Molecular characterization and evolution of the resident population of some alfalfa mosaic virus (AMV) isolates in Egypt.

BACKGROUND: Alfalfa mosaic virus (AMV) is an important virus affecting many vegetable crops in Egypt. In this study, virus isolates were collected from naturally infected potato, tomato, alfalfa and clover plants that showed suspected symptoms of AMV in different locations of Beheira and Alexandria governorates during the 2019-2020 growing season. The relative incidence of the virus ranged from 11-25% based on visual observations of symptoms and ELISA testing. A total of 41 samples were tested by ELISA using polyclonal antisera for AMV. Four AMV isolates collected from different host plants, named AM1 from potato, AM2 from tomato, AM3 from alfalfa and AM4 from alfalfa, were maintained on Nicotiana glutinosa plants for further characterization of AMV. RESULTS: Electron micrographs of the purified viral preparation showed spheroidal particles with a diameter of 18 nm and three bacilliform particles with lengths of roughly 55, 68, and 110 nm and diameters identical to those of the spheroidal particles. The CP gene sequence comparisons of four AMV isolates (AM1, AM2, AM3 and AM4) showed the highest nucleotide identity of 99.7% with the Gomchi isolate from South Korea infecting Gomchi (Ligularia fischeri) plants. Phylogenetic analysis showed that the present isolates were grouped together into a distinct separate clade (GPI) along with the Gomchi isolate from South Korea. Similarly, the deduced amino acid sequence comparisons of Egyptian AMV isolates revealed that amino acids Q29, S30, T34, V92 and V175 were conserved among the Egyptian isolates in GPI. CONCLUSION: The present study found strong evolutionary evidence for the genetic diversity of AMV isolates by the identification of potential recombination events involving parents from GPI and GPII lineages. Additionally, the study found that Egyptian AMV isolates are genetically stable with low nucleotide diversity. Genetic analysis of the AMV population suggested that the AMV populations differ geographically, and AMV CP gene is under mild purifying selection. Furthermore, the study proposed that the Egyptian AMV population had common evolutionary ancestors with the Asian AMV population. Antioxidant enzymes activity was assessed on N. glutinosa plants in response to infection with each AMV isolate studied, and the results revealed that the enzyme activity varied.

Rhizopus stolonifer biomass catalytic transesterification capability: optimization of cultivation conditions.

BACKGROUND: Using fungal biomass for biocatalysis is a potential solution for the expensive cost of the use o enzymes. Production of fungal biomass with effective activity requires optimizing the cultivation conditions. RESULTS: Rhizopus stolonifer biomass was optimized for transesterification and hydrolysis of waste frying oil (WFO). Growth and biomass lipolytic activities of R. stolonifer improved under shaking conditions compared to static conditions, and 200 rpm was optimum. As biomass lipase and transesterification activities inducer, olive oil was superior to soybean, rapeseed, and waste frying oils. Biomass produced in culture media containing fishmeal as an N-source feedstock had higher lipolytic capabilities than corn-steep liquor and urea. Plackett Burman screening of 9 factors showed that pH (5-9), fishmeal (0.25-1.7%, w/v), and KH2PO4 (0.1-0.9%, w/v) were significant factors with the highest main effect estimates 11.46, 10.42, 14.90, respectively. These factors were selected for response surface methodology (RSM) optimization using central composite design (CCD). CCD models for growth, biomass lipase activity, and transesterification capability were significant. The optimum conditions for growth and lipid modification catalytic activities were pH 7.4, fishmeal (2.62%, w/v), and KH2PO4 (2.99%, w/v). CONCLUSION: Optimized culture conditions improved the whole cell transesterification capability of Rhizopus stolonifer biomass in terms of fatty acid methyl ester (FAME) concentration by 67.65% to a final FAME concentration of 85.5%, w/w.

Biocatalysis of triglycerides transesterification using fungal biomass: a biorefinery approach.

