Study on Separation and Immunocompetence of Water Extract of Polysaccharide from Cistanche tubulosa / 中国药房

OBJECTIVE：To isolate the water extract of polysaccharide from Cistanche tubulosa ，and to investigate their immunocompetence in vitro . METHODS ：AB-8 macroporous adsorption resin was used to decolorize C. tubulosa polysaccharide. The decolorization process was optimized by orthogonal test with retention rate and decolorization rate of polysaccharide as comprehensive score ，and using adsorption rate ，decolorization time ，sample concentration as factors. The verification tests were conducted. DEAE- 650M ion exchange column was used to separate the water extract of decolorized C. tubulosa polysaccharide. CCK-8 assay was used to detect the effect s of different concentration of polysaccharide （6.25-100 μg/mL）before and after isolation on the proliferation rate of mice macrophage RAW 264.7. Griess method and ELISA assay were adopted to detect the effects of low ， medium and high concentration of polysaccharide （12.5，25，50 μg/mL）on the release of NO ，IL-6 and TNF-α in LPS-induced RAW264.7 cells. RESULTS ：In the optimal decolorization process of AB- 8 macroporous adsorption resin ，the adsorption flow rate was 1.2 BV/h，the decolorization time was 9 h，and sample concentration was 25 mg/mL. The comprehensive scores of 3 times of verification tests were 63.43％，63.29％ and 63.34％，respectively，with an average of 63.35％（RSD＝0.11％，n＝3）. One neutral polysaccharide （CTZ）and 5 acid polysaccharides （CT1，CT2，CT3，CT4，CT5）were isolated from the polysaccharide of C. cistanche ，the contents were 299.2，168.0，123.2，121.6，54.4，11.2 mg/g. Compared with control group ，6.25-100 μg/mL CTZ （except for 6.25 μg/mL），CT2，CT4，CT5 and 6.25 μg/mL CTC（the polysaccharide before seperation ）could significantly increase the proliferation rate of RAW 264.7 cells（P＜0.05），while 6.25-100 μg/mL CT1，CT3 and 50 μg/mL CTC could decrease te proliferation rate of RAW 264.7 cells（P＜0.05）. Compared with LPS group ，the release of NO were decreased significantly in low，medium and high concentration groups of CTC ，CT2，CT3 and CT 5，CTZ low concentration group （P＜0.05），while were increased significantly in high concentration groups of CT 1 and CT 4 （P＜0.05）. The release of IL- 6 （except for CT 1 high concentration group and CT 5 low concentration group ）and TNF-α（except for CT 1 medium concentration group ）were decreased significantly in low ，medium and high concentration groups （P＜0.05）. CONCLUSIONS ：The optimized decolorization technology of macroporous adsorption resin is stable and feasible in the study. One neutral polysaccharide and 5 acidic polysaccharides can be isolated from water extract of C. tubulosa polysaccharides，among which CT 2 polysaccharide has stronger anti-inflammatory ability.

Factors and mass ratio analyses for Reactive Blue 19 dye decolorization using ozone: an experimental and analytical modelling approach / Análises de atributos e taxa mássica da descolorização do corante Azul Reativo 19 usando ozônio: modelagem experimental e analítica

ABSTRACT Dyes highly reduce sunlight penetration into the stream, and consequently affect photosynthesis and oxygen transfer into water bodies. An experimental and analytical modelling approach to Reactive Blue 19 (RB19) removal using ozone was carried out. For this purpose, factors and mass ratio analyses were assessed based on batch assays experiments. Removal efficiency increased from 64 to 94% when the dosage increased from 38.4 to 153.6 mg O3.L-1. Results showed that RB19 is more efficiently removed when initial pH is 7. The rate of RB19 removal decreased as the initial dye concentration increased. Kinetic studies showed that the ozonation of RB19 was a pseudo first-order reaction with respect to the dye, and the apparent rate constant declined logarithmically with the initial dye concentration. Mass ratio studies showed that, for the empirical analysis, the power law equation was adequate to describe mass ratio over time and the analytical analysis suggests that the process is influenced by mass transfer in the liquid film as well as in the bulk fluid.

