Improvement of violacein production using abiotic stresses and microbial adaptation.

The aim of the current research was to improve violacein production with Janthinobacterium lividum using abiotic stresses and bacterial adaptation against stress. Initially, the effect of carbon sources and the medium volume: air ratio on violacein production was assessed. Then, the production of violacein under hydrogen peroxide (H2O2) and ampicillin (Amp) stresses and acyl homoserine lactone (AHL) was evaluated. In the next step, J. lividum was adapted against increased concentrations of Amp. Finally, the production of violacein was analyzed in adapted bacterium cultivated in the presence of optimal amounts of H2O2, Amp, and AHL. The alterations in the expression of some of genes involved in violacein production was evaluated using Real-time PCR (RT-PCR). The highest amount of violacein was achieved using medium volume: air ratio of 10% v/v (in 100 ml flasks) and glycerol as carbon source. Also, H2O2 (103 mg/l) and Amp (130 mg/l) stresses increased the production of violacein significantly compared to normal conditions (57 mg/l) and violacein production in the presence of crude AHL increased from 56 mg/l to 210 mg/l. The production of violacein with adapted bacterium under the above-mentioned stresses and AHL was about 1.3 g/l. RT-PCR results showed that the expression of the AHL encoding gene (luxI) was repressed in the presence of stresses and glycerol. Also, the expression of vioA increased in the presence of Amp but H2O2 had no significant effect on vioA expression. Totally, we showed that microbial adaptation and abiotic stresses are cost-effective methods to generate significant improvement in violacein production.

Antibacterial activity of epsilon-poly-l-lysine produced by Stenotrophomonas maltophilia HS4 and Paenibacillus polymyxa HS5, alone and in combination with bacteriophages.

Over the past decades, antibiotic resistance has become a major clinical problem, and searching for new therapeutic strategies seems to be necessary. Using novel natural compounds, antimicrobial peptides, and bacteriophages is the most promising solution. In this study, various cationic metabolite-producer bacteria were isolated from different soil samples. Two isolates were identified as Stenotrophomonas maltophilia HS4 (accession number: MW791428) and Paenibacillus polymyxa HS5 (accession number: MW791430) based on biochemical characteristics and phylogenetic analysis using 16S rRNA gene sequences. The cationic compound in the fermentation broth was precipitated and purified with sodium tetraphenylborate salt. The purified cationic peptide was confirmed to be epsilon-poly-l-lysine by structural and molecular analysis using High-Performance Liquid Chromatography, Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, and Fourier-transform infrared spectroscopy. The antibacterial activity of epsilon-poly-l-lysine was evaluated against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Serratia marcescens ATCC 13880, and Klebsiella pneumoniae ATCC 13883 by microdilution method. Furthermore, the antibacterial effects of purified epsilon-poly-l-lysine in combination with two long non-contractile tail bacteriophages against vancomycin-resistant Enterococcus faecalis and colistin-resistant Klebsiella pneumoniae were investigated. The results indicated great antibacterial activity of epsilon-poly-l-lysine which was produced by two novel bacteria. The epsilon-poly-l-lysine as a potent cationic antimicrobial peptide is demonstrated to possess great antimicrobial activity against pathogenic and also antibiotic-resistant bacteria.

Biosynthesis of Copper Oxide and Silver Nanoparticles by Bacillus Spores and Evaluation of the Feasibility of Their Use in Antimicrobial Paints.

Modification of paint with nanoparticles (NPs) provides self-cleaning, water/dirt-repellent, and other properties. Therefore, the aim of the present study was to biosynthesize silver (Ag) and copper oxide (CuO) NPs and to prepare NP-modified paint. To this end, AgNPs and CuONPs were biosynthesized using Bacillus atrophaeus spores and commercial and crude dipicolinic acid (DPA) extracted from the spore of this bacterium. The synthesized NPs were characterized using electron microscopy, Fourier-transform infrared (FTIR), X-ray diffraction analysis (XRD), and energy-dispersive X-ray spectroscopy (EDS) methods. A minimum inhibitory concentration (MIC) assay of NPs against Escherichia coli ATCC8739 and Staphylococcus aureus ATCC6538 was carried out. The antibacterial effects of prepared NP-paint complexes were assessed using an optical density (OD) comparison before and after adding metal sheets coated with NP-paint complexes into the nutrient broth medium. Four different types of NPs were synthesized in this research: AgNPs synthesized by spore (A), AgNPs synthesized by commercial DPA (B), AgNPs synthesized by crude DPA (C), and CuONPs synthesized by spore (D). SEM analysis confirmed the spherical shape of NPs. According to the results, NPs A, B, and D showed higher antibacterial activity against S. aureus compared to E. coli. Furthermore, the analysis of the antibacterial effects of NP-paint complexes suggested that paint-NPs A, B, and C displayed higher activity on E. coli compared to S. aureus. Moreover, the antibacterial effect of paint-NP D was significantly lower than other NPs. According to this robust antibacterial effect on pathogenic bacteria, it seems that these NP-paint complexes could be useful in public places such as hospitals, airports, dormitories, schools, and office buildings, where the rate of transmission of infection is high.

