Enhanced toughness and strength of 3D printed carbide-oxide composite for biomedical applications.

Natural materials derived/extracted Ceramics is an excellent material for developing ceramic-based orthopedic implants. Recently, we have demonstrated an easily scalable, energy-efficient green method to extract ceramic particles from bio-waste i.e. chicken bone. Though the chicken bone extract (CBE) has good biocompatibility, it lacks good mechanical properties in the 3D printed condition as that of human bones. Here, we have reinforced CBE with different weight proportions of silicon carbide to improve the mechanical characteristics of the composite. The hybrid of CBE (oxide) and carbide (SiC) is sintered at different temperatures to understand the effect of the interface of the two ceramics. It is observed that temperature has minimal effect and composition has a noticeable effect on mechanical strength as well as bio-toxicity. The toughness (∼3.58 MJ/m3) and compressive strength (∼64.64 MPa) of the 90:10 composition sintered at 1250 °C show the maximum optimum values. A mathematical model has also been developed to predict and correlate the toughness with porosity, volumetric loading, and elastic modulus of the 3D-printed ceramic composite.

Enhanced remediation of polyaromatic hydrocarbon using agro-industrial waste for biofuel production and environmental pollution mitigation.

In the present study, attention has been paid to the development of economically feasible strategies for enhanced remediation of anthracene and its conversion into biofuels. The strategies developed (B1, B2, B3, and B4) include bagasse and lipid-producing strain Rhodotorula mucilagenosa IIPL32 synthesizing surface active metabolites. The results indicate the highest production of surface-active metabolites in strategies B2, B3, and B4 along with a maximum biodegradation rate. GC-MS analysis affirmed the conversion of anthracene into phthalic acid in all the strategies. Biofuel quality of the lipid produced by the strain showed higher cetane number and improved cold flow property indicating the efficiency of the developed strategies for the production of commercial grade biodiesel. Furthermore, the phytotoxicity study of the spent wash revealed that 50% and 75% diluted spent wash were non-toxic and can be employed for ferti-irrigation. Thus, the study signifies the development of an economically feasible process that can be commercially employed in biofuel industries.

Extraction of chitosan from biologically-derived chitin by bacterial chitin deacetylase: Process optimization and product quality assessment.

Commercial chitosan manufacturing process relies on strong chemical treatment on chitin that generates chitosan with undesirable properties and leads to environmental pollution. To overcome the adverse consequences, enzymatic chitosan preparation from chitin was undertaken in the current study. A potent chitin deacetylase (CDA) producing bacterial strain was screened and subsequently identified as Alcaligens faecalis CS4. After optimization 40.69 U/mL of CDA production was achieved. By treating the organically extracted chitin with partially purified CDA chitosan yield of 19.04 % was attained having 71 % solubility, 74.9 % degree of deacetylation, 21.16 % crystallinity index, 246.4 kDa molecular weight and 298 °C highest-decomposition temperature. FTIR and XRD analysis revealed characteristics peaks respectively within 870-3425 cm-1 wavenumber and 10°-20°, for enzymatically and chemically extracted (commercial) chitosan that endorses their structural similarity which validated through electron microscopic study. At 10 mg/mL chitosan concentration 65.49 % DPPH radical scavenging activity endorsed its antioxidant potential. Minimum inhibitory concentration of chitosan was 0.675, 1.75, 0.33 and 0.75 mg/mL for Streptococcus mutans, Enterococcus faecalis, Escherichia coli and Vibrio sp., respectively. Mucoadhesiveness and cholesterol binding properties were also exhibited by extracted chitosan. The present study opens a new vista for eco-friendly extraction of chitosan from chitin that is proficient and sustainable in environmental perspective.

A luminescent Cu4 cluster film grown by electrospray deposition: a nitroaromatic vapour sensor.

We present the fabrication and use of a film of a carborane-thiol-protected tetranuclear copper cluster with characteristic orange luminescence using ambient electrospray deposition (ESD). Charged microdroplets of the clusters produced by an electrospray tip deposit the clusters at an air-water interface to form a film. Different microscopic and spectroscopic techniques characterized the porous surface structure of the film. Visible and rapid quenching of the emission of the film upon exposure to 2-nitrotoluene (2-NT) vapours under ambient conditions was observed. Density functional theory (DFT) calculations established the favourable binding sites of 2-NT with the cluster. Desorption of 2-NT upon heating recovered the original luminescence, demonstrating the reusability of the sensor. Stable emission upon exposure to different organic solvents and its quenching upon exposure to 2,4-dinitrotoluene and picric acid showed selectivity of the film to nitroaromatic species.

