Untargeted metabolomics and molecular docking studies on green silver nanoparticles synthesized by Sarocladium subulatum: Exploring antibacterial and antioxidant properties.

The biological synthesis of silver nanoparticles (Ag-NPs) with fungi has shown promising results in antibacterial and antioxidant properties. Fungi generate metabolites (both primary and secondary) and proteins, which aid in the formation of metal nanoparticles as reducing or capping agents. While several studies have been conducted on the biological production of Ag-NPs, the exact mechanisms still need to be clarified. In this study, Ag-NPs are synthesized greenly using an unstudied fungal strain, Sarocladium subulatum AS4D. Three silver salts were used to synthesize the Ag-NPs for the first time, optimized using a cell-free extract (CFE) strategy. Additionally, these NPs were assessed for their antimicrobial and antioxidant properties. Various spectroscopic and microscopy techniques were utilized to confirm Ag-NP formation and analyze their morphology, crystalline properties, functional groups, size, stability, and concentrations. Untargeted metabolomics and proteome disruption were employed to explore the synthesis mechanism. Computational tools were applied to predict metabolite toxicity and antibacterial activity. The study identified 40 fungal metabolites capable of reducing silver ions, with COOH and OH functional groups playing a pivotal role. The silver salt type impacted the NPs' size and stability, with sizes ranging from 40 to 52 nm and zeta potentials from -0.9 to -30.4 mV. Proteome disruption affected size and stability but not shape. Biosynthesized Ag-NPs using protein-free extracts ranged from 55 to 62 nm, and zeta potentials varied from -18 to -27 mV. Molecular docking studies and PASS results found no role for the metabolome in antibacterial activity. This suggests the antibacterial activity comes from Ag-NPs, not capping or reducing agents. Overall, the research affirmed the vital role of specific reducing metabolites in the biosynthesis of Ag-NPs, while proteins derived from biological extracts were found to solely affect their size and stability.

Comparative molecular docking and toxicity between carbon-capped metal oxide nanoparticles and standard drugs in cancer and bacterial infections.

Introduction: Nanoparticles (NPs) are of great interest in the design of various drugs due to their high surface-to-volume ratio, which result from their unique physicochemical properties. Because of the importance of examining the interactions between newly designed particles with different targets in the case of various diseases, techniques for examining the interactions between these particles with different targets, many of which are proteins, are now very common. Methods: In this study, the interactions between metal oxide nanoparticles (MONPs) covered with a carbon layer (Ag2O3, CdO, CuO, Fe2O3, FeO, MgO, MnO, and ZnO NPs) and standard drugs related to the targets of Cancer and bacterial infections were investigated using the molecular docking technique with AutoDock 4.2.6 software tool. Finally, the PRO TOX-II online tool was used to compare the toxicity (LD50) and molecular weight of these MONPs to standard drugs. Results: According to the data obtained from the semi flexible molecular docking process, MgO and Fe2O3 NPs performed better than standard drugs in several cases. MONPs typically have a lower 50% lethal dose (LD50) and a higher molecular weight than standard drugs. MONPs have shown a minor difference in binding energy for different targets in three diseases, which probably can be attributed to the specific physicochemical and pharmacophoric properties of MONPs. Conclusion: The toxicity of MONPs is one of the major challenges in the development of drugs based on them. According to the results of these molecular docking studies, MgO and Fe2O3 NPs had the highest efficiency among the investigated MONPs.

Sulfonamides stimulate ROS formation upon glycation of human carbonic anhydrase II.

Advanced glycation end products are the most important species of glycation pathway, and cause disorders such as oxidative stress and diabetes. Sulfonamide compounds, which are generally known as widespread inhibitors, are potential agents used in different drug products, which can readily enter biological matrices. In the present work, the structure and activity of human carbonic anhydrase II studied in the presence of glucose as well as four sulfonamide agents from different views. These included enzyme kinetics, free lysine content, fluorescence spectroscopy, circular dichroism, and ROS measurement. Our results indicated that upon glycation, the structure of HCA II collapses and 8 to 13 lysine residues will be more available based on ligand incubation. Secondary and tertiary structural changes were also observed in the presence and absence of sulfonamide agents using fluorescence and circular dichroism methods, respectively. These spectroscopic data also showed a remarkable increase in hydrophobicity and decrease in α-helix contents during glycation, especially after 35 days of incubation. ROS assay was studied in the presence of glucose and sulfonamide compounds, that demonstrated the role of sulfonamide compounds in ROS formation in the presence of glucose in a synergistic manner. Overall, our data indicated that sulfonamides act as a stimulant factor upon prolonged interaction with HCA II and may intensify the complications of some disorders, such as diabetes and other conformational diseases.