Bacillus subtilis isolates from camel milk as probiotic candidates.

Recently Bacillus spp. has gained much attention as potential probiotics due to the production of resistant cells. So, this research is purposeful for evaluation of probiotic characteristics of Bacillus isolates from camel milk as a suitable source for growth and isolation of microorganisms that can be candidate to be used as probiotic. First, forty-eight colonies were screened by using morphological and biochemical analysis. Among the isolates, two of them were recognized as Bacillus subtilis CM1 and CM2 by partial 16SrRNA sequencing that, probiotic potentials of them were evaluated. Both of them, in the preliminary safety screening, were found negative for hemolysis and lecithinase activity. Also, in vitro characteristics such as acid, bile salts and artificial gastric juice resistant, cell surface hydrophobicity, auto-aggregation, antioxidant characteristics, and adherent capability to HT-29 cells were determined for them approximately in the range of other probiotic strains. Two strains were susceptible to various antibiotics and enterotoxigenic activities were not detected by PCR which means isolated Bacillus strains could be classified as safe. Altogether, results demonstrate that Bacillus CM1 and CM2 strains could have the potential of consideration as probiotics, however more extensive in vitro/vivo studies are needed.

Investigation of bioactivity of unsaturated oligo­galacturonic acids produced from apple waste by Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 Pectinases.

Pectin is one of the main structural components in fruits and an indigestible fiber made of D-galacturonic acid units with α (1-4) linkage. This study investigates the microbial degradation of pectin in apple waste and the production of bioactive compounds. Firstly, pectin-degrading bacteria were isolated and identified, then pectinolytic activity was assessed by DNS. The products were evaluated by TLC and LC-MS-ESI. The antioxidative effects were investigated using DPPH and anti-cancer effects and cytotoxicity were analyzed by MTT and flow cytometry. In this study two new bacterial isolates, Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 with the pectinolytic enzyme were introduced. Structure analysis showed that the products of enzymatic degradation include unsaturated mono, di, tri, and penta galacturonic acids with 74% and 69% RSA at 40 mg/mL for A. faecalis and P. polymyxa S4, respectively. The results of anti-tumor properties on MCF-7 cells by MTT assay, for products of AGS3 and S4 at 40 mg/mL after 48 h, showed 7% and 9% survival, respectively. In the flow cytometric assessment, the compounds of AGS3 at 40 mg/mL were 100% lethal in 48 h and regarding S4 isolate caused 98% death. Cytotoxicity evaluation on L-929 cells showed no significant toxicity on living cells.

Synthesis and characterization of Ag-ion-exchanged zeolite/TiO2 nanocomposites for antibacterial applications and photocatalytic degradation of antibiotics.

This paper investigates the synthesis, antibacterial, and photocatalytic properties of silver ion-exchanged natural zeolite/TiO2 photocatalyst nanocomposite. Zeolite is known to have a porous surface structure, making it an ideal substrate and framework in different nanocomposites. Moreover, natural zeolite has a superior thermal and chemical stability, with hardly any reactivity with chemicals. Finding an effective and low-cost method to remove both antibiotics and bacteria from water resources has become a vital global issue due to the worldwide excessive use of chemicals and antibiotics. This research aims to propose a facile method to synthesize Ag-ion-exchanged zeolite/TiO2 catalyst for anti-bacterial purposes and photocatalytic removal of atibiotics from wastewaters. TiO2 particles were deposited on the surface of natural zeolite. Ag ion exchanging was performed via a liquid ion-exchange method using 0.1 M AgNO3 solution. X-ray diffractometry (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and Fourier-transform infrared spectroscopy (FTIR) were used to evaluate the structure of synthesized powders. Antibacterial activities of samples were assessed, using Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 by disc diffusion method. It was shown that Ag-containing nanocomposite samples have an improved antibacterial performance in both cases. Results showed that the synthesized catalyst has promising potentials in wastewater treatment.

