Mandels Medium Research Articles

Valorisation of slaughter house and deinking paper waste streams for the production of enzyme by Trichoderma reesei

The study investigates for the first time the possibility of using carbon rich paper recovery sludge, and nitrogen rich meat processing industry waste as cultivation medium for the production of high value enzymes needed in the respective industries. The complex cellulose rich deinking sludge was able to support the growth of many industrially relevant enzyme producing microorganisms (Bacillus licheniformis, Candida cylindracea, Aspergillus oryzae, Trichoderma reesei) and of recombinant enzyme producers (Escherichia coli and Pichia pastoris). Further detailed studies with Trichoderma reesei as model organism demonstrated that the organism was able to grow optimally in the presence of 40gL-1 paper sludge as carbon source and 67.5 gL-1 pasteurised blood as nitrogen source substituted in Mandels medium. Under these conditions cellulase activities up to 28.1 nkat FPU were achieved. Anyhow, to achieve these results pretreatment of both waste streams is inevitable. In summary, this study provides the practical basis for a valorisation systems of paper industry waste to produce valuable enzymes to be used on-site in paper processing or for other purposes.

Optimisation of fungal cellulase production from textile waste using experimental design

Abstract Abundant textile waste has raised increasing concerns worldwide in developing novel circular textiles approach. This study investigated the optimum cellulase production from textile waste by Aspergillus niger CKB. Textile wastes consisting of cotton and polyester in various ratios were used as low-cost feedstock. Three types of cultivation media were compared in solid state fermentation and Mandels medium with yeast extract selected due to its superior cellulase production. Conditions including moisture, pH, inoculum size and organic nitrogen were evaluated and optimised via response surface methodology. Supplementary carbon sources and cellulase inducers were also employed to enhance fungal growth and cellulase production. The results indicated that the optimised fermentation method significantly improved cellulase producing efficiency and enzyme activity by 88.7% and 25.8%, respectively. The maximum cellulase activity reached 1.56 FPU g−1 in 6 days. The outcomes led to the efficient recovery of glucose and polyester, which could contribute to a closed-loop recycling strategy for the textile industry, and enable the transition to an advanced circular textiles economy.

English

  The indigenous cellulolytic fungus Trichoderma longibrachiatum (GHL) isolated from soil near an Algerian hot spring was used for the production of cellulases by submerged fermentation on Mandels medium with cellulose Avicel (1%) as the sole carbon source. Endoglucanase and filter paper activities of the wild-type strain ofTrichoderma were compared to the hypercellulolytic mutated Trichoderma reesei Rut C-30, in shake flask cultures at 35°C. After seven days of fermentation, T. longibrachiatum show equivalent activities than T. reesei (10.61 IU/ml of endoglucanase (CMCase) and 2.04 IU/ml of filter paper activity (FPA)). On the other hand, the ß-glucosidase activity of Trichoderma GHL was twice more important than that of T. reseei. The influence of inoculum size on cellulase activities did not prove significant differences in enzymatic activities for spore concentrations between 105and 108 spores/ml. The cellulases produced by the isolated strain were also characterized. The optimum temperatures were 55 and 60°C for endoglucanase and FPA, respectively. The endoglucanase was thermostable at 70°C after 5 h incubation, and it preserved 80% of the original activity. The half-life of the FPA appeared to be 3 h at 60°C. The endoglucanase was optimally active at pH 4.0, and the FPA was optimal at pH 4.0 and 5.0. These activities were stable at 50°C after 5 h incubation in a pH range of 3.0 to 6.0 and 4.0 to 6.0, respectively. These results suggest that the non-mutated strain T. longibrachiatum (GHL) should be an attractive producer for cellulases production.   Key words: Cellulase, Trichoderma reesei, Trichoderma longibrachiatum, submerged fermentation, characterization.

Optimization of media and submerged fermentation conditions using central composite design for increased endoglucanase production by Cladosporium sp. NCIM 901

Objective: Present work was focused on utilization of lignocellulosic substrates like bagasse and citrus peel powder for CMCase (endoglucanase) production through submerged fermentation by Cladosporium sp. NCIM 901. Methods: Fungal strain Cladosporium sp. was procured from NCIM, Pune. Peracetic acid pretreated bagasse was used in order to reduce the crystallinity of bagasse. Mary Mandels medium was used for enzyme production. Central composite design was evaluated to study the effect of five variables on CMCase production and to optimize the medium composition. Results: Experimental results showed the optimal medium for higher enzyme production was composed of 10% (w/v) bagasse substrate, 4.5% (w/v) citrus peel powder, 0.17% (w/v) MgSO 4 , 0.17% (w/v) urea with the initial pH of medium at 5.5. Maximum enzyme yield (15.02 IU/ml) was obtained on by the interaction of optimal level of process involved parameters. There is a good correlation between actual and predicted results. The coefficient of determination (R2) is 0.9414. Conclusion: Use of cheap and environment friendly available agricultural and fruit waste substrates for enzyme production eventually reduces the production cost of enzymes. Peracetic acid pretreated bagasse was used with citrus peel powder as fermentation medium constituent to induce the enzyme production. Observed actual enzyme activity was well agreed with the predicted enzymatic activity, shown the model was applicable for enzyme production through submerged fermentation.

