Application of microbial resources in biorefineries: Current trend and future prospects.

The recent growing interest in sustainable and alternative sources of energy and bio-based products has driven the paradigm shift to an integrated model termed "biorefinery." Biorefinery framework implements the concepts of novel eco-technologies and eco-efficient processes for the sustainable production of energy and value-added biomolecules. The utilization of microbial resources for the production of various value-added products has been documented in the literatures. However, the appointment of these microbial resources in integrated resource management requires a better understanding of their status. The main of aim of this review is to provide an overview on the defined positioning and overall contribution of the microbial resources, i.e., algae, fungi and bacteria, for various bioprocesses and generation of multiple products from a single biorefinery. By utilizing waste material as a feedstock, biofuels can be generated by microalgae while sequestering environmental carbon and producing value added compounds as by-products. In parallel, fungal biorefineries are prolific producers of lignocellulose degrading enzymes along with pharmaceutically important novel products. Conversely, bacterial biorefineries emerge as a preferred platform for the transformation of standard cells into proficient bio-factories, developing chassis and turbo cells for enhanced target compound production. This comprehensive review is poised to offer an intricate exploration of the current trends, obstacles, and prospective pathways of microbial biorefineries, for the development of future biorefineries.

Recent advances in research for potential utilization of unexplored lichen metabolites.

Several research studies have shown that lichens are productive organisms for the synthesis of a broad range of secondary metabolites. Lichens are a self-sustainable stable microbial ecosystem comprising an exhabitant fungal partner (mycobiont) and at least one or more photosynthetic partners (photobiont). The successful symbiosis is responsible for their persistence throughout time and allows all the partners (holobionts) to thrive in many extreme habitats, where without the synergistic relationship they would be rare or non-existent. The ability to survive in harsh conditions can be directly correlated with the production of some unique metabolites. Despite the potential applications, these unique metabolites have been underutilised by pharmaceutical and agrochemical industries due to their slow growth, low biomass availability and technical challenges involved in their artificial cultivation. However, recent development of biotechnological tools such as molecular phylogenetics, modern tissue culture techniques, metabolomics and molecular engineering are opening up a new opportunity to exploit these compounds within the lichen holobiome for industrial applications. This review also highlights the recent advances in culturing the symbionts and the computational and molecular genetics approaches of lichen gene regulation recognized for the enhanced production of target metabolites. The recent development of multi-omics novel biodiscovery strategies aided by synthetic biology in order to study the heterologous expressed lichen-derived biosynthetic gene clusters in a cultivatable host offers a promising means for a sustainable supply of specialized metabolites.

Antimicrobial Agents Based on Metal Complexes: Present Situation and Future Prospects.

The rise in antimicrobial resistance is a cause of serious concern since the ages. Therefore, a dire need to explore new antimicrobial entities that can combat against the increasing threat of antibiotic resistance is realized. Studies have shown that the activity of the strongest antibiotics has reduced drastically against many microbes such as microfungi and bacteria (Gram-positive and Gram-negative). A ray of hope, however, was witnessed in early 1940s with the development of new drug discovery and use of metal complexes as antibiotics. Many new metal-based drugs were developed from the metal complexes which are potentially active against a number of ailments such as cancer, malaria, and neurodegenerative diseases. Therefore, this review is an attempt to describe the present scenario and future development of metal complexes as antibiotics against wide array of microbes.

Bio-Synthesis of Aspergillus terreus Mediated Gold Nanoparticle: Antimicrobial, Antioxidant, Antifungal and In Vitro Cytotoxicity Studies.