3D Printing of a Biocompatible Nanoink Derived from Waste Animal Bones.

Direct ink writing (DIW) additive manufacturing is a versatile 3D printing technique for a broad range of materials. DIW can print a variety of materials provided that the ink is well-engineered with appropriate rheological properties. DIW could be an ideal technique in tissue engineering to repair and regenerate deformed or missing organs or tissues, for example, bone and tooth fracture that is a common problem that needs surgeon attention. A critical criterion in tissue engineering is that inserts must be compatible with their surrounding environment. Chemically produced calcium-rich materials are dominant in this application, especially for bone-related applications. These materials may be toxic leading to a rejection by the body that may need secondary surgery to repair. On the other hand, there is an abundance of biowaste building blocks that can be used for grafting with little adverse effect on the body. In this work, we report a bioderived ink made entirely of calcium derived from waste animal bones using a benign process. Calcium nanoparticles are extracted from the bones and the ink prepared by mixing with different biocompatible binders. The ink is used to print scaffolds with controlled porosity that allows better growth of cells. DIW printed parts show better mechanical properties and biocompatibility that are important for the grafting application. Degradation tests and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay study were done to examine the biocompatibility of the extracted materials. In addition, discrete element modeling and computational fluid dynamics numerical methods are used in Rocky and Ansys software programs. This work shows that biowaste materials if well-engineered can be a never-ending source of raw materials for advanced application in orthopedic grafting.

Carborane-thiol protected copper nanoclusters: stimuli-responsive materials with tunable phosphorescence.

Atomically precise nanomaterials with tunable solid-state luminescence attract global interest. In this work, we present a new class of thermally stable isostructural tetranuclear copper nanoclusters (NCs), shortly Cu4@oCBT, Cu4@mCBT and Cu4@ICBT, protected by nearly isomeric carborane thiols: ortho-carborane-9-thiol, meta-carborane-9-thiol and ortho-carborane 12-iodo 9-thiol, respectively. They have a square planar Cu4 core and a butterfly-shaped Cu4S4 staple, which is appended with four respective carboranes. For Cu4@ICBT, strain generated by the bulky iodine substituents on the carboranes makes the Cu4S4 staple flatter in comparison to other clusters. High-resolution electrospray ionization mass spectrometry (HR ESI-MS) and collision energy-dependent fragmentation, along with other spectroscopic and microscopic studies, confirm their molecular structure. Although none of these clusters show any visible luminescence in solution, bright µs-long phosphorescence is observed in their crystalline forms. The Cu4@oCBT and Cu4@mCBT NCs are green emitting with quantum yields (Φ) of 81 and 59%, respectively, whereas Cu4@ICBT is orange emitting with a Φ of 18%. Density functional theory (DFT) calculations reveal the nature of their respective electronic transitions. The green luminescence of Cu4@oCBT and Cu4@mCBT clusters gets shifted to yellow after mechanical grinding, but it is regenerated after exposure to solvent vapour, whereas the orange emission of Cu4@ICBT is not affected by mechanical grinding. Structurally flattened Cu4@ICBT didn't show mechanoresponsive luminescence in contrast to other clusters, having bent Cu4S4 structures. Cu4@oCBT and Cu4@mCBT are thermally stable up to 400 °C. Cu4@oCBT retained green emission even upon heating to 200 °C under ambient conditions, while Cu4@mCBT changed from green to yellow in the same window. This is the first report on structurally flexible carborane thiol appended Cu4 NCs having stimuli-responsive tunable solid-state phosphorescence.

Efficient valorization of feather waste by Bacillus cereus IIPK35 for concomitant production of antioxidant keratin hydrolysate and milk-clotting metallo-serine keratinase.