Interaction of zincite, alpha-terpineol, geranyl acetate, linalool, myrcenol, terpinolene, and thymol with virulence factors of Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Staphylococcus aureus.

BACKGROUND: Based on gas chromatography - mass spectrometry (GC-MS) results of a previous study, six metabolites including alpha-terpineol, geranyl acetate, linalool, myrcenol, terpinolene, and thymol showed significantly higher amounts relative to other metabolites. METHODS: A continuation of the previous study, the interaction of these metabolites with the main virulence factors of P. aeruginosa (pseudomonas elastase and exotoxin A), Staphylococcus aureus (alpha-hemolysin and protein 2a), Mycobacterium tuberculosis (ESX-secreted protein B and the serine/threonine protein kinase), and Escherichia coli (heat-labile enterotoxin and Shiga toxin) were evaluated by molecular docking study and molecular simulation. RESULTS: In the case of Shiga toxin, higher and lower binding affinities were related to alpha-terpinolene and zincite with values of -5.8 and -2.6 kcal/mol, respectively. For alpha-hemolysin, terpinolene and alpha-terpinolene demonstrated higher binding affinities with similar energies of -5.9 kcal/mol. Thymol and geranyl acetate showed lower binding energy of -5.7 kcal/mol toward protein 2a. Furthermore, thymol had a higher binding affinity toward heat-labile enterotoxin and ESX-secreted protein B with values of -5.9 and -6.1 kcal/mol, respectively. CONCLUSIONS: It is concluded that the availability of secondary metabolites of A. haussknechtii surrounding zinc oxide (ZnO) NPs can hinder P. aeruginosa by inactivating Pseudomonas elastase and exotoxin.

Biosynthesis of Quantum Dots and Their Therapeutic Applications in the Diagnosis and Treatment of Cancer and SARS-CoV-2.

Quantum dots (QDs) are semiconductor materials that range from 2 nm to 10 nm. These nanomaterials (NMs) are smaller and have more unique properties compared to conventional nanoparticles (NPs). One of the unique properties of QDs is their special optoelectronic properties, making it possible to apply these NMs in bioimaging. Different size and shape QDs, which are used in various fields such as bioimaging, biosensing, cancer therapy, and drug delivery, have so far been produced by chemical methods. However, chemical synthesis provides expensive routes and causes serious environmental and health issues. Therefore, various biological systems such as bacteria, fungi, yeasts, algae, and plants are considered as potent eco-friendly green nanofactories for the biosynthesis of QDs, which are both economic and environmentally safe. The review aims to provide a descriptive overview of the various microbial agents for the synthesis of QDs and their biomedical applications for the diagnosis and treatment of cancer and SARS-CoV-2.

Biosynthesis of silver nanoparticles using Pseudomonas canadensis, and its antivirulence effects against Pseudomonas tolaasii, mushroom brown blotch agent.

This study reports the biosynthesis of silver nanoparticles (AgNPs) using a Pseudomonas canadensis Ma1 strain isolated from wild-growing mushrooms. Freshly prepared cells of P. canadensis Ma1 incubated at 26-28 °C with a silver nitrate solution changed to a yellowish brown color, indicating the formation of AgNPs, which was confirmed by UV-Vis spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction. SEM analysis showed spherical nanoparticles with a distributed size mainly between 21 and 52 nm, and the XRD pattern revealed the crystalline nature of AgNPs. Also, it provides an evaluation of the antimicrobial activity of the biosynthesized AgNPs against Pseudomonas tolaasii Pt18, the causal agent of mushroom brown blotch disease. AgNPs were found to be bioactive at 7.8 µg/ml showing a minimum inhibitory concentration (MIC) effect against P. tolaasii Pt18 strain. AgNPs at the MIC level significantly reduced virulence traits of P. tolaasii Pt18 such as detoxification of tolaasin, various motility behavior, chemotaxis, and biofilm formation which is important for pathogenicity. Scanning electron microscopy (SEM) revealed that bacterial cells treated with AgNPs showed a significant structural abnormality. Results showed that AgNPs reduced brown blotch symptoms in vivo. This research demonstrates the first helpful use of biosynthesized AgNPs as a bactericidal agent against P. tolaasii.