Cellobiose dehydrogenase influences the production of S. microspora β-glucosidase

BglG, a Stachybotrys microspora β-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5min at 100°C) of the aliquots, which supported the non-proteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a δ-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of δ-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.

The Character of Normal Temperature Straw-Rotting Microbial Community

In order to study degradation capability and optimal condition of produced endoglucanase (CMCcase), the microbial community with efficient cellulose degrading ability in 28°C was studied. Microbial community came from rotted rice straw which was enriched and domesticated by improved Mandels medium. The standard cellulase activity assays were used to determine cellulase activity, degradation products were analyzed by gas chromatography mass spectrometry (GC/MS), and denaturing gradient gel electrophoresis (DGGE) was used to identify the composition dynamic of the community. The results showed that the microbial community could degrade 39.6% of rice straw gross weight within 6 d. When culture medium volume was 1/5 and pH = 6 on day 5, the CMCcase activity reached the highest of 14 IU mL −1. During the fermentation, more than ten products were detected using GCMS. The microbial changed lots in different fermentation periods which detected by DGGE. Phylogenetic tree derived from 16S rDNA sequence found that the community composed of bacteria, and the closest relative were belong to Clostridium sp., Brevibacillus sp. and Bacterium sp. This microbial community could accelerate rice straw rotting and decomposed products could promote organic matter increase of soil.

Production and use of mutanase from Trichoderma harzianum for effective degradation of streptococcal mutans

Basic cultural parameters affecting mutanase production by Trichoderma harzianum F-340 in shaken flasks and aerated fermenter cultures have been standardized. The best medium for enzyme production was Mandels medium A with initial pH 5.3, supplemented with 0.3% mutan and 0.05% peptone and inoculated with 20% of the 72-h mycelium as inoculum. It was shown that mycelial mass, used in the culture medium as a sole carbon source, induced mutanase synthesis and could be utilized as an inexpensive and easily available substitute for bacterial mutan. Application of optimized medium and cultural conditions enabled us to obtain a high mutanase yield (0.6-0.7 U/mL, 2.0-2.5 U/mg protein) in a short period of time (3-5 days), which was much higher than the best reported in literature. The enzyme in crude state was stable in the pH range of 4.5-6.0, and at temperatures of up to 40oC; its maximum activity was recorded at 45oC and at pH 5.5. The mutanase preparation obtained from the T. harzianum fungus was relatively stable under storage conditions, and showed a high hydrolytic potential in reaction with a mixed-linkage (a-1,3, a-1,6) water-insoluble mutan of streptococcal origin (hydrolysis yield reached a value of 69% in 24 h). Steady-state measurement of the enzymic reaction products during the hydrolysis revealed that mutanase exhibited an exo type of action on mutan. Thin-layer chromatographic analysis showed that glucose was the primary final product of mutan hydrolysis with mutanase. The potential application of mutanase in dentistry is discussed.

The comparison of cellulase activities from bacteria and molds isolated from termites

Screening for choosing the best bacterium and mold producing cellulase was carried out from eight xylanolytic bacteria and three cellulolytic molds isolated from termites. Each bacterium and mold was inoculated on the agar medium containing minerals and carboxymethylcellulose. The diameters of colony and clearing zones were measured after stained with Congo red. Four bacteria: Bacillus larvae XB 1-1, B. larvae XU 2-2, Bacillus PU 2-2, and B. pumilus PU 4-2 showing higher clearing zone ratio were further submerged cultured in PM medium containing 2% wheat pollard in the shaker incubator at room temperature, 150 rpm for 36 and 48 hours. The three molds: Aspergillus flavus S 13, MS 21, and Penicillium nalgiovense S 11 were also cultured in the same way as bacteria, but in Mandels medium containing 2% wheat pollard for three and five days incubation time. B. pumilus PU 4-2 and A. flavus S 13 produced the highest CMCase in each group respectively. All enzyme assays of A. flavus S 13 had higher activities than that of B. pumilus PU 4-2. The specific activity of CMCase, avicelase, FPase, and cellobiohidrolase were the highest on the enzyme of A. flavus S 13 produced from 3 days incubation time, while its β- glucosidase was the best for five days incubation. In term of activities, A. flavus S 13 had the highest ability to produce cellulase. The ability of B. pumilus PU 4-2 to produce cellulase and xylanase at the same time was an additional value. Key words: Bacillus pumilus, Aspergillus flavus , cellulases