Investigation of antioxidant and anticancer activities of unsaturated oligo-galacturonic acids produced by pectinase of Streptomyces hydrogenans YAM1.

Pectin, a diverse carbohydrate polymer in plants consists of a core of α-1,4-linked D-galacturonic acid units, includes a vast portion of fruit and agricultural wastes. Using the wastes to produce beneficial compounds is a new approach to control the negative environmental impacts of the accumulated wastes. In the present study, we report a pectinase producing bacterium Streptomyces hydrogenans YAM1 and evaluate antioxidative and anticancer effects of the oligosaccharides obtained from pectin degradation. The production of oligosaccharides due to pectinase activity was detected by thin layer chromatography (TLC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results revealed that S. hydrogenans YAM1 can degrade pectin to unsaturated pectic oligo-galacturonic acids (POS) with approximately 93% radical scavenging activity in 20 mg/mL which it is more than 50% of the same concentration of pectin. Flow cytometric analysis revealed that MCF-7 cells viability decreased more than 32 and 92% following treatment with 6 and 20 mg/mL POS after 24 h, respectively. It is suggested that pectin degradation by S. hydrogenans YAM1 is not only a new approach to produce highly active compounds from fruit wastes, but also is an effective method to remove fibrous pollutants from different environments.

Chitooligosaccharides and Thermostable Chitinase Against Vulvovaginal Candidiasis and Saprophyte Fungi: LC Mass Studies of Shrimp Shell Fermentation by Bacillus altitudinis.

Some patients with candidiasis seek alternatives drug to treat vaginal yeast infection like herbal preparations and probiotics. However, the effectiveness of such treatments has not received much study. In this research, the unique chitinotrophic Bacillus was isolated on shrimp shell from salt lakes and identified as Bacillus altitudinis by 16SRNA sequencing. This strain produced a novel chitin-oligosaccharide material and thermostable chitinase (5.1 units/ml) during 4 days incubation on shrimp shell medium; nevertheless, its growth on nutrient agar was negative. The zymogram showed less than 50 kD protein responsible for chitinase activities. The LC/MS detection of concentrate fermented products showed the production of oligosaccharide during chitin fermentation. As results of shrimp shell degradation, 65.6 mg/l protein, 73.4 mg/l N-acetyl glucose amine, and oligosaccharide were produced. Synergism activities of chitooligosaccharide and chitinase from this strain against fungi and pathogen candida (staining with methylene blue showed that almost 50% of 106 cells were died during 6 h) are promising for new anti-fungal drug with no side effect.

Novel Bacillus subtilis Spore-Displayed Tyrosinase Kit for Rapid Detection of Tyrosine in Urine: Pharmaceutical Applications for the Early Diagnosis of Kidney-Related Diseases.

Purpose: Simple and cheap diagnostic kit development is one of the important aims of pharmaceutical developers and companies focused on public health improvement. The Bacillus subtilis spore surface-display technique is a genetic engineering method that is used to develop new-generation diagnostic kits applicable for the early detection of various types of diseases. In this study, we developed a novel simple, rapid, and inexpensive diagnostic paper-based kit to detect tyrosine in urine samples of humans and animals that is applicable for home or laboratory use. Methods: The B. subtilis spore-displayed tyrosinase system developed by genetic engineering methods was used to prepare a paper-based kit to detect tyrosine in urine samples of different groups of patients (i.e., patients with diabetes, diabetes with chronic kidney disease (CKD), and chronic kidney disease) for the detection of tyrosine during the acute disease phase. To confirm the sensitivity and specificity of the kit, tyrosine was also detected in urine samples using conventional liquid chromatography/mass spectroscopy. Results: Different concentrations of tyrosine (0.1-1 mM) were detected in urine samples based on visible changes of color from bright brownish-gray to dark brownish-gray within 1 hour. The kit could screen samples to distinguish the three groups of patients based on formation of a broad spectrum of colors reflecting the concentration of tyrosine. Conclusion: To the best of our knowledge, this is the first diagnostic kit with potential to rapidly diagnose various diseases related to the production of tyrosine in biological samples. This kit is not only widely applicable, including for personal use in the home, but is also appropriate as a part of standard screening tests and health protection programs in countries with limited resources.