BACKGROUND: The use of microbial biomasses, such as fungal biomass, to catalyze the transesterification of triglycerides (TG) for biodiesel production provides a sustainable, economical alternative while still having the main advantages of expensive immobilized enzymes. RESULTS: Biomasses of Aspergillus flavus and Rhizopus stolonifera were used to catalyze the transesterification of TG in waste frying oil (WFO). Isopropanol as an acyl-acceptor reduced the catalytic capability of the biomasses, while methanol was the most potent acyl-acceptor with a final fatty acid methyl ester (FAME) concentration of 85.5 and 89.7%, w/w, for R. stolonifer and A. flavus, respectively. Different mixtures of the fungal biomasses were tested, and higher proportions of A. flavus biomass improved the mixture's catalytic capability. C. sorokiniana cultivated in synthetic wastewater was used as feedstock to cultivate A. flavus. The biomass produced had the same catalytic capability as the biomass produced in the control culture medium. Response surface methodology (RSM) was adopted using central composite design (CCD) to optimize the A. flavus biomass catalytic transesterification reaction, where temperature, methanol concentration, and biomass concentration were selected for optimization. The significance of the model was verified, and the suggested optimum reaction conditions were 25.5 °C, 250 RPM agitation with 14%, w/w, biomass, 3 mol/L methanol, and a reaction duration of 24 h. The suggested optimum conditions were tested to validate the model and a final FAME concentration of 95.53%. w/w was detected. CONCLUSION: Biomasses cocktails might be a legitimate possibility to provide a cheaper technical solution for industrial applications than immobilized enzymes. The use of fungal biomass cultivated on the microalgae recovered from wastewater treatment for the catalysis of transesterification reaction provides an additional piece of the puzzle of biorefinery. Optimizing the transesterification reaction led to a valid prediction model with a final FAME concentration of 95.53%, w/w.

Semi-pilot scale production of biodiesel from waste frying oil by genetically improved fungal lipases.

This paper addresses the issue of combining the usage of waste frying oil (WFO), as a feedstock, and a lipase produced in solid-state fermentation (SSF), as a biocatalyst, for semi-pilot scale production of biodiesel as fatty acid methyl esters (FAME). Two fungal mutants namely; Rhizopus stolonifer 1aNRC11 mutant F (1F) and Aspergillus tamarii NDA03a mutant G (3G) were used as a cocatalyst. The two mutants were cultivated separately by SSF in a tray bioreactor. The dried fermented solid of 1F and 3G mutants were used in a ratio of 3:1, respectively, for WFO transesterification. Optimization of several semi-pilot process stages including SSF and WFO transesterification reaction conditions resulted in 92.3% conversion of WFO to FAME. This FAME yield was obtained after 48 h using 10% cocatalyst (w/w of WFO), 10% water (w/w of WFO) and 3:1 methanol/ WFO molar ratio at 30 °C and 250 rpm. A preliminary economic evaluation of produced biodiesel price (190 $/Ton) is less than half the price of petroleum diesel in Egypt (401$/Ton) and is about 40.3% the price of biodiesel produced using a pure enzyme, which is a promising result. This strategy makes the biodiesel synthesis process greener, economical and sustainable.

Improvement of Aspergillus flavus saponin hydrolase thermal stability and productivity via immobilization on a novel carrier based on sugarcane bagasse.

Soyasapogenol B (SB) is known to have many biological activities such as hepatoprotective, anti-inflammatory, anti-mutagenic, antiviral and anticancer activities. Enzymatic conversion of soyasaponins to SB was carried out using saponin hydrolase (SH) extracted from Aspergillus flavus. The partially purified enzyme was immobilized on different carriers by physical adsorption, covalent binding or entrapment. Among the investigated carriers, Eupergit C and sugarcane bagasse (SCB) activated by DIC and NHS were the most suitable two carriers for immobilization (the immobilized forms recovered 46.5 and 37.1% of the loaded enzyme activity, respectively). Under optimized immobilization conditions, immobilized SH on Eupergit C and on activated SBC recovered 87.7 and 83.3% of its original activity, respectively. Compared to free SH, immobilized SH on Eupergit C and on activated SCB showed higher optimum pH, activation energy, half-lives and lower deactivation constant rate. Also, their SB productivities were improved by 2.3- and 2.2-folds compared to free SH (87.7 and 83.3 vs. 37.5%, respectively). Hence, being SCB more sustainable and an inexpensive material, it can be considered a good alternative to Eupergit C as a support for SH immobilization. SH immobilization on industrially applicable and inexpensive carrier is necessary to improve SB yield and reduce its production cost. The chemical structure of SCB and the resulting cellulose derivatives were studied by ATR-IR spectroscopy. The thermal analysis technique was used to study the chemical treatment of SCB and coupling with the enzyme. This technique confirmed the removal of lignin and hemicellulose by chemical treatment of SCB.