Mycosynthesis of AgNPs: mechanisms of nanoparticle formation and antimicrobial activities.

INTRODUCTION: The inactivation and eradication of multidrug-resistant bacteria, fungi, and viruses by conventional antibiotics and drugs have not been effective. The hindering of these pathogens in hospital-acquired infections caused by Gram-positive bacteria, particularly strains of S. aureus including community-acquired methicillin-resistant (CA-MRSA) and hospital-acquired MRSA (HA-MRSA), is more complicated, specifically in patients having immunodeficiency syndrome. RESEARCH AREA: Bare and functionalized metal and metal oxide nanoparticles (NPs) specifically silver (Ag) NPs have shown significant antibacterial, antifungal, and antiviral activities. Biosynthesis of AgNPs by fungal species in media of cell-free filtrate and culture supernatant can provide new therapeutic properties compared to physical and chemical methods. EXPERT OPINION: Various primary and secondary metabolites of fungi such as phytochelatin, trichodin, primin, altersolanol A, periconicin A, brefeldin A, graphislactone A, phomol, polysaccharides (chitin, glucans, and galactomannans), and enzymes can contribute to reducing Ag+ ions and stabilizing NPs in one-pot method. These natural compounds can augment antimicrobial activity by bypassing multidrug-resistance barriers in viruses, bacteria, and fungi. Controlling physicochemical properties and effective therapeutic concentration of fungal AgNPs can be the determinative parameters for the antimicrobial strength of AgNPs. Therefore, in this review, we have tried to address the antimicrobial mechanisms and physicochemical properties of fungal synthesized AgNPs.

Interaction of Epigallocatechin Gallate and Quercetin with Spike Glycoprotein (S-Glycoprotein) of SARS-CoV-2: In Silico Study.

Severe acute respiratory syndrome (SARS)-CoV-2 from the family Coronaviridae is the cause of the outbreak of severe pneumonia, known as coronavirus disease 2019 (COVID-19), which was first recognized in 2019. Various potential antiviral drugs have been presented to hinder SARS-CoV-2 or treat COVID-19 disease. Side effects of these drugs are among the main complicated issues for patients. Natural compounds, specifically primary and secondary herbal metabolites, may be considered as alternative options to provide therapeutic activity and reduce cytotoxicity. Phenolic materials such as epigallocatechin gallate (EGCG, polyphenol) and quercetin have shown antibacterial, antifungal, antiviral, anticancer, and anti-inflammatory effects in vitro and in vivo. Therefore, in this study, molecular docking was applied to measure the docking property of epigallocatechin gallate and quercetin towards the transmembrane spike (S) glycoprotein of SARS-CoV-2. Results of the present study showed Vina scores of -9.9 and -8.3 obtained for EGCG and quercetin by CB-Dock. In the case of EGCG, four hydrogen bonds of OG1, OD2, O3, and O13 atoms interacted with the Threonine (THR778) and Aspartic acid (ASP867) amino acids of the spike glycoprotein (6VSB). According to these results, epigallocatechin gallate and quercetin can be considered potent therapeutic compounds for addressing viral diseases.

Antibacterial and wound healing applications of curcumin in micro and nano-scaffolds based on chitosan, cellulose, and collagen.

About 80% higher risk of amputation resulted from microbial infection was indicated for patients with diabetic foot ulcers (DFUs). Micro and nano-scaffolds made of natural polymers specifically cellulose, chitosan, and collagen can donate the biocompatibility, biodegradability, and bioavailability properties appropriate to  accelerate wound closure before microbial biofilm formation. Antimicrobial activity of these wound dressings can be improved by incorporation of bioactive compounds extracted from medicinal plant species such as curcumin. Low water solubility and poor bioavailability are recognized as two main disadvantages of curcumin, lipophilic phytopolyphenol, which could be controlled by targeted polymeric micro and nano-scaffolds. Consequently, this review has discussed the capacity and challenges of these types of formulations according to recent investigations.

Antifungal effects of volatile compounds produced by Tetrapisispora sp. strain 111A-NL1 as a new biocontrol agent on the strawberry grey mold disease.