RESUMO Os corantes reduzem significativamente a penetração da luz solar no corpo d'água e, consequentemente, afetam a fotossíntese e a transferência de oxigênio. Realizaram-se modelagens experimental e analítica da remoção do azul reativo 19 por ozônio. Para isso, avaliaram-se os atributos e a taxa mássica por ensaios em batelada. A eficiência de remoção foi de 64 para 94% quando a dose de ozônio aumentou de 38,4 para 153,6 mg O3.L-1. Os resultados mostraram que o azul reativo 19 é removido mais eficientemente em pH inicial da solução de 7. A taxa de remoção do azul reativo 19 reduziu à medida que a sua concentração inicial aumentou. Os estudos cinéticos mostraram que a ozonização do azul reativo 19 é uma reação de pseudoprimeira ordem em relação ao corante e a constante cinética aparente decai logaritmicamente com a concentração inicial de corante. A análise empírica indica que a taxa mássica ao longo do tempo pode ser descrita adequadamente por uma equação de potência, e os estudos analíticos sugerem que o processo é influenciado pela transferência de massa tanto no filme líquido quanto na massa líquida.

Two Manganese Peroxidases and a Laccase of Trametes polyzona KU-RNW027 with Novel Properties for Dye and Pharmaceutical Product Degradation in Redox Mediator-Free System

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

Decolorization of Synthetic Dyes by Ficus carica Latex Peroxidase Isoenzymes

Aims: The current study aims to elucidate the potential of Ficus carica latex peroxidase isoenzymes for decolorizing different synthetic dyes in comparison to the commercial horseradish peroxidase. Study Design: The decolorization of 20 dyes was investigated using the purified F. carica latex peroxidase isoenzymes (purified FP1 and partially purified FP2, and FP3), and horseradish peroxidase (HRP) as a control. Place and Duration of Study: Molecular Biology Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, Egypt, between January 2017 and March 2018. Methodology: The purified and partially fractions of peroxidase isolated from latex of F. carica were used for the present study. Stock solutions of the dyes were prepared in 0.05 M sodium acetate buffer (pH 5.5) and diluted to the requested concentrations ranged from 12 to 330 µM in order to get maximum absorbance does not exceed 1.5 as initial reading. The efficiency of decolorization was expressed in terms of percentage. All experiments were performed in triplicate. Results: F. carica latex peroxidase isoenzymes and commercial horseradish peroxidase were able to decolorize some of tested dyes and the extent of decolorization achieved with different dyes classes were varied according to different chemical structure of each dye. The decolorization efficiency after 3 h of incubation at 40°C using 6.4 U/ml of peroxidase activity of FP1, FP2, FP3 and HRP, was found to be extremely efficient in decolorizing some dyes and relatively low in other dyes. Conclusion: The efficiency of F. carica latex peroxidase isoenzymes toward different synthetic dyes meet the prerequisites needed for environmental and industrial applications.

Efficiency of decolorization of different dyes using fungal biomass immobilized on different solid supports

Abstract Different technologies may be used for decolorization of wastewater containing dyes. Among them, biological processes are the most promising because they seem to be environmentally safe. The aim of this study was to determine the efficiency of decolorization of two dyes belonging to different classes (azo and triphenylmethane dyes) by immobilized biomass of strains of fungi (Pleurotus ostreatus - BWPH, Gleophyllum odoratum - DCa and Polyporus picipes - RWP17). Different solid supports were tested for biomass immobilization. The best growth of fungal strains was observed on the washer, brush, grid and sawdust supports. Based on the results of dye adsorption, the brush and the washer were selected for further study. These solid supports adsorbed dyes at a negligible level, while the sawdust adsorbed 82.5% of brilliant green and 19.1% of Evans blue. Immobilization of biomass improved dye removal. Almost complete decolorization of diazo dye Evans blue was reached after 24 h in samples of all strains immobilized on the washer. The process was slower when the brush was used for biomass immobilization. Comparable results were reached for brilliant green in samples with biomass of strains BWPH and RWP17. High decolorization effectiveness was reached in samples with dead fungal biomass. Intensive removal of the dyes by biomass immobilized on the washer corresponded to a significant decrease in phytotoxicity and a slight decrease in zootoxicity of the dye solutions. The best decolorization results as well as reduction in toxicity were observed for the strain P. picipes (RWP17).

