is often overlooked when utilizing microbial-induced carbonate precipitation technology for removing barium, calcium, and magnesium ions from oilfield wastewater. In this study, Bacillus licheniformis was used to bio-precipitate calcium, magnesium, and barium ions. The effects of barium ions on the physiological and biochemical characteristics of bacteria, as well as the components of extracellular polymers and mineral characteristics, were also studied in systems containing coexisting barium, calcium, and magnesium ions. The results show that the increasing concentrations of barium ions decreased pH, carbonic anhydrase activity, and concentrations of bicarbonate and carbonate ions, while it increased the contents of humic acids, proteins, polysaccharides, and DNA in extracellular polymers in the systems containing all three types of ions. With increasing concentrations of barium ions, the content of magnesium within magnesium-rich calcite and the size of minerals precipitated decreased, while the full width at half maximum of magnesium-rich calcite, the content of O-C=O and N-C=O, and the diversity of protein secondary structures in the minerals increased in systems containing all three coexisting ions. Barium ions does inhibit the precipitation of calcium and magnesium ions, but the immobilized bacteria can mitigate the inhibitory effect. The precipitation ratios of calcium, magnesium, and barium ions reached 81-94%, 68-82%, and 90-97%. This research provides insights into the formation of barium-enriched carbonate minerals and offers improvements for treating oilfield wastewater.

Comparative negation of amphiphile production using nutrition factors: Amyloids versus biosurfactants.

Microbial amphiphiles play an important role in environmental activities such as microbial signaling, bioremediation, and biofilm formation. Microorganisms rely on their unique characteristics of interfaces to carry out critical biological functions, which are helped by amphipathic biomolecules known as amphiphiles. Bacillus amyloids aid in cell adhesion and biofilm formation. Pseudomonas sp. are essential in biofilm development and are a vital survival strategy for many bacteria. Furthermore, Pseudomonas and Bacillus are well-known for their ability to produce biosurfactants with a range of applications, including bioremediation and removing biological pollutants from different environments. The study employed 31 different media types and a range of analytical techniques to assess the presence of amyloid proteins and the absence of biosurfactants in Bacillus licheniformis K125 (GQ850525.1) and Pseudomonas fluorescens CHA0. The presence of amyloid proteins was confirmed through Congo red and thioflavin T staining. The carefully constructed medium also efficiently inhibited the synthesis of biosurfactants by these bacteria. Additionally, surface tension measurements, emulsification index, thin-layer chromatography, and high-performance thin-layer chromatography analyses indicated the absence of biosurfactants in the tested media.

Improved production of RNA-inhibiting antimicrobial peptide by Bacillus licheniformis MCC 2514 facilitated by a genetic algorithm optimized medium.

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH4Cl and NaNO3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup.

Regulator DegU can remarkably influence alkaline protease AprE biosynthesis in Bacillus licheniformis 2709.

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis, which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5'-TAAAT……AAAAT…….AACAT…TAAAA-3') located upstream -91 to -87 bp, -101 to -97 bp, -195 to -191 bp, -215 to -211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE, which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism.

Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway.

Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production. ONE-SENTENCE SUMMARY: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.

A newly synthesized magnetic nanoparticle coated with glycidyl methacrylate monomer and 1,2,4-Triazole: Immobilization of α-Amylase from Bacillus licheniformis for more reuse, stability, and activity in the presence of H2O2.

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1-3% H2O2.

A new ROS response factor YvmB protects Bacillus licheniformis against oxidative stress under adverse environment.