An antagonistic yeast strain was isolated from the strawberry fruit cv. Paros, and its antifungal properties against Botrytis cinerea causal agent of strawberry grey mold disease were evaluated under in vitro and in vivo experiments. The isolate was tentatively identified as Tetrapisispora sp. strain 111A-NL1 based on phenotypic characteristics and sequence analysis of D1/D2 domains of the 26S rRNA gene. Volatile organic compounds (VOCs) produced by the 111A-NL1 strain inhibited the mycelial growth of fungal pathogen (75.19%) and conidial germination (63.34%); however, inhibition percentage of mycelial growth of pathogen by dual culture test was less (19.49%). Also, the strain produced pectinase, siderophore, chitinase, IAA, as well as gibberellin, and could solubilize phosphate. Additionally, the disease severity of strawberry grey mold was decreased by employing living cells and volatile metabolites methods by 47.61% and 74.05%, respectively, in comparison with untreated control seven days after inoculation. Therefore, its mode of action might consist of antibiosis and VOCs production by yeast strain 111A-NL1 against B. cinerea. The VOCs released by strain 111A-NL1 were analyzed, and thirty-three chemical compounds were determined by gas chromatography-mass spectroscopy (GC-MS). Out of them, Decane (12.79%), Squalene (9.60%), Undecane (7.98%), Benzene, 1,2,3-trimethyl- (7.67%), Nonane, 2,6-dimethyl- (5.69%), Benzene, 1-ethyl-3-methyl- (5.55%), Mesitylene (4.17%), and Phenylethyl Alcohol (3.33%) were the major components. In addition, the selected strain reduced natural decay incidence and weight loss of fruit, and preserved quality parameters of strawberry fruit including firmness, soluble solids content, and titratable acidity. This research averred, for the first time, that the creation of VOCs by Tetrapisispora sp. strain 111A-NL1 could play an essential role as a biofumigant in the antifungal activity against strawberry grey mold.

The concept of protein folding/unfolding and its impacts on human health.

Proteins have evolved in specific 3D structures and play different functions in cells and determine various reactions and pathways. The newly synthesized amino acid chains once depart ribosome must crumple into three-dimensional structures so can be biologically active. This process of protein that makes a functional molecule is called protein folding. The protein folding is both a biological and a physicochemical process that depends on the sequence of it. In fact, this process occurs more complicated and in some cases and in exposure to some molecules like glucose (glycation), mistaken folding leads to amyloid structures and fatal disorders called conformational diseases. Such conditions are detected by the quality control system of the cell and these abnormal proteins undergo renovation or degradation. This scenario takes place by the chaperones, chaperonins, and Ubiquitin-proteasome complex. Understanding of protein folding mechanisms from different views including experimental and computational approaches has revealed some intermediate ensembles such as molten globule and has been subjected to biophysical and molecular biology attempts to know more about prevalent conformational diseases.

A newly isolated Bacillus amyloliquefaciens SRB04 for the synthesis of selenium nanoparticles with potential antibacterial properties.

The aim of this study was to isolate and characterize marine bacterial strains capable of converting selenite to elemental selenium with the formation of Se nanoparticles (SeNPs). For the first time, a novel marine strain belonging to Bacillus amyloliquefaciens (GenBank accession no. MK392020) was isolated from the coast of the Caspian Sea and characterized based on its ability for transformation of selenite to SeNPs under aerobic conditions. The preliminary formation of SeNPs was confirmed via color changes and the products characterized by UV-Vis spectroscopy. The field-emission scanning electron microscopy (FESEM) together with energy-dispersive X-ray (EDX) analysis showed the presence of the spherical SeNPs on both the surface of the bacterial biomass and in the supernatant solution. Dynamic light scattering (DLS) analysis showed the SeNPs to have an average particle size (Z-average) around 45.4-68.3 nm. The X-ray diffraction (XRD) studies substantiated the amorphous nature of the biosynthesized SeNPs. Fourier-transform infrared spectroscopic (FTIR) studies of the SeNPs indicated typical proteinaceous and lipid-related bands as capping agents on the SeNPs. Different effective parameters corresponding the yield of SeNPs by B. amyloliquefaciens strain SRB04 were optimized under resting cell strategy. Results showed that the optimal process conditions for SeNP production were 2 mM of selenite oxyanion, 20 g/L of cell biomass, and 60 h reaction time. The synthesized SeNPs had a remarkable antibacterial activity on Staphylococcus aureus compared with chloramphenicol as a broad-spectrum antibiotic.