Decolorization of Cibacron Blue 3G-A Dye by Agaricus bisporus CU13 Laccase - Mediator System: A Statistical Study for Optimization via Response Surface Methodology

Aim: the present study aims to optimize Cibacron Blue 3G-A decolorization as a model dye through laccase ‎enzymatic biocatalysis presenting the role of HBT as a redox mediator via ‎RSM approach.‎ Study Design: RSM using Central Composite Design (CCD) was used in order to determine the most effective variables levels in Cibacron Blue 3G-A decolorization and to investigate their interactions. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Research Division, National Research Centre (NRC), Cairo, Egypt, between August 2017 and January 2018. Methodology: The evaluation of Cibacron Blue 3G-A decolorization by A. bisporus CU13 crude laccase was conducted through different trials using a 1.5 mL reaction mixture containing different concentration of crude laccase, Cibacron Blue 3G-A, and HBT in 0.1 M sodium citrate buffer (pH 4.5) at room temperature for different incubation periods. Results: Hydroxybenzotriazole (HBT) as a mediator enhanced Cibacron Blue 3G-A decolorization levels significantly, where decolorization percentage caused by laccase enzyme alone were ‎11.92 and ‎23.78%, ‎whereas that caused by laccase HBT mediator system under the same conditions were 43.43 and ‎76.34% after 1 and 22 h of incubation, respectively. HBT concentration, dye concentration, enzyme activity, and incubation time were chosen as study variables to optimize Cibacron Blue 3G-A dye decolorization through RSM approach via central composite design (CCD). The optimum conditions for Cibacron Blue 3G-A decolorization were found to be under using 0.50 U/mL of Agaricus bisporus CU13 laccase, 92.19 ppm of Cibacron Blue 3G-A, and 1 mM of HBT in order to get decolorization percentage of 29.29% in 35 min. Conclusion: Agaricus bisporus CU13 crude laccase was used as a biocatalyst to decolorize Cibacron Blue 3G-A in presence of HBT as a mediator through utilizing the response surface methodology approach. HBT concentration, dye concentration, enzyme activity, ‎and incubation time affects the decolorization levels considerably.

Decolorization technique optimization for Boletus edulis polysaccharides and the antioxidant activity / 中成药

AIM To optimize the decolorization technique for Boletus edulis polysaccharides and to evaluate the antioxidant activity.METHODS With decolorization temperature,resin consumption,polysaccharide concentration and decolorization time as influencing factors,decolorization rate and polysaccharide recovery rate as evaluation indices,orthogonal test was applied to optimizing the decolorization technique on the basis of single factor test.Then the antioxidant activity was determined by Fe2+ chelating power,hydroxyl free radical scavenging activity and reducing power tests.RESULTS The optimal conditions were determined to be 30 ℃ for decolorization temperature,7.5 g/100 mg polysaccharides for S8 resin consumption,1 g/L for polysaccharide concentration,and 2 h for decolorization time,the decolorization rate and polysaccharide recovery rate were 89.34％ and 49.26％,respectively.The polysaccharides demonstrated strong Fe2+ chelating power and hydroxyl free radical scavenging activity with weak Fe3+ reduction power.CONCLUSION This stable and reliable method can be used for the decolorization for Boletus edulis polysaccharides with good antioxidant activity.