Bacillus spp., a class of aerobic bacteria, is widely used as a biocontrol microbe in the world. However, the reactive oxygen species (ROS) will accumulate once the aerobic bacteria are exposed to environmental stresses, which can decrease cell activity or lead to cell death. Hydroxyl radical (·OH), the strongest oxide in the ROS, can damage DNA directly, which is generated through Fenton Reaction by H2O2 and free iron. Here, we proved that the synthesis of pulcherriminic acid (PA), an iron chelator produced by Bacillus spp., could reduce DNA damage to protect cells from oxidative stress by sequestrating excess free iron, which enhanced the cell survival rates in stressful conditions (salt, antibiotic, and high temperature). It was worth noting that the synthesis of PA was found to be increased under oxidative stress. Thus, we demonstrated that the YvmB, a direct negative regulator of PA synthesis cluster yvmC-cypX, could be oxidized at cysteine residue (C57) to form a dimer losing the DNA-binding activity, which led to an improvement in PA production. Collectively, our findings highlight that YvmB senses ROS to regulate PA synthesis is one of the evolved proactive defense systems in bacteria against adverse environments.IMPORTANCEUnder environment stress, the electron transfer chain will be perturbed resulting in the accumulation of H2O2 and rapidly transform to ·OH through Fenton Reaction. How do bacteria deal with oxidative stress? At present, several iron chelators have been reported to decrease the ·OH generation by sequestrating iron, while how bacteria control the synthesis of iron chelators to resist oxidative stress is still unclear. Our study found that the synthesis of iron chelator PA is induced by reactive oxygen species (ROS), which means that the synthesis of iron chelator is a proactive defense mechanism against environment stress. Importantly, YvmB is the first response factor found to protect cells by reducing the ROS generation, which present a new perspective in antioxidation studies.

Characterization, modeling, and anticancer activity of L.arginase production from marine Bacillus licheniformis OF2.

BACKGROUND: L-arginase, is a powerful anticancer that hydrolyzes L-arginine to L-ornithine and urea. This enzyme is widely distributed and expressed in organisms like plants, fungi, however very scarce from bacteria. Our study is based on isolating, purifying, and screening the marine bacteria that can produce arginase. RESULTS: The highest arginase producing bacteria will be identified by using microbiological and molecular biology methods as Bacillus licheniformis OF2. Characterization of arginase is the objective of this study. The activity of enzyme was screened, and estimated beside partial sequencing of arginase gene was analyzed. In silico homology modeling was applied to generate the protein's 3D structure, and COACH and COFACTOR were applied to determine the protein's binding sites and biological annotations based on the I-TASSER structure prediction. The purified enzyme was undergone an in vitro anticancer test. CONCLUSIONS: L-arginase demonstrated more strong anti-cancer cells with an IC50 of 21.4 ug/ml in a dose-dependent manner. L-arginase underwent another investigation for its impact on the caspase 7 and BCL2 family of proteins (BCL2, Bax, and Bax/Bcl2). Through cell arrest in the G1/S phase, L-arginase signals the apoptotic cascade, which is supported by a flow cytometry analysis of cell cycle phases.

Rational engineering and insight for a L-glutaminase activity reduced type II L-asparaginase from Bacillus licheniformis and its antileukemic activity in vitro.

Type II L-asparaginase (ASNase) has been approved by the FDA for treating acute lymphoid leukemia (ALL), but its therapeutic effect is limited by low catalytic efficiency and L-glutaminase (L-Gln) activity. This study utilized free energy based molecular dynamics calculations to identify residues associated with substrate binding in Bacillus licheniformis L-asparaginase II (BLASNase) with high catalytical activity. After saturation and combination mutagenesis, the mutant LGT (74 L/75G/111 T) with intensively reduced l-glutamine catalytic activity was generated. The l-glutamine/L-asparagine activity (L-Gln/L-Asn) of LGT was only 6.6 % of parent BLASNase, whereas the L-asparagine (L-Asn) activity was preserved >90 %. Furthermore, structural comparison and molecular dynamics calculations indicated that the mutant LGT had reduced binding ability and affinity towards l-glutamine. To evaluate its effect on acute leukemic cells, LGT was supplied in treating MOLT-4 cells. The experimental results demonstrated that LGT was more cytotoxic and promoted apoptosis compared with commercial Escherichia coli ASNase. Overall, our findings firstly provide insights into reducing l-glutamine activity without impacting L-asparagine activity for BLASNase to possess remarkable potential for anti-leukemia therapy.

Development of an Inosine Hyperproducer from Bacillus licheniformis by Systems Metabolic Engineering.

Inosine is widely used in food, chemical, and medicine. This study developed Bacillus licheniformis into an inosine hyperproducer through systems metabolic engineering. First, purine metabolism was activated by deleting inhibitors PurR and YabJ and overexpressing the pur operon. Then, the 5-phosphoribosyl-1-pyrophosphate (PRPP) supply was increased by optimizing the glucose transport system and pentose phosphate pathway, increasing the inosine titer by 97% and decreasing the titers of byproducts by 36%. Next, to prevent the degradation of inosine, genes deoD and pupG coding purine nucleoside phosphorylase were deleted, accumulating 0.91 g/L inosine in the culture medium. Additionally, the downregulation of adenosine 5'-monophosphate (AMP) synthesis pathway increased the inosine titer by 409%. Importantly, enhancing the glycine and aspartate supply increased the inosine titer by 298%. Finally, the guanosine synthesis pathway was blocked, leading to strain IR-8-2 producing 27.41 g/L inosine with a 0.46 g inosine/g glucose yield and a 0.38 g/(L·h) productivity in a shake flask.