Antifungal activity of volatile compounds produced by Staphylococcus sciuri strain MarR44 and its potential for the biocontrol of Colletotrichum nymphaeae, causal agent strawberry anthracnose.

A nonpathogenic endophytic bacterial isolate, recovered from Fragaria × ananassa stolon, and its antifungal activity against Colletotrichum nymphaeae was evaluated under in vitro, in vivo, and greenhouse conditions. Bacterial isolate was identified as Staphylococcus sciuri MarR44 (Strain ID: WDCM 891 = CCSM-B 00640) using phenotypic and biochemical properties and molecular phylogenetic analysis of the 16S rDNA gene sequences. The living cells of strain MarR44 inhibited mycelial growth of C. nymphaeae (52.46%) using dual-culture method. The volatile compounds (VOCs) produced by MarR44 inhibited mycelial growth and conidial germination of C. nymphaeae by 34.52% and 82.81%, respectively. However, inhibition percentage of mycelial growth of pathogen by culture filtrates of the strain MarR44 was lower (23.07%) than that for the two dual culture and volatile compounds assay tests. Moreover, the cell-free-culture filtrates of this strain reduced the biomass and conidial germination of pathogen by 91.89% and 41.10%, respectively. Also, the strain MarR44 was capable of producing protease, chitinase, HCN, siderophore, IAA, gibberellin, and biofilm. The living cells and volatile compounds of the strain MarR44 reduced anthracnose disease at post-harvest on fruit by 52.45% and 72.17%, respectively. Furthermore, disease severity of strawberry anthracnose was reduced using drenching soil and inoculated plants methods by 77.77 and 72.22%, respectively, 60 days after inoculation. The VOCs released by strain MarR44 were analyzed by Gas chromatography-mass spectroscopy (GC-MS). Out of 24 identified VOCs, Mesityl oxide (81.436%), Acetic acid, 2-methylpropyl ester (3.442%), 4-Methyldecane (1.837%), 4-Penten-2-one,4-methyl- (1.739%), Toluene (1.248%), and o-Xylene (1.24%) were the major components. The mode of action of S. sciuri MarR44 on the C. nymphaeae was through the production of antifungal volatile compounds (Antibiosis), which inhibited mycelial growth and conidial germination of pathogen in vitro and fruit decay development in vivo. To the best of our knowledge, this is the first report of S. sciuri having antifungal activity against causal agent strawberry anthracnose. These results indicated that the VOCs of S. sciuri strain MarR44 are promising biofumigant for management of strawberry anthracnose.

Bioconversion of isoeugenol to vanillin and vanillic acid using the resting cells of Trichosporon asahii.

40 isoeugenol-tolerant yeasts were isolated from the rhizosphere soil samples which in turn were collected from aromatic plants in different regions of Iran, and further tested for their ability to grow on a minimal medium containing isoeugenol as the sole carbon and energy source. Nine isolates which were able to grow on isoeugenol were examined for their ability to convert isoeugenol into vanillin under growing cell experiments. Of the tested yeasts, the highest conversion efficiency was observed in isolate MP24. The isolate was identified as Trichosporon asahii based on morphological, biochemical and molecular (ITS region) characters and tested to effectively convert isoeugenol into vanillin under resting cell system. A comparative analysis of thin layer chromatography (TLC), UV-Vis spectrometry, and high-performance liquid chromatography (HPLC) verified that vanillin and vanillic acid are accumulated as two major metabolites using T. asahii strain MP24 resting cells. In the presence of 7.5 g/l of wet weight cells of the strain MP24 pre-grown on isoeugenol and harvested at the end of the exponential growth phase, the optimal concentration of vanillin reached 2.4 g/l with a molar conversion of 52.5% in the potassium phosphate buffer (100 mM, pH 5.8) supplemented with 5 g/l of isoeugenol and 2% (v/v) N,N-dimethylformamide (DMF). The total concentration of vanillin and vanillic acid obtained from the bioconversion process was 4.2 g/l (total molar yield of 88.3%). Until now, no data has been published on the conversion of isoeugenol into vanillin by the strains of the genus Trichosporon.

A novel strain of Aureobasidium sp. TeO12 for theophylline production from caffeine.