Combined strategies for improving production of a thermo-alkali stable laccase in Pichia pastoris

Background: Laccases are copper-containing enzymes which have been used as green biocatalysts for many industrial processes. Although bacterial laccases have high stabilities which facilitate their application under harsh conditions, their activities and production yields are usually very low. In this work, we attempt to use a combinatorial strategy, including site-directed mutagenesis, codon and cultivation optimization, for improving the productivity of a thermo-alkali stable bacterial laccase in Pichia pastoris. Results: A D500G mutant of Bacillus licheniformis LS04 laccase, which was constructed by site-directed mutagenesis, demonstrated 2.1-fold higher activity when expressed in P. pastoris. The D500G variant retained similar catalytic characteristics to the wild-type laccase, and could efficiently decolorize synthetic dyes at alkaline conditions. Various cultivation factors such as medium components, pH and temperature were investigated for their effects on laccase expression. After cultivation optimization, a laccase activity of 347 ± 7 U/L was finally achieved for D500G after 3 d of induction, which was about 9.3 times higher than that of wild-type enzyme. The protein yield under the optimized conditions was about 59 mg/L for D500G. Conclusions: The productivity of the thermo-alkali stable laccase from B. licheniformis expressed in P. pastoris was significantly improved through the combination of site-directed mutagenesis and optimization of the cultivation process. The mutant enzyme retains good stability under high temperature and alkaline conditions, and is a good candidate for industrial application in dye decolorization.

Optimization of Activated Carbon Decolorization Technology for Bletilla Striata Polysaccharide by Orthogonal Test / 中国药师

Objective: To optimize the decolorization technology of activated carbon for the plant pigment in Bletilla striata polysaccharide.Methods: Using L 9 (3 4) orthogonal test with activated carbon as the decolorizer, the amount of activated carbon, decolorization temperature, decolorization liquid pH and decolorization time were investigated.The decolorization rate and polysaccharide retention rate were investigated.The decolorization rate and polysaccharide retention rate were taken as the indices.Results: The optimum decolorization technology was as follows: the amount of activated carbon was 1.0%, the decolorization temperature was 40 ℃, the pH value was 5 and the decolorization time was 30 min.Under those conditions, the decolorization rate of Bletilla striata polysaccharide was 91.3% and the retention rate of polysaccharide was 80.6%.Conclusion: The selected decolorization technology of activated carbon can make Bletilla striata polysaccharide get the best decolorizing effect.

Research Progress in Decolorization Methods for Polysaccharide Extracts of Traditional Chinese Medicines / 中国药师

The relevant literatures were looked up, summarized, classified and reviewed, and the research progress in decolorization methods for the extracts of traditional Chinese medicines containing polysaccharide was introduced.The decolorization methods commonly used for polysaccharide extracts of traditional Chinese medicines were activated carbon decolorization, macroporous resin decolorization, hydrogen peroxide decolorization, sodium hypochlorite decolorization and so on.According to the combination form of polysaccharide and pigment, the suitable decolorization method should be selected to improve the purity of polysaccharide and the quality of related preparations, which shows guidance effect on fine finishing of traditional Chinese medicines.

Purification of macroporous adsorption resin and decolorization of ion exchange resin of total saponins of Panax japonicus / 中草药

Objective: To establish a simple and effective extraction method for the preparation of total saponins of Panax japonicas (TSPJ). Methods: Combination of macroporous adsorption and ion exchange resin chromatography was adopted in the present study. For quality evaluation, chikusetsusaponin IVa was used as reference, and vanillin-perchloric acid was applied as chromogenic reagent to determine total saponin content at 545 nm. Results: X-5 macrophous resin offered better adsorption and desorption capacities for TSPJ than other macrophous resins. The optimum purification process was confirmed as follows: The sample solution concentration was 0.2 mg/L; The sample volume was 10 g/g, and eluting with 5 mL of 70% aqueous ethanol solutions on 1 g wet macrophous resin column. Followed this step, decoloring of TSPJ was studied and the decoloring capacity of two different types of ion exchange resins was evaluated. The result showed that 732-type cation exchange resin was the better resin for decolorization of the TSPJ. The total saponin products with higher purity and quality were obtained, with the mass fraction more than 85.0%, and the transfer rate of TSPJ was more than 70.0%. Conclusion: The results show that the total saponins can be separated and purified effectively from P. japonicus. The preparation method is simple, effective, and efficient for large-scale preparation of TSPJ.