Design of a sorbitol-activated nitrogen metabolism-dependent regulatory system for redirection of carbon metabolism flow in Bacillus licheniformis.

Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.

Extracellular nanovesicles produced by Bacillus licheniformis: A potential anticancer agent for breast and lung cancer.

Cancer is a major public burden and leading cause of death worldwide; furthermore, it is a significant barrier to increasing life expectancy in most countries of the world. Among various types of cancers, breast and lung cancers lead to significant mortality in both males and females annually. Bacteria-derived products have been explored for their use in cancer therapy. Although bacteria contain significant amounts of anticancer substances, attenuated bacteria may still pose a potential risk for infection owing to the variety of immunomodulatory molecules present in the parental bacteria; therefore, non-cellular bacterial extracellular vesicles (BEVs), which are naturally non-replicating, safer, and are considered to be potential anticancer agents, are preferred for cancer therapy. Gram-positive bacteria actively secrete cytoplasmic membrane vesicles that are spherical and vary between 10 and 400 nm in size. However, no studies have considered cytoplasmic membrane vesicles derived from Bacillus licheniformisin cancer treatment. In this study, we investigated the potential use of B. licheniformis extracellular nanovesicles (BENVs) as therapeutic agents to treat cancer. Purified BENVs from the culture supernatant of B. licheniformis using ultracentrifugation and ExoQuick were characterized using a series of analytical techniques. Human breast cancer cells (MDA-MB-231) and lung cancer cells (A549) were treated with different concentrations of purified BENVs, which inhibited the cell viability and proliferation, and increased cytotoxicity in a dose-dependent manner. To elucidate the mechanism underlying the anticancer activity of BENVs, the oxidative stress markers such as reactive oxygen species (ROS) and glutathione (GSH) levels were measured. The ROS levels were significantly higher in BENV-treated cells, whereas the GSH levels were markedly reduced. Cells treated with BENVs, doxorubicin (DOX), or a combination of BENVs and DOX showed significantly increased expression of p53, p21, caspase-9/3, and Bax, and concomitantly decreased expression of Bcl-2. The combination of BENVs and doxorubicin enhanced mitochondrial dysfunction, DNA damage, and apoptosis. To our knowledge, this is the first study to determine the anticancer properties of BENVs derived from industrially significant probacteria on breast and lung cancer cells.

Influence of pH on Inulin Conversion to 2,3-Butanediol by Bacillus licheniformis 24: A Gene Expression Assay.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

Bioconversion of feather waste into bioactive nutrients in water by Bacillus licheniformis WHU.

Feathers become hazardous pollutants when deposited directly into the environment. The rapid expansion of the poultry industry has significantly increased feather waste, necessitating the development of new ways to degrade and utilize feathers. This study investigated the ability of Bacillus licheniformis WHU to digest intact chicken feathers in water. The results indicated that yields of free amino acids, bioactive peptides, and keratin-derived nano-/micro-particles were improved in bacteria- versus purified keratinase-derived feather hydrolysate. Bacteria-derived feather hydrolysate supplementation induced health benefits in mice, including significantly increased intestinal villus height and zonula occludens-1 protein expression, as well as increased secretory immunoglobulin A levels in the intestinal mucosa and superoxide dismutase activity in serum. Additionally, feather hydrolysate supplementation modulated the mouse gut microbiota, reflected by increased relative abundance of probiotics such as Lactobacillus spp., decreased relative abundance of Proteobacteria at the phylum level and pathogens such as Staphylococcus spp., and increased Bacteroidota/Firmicutes ratio. This study developed a simple, cost-effective method to degrade feathers by B. licheniformis WHU digestion, yielding a hydrolysate that can be directly used as a bioactive nutrient resource. The study findings have applications in the livestock, poultry, and aquaculture industries, which have high demands for cheap protein. KEY POINTS: • Bacillus licheniformis could degrade intact feather in water. • The resulting feather hydrolysate shows prebiotic effects on mouse.