Keratinase production by Bacillus cereus IIPK35 was investigated under solid-state fermentation (SSF) and the maximum titer of 648.28 U/gds was revealed. Feather hydrolysates obtained from SSF exhibited paramount antioxidant properties in ABTS [2,2'-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid], FRAP [Ferric ion reducing antioxidant power], and DPPH [2,2,-Diphenyl-1-picrylhydrazyl] assay. The keratinase was purified up to homogeneity have a molecular weight of 42 kDa, and showed its stability between pH 6.5-10.0 and temperature 35-60 °C with optimum enzyme activity at pH 9.0 and 55 °C. The catalytic indices viz. Km of 9.8 mg/ml and Vmax of 307.7 µmol/min for keratin were determined. Besides keratin, the enzyme displayed broad and proteolytic activity towards other proteinaceous substrates such as casein, skim milk, gelatin, and bovine serum albumin. Pure keratinase activity was stimulated in presence of Ca2+ and Mg2+ ions, while it was strongly inhibited by both iodoacetamide and EDTA, indicating it to be a metallo-serine protease in nature. Circular dichroism study endorses the structural stability of the secondary structure at the said range of pH and temperature. The IIPK35 keratinase is non-cytotoxic in nature, shows remarkable storage stability and is stable in presence of Tween 80, Triton X 100, and sodium sulfite. Furthermore, it showed excellent milk clotting potential (107.6 Soxhlet Unit), suggesting its usefulness as an alternative milk clotting agent in the dairy industry. This study unlocks a new gateway for keratinase investigation in SSF using chicken feathers as substrate and biochemical and biophysical characterization of keratinase for better understanding and implication in industrial applications.

Carboranethiol-Protected Propeller-Shaped Photoresponsive Silver Nanomolecule.

We report the synthesis, structural characterization, and photophysical properties of a propeller-shaped Ag21 nanomolecule with six rotary arms, protected with m-carborane-9-thiol (MCT) and triphenylphosphine (TPP) ligands. Structural analysis reveals that the nanomolecule has an Ag13 central icosahedral core with six directly connected silver atoms and two more silver atoms connected through three Ag-S-Ag bridging motifs. While 12 MCT ligands protect the core through metal-thiolate bonds in a 3-6-3-layered fashion, two TPP ligands solely protect the two bridging silver atoms. Interestingly, the rotational orientation of a silver sulfide staple motif is opposite to the orientation of carborane ligands, resembling the existence of a bidirectional rotational orientation in the nanomolecule. Careful analysis reveals that the orientation of carborane ligands on the cluster's surface resembles an assembly of double rotors. The zero circular dichroism signal indicates its achiral nature in solution. There are multiple absorption peaks in its UV-vis absorption spectrum, characteristic of a quantized electronic structure. The spectrum appears as a fingerprint for the cluster. High-resolution electrospray ionization mass spectrometry proves the structure and composition of the nanocluster in solution, and systematic fragmentation of the molecular ion starts with the loss of surface-bound ligands with increasing collision energy. Its multiple optical absorption features are in good agreement with the theoretically calculated spectrum. The cluster shows a narrow near-IR emission at 814 nm. The Ag21 nanomolecule is thermally stable at ambient conditions up to 100 °C. However, white-light illumination (lamp power = 120-160 W) shows photosensitivity, and this induces structural distortion, as confirmed by changes in the Raman and electronic absorption spectra. Femtosecond and nanosecond transient absorption studies reveal an exceptionally stable excited state having a lifetime of 3.26 ± 0.02 µs for the carriers, spread over a broad wavelength region of 520-650 nm. The formation of core-centered long-lived carriers in the excited state is responsible for the observed light-activated structural distortion.

Light-Activated Intercluster Conversion of an Atomically Precise Silver Nanocluster.