Extraction and Content Determination of Polysaccharide in Mongolian Medicine Vicia amoena / 中国药房

OBJECTIVE:To optimize the decolorization condition for polysaccharide extract of Mongolian medicine Vicia amoena,and to establish the method for its extraction and content determination. METHODS:The water extract-alcohol precipita-tion was used to extract polysaccharide from Mongolian medicine V. amoena. Using decolorization rate as index,orthogonal test was designed to investigate the effects of the dosage of activated carbon,decolorization temperature,decolorization time on the de-colorization of polysaccharide,so as to optimize the conditions for the decoloration of polysaccharide. Using glucose as control, phenol sulfuric acid method was adopted,and the content of polysaccharide in crude polysaccharide was determined by UV spectro-photometry at 490 nm. RESULTS:The optimal decoloration condition was as follows as actived carbon of 3%,decoloration time of 40 min,decoloration temperature of 60 ℃. On this basis,the average decolorization rate reached 19.77%(RSD=1.85%,n=3) by the verification test of the decoloration. The average extraction yield for the crude polysaccharide was 4.56%(RSD=2.38%,n=3),of which the polysaccharide content was 1.98%(RSD=2.18%,n=4). CONCLUSIONS:This experiment has relatively good reproducibility in polysaccharide yield;established method for content determination of polysaccharide is stable and feasible.

Biosorption of anionic textile dyes from aqueous solution by yeast slurry from brewery

ABSTRACT This study investigated the biosorption of the anionic textile dyes: Reactive Red 239 (RR239), Reactive Black B (RBB) and Direct Blue 85 (DB85) according to pH, biomass dosage, contact time and dye concentration onto waste beer yeast slurry. The kinetics and isotherm of the removal of dyes were also studied. The equilibrium of biosorption reaction was reached after 30 min for the reactive dyes and after 60 min for the direct dye. Optimum decolorization was observed at pH 2 and 0.63 g/L of biomass dosage. The kinetic data of the three dyes were better described by the pseudo second-order model. The adsorption process followed the Langmuir isotherm model and the biosorption capacity being estimated to be 152.9, 162.7 and 139.2 mg/g for RR239, RBB and DB85, respectively. Our findings indicated that the waste beer yeast slurry was an attractive low-cost biosorbent for the removal of anionic textile dyes from aqueous solution.

Partial removal and detoxification of Malachite Green and Crystal Violet from laboratory artificially contaminated water by Pleurotus ostreatus / Eliminación parcial y desintoxicación de Verde Malaquita y Cristal Violeta del laboratorio con agua contaminada artificialmente por Pleurotus ostreatus / Extração e desintoxicação de Verde Malaquita e Cristal Violeta de água contaminada artificialmente em laboratório por Pleurotus ostreatus

Abstract The triphenylmethane Malachite Green (MG) and Crystal Violet (CV) dyes are cationic dyes and mix with domestic wastewater when dumped; increasing, among others, the chemical and biological oxygen demand and can cause acute toxicity at different trophic levels. Promoting the removal (decolorization) of MG and CV, and laccase activity (54.8 ± 8.9 and 30.6 ± 2.9 UL-1 respectively) by using P. ostreatus viable biomass needed parameters such as pH (4.5 and 6.0), temperature (25 to 30 °C), stirring speed (120 rpm), percentage of inoculum (2% v/v), and dye concentration (20 and 10 mg L-1). In adsorption studies, it was showed that an acidic pH favors the adsorption of both dyes and the model of pseudo-second order describes best the phenomenon of adsorption. Finally, the germination index (GI), using Lactuca sativa seeds for the initial dyes solutions, was < 50%; demonstrating its high phytotoxic effect. When dye solutions were treated with viable biomass, the GI increased, leaving open the possibility to perform future research to determine if the aqueous solutions, post-treated with P. ostreatus, could be used in treatments that generate less toxic water which could be used in processes that do not require potable water.