Gold nanoparticles (GNP) were bio-fabricated utilizing the methanolic extract of the endophytic isolate Aspergillus terreus. The biosynthesised gold nanoparticles (GNP023) were characterised using UV-visible spectroscopy (UV-Vis); transmission electron microscopy (TEM), Fourier-transform nfrared spectroscopy (FTIR) and X-ray diffraction (XRD) studies. The bio-fabricated GNP023 displayed a sharp SPR peak at 536 nm, were spherically shaped, and had an average size between 10-16 nm. The EDX profile confirmed the presence of gold (Au), and XRD analysis confirmed the crystalline nature of GNP023. The antimicrobial activity of GNP023 was investigated against several food-borne and phytopathogens, using in vitro antibacterial and antifungal assays. The maximum zone of inhibition was observed for S. aureus and V. cholera at 400 µg /mL, whereas inhibition in radial mycelial growth was observed against Fusarium oxysporum and Rhizoctonia solani at 52.5% and 65.46%, respectively, when challenged with GNP023 (200 µg/mL). Moreover, the gold nanoparticles displayed significant antioxidant activity against the ABTS radical, with an IC50 of 38.61 µg/mL, and were non-toxic when tested against human kidney embryonic 293 (HEK293) cells. Thus, the current work supports the application of myco-synthesised gold nanoparticles as a versatile antimicrobial candidate against food-borne pathogens.

Evaluation of the anticancer potential of secondary metabolites from Pseudevernia furfuracea based on epidermal growth factor receptor inhibition.

UHPLC/ESI/MS/MS profiling followed by bioactivity guided isolation of Pseudevernia furfuracea (P. furfuracea) extract yielded two polyphenolic molecules, Methyl haematommate (PF-1) and Atraric acid (PF-2). These molecules were evaluated for bioactivity against five cancerous cell lines. The results revealed that atraric acid showed significant activity against ovarian cancer cell line (PA-1) having GI50 at 16.42 µg/mL and moderate activity against the breast cancer cell line (MCF-7), having GI50 at 64.35 µg/mL. The results were further supported by in silico molecular docking studies of atraric acid with the epidermal growth factor receptor (EGFR) tyrosine kinase protein. The study revealed that atraric acid has the capacity to act as a potential EGFR inhibitor via occupying the ATP binding pocket of EGFR and making favourable electrostatic interactions and van der Waals interaction with its key residues. Our results highlight P. furfuracea and its polyphenolic compound, atraric acid as a promising candidate for ovarian cancer management.

Characterization of an Endophytic Strain Talaromyces assiutensis, CPEF04 With Evaluation of Production Medium for Extracellular Red Pigments Having Antimicrobial and Anticancer Properties.

Considering the worldwide demand for colorants of natural origin, the utilization of ascomycete fungi as a prolific pigment producer unfolds a novel way to obtain these pigments for various applications, including food, cosmetic, and medical use. The presence of very few natural red pigment alternatives in the market also attracts research and industry priorities to unearth novel and sustainable red pigment producers. The present work is an attempt to identify a novel source of red color obtained from endophytic fungi isolated from terrestrial and marine habitats. Based upon the fungal capacity for pigment production, seven isolates of endophytic fungi were recognized as prospective pigment producers. Out of all, fungal isolate CPE04 was selected based upon its capacity to produce profuse extracellular red pigment. The isolate was identified as Talaromyces assiutensis, employing morphological features and phylogenetic characterization by internal transcribed spacer (ITS) sequences. To understand the chemical behavior of pigment molecules, an investigation of the chemical profile of fungal culture filtrate dried powder (CFDP) was performed using ultra-high-performance liquid chromatography-diode array detector-mass spectrometry (UPLC-DAD-MS). In total, eight compounds having pigment and pharmaceutical application were tentatively identified using UPLC-DAD-MS. Considering the commercial aspect of the stated work, an effort was also made for standardizing the upscaling of the pigment molecule. Investigations were performed for optimum medium and culturing conditions for maximum pigment production. CFDP was found to have a significant antibacterial activity against the bacterial pathogens Staphylococcus aureus (MTCC737), Vibrio cholerae (N16961), and methicillin-resistant S. aureus (MRSA) (ATCC BAA811). The CFDP showed a minimum inhibitory concentration at 64, 128, and 256 µg/ml against S. aureus, MRSA, and V. cholerae. A concentration-dependent (50-400 µg/ml) anticancer effect on HeLa cancer line was also observed, having a half-maximal inhibitory concentration (IC50) at 300 µg/ml. The antioxidant potential of CFDP has also been proven with the help of an antioxidant assay against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (IC50, 32.01 µg/ml); DNA nicking assay and reactive oxygen species were generated in HeLa cancer line cells. The CFDP was also found to have no cytotoxicity toward HEK 293 T cell line using alamar blue (resazurin), a cell metabolic activity reagent.