Properties of the cellulolytic system from Aspergillus niveus

The thermotolerant Aspergillus niveus strain RMF 7883, was grown in media containing insoluble crystalline cellulose as carbon source and produced more enzyme at 35 °C than 25 °C. The cell growth, expressed as cellular protein content and cellulase production using Czapek medium was superior to that produced on Mandels medium. Maximum enzyme activities were recorded at pH 5, 4·8 and 5·2 for β-glucosidase, endoglucanase and filter paper degrading activity (FPase), respectively. Activation of enzymes was observed after exposure to elevated temperature for 15 min. Optimum temperatures and heat stability were determined. Activity of the enzymes was improved in cultures where buffering capacity was controlled through the addition of K-phthalate. Alkali pretreatment of lignocellulosic substrate increases accessibility of the substrate towards the enzyme very effectively, the treated was 11·2% saccharified vs 0·98% for the untreated substrate. The cellulolytic enzyme system from A. niveus was characterized by its high β-glucosidase activity, ability to degrade highly ordered cellulose and saccharification of biomass.

Isolation of a Cellulolytic Enzyme Producing Microorganism, Culture Conditions and Some Properties of the Enzymes

A fungus was isolated from soil as a cellulolytic enzyme-producing microorganism. This wild strain, named fungal strain Y-94, secreted a large amount of cellulolytic enzyme components consisting of 0.8 units of avicelase, 9.2 units of carboxymethyl cellulose (CMC)-hydrolyzing enzyme (CMCase) and 2.0 units of β-glucosidase per ml of culture broth on cultivation in Mandels medium for 7 to 10 days at 30°C, and also produced in the culture filtrate various other polysaccharide- hydrolyzing enzymes such as xylanase, β-1,3-glucanase, amylase, etc. Higher productivity of the cellulolytic enzymes (avicelase, 6.5 units/ml; CMCase, 82 units/ml; β-glucosidase, 25 units/ml; and filter paper units (F.P.U.),* 5.0 units/ml) was obtained by altering the medium composition. The maximum production of the cellulolytic enzymes was observed when this strain was cultured in a medium containing 4% cellulose powder, 1% peptone, 0.6% KNO3, 0.2% urea, 1.2% KH2PO4, 0.16% KCl, 0.12% MgSO4 7H20, 0.001% MnSO4 6H20, 0.001% CuSO4 7H20 and 0.1% Tween 80 or polyethylene glycol 1000, pH 4.0, under the same culture conditions.

Isolation of a cellulolytic enzyme producing microorganism, culture conditions and some properties of the enzymes.

A fungus was isolated from soil as a cellulolytic enzyme-producing microorganism. This wild strain, named fungal strain Y-94, secreted a large amount of cellulolytic enzyme components consisting of 0.8 units of avicelase, 9.2 units of carboxymethyl cellulose (CMC)-hydrolyzing enzyme (CMCase) and 2.0 units of β-glucosidase per ml of culture broth on cultivation in Mandels medium for 7 to 10 days at 30°C, and also produced in the culture filtrate various other polysaccharide- hydrolyzing enzymes such as xylanase, β-1,3-glucanase, amylase, etc. Higher productivity of the cellulolytic enzymes (avicelase, 6.5 units/ml; CMCase, 82 units/ml; β-glucosidase, 25 units/ml; and filter paper units (F.P.U.),* 5.0 units/ml) was obtained by altering the medium composition. The maximum production of the cellulolytic enzymes was observed when this strain was cultured in a medium containing 4% cellulose powder, 1% peptone, 0.6% KNO3, 0.2% urea, 1.2% KH2PO4, 0.16% KCl, 0.12% MgSO4 7H20, 0.001% MnSO4 6H20, 0.001% CuSO4 7H20 a...

Effect of fermentation variables on cellulase production by Trichoderma Sp.

Batch cultivation ofTrichodermma reesei QM9414 was carried out in Mandels medium containing(w/v) 1% beech wood cellulose and 0.05% yeast extract at 29°C. Use of 36 hours old inoculum(10% v/v),3.2 1/min aeration rate at 400 rpm(KLa 220/h) and pH cycling strategy produced 4 g/1 cell mass and 21.5 IU/1/h FPA cellulase.