Resumen Los colorantes trifenilmetánicos Verde Malaquita (MG) y Crystal Violeta (CV) son catiónicos y al ser vertidos se mezclan con aguas residuales domésticas, incrementando, entre otros, la demanda química y biológica de oxígeno; pudiendo causar toxicidad aguda en diferentes niveles tróficos. En este estudio se encontró que los parámetros pH (4,5 y 6,0), temperatura (25 y 30 °C), velocidad de agitación (120 r.p.m.), porcentaje de inóculo (2 % v/v) y concentración de colorante (20 y 10 mgL-1), presentaron un efecto significativo (p < 0.05) para favorecer la remoción (decoloración) de MG y CV, así como la actividad lacasa (54,76 ± 8,91 y 30,59 ± 2,89 UL-1 respectivamente) al utilizar biomasa viable de P. ostreatus. En los estudios de adsorción se evidenció que pH ácidos favorecen la adsorción de ambos colorantes y que el modelo de Pseudo-segundo orden describe mejor el fenómeno de quimisorción. Finalmente los índices de germinación (IG) empleando semillas de Lactuca sativa, para los colorantes iniciales fueron < 50 %; demostrando su efecto fitotóxico elevado. Cuando las soluciones de colorantes fueron tratadas con biomasa viable, el IG aumentó, dejando abierta la puerta para la realización de investigaciones futuras con la intensión de determinar si las soluciones acuosas, postratadas con P ostreatus, pueden ser utilizadas en tratamientos que generen aguas menos tóxicas y que estas puedan ser empleadas en otros procesos que no requieran agua potable.

Resumen Os corantes de tipo trifenilmetano Verde Malaquita (VM) e Cristal Violeta (CV) são corantes catiônicos e se misturam com águas residuais domésticas quando descartadas; aumentando, entre outros, as demandas químicas e biológicas de oxigênio, podendo causar toxicidade aguda em diferentes níveis tróficos. Promoveu-se a remoção (descoloração) de VM e CV, e atividade da lacase (54.8 ± 8.9 e 30.6 ± 2.9 UL-1 respectivamente) utilizando como parâmetros necessários para a biomassa viável de P. ostreatus como pH (4,5 e 6,0), temperatura (25 a 30 °C), velocidade de agitação (120 RPM), porcentagem de inócuo (2 % v/v), e concentração de corante (20 e 10 mg L-1). Em estudos de absorção, se demonstrou que um pH mais ácido favorece a absorção de ambos corantes e o modelo de pseudo-segunda ordem descreve melhor o fenômeno da absorção. Finalmente, o índice de germinação (IG), utilizando sementes de Lactuca sativa para as soluções iniciais dos corantes, foi < 50 %; demonstrando assim seu alto efeito fitotóxico. Quando as soluções de corante foram tratadas com a biomassa viável, o IG aumentou, deixando em aberto a possibilidade de realizar futuras investigações para determinar se as soluções aquosas, tratadas com P. ostreatus, poderiam ser utilizadas em tratamentos que gerem águas menos tóxicas, que poderia ser utilizada em processos que não requerem água potável.

Screening of freshwater fungi for decolorizing multiple synthetic dyes

Abstract The biodegradation of synthetic dyes by fungi is emerging as an effective and promising approach. In the present study, freshwater fungal strains isolated from submerged woods were screened for the decolorization of 7 synthetic dyes. Subsequently, 13 isolates with high decolorization capability were assessed in a liquid system; they belonged to 9 different fungal species. Several strains exhibited a highly effective decolorization of multiple types of dyes. New absorbance peaks appeared after the treatment with 3 fungal strains, which suggests that a biotransformation process occurred through fungal biodegradation. These results showed the unexploited and valuable capability of freshwater fungi for the treatment of dye-containing effluents. The ability of certain fungi to decolorize dyes is reported here for the first time.

Evaluation of crystal violet decolorization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.

Induction and enhancement of the novel marine-derived Alternaria tenuissima KM651985 laccase enzyme using response surface methodology: Application to Azo and Triphenylmethane dyes decolorization.