Metabolite Profiling of the Indian Food Spice Lichen, Pseudevernia furfuracea Combined With Optimised Extraction Methodology to Obtain Bioactive Phenolic Compounds.

Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) is a well-known epiphytic lichen commonly used in Indian spice mixtures and food preparations such as curries. This study is an attempt to find the best extraction methodology with respect to extractive yield, total polyphenolic content (TPC), total flavonoid content and antioxidant activities of lichen P. furfuracea. Two phenolic compounds, atraric acid and olivetoric acid were isolated and quantified in their respective extracts with the aid of reverse phase high performance liquid chromatography (RP-HPLC). The highest concentration of both the compounds, atraric acid (4.89 mg/g DW) and olivetoric acid (11.46 mg/g DW) were found in 70% methanol extract. A direct correlation was also observed between the concentrations of these compounds with the free radical scavenging potential of the extracts which might contribute towards the antioxidant potential of the extract. Moreover, scanning electron microscopy and HPLC analysis which was used to study the effect of pre-processing on extraction process highlighted the capacity of a mixer grinder technique for improved separation of surface localized metabolites and enrichment of the fraction. An investigation of the chemical profile of the bioactive extract 70% methanol extract using UHPLC-DAD-MS lead to tentative identification of forty nine compounds. This extract was also assessed towards HEK 293 T cell line for cytotoxicity analysis. Concentration range of 0.156 to 100 µg/ml of PF70M extract exhibited no significant cell death as compared to control. Further, the active extract showed protective effect against hydroxyl radical's destructive effects on DNA when assessed using DNA nicking assay. Based upon this, it can be concluded that optimization of extraction solvent, sample pre-proceesing and extraction techniques can be useful in extraction of specific antioxidant metabolites.

Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of oxacillin and a bioactive compound from Ramalinaroesleri.

Lichens are known to be useful and important in ethanopharmacology since ages and still possess substantial interest in alternative medical practices around the world. The intent of this investigation was to evaluate and to understand the antibacterial potential of usnic acid which was isolated from Himalyan fruticose lichen Ramalina roesleri. Usnic acid is predicted for its pharmaceutical properties through in -silico studies. Binding efficiency of usnic acid with Penicillin binding protein-PBP2a, a protein which is responsible for conferring resistance in Staphylococcus aureus was accessed using in-silico interaction assays comparing with oxacillin and ceftaroline. Further, the validation of in-silico modelling was checked by determining the antibacterial potential of usnic acid against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. In total, 28 clinical isolates collected from hospitals/medical students were included in the study and the anti-Staphylococcal activity was determined using agar plate dilution method followed by time-kill kinetics and synergistic studies. The scanning electron microscopic (SEM) pictures were obtained to show the cell wall disruption of MRSA by usnic acid. Docking results clearly indicated the enhanced binding potential of usnic acid (Glide XP G Score: 10.968; Glide energy -64.869) with PBP2a which is better than the energy range of reference compound, oxacillin (Glide XP G Score: 6.596; Glide energy -53.285) and roughly comparable to the co-crystallized ligand ceftaroline (Glide XP G Score: 12.20; Glide energy -70.322). Cefteroline is known to be more active against MRSA compared to oxacillin. The minimum inhibitory concentrations (MICs) of usnic acid against the clinical isolates of MRSA and reference strain (NCTC-6571) were in the range of 32-128 µg/ml. The high affinity of usnic acid to bind with PBP2a which is demonstrated via in-silico studies is further confirmed by the impressive inhibitory activity of usnic acid on MRSA clinical isolates.

Fungi as a Potential Source of Pigments: Harnessing Filamentous Fungi.