Noble metal nanoclusters protected with carboranes, a 12-vertex, nearly icosahedral boron-carbon framework system, have received immense attention due to their different physicochemical properties. We have synthesized ortho-carborane-1,2-dithiol (CBDT) and triphenylphosphine (TPP) coprotected [Ag42(CBDT)15(TPP)4]2- (shortly Ag42) using a ligand-exchange induced structural transformation reaction starting from [Ag18H16(TPP)10]2+ (shortly Ag18). The formation of Ag42 was confirmed using UV-vis absorption spectroscopy, mass spectrometry, transmission electron microscopy, X-ray photoelectron spectroscopy, infrared spectroscopy, and multinuclear magnetic resonance spectroscopy. Multiple UV-vis optical absorption features, which exhibit characteristic patterns, confirmed its molecular nature. Ag42 is the highest nuclearity silver nanocluster protected with carboranes reported so far. Although these clusters are thermally stable up to 200 °C in their solid state, light-irradiation of its solutions in dichloromethane results in its structural conversion to [Ag14(CBDT)6(TPP)6] (shortly Ag14). Single crystal X-ray diffraction of Ag14 exhibits Ag8-Ag6 core-shell structure of this nanocluster. Other spectroscopic and microscopic studies also confirm the formation of Ag14. Time-dependent mass spectrometry revealed that this light-activated intercluster conversion went through two sets of intermediate clusters. The first set of intermediates, [Ag37(CBDT)12(TPP)4]3- and [Ag35(CBDT)8(TPP)4]2- were formed after 8 h of light irradiation, and the second set comprised of [Ag30(CBDT)8(TPP)4]2-, [Ag26(CBDT)11(TPP)4]2-, and [Ag26(CBDT)7(TPP)7]2- were formed after 16 h of irradiation. After 24 h, the conversion to Ag14 was complete. Density functional theory calculations reveal that the kernel-centered excited state molecular orbitals of Ag42 are responsible for light-activated transformation. Interestingly, Ag42 showed near-infrared emission at 980 nm (1.26 eV) with a lifetime of >1.5 µs, indicating phosphorescence, while Ag14 shows red luminescence at 626 nm (1.98 eV) with a lifetime of 550 ps, indicating fluorescence. Femtosecond and nanosecond transient absorption showed the transitions between their electronic energy levels and associated carrier dynamics. Formation of the stable excited states of Ag42 is shown to be responsible for the core transformation.

Extraction of chitin from Litopenaeus vannamei shell and its subsequent characterization: an approach of waste valorization through microbial bioprocessing.

Chemical extraction of chitin is very hazardous and costly which can be overwhelmed by microbial bioprocessing. In this study, potent protease and lactic acid-producing bacteria were screened and identified as Alcaligens faecalis S3 and Bacillus coagulans L2, respectively. Productions of protease and lactic acid by the respective bacterial strains were optimized. The shell of Litopenaeus vannamei was sequentially treated with the partially purified protease and lactic acid and the treatment conditions were optimized for betterment of chitin yield. Spectral characterization by SEM-EDS, IR, XRD, NMR, XPS and thermal characterization by TG and DTG analysis of the extracted chitin was made and compared with commercial one. It was revealed that both the chitin have similar characteristics. Therefore, it can be articulated that chitin can be extracted from crustacean shells in pure form by microbial bioprocessing which will be a good catch for biorefinary industries for chitin extraction through greener route.

Smartphone-based Fluoride-specific Sensor for Rapid and Affordable Colorimetric Detection and Precise Quantification at Sub-ppm Levels for Field Applications.

Higher levels of fluoride (F-) in groundwater constitute a severe problem that affects more than 200 million people spread over 25 countries. It is essential not only to detect but also to accurately quantify aqueous F- to ensure safety. The need of the hour is to develop smart water quality testing systems that would be effective in location-based real-time water quality data collection, devoid of professional expertise for handling. We report a cheap, handheld, portable mobile device for colorimetric detection and rapid estimation of F- in water by the application of the synthesized core-shell nanoparticles (near-cubic ceria@zirconia nanocages) and a chemoresponsive dye (xylenol orange). The nanomaterial has been characterized thoroughly, and the mechanism of sensing has been studied in detail. The sensor system is highly selective toward F- and shows unprecedented sensitivity in the range of 0.1-5 ppm of F-, in field water samples, which is the transition regime, where remedial measures may be needed. It addresses multiple issues expressed by indicator-based metal complexes used to determine F- previously. Consistency in the performance of the sensing material has been tested with synthetic F- standards, water samples from F- affected regions, and dental care products like toothpastes and mouthwash using a smartphone attachment and by the naked eye. The sensor performs better than what was reported by prior works on aqueous F- sensing.

Dual emitting Ag35 nanocluster protected by 2-pyrene imine thiol.