In our search for a laccase producer from unconventional sources, the marine derived fungus Alternaria tenuissima KM651985 was isolated from decayed wood. It was identified on the basis of morphological and molecular taxonomy and got the Genbank accession number KM651985. Two statistical experimental designs were employed to enhance laccase production. At first, a two level Plackett-Burman design was employed to screen the medium constituents and inducers that significantly affect the enzyme production. Second experiment was important for optimization of the most effective constituents and inducers using central composite design. Applying the above methods revealed that guaiacol (2 mM), copper sulphate (3 mM) and wheat bran (46.82 g/l) were the most effective factors affecting laccase production. A maximal enzyme activity of 91.84 U/ml was more than 6.33-folds the activity obtained using the basal medium. Application of A. tenuissima KM651985 culture medium containing laccase to decolorize two structurally different synthetic dyes was done successfully. This is the first report on the statistical optimization of the marine-derived A. tenuissima KM651985 laccase enzyme and its applications for dyes decolorization.

Decolorization of azo dyes (Direct Blue 151 and Direct Red 31) by moderately alkaliphilic bacterial consortium

Abstract Removal of synthetic dyes is one of the main challenges before releasing the wastes discharged by textile industries. Biodegradation of azo dyes by alkaliphilic bacterial consortium is one of the environmental-friendly methods used for the removal of dyes from textile effluents. Hence, this study presents isolation of a bacterial consortium from soil samples of saline environment and its use for the decolorization of azo dyes, Direct Blue 151 (DB 151) and Direct Red 31 (DR 31). The decolorization of azo dyes was studied at various concentrations (100–300 mg/L). The bacterial consortium, when subjected to an application of 200 mg/L of the dyes, decolorized DB 151 and DR 31 by 97.57% and 95.25% respectively, within 5 days. The growth of the bacterial consortium was optimized with pH, temperature, and carbon and nitrogen sources; and decolorization of azo dyes was analyzed. In this study, the decolorization efficiency of mixed dyes was improved with yeast extract and sucrose, which were used as nitrogen and carbon sources, respectively. Such an alkaliphilic bacterial consortium can be used in the removal of azo dyes from contaminated saline environment.

Efficacy of fungal decolorization of a mixture of dyes belonging to different classes

Dyes are the most difficult constituents to remove by conventional biological wastewater treatment. Colored wastewater is mainly eliminated by physical and chemical procedures, which are very expensive and have drawbacks. Therefore, the advantage of using biological processes, such as the biotransformation of dyes, is that they may lead to complete mineralization or formation of less toxic products. To prove the possibility of using fungal processes for decolorization and other applications, the analysis of the toxicity of the processes' products is required. The decolorization of the mixture of two dyes from different classes - triphenylmethane brilliant green and azo Evans blue (GB - total concentration 0.08 g/L, proportion 1:1 w/w) - by Pleurotus ostreatus (BWPH and MB), Gloeophyllum odoratum (DCa), RWP17 (Polyporus picipes) and Fusarium oxysporum (G1) was studied. Zootoxicity (Daphnia magna) and phytotoxicity (Lemna minor) changes were estimated at the end of the experiment. The mixture of dyes was significantly removed by all the strains that were tested with 96 h of experimental time. However, differences among strains from the same species (P. ostreatus) were noted. Shaking improved the efficacy and rate of the dye removal. In static samples, the removal of the mixture reached more than 51.9% and in shaken samples, more than 79.2%. Tests using the dead biomass of the fungi only adsorbed up to 37% of the dye mixture (strain BWPH), which suggests that the process with the living biomass involves the biotransformation of the dyes. The best results were reached for the MB strain, which removed 90% of the tested mixture under shaking conditions. Regardless of the efficacy of the dye removal, toxicity decreased from class V to class III in tests with D. magna. Tests with L. minor control samples were classified as class IV, and samples with certain strains were non-toxic. The highest phytotoxicity decrease was noted in shaken samples where the elimination of dye mixture was the best.

Copper induction and differential expression of laccase in Aspergillus flavus

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.