LC/MS detection of oligogalacturonic acids obtained from tragacanth degradation by pectinase producing bacteria.

Tragacanth, a highly branched carbohydrate polymer isolated from Astragalus, is one of the most commonly used gums in food industry. The primary structure of tragacanth is composed of galacturonic acid monomers connected with α 1-4 links, and it is very similar to the pectin. Tragacanth degradation by microorganisms is significant in two aspects: first, food preservation and microbial growth control due to too much use of tragacanth in the food industry, second, therapeutic and pharmaceutical potential of obtained oligosaccharides. In the present study, we report three new strains of bacteria, Acinetobacter guillouiae strain TD1, Kosakonia sacchari strain TD2, and Bacillus vallismortis strain PD1 with the capability of growing in tragacanth as an only source of carbon and energy. The evolutionary history of the isolated strains was analyzed based on 16S rRNA gene sequences in MEGA7 using the neighbor-joining method. The production of di and tri galacturonic acid due to pectinase activities of the strains were detected by thin layer chromatography (TLC) and liquid chromatography/Mass spectroscopy (LC/MS) analysis. Here is the first report of the ability to grow in tragacanth and pectinase activity monitoring in bacteria. Our results revealed that all of the isolated strains are capable of degrading pectin and tragacanth to oligo-galacturonic acids. The obtained products, which have different structures depending on the tragacanth structures and types of pectinolytic enzymes, would show therapeutic and pharmaceutical potentials.

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications.

Biosynthesis of silver nanoparticles by Bacillus stratosphericus spores and the role of dipicolinic acid in this process.

Seeking for simple, rapid, and environmental-friendly routes to produce metal nanoparticles is quite attractive for various biotechnological applications. Biological synthesis method of silver nanoparticles has been found very promising due to their non-toxicity and simplicity. Here, the spores of Bacillus stratosphericus isolated from soil enriched with 30 % H2O2 were used for the production of silver nanoparticles. Furthermore, the possible mechanism of silver nanoparticle synthesis by the spores was elucidated for the first time. In this regard, dipicolinic acid (DPA) was shown to play a critical role as a nanoparticle-producing agent. UV-Vis absorption spectroscopy, X-ray diffraction technique, energy-dispersive spectroscopy, and transmission electron microscopy were used to characterize the nanoparticles. Unlike vegetative cells of B. stratosphericus, the spores and the purified DPA were capable of producing nanoparticles from silver nitrate (AgNO3). These biogenic nanoparticles, which were highly toxic against different pathogenic bacteria, showed mixed structures including spherical, triangular, cubic, and hexagonal with the approximate size between 2 and 20 nm in diameter. Our results illustrated the role of dipicolinic acid as a main factor for the synthesis of nanoparticles by the bacterial spores.

Development of an eco-friendly approach for biogenesis of silver nanoparticles using spores of Bacillus athrophaeus.

The biological synthesis methods have been emerging as a promising new approach for production of nanoparticles due to their simplicity and non-toxicity. In the present study, spores of Bacillus athrophaeus were used to achieve the objective of developing a green synthesis method of silver nanoparticles. Enzyme assay revealed that the spores and their heat inactivated forms (microcapsules) were highly active and their enzymatic contents differed from the vegetative cells. Laccase, glucose oxidase, and alkaline phosphatase activities were detected in the dormant forms, but not in the vegetative cells. Although no nanoparticle was produced by active cells of B. athrophaeus, both spores and microcapsules were efficiently capable of reducing the silver ions (Ag⁺) to elemental silver (Ag°) leading to the formation of nanoparticles from silver nitrate (AgNO3). The presence of biologically synthesized silver nanoparticles was determined by obtaining broad spectra with maximum absorbance at 400 nm in UV-visible spectroscopy. The X-ray diffraction analysis pattern revealed that the nanoscale particles have crystalline nature with various topologies, as confirmed by transmission electron microscopy (TEM). The TEM micrograph showed the nanocrystal structures with dimensions ranging from 5 to 30 nm. Accordingly, the spore mixture could be employed as a factory for detoxification of heavy metals and subsequent production of nanoparticles. This research introduces an environmental friendly and cost effective biotechnological process for the extracellular synthesis of silver nanoparticles using the bacterial spores.