A total of 40 fungal cultures were isolated for their ability to grow on caffeine as a sole source carbon and nitrogen, and further screened for theophylline-producing activities under the growing cell system. Based on thin-layer chromatography and high-performance liquid chromatography analyses, the potent strain Aureobasidium sp. TeO12 was chosen for its capability to generate theophylline via biotransformation of caffeine. It was identified based on phenotypic characteristics and its ITS1-5.8S-ITS2 rDNA sequencing data (GenBank accession number no. KT439072). To improve theophylline yield, the effects of various factors, such as resting cell density, Fe(II) concentration, and course of the transformation of caffeine, were studied in a biotransformation reaction containing 0.1 M sodium phosphate buffer (pH 7), Aureobasidium sp. TeO12 resting cells as the whole-cell catalyst and caffeine (2.5 g/L) as the substrate, and the reaction was incubated at 30 °C on an orbital shaker (200 rpm). The results indicated that optimal combination included resting cell density 6 g/L, Fe(II) concentration 75 mg/L, and the biotransformation time 72 h. Under these optimal reaction conditions, the highest theophylline concentration of 1.55 g/L (molar yield of 67%) with an average degradation yield of the substrate of about 83% was obtained in the biotransformation process. This is the first report on the biotransformation of caffeine into theophylline by a novel strain of the genus Aureobasidium.

Salinivibrio costicola GL6, a Novel Isolated Strain for Biotransformation of Caffeine to Theobromine Under Hypersaline Conditions.

The present study has been conducted towards isolation of moderately halophilic bacteria capable of transforming caffeine into theobromine. A total of 45 caffeine-degrading moderate halophiles were enriched from hypersaline lakes and examined for the biotransformation of caffeine to theobromine by thin-layer chromatography (TLC) and high-performance liquid chromatography analyses. Strain GL6, giving the highest yield of theobromine, was isolated from the Hoz Soltan Lake, 20 % w/v salinity, central Iran, and identified as Salinivibrio costicola based on morphological and biochemical features as well as its 16S rRNA gene sequence analysis (GeneBank Accession No. KT378066) and DNA-DNA relatedness. The biotransformation of caffeine with strain GL6 leads to the formation of two metabolites, identified as theobromine and paraxanthine, but the yield of paraxanthine was much lower. Further study on the production of theobromine from caffeine under resting cell experiment was carried out subsequently. The optimal yield of theobromine (56 %) was obtained after a 32-h incubation using 5 mM of caffeine and 15 g l-1 (wet weight) of biomass in 0.1 M saline phosphate buffer (pH 7.0 and 10 % w/v NaCl) under agitation 180 rpm at 30 °C. The biotransformed theobromine was purified by preparative TLC and subjected to FTIR and mass spectroscopy for chemical identification. This is the first evidence for biotransformation of caffeine into theobromine by strains of the genus Salinivibrio.

Sesquiterpene lactone engineering in microbial and plant platforms: parthenolide and artemisinin as case studies.

Sesquiterpene lactones (SLs) are one of the most diverse groups of secondary metabolites that mainly have been observed in the Asteraceae. They are composed of a C15 skeleton bearing functional groups, e.g., hydroxy, keto, or epoxy. Sesquiterpene lactones have been shown to display several biological activities; hence, their therapeutic effects are indispensable. To overcome low yield of sesquiterpene lactone content in native plants, manipulation of their biosynthetic pathway(s) has become an interesting approach for many researchers. Several genetic engineering strategies have been used in plants or microbial systems for elucidation of the biosynthetic pathway and high-level production of sesquiterpene lactones. Here, we will introduce ongoing research and perspectives about the manipulation of sesquiterpene lactone biosynthesis by various non-traditional metabolic engineering strategies, along with successful examples of high-yield production of sesquiterpene lactones mainly focused on parthenolide and artemisinin in plants and microorganisms.

Use of Taguchi methodology to enhance the yield of caffeine removal with growing cultures of Pseudomonas pseudoalcaligenes.