The growing concern over the harmful effects of synthetic colorants on both the consumer and the environment has raised a strong interest in natural coloring alternatives. As a result the worldwide demand for colorants of natural origin is rapidly increasing in the food, cosmetic and textile sectors. Natural colorants have the capacity to be used for a variety of industrial applications, for instance, as dyes for textile and non-textile substrates such as leather, paper, within paints and coatings, in cosmetics, and in food additives. Currently, pigments and colorants produced through plants and microbes are the primary source exploited by modern industries. Among the other non-conventional sources, filamentous fungi particularly ascomycetous and basidiomycetous fungi (mushrooms), and lichens (symbiotic association of a fungus with a green alga or cyanobacterium) are known to produce an extraordinary range of colors including several chemical classes of pigments such as melanins, azaphilones, flavins, phenazines, and quinines. This review seeks to emphasize the opportunity afforded by pigments naturally found in fungi as a viable green alternative to current sources. This review presents a comprehensive discussion on the capacity of fungal resources such as endophytes, halophytes, and fungi obtained from a range or sources such as soil, sediments, mangroves, and marine environments. A key driver of the interest in fungi as a source of pigments stems from environmental factors and discussion here will extend on the advancement of greener extraction techniques used for the extraction of intracellular and extracellular pigments. The search for compounds of interest requires a multidisciplinary approach and techniques such as metabolomics, metabolic engineering and biotechnological approaches that have potential to deal with various challenges faced by pigment industry.

Isolation, characterization and antifungal activity of major constituents of the Himalayan lichen Parmelia reticulata Tayl.

Antifungal activity of hexane, ethyl acetate and methanol extracts of Parmelia reticulata was evaluated against soilborne pathogenic fungi, namely, Sclerotium rolfsii, Rhizoctonia solani, R. bataticola, Fusarium udum, Pythium aphanidermatum and P. debaryanum by poisoned food technique. Maximum antifungal activity was exhibited by hexane and ethyl acetate extracts against most of the test pathogens. Secondary metabolites, namely, (±)-isousnic acid, (±)-protolichesterinic acid, atranorin, evernyl, ethyl hematommate, ethyl orsellinate, methyl hematommate (3-formyl-2,4-dihydroxy-6-methylbenzoic acid methyl ester), 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid, 1-hydroxy-3,6-dimethoxy-8-methyl-xanthen-9-one, baeomycesic acid and salazinic acid, were isolated from the above extracts and identified by 1H NMR, 13C NMR and mass spectroscopic methods. When these metabolites were tested for antifungal activity against test pathogens, maximum antifungal activity was exhibited by (±)-protolichesterinic acid against R. solani (ED50=23.09 µg mL(-1)) and P. debaryanum (ED50=16.07 µg mL(-1)) and by atranorin against S. rolfsii (ED50=39.70 µg mL(-1)). The antifungal activity of protolichesterinic acid was found to be comparable to that of hexaconazole, a commercial fungicide.

Metabolism of 14C-azoxystrobin in water at different pH.

Metabolism of (14)C-azoxystrobin was studied in water at pH 4, 7 and 9. The study suggested that volatilization losses of azoxystrobin were very low (3%) during 130 days of incubation. Only 2.5-4.2% of azoxystrobin was mineralised to CO(2) and pH of water did not have much effect on rate of mineralisation. The dissipation of azoxystrobin in water of all the three pHs followed first order kinetic with half-life values ranging from 143 to 158 d; degradation was the fastest at pH 9. Azoxystrobin acid, a major metabolite, was detected 4-7 day onwards and its concentration increased up to 130 days. The formation of azoxystrobin acid was more and faster under alkaline (pH 9) condition than neutral (pH 7) or acidic (pH 4) conditions.

Spectrophotometric estimation of functional groups on microslides for preparation of biochips.

A universal reagent 1-O-(4,4'-dimethoxytrityl)-6-aminohexanol (DTAH) is described for the estimation of surface-bound functionalities (epoxy, aldehyde, and carboxyl) required for preparation of oligonucleotide arrays (biochips). The method involves the reaction of universal reagent DTAH with surface-bound functionality under microwaves for 10 min, followed by washings to remove the excess reagent. In the subsequent step, a weighed amount of DTAH-treated surface is exposed to acid to liberate 4,4'-dimethoxytrityl cation, which is measured at 505 nm to determine the functional group loading on the surface.