Mohamed Neifar

Biotechnological applications of extremophiles, extremozymes and extremolytes

In the last decade, attention to extreme environments has increased because of interests to isolate previously unknown extremophilic microorganisms in pure culture and to profile their metabolites. Microorganisms that live in extreme environments produce extremozymes and extremolytes that have the potential to be valuable resources for the development of a bio-based economy through their application to white, red, and grey biotechnologies. Here, we provide an overview of extremophile ecology, and we review the most recent applications of microbial extremophiles and the extremozymes and extremolytes they produce to biotechnology.

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117.

Aim:  To produce high laccase activities from the white‐rot fungus Fomes fomentarius.

Application of Asymetrical and Hoke Designs for Optimization of Laccase Production by the White-Rot Fungus Fomes fomentarius in Solid-State Fermentation

Statistical approaches were employed for the optimization of different cultural parameters for the production of laccase by the white rot fungus Fomes fomentarius MUCL 35117 in wheat bran-based solid medium. first, screening of production parameters was performed using an asymmetrical design 2533//16, and the variables with statistically significant effects on laccase production were identified. Second, inoculum size, CaCl2 concentration, CuSO4 concentration, and incubation time were selected for further optimization studies using a Hoke design. The application of the response surface methodology allows us to determine a set of optimal conditions (CaCl2, 5.5 mg/gs, CuSO4, 2.5 mg/gs, inoculum size, 3 fungal discs (6 mm Ø), and 13 days of static cultivation). Experiments carried out under these conditions led to a laccase production yield of 150 U/g dry substrate.

Purification and characterization of a highly thermostable esterase from the actinobacterium Geodermatophilus obscurus strain G20

Intracellular thermostable esterase produced by the extremophilic Geodermatophilus obscurus G20 was purified to homogeneity by a heat treatment, followed by an anion‐exchange chromatography, and then characterized. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was shown to be approximatively 55 kDa. The enzyme showed an optimal activity between pH 8.0 and 9.0 and was stable in the pH range 7.0–10.0. Moreover, it is highly thermostable, with a residual activity greater than 90% after incubation at 80 °C for more than 10 h. The enzyme showed preference for esters of p ‐nitrophenol with short chain fatty acid. When the p ‐nitrophenyl acetate (C2) was used as substrate, the Michaelis–Menten constant (Km) and maximum velocity for the reaction (Vmax) of esterase were 400 μM and 2500 U/mg protein, respectively. The effect of phenylmethanesulphonyl fluoride (PMSF), a serine‐specific inhibitor, on the enzyme activity suggested that the thermostable esterase belong to the serine hydrolase group. Because of its high thermostability, activity at alkaline pH, tolerance to methanol and various metal ions and specificity for short chain fatty acids, this enzyme showed high potential for use in biocatalysis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Decolorization of Solophenyl Red 3BL Polyazo Dye by Laccase-Mediator System: Optimization through Response Surface Methodology

The decolorization of direct Solophenyl red 3BL (SR), a polyazo dye extensively used in textile industry was studied. The Fomes fomentarius laccase alone did not decolorize SR. The natural redox mediator, acetosyringone (AS), was necessary for decolorization to occur. Box-Behnken design was used to evaluate the effects of three parameters, namely, enzyme concentration (0.5–2.5 U mL−1), redox mediator concentration (3–30 μM), and incubation time (1–24 h), on the SR decolorization yield. The fitted mathematical model allowed us to plot response surfaces as well as isoresponse curves and to determine optimal decolorization conditions. The results clearly indicated that the AS concentration was the main factor influencing the SR decolorization yield. The selected optimal conditions were enzyme concentration 0.8 U mL−1, mediator concentration 33 μM, and time 14 h 30 min. These conditions allowed 79.66% of SR decolorization versus 80.70% for the predicted value. These results showed a promising future of applying laccase-AS system for industrial wastewater bioremediation.