BACKGROUND AND OBJECTIVES: Microbial caffeine removal is a green solution for treatment of caffeinated products and agro-industrial effluents. We directed this investigation to optimizing a bio-decaffeination process with growing cultures of Pseudomonas pseudoalcaligenes through Taguchi methodology which is a structured statistical approach that can be lowered variations in a process through Design of Experiments (DOE). MATERIAL AND METHODS: Five parameters, i.e. initial fructose, tryptone, Zn(+2) ion and caffeine concentrations and also incubation time selected and an L16 orthogonal array was applied to design experiments with four 4-level factors and one 3-level factor (4(4) × 1(3)). Data analysis was performed using the statistical analysis of variance (ANOVA) method. Furthermore, the optimal conditions were determined by combining the optimal levels of the significant factors and verified by a confirming experiment. Measurement of residual caffeine concentration in the reaction mixture was performed using high-performance liquid chromatography (HPLC). RESULTS: Use of Taguchi methodology for optimization of design parameters resulted in about 86.14% reduction of caffeine in 48 h incubation when 5g/l fructose, 3 mM Zn(+2) ion and 4.5 g/l of caffeine are present in the designed media. Under the optimized conditions, the yield of degradation of caffeine (4.5 g/l) by the native strain of Pseudomonas pseudoalcaligenes TPS8 has been increased from 15.8% to 86.14% which is 5.4 fold higher than the normal yield. CONCLUSION: According to the experimental results, Taguchi methodology provides a powerful methodology for identifying the favorable parameters on caffeine removal using strain TPS8 which suggests that the approach also has potential application with similar strains to improve the yield of caffeine removal from caffeine containing solutions.

Application of Taguchi Design and Response Surface Methodology for Improving Conversion of Isoeugenol into Vanillin by Resting Cells of Psychrobacter sp. CSW4.

For all industrial processes, modelling, optimisation and control are the keys to enhance productivity and ensure product quality. In the current study, the optimization of process parameters for improving the conversion of isoeugenol to vanillin by Psychrobacter sp. CSW4 was investigated by means of Taguchi approach and Box-Behnken statistical design under resting cell conditions. Taguchi design was employed for screening the significant variables in the bioconversion medium. Sequentially, Box-Behnken design experiments under Response Surface Methodology (RSM) was used for further optimization. Four factors (isoeugenol, NaCl, biomass and tween 80 initial concentrations), which have significant effects on vanillin yield, were selected from ten variables by Taguchi experimental design. With the regression coefficient analysis in the Box-Behnken design, a relationship between vanillin production and four significant variables was obtained, and the optimum levels of the four variables were as follows: initial isoeugenol concentration 6.5 g/L, initial tween 80 concentration 0.89 g/L, initial NaCl concentration 113.2 g/L and initial biomass concentration 6.27 g/L. Under these optimized conditions, the maximum predicted concentration of vanillin was 2.25 g/L. These optimized values of the factors were validated in a triplicate shaking flask study and an average of 2.19 g/L for vanillin, which corresponded to a molar yield 36.3%, after a 24 h bioconversion was obtained. The present work is the first one reporting the application of Taguchi design and Response surface methodology for optimizing bioconversion of isoeugenol into vanillin under resting cell conditions.

Conversion of isoeugenol to vanillin by Psychrobacter sp. strain CSW4.

To screen strains of halotolerant or halophile bacteria which are able to convert isoeugenol to vanillin, 36 different strains of bacteria isolated from the salty environments in Iran were investigated. During growth on isoeugenol, a moderately halotolerant Gram-negative coccobacil showed capability of converting isoeugenol to vanillin. Based on morphological, physiological, and phylogenetic studies, strain CSW4 was classified as a bacterium belonging to the genus Psychrobacter. The bioconversion products were confirmed by thin-layer chromatography, high-performance liquid chromatography, and spectral data obtained from UV/Vis spectroscopy, FTIR, and mass-spectroscopy. Using growing cells, vanillin reached its maximum level of 88.18 mg L(-1) after 24 h of reaction time in the presence of 1 g L(-1) isoeugenol, resulting in a molar yield of 10.2%. The use of resting cells led to the optimal yield of vanillin (16.4%) which was obtained after 18-h reaction using 1 g L(-1) isoeugenol and 3.1 g of dry weight of cells per liter harvested at the end of the exponential growth phase. To improve vanillin yield, the effect of substrate concentration on vanillin production under resting cells conditions was also investigated. Using 10 g L(-1) isoeugenol, the maximal vanillin concentration (1.28 g L(-1)) was achieved after a 48-h reaction, without further optimization. The present study brings the first evidence for biotransformation of isoeugenol to vanillin in the genus Psychrobacter.