In this communication, we present the synthesis of 2-pyrene imine thiol (2-PIT)-protected Ag35 nanoclusters using a ligand exchange-induced structural transformation reaction. The formation of the nanocluster and its composition were confirmed through several spectroscopic and electron microscopic studies. The UV-vis absorption spectrum showed a set of characteristic features of the nanocluster. This nanocluster showed blue emission under UV light due to pyrene to metal core charge-transfer, and NIR emission due to charge-transfer within the metal core. This is the first report on dual emitting pyrene protected atomically precise silver nanoclusters.

Xylitol Production from Lignocellulosic Pentosans: A Rational Strain Engineering Approach toward a Multiproduct Biorefinery.

Kluyveromyces marxianus IIPE453 can utilize biomass-derived fermentable sugars for xylitol and ethanol fermentation. In this study, the xylitol production in the native strain was improved by overexpression of endogenous d-xylose reductase gene. A suitable expression cassette harboring the gene of interest was constructed and incorporated in the native yeast. qPCR analysis demonstrated the 2.1-fold enhancement in d-xylose reductase transcript levels in the modified strain with 1.62-fold enhancement in overall xylitol yield without affecting its ethanol fermenting capacity. Material balance analysis on 2 kg of sugar cane bagasse-derived fermentable sugars illustrated an excess of 58.62 ± 0.15 g of xylitol production by transformed strain in comparison to the wild variety with similar ethanol yield. The modified strain can be suitably used as a single biocatalyst for multiproduct biorefinery application.

Phenothiazine-Based Oligo(p-phenylenevinylene)s: Substituents Affected Self-Assembly, Optical Properties, and Morphology-Induced Transport.

Designing intramolecular charge-transfer (ICT)-based luminogenic ordered assemblies exhibiting significant electrical transport is a challenging task in the field of organic optoelectronics. In this context, a series of novel phenothiazine-based oligo(p-phenylenevinylene) (OPV1-6) derivatives were designed and their structure-property relationship was investigated. Upon examining their photophysical properties, all the OPVs were found to exhibit significant intramolecular charge-transfer characteristics in organic solvents. While inspecting the self-assembly behaviour, the OPV with a long alkyl chain on the central phenyl core (OPV4) underwent gelation in organic solvent mixtures through strong hydrophobic interactions of the long hexadecyl chains and π-interactions from their aromatic counterparts. Computational studies revealed a lamellar packing of molecules in the assembly. Interestingly, the degree of ICT and the gelation abilities of OPVs were significantly influenced by the electronic nature of the substituents appended to the peripheral phenothiazines. Further, the AC impedance results revealed an increase in storage and electronic transport for the fluorescent thin films prepared by an increase in the content of OPV4 in PMMA.

Phenothiazinyl Boranes: A New Class of AIE Luminogens with Mega Stokes Shift, Mechanochromism, and Mechanoluminescence.

Phenothiazines with a dimesityl boron moiety, a new class of aminoboranes with B-N linkage, were synthesized. These aminoboranes exhibited interesting photophysical behavior including aggregation-induced emission (AIE), mechanochromism (MC), mechanoluminescence (ML), and a mega Stokes shift (up to 312 nm in hexane). The solid-state emission of the aminoboranes could be switched reversibly by grinding-fuming processes. Furthermore, the phenothiazine derivative with a bromo and an arylborane group at 3- and 7-positions exhibited bright mechanoluminescence.

Production of α-Amylase by Aspergillus terreus NCFT 4269.10 Using Pearl Millet and Its Structural Characterization.

In this investigation, Aspergillus terreus NCFT4269.10 was employed in liquid static surface (LSSF) and solid state (SSF) fermentation to assess the optimal conditions for α-amylase biosynthesis. One-variable-at-a-time approach (quasi-optimum protocol) was primarily used to investigate the effect of each parameter on production of amylase. The maximum amylase production was achieved using pearl millet (PM) as substrate by SSF (19.19 ± 0.9 Ug(-1)) and also in presence of 1 mM magnesium sulfate, 0.025% (w/v) gibberellic acid, and 30 mg/100 ml (w/v) of vitamin E (~60-fold higher production of amylase) with the initial medium pH of 7.0 and incubation at 30 °C for 96 h. In addition, maltose, gelatin and isoleucine also influenced the α-amylase production. Amylase was purified to homogeneity with molecular mass around 15.3 kDa. The enzyme comprised of a typical secondary structure containing α-helix (12.2%), ß-pleated sheet (23.6%), and ß-turn (27.4%). Exploitation of PM for α-amylase production with better downstream makes it the unique enzyme for various biotechnological applications.