Improved Mannanase Production from Penicillium occitanis by Fed-Batch Fermentation Using Acacia Seeds

By applying a fed-batch strategy, production of Penicillium occitanis mannanases could be almost doubled as compared to a batch cultivation on acacia seeds (76 versus 41 U/mL). Also, a 10-fold increase of enzyme activities was observed from shake flask fermentation to the fed-batch fermentation. These production levels were 3-fold higher than those obtained on coconut meal. The high mannanase production using acacia seeds powder as inducer substrate showed the suitability of this culture process for industrial-scale development.

Improving the nutritive value of Olive Cake by solid state cultivation of the medicinal mushroom Fomes fomentarius

Olive Cake (OC) generated by the olive oil industries, well implanted in Tunisia, represents a major disposal and potentially severe pollution problem. This work presents the study of bioconversion of OC in solid state fermentation with the medicinal mushroom, Fomes fomentarius so as to upgrade its nutritional values and digestibility for its use as ruminants feed. The fungus was cultured on OC for 7-30 d, and subsequently the chemical composition, lignocellulolytic enzyme activities and in vitro digestibility of the resultant substrate were determined. The results obtained showed an increase in the crude protein ranging from 6% to 22% for the control and for treated OC, respectively. Significant (P<0.05) decreases in the values of neutral detergent fiber (hemicelluloses, cellulose and lignin), acid detergent fiber (lignin and cellulose) and acid detergent lignin were detected (23%, 13% and 10%, respectively). The estimated in vitro digestibility improved from 9% (control) to 25% (treated OC). The present findings revealed F. fomentarius to be an efficient organism for lignocellulolytic enzymes production and simultaneous enhancement in crude protein and in vitro digestibility of OC.

Diversity and Enzymatic Profiling of Halotolerant Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia

Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah, a Saharan salt flat located in southern Tunisia. Based on morphology and sequence inference from the internal transcribed spacer regions, 28S rRNA gene and other specific genes such as β-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase, the isolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes: Cladosporium (n = 3), Alternaria (n = 4), Aspergillus (n = 3), Penicillium (n = 5), Ulocladium (n = 2), and Engyodontium (n = 2). Their tolerance to different concentrations of salt in solid and liquid media was examined. Excepting Cladosporium cladosporioides JA18, all isolates were considered as alkali-halotolerant since they were able to grow in media containing 10% of salt with an initial pH 10. All isolates were resistant to oxidative stresses and low temperature whereas 5 strains belonging to Alternaria, Ulocladium, and Aspergillus genera were able to grow at 45°C. The screening of fungal strains for sets of enzyme production, namely, cellulase (CMCase), amylase, protease, lipase, and laccase, in presence of 10% NaCl, showed a variety of extracellular hydrolytic and oxidative profiles. Protease was the most abundant enzyme produced whereas laccase producers were members of the genus Cladosporium.

Comparative study of olive oil mill wastewater treatment using free and immobilized Coriolopsis polyzona and Pycnoporus coccineus

The efficiency of the two white-rot fungi Pycnoporus coccineus and Coriolopsis polyzona in the Olive Oil Mill Wastewater (OOMW) treatment was investigated. Both fungi were active in the decolourisation and COD removal of OOMW at 50 g/L COD, but only the first fungus remains effective on the crude effluent (COD=100 g/L). Moreover P. coccineus was less affected by oxygen supplementation and exhibited a high tolerance to agitation in comparison to C. polyzona. However, it required a nitrogen supplementation to obtain faster and higher COD removal. To overcome the negative effect of agitation on fungi growth and efficiency, immobilisation of C. polyzona and P. coccineus in polyurethane foam was applied. The immobilized system showed better COD decreases during three consecutive batches without remarkable loss of performances. The results obtained in this study suggested that immobilized C. polyzona and especially immobilized P. coccineus might be applicable to a large scale for the removal colour and COD of OOMW.