Role of probiotic Lactobacillus fermentum KKL1 in the preparation of a rice based fermented beverage.

A dominant lactic acid bacteria, Lactobacillus fermentum KKL1 was isolated from an Indian rice based fermented beverage and its fermentative behavior on rice was evaluated. The isolate grown well in rice and decreased the pH, with an increase of total titratable acidity on account of high yield in lactic acid and acetic acid. The production of α-amylase and glucoamylase by the strain reached plateau on 1st and 2nd day of fermentation respectively. The accumulation of malto-oligosaccharides of different degrees of polymerization was also found highest on 4th day. Besides, phytase activity along with accumulation of free minerals also unremittingly increased throughout the fermentation. The fermented materials showed free radical scavenging activity against DPPH radicals. In-vitro characteristics revealed the suitability of the isolate as probiotic organism. The above profiling revealed that probiotic L. fermentum KKL1 have the significant impact in preparation of rice beer and improves its functional characteristics.

Microbial, saccharifying and antioxidant properties of an Indian rice based fermented beverage.

Haria, a popular rice based ethnic fermented beverage, is consumed as a staple food and refreshing drink by the vast number of Indian tribal people. In this study, the composition of microbial consortia and the occurrence of some important nutraceuticals during haria preparation were investigated. The quantities of moulds and yeasts were highest at 2nd day, and then declined, but, on the contrary, the quantity of Lactic Acid Bacteria and Bifidobacterium sp. increased concurrently during the course of fermentation. Accumulation of starch hydrolytic enzymes along with different types of malto-oligosaccharides like maltotetrose (26.18µg/gm), maltotriose (28.16µg/gm), and maltose (26.94µg/gm) were also noted. Furthermore, GC-MS analysis indicated the occurrence of pyranose derivatives in the fermented products. The fermented materials showed higher free radicals scavenging activity (82.54%, 4th day) against DPPH radicals. These studies clearly demonstrated that the microbial interaction during fermentation of rice makes it more nutritious, and most likely more beneficial for health.

Appraisal of antioxidant, anti-hemolytic and DNA shielding potentialities of chitosaccharides produced innovatively from shrimp shell by sequential treatment with immobilized enzymes.

Chitosaccharides (CS) of varied size were prepared from shrimp shell through sequential catalysis, using crude protease and chitinase enzymes immobilized on agar beads. In the optimized state, immobilization yield and activity yield for protease were 84% and 62%, and for chitinase were 75% and 57%, respectively. Immobilized protease and chitinase treatment improved CS yields (101 µg/ml) and retained 63% and 52% of activities after 10 reuses, respectively. Stronger radical-scavenging activity (RSA) of CS against ABTS, DPPH and hydroxyl radical was noted with EC50 values 19.1, 26.4 and 29.6 µg/ml, respectively. Peroxyl and superoxide RSAs of 96.8% and 88.6% were noticed at 70 µg/ml of CS. Singlet oxygen quenching, reducing power and ferrous ion-chelating activities of CS were also pronounced. CS reasonably reduced oxidative damage of DNA, protein and RBC by inhibiting H2O2 and AAPH radicals. Reversible CS-DNA condensation leads to DNA stabilization without changing its conformation and advocates its employment in gene therapy.

Biosynthesis, structural architecture and biotechnological potential of bacterial tannase: a molecular advancement.

Tannin-rich materials are abundantly generated as wastes from several agroindustrial activities. Therefore, tannase is an interesting hydrolase, for bioconversion of tannin-rich materials into value added products by catalyzing the hydrolysis of ester and depside bonds and unlocked a new prospect in different industrial sectors like food, beverages, pharmaceuticals, etc. Microorganisms, particularly bacteria are one of the major sources of tannase. In the last decade, cloning and heterologous expression of novel tannase genes and structural study has gained momentum. In this article, we have emphasized critically on bacterial tannase that have gained worldwide research interest for their diverse properties. The present paper delineate the developments that have taken place in understanding the role of tannase action, microbial sources, various cultivation aspects, downstream processing, salient biochemical properties, structure and active sites, immobilization, efforts in cloning and overexpression and with special emphasis on recent molecular and biotechnological achievements.