黄孢原毛平革菌木质素过氧化物酶基因在甲醇毕赤酵母中的表达

将编码黄孢原毛平革菌木质素过氧化物酶(lip)的cDNA克隆到酵母整合型质粒pMETA上,电转化Ade缺陷型甲醇毕赤酵母(Pichiamethanolica)PMAD16,通过MD平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDSPAGE分析和木质素过氧化物酶活力测定等方法鉴定,表明带自身信号肽的黄孢原毛平革菌木质素过氧化物酶基因(lip)在甲醇毕赤酵母中得到表达。优化其发酵培养条件,以藜芦醇为底物进行酶活测定,其酶活可达932U/L。相应发酵指数为12.94U/h·L。比出发菌株提高了24.18%。     

Biodegradation of Grape Cluster Stems and Ligninolytic Enzyme Production by Phanerochaete chrysosporium during Semi‐Solid‐State Cultivation

The degradation undergone by grape cluster stems (woody component of vine bagasse), an agroindustrial waste, was investigated during the semi‐solid‐state cultivation of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725). For this, the content of lignin, cellulose and hemicellulose in grape cluster stems was determined before and after the enzymatic process. It was found that about 20% of Klason lignin, 48% of hemicellulose and 5% of cellulose were degraded during the process, being the ligninolytic enzymes (manganese‐dependent peroxidase and lignin peroxidase) produced by such cultures responsible for the degradation of grape cluster stems. In parallel, semi‐solid‐state cultures of P. chrysosporium grown on an inert support (cubes of nylon sponge), which is not susceptible to undergoing degradation during the enzymatic process, were used as reference cultures. In addition, the in vivo decolourisation of a model dye, the polymeric dye Poly R‐478, by both grape cluster stem and nylon cultures was studied in order to assess their degradative ability. A percentage of biological decolourisation higher than 90% after four days of dye addition was obtained using nylon sponge cultures, whereas grape cluster stem cultures led to a decolourisation of around 70% after eight days of dye incubation. The lower percentage of dye degradation achieved by the cultures grown on grape cluster stems was due to the enzymes produced, which were not only employed in the decolourisation of the dye but also in the degradation of the support, as indicated by the data mentioned above.     

Adsorption with biodegradation for decolorization of reactive black 5 by Funalia trogii 200800 on a fly ash-chitosan medium in a fluidized bed bioreactor-kinetic model and reactor performance

A non-steady-state mathematical model system for the kinetics of adsorption and biodegradation of reactive black 5 (RB5) by Funalia trogii (F. trogii) ATCC 200800 biofilm on fly ash-chitosan bead in the fluidized bed process was derived. The mechanisms in the model system included adsorption by fly ash-chitosan beads, biodegradation by F. trogii cells and mass transport diffusion. Batch kinetic tests were independently performed to determine surface diffusivity of RB5, adsorption parameters for RB5 and biokinetic parameters of F. trogii ATCC 200800. A column test was conducted using a continuous-flow fluidized bed reactor with a recycling pump to approximate a completely-mixed flow reactor for model verification. The experimental results indicated that F. trogii biofilm bioregenerated the fly ash-chitosan beads after attached F. trogii has grown significantly. The removal efficiency of RB5 was about 95 % when RB5 concentration in the effluent was approximately 0.34 mg/L at a steady-state condition. The concentration of suspended F. trogii cells reached up to about 1.74 mg/L while the thickness of attached F. trogii cells was estimated to be 80 μm at a steady-state condition by model prediction. The comparisons of experimental data and model prediction show that the model system for adsorption and biodegradation of RB5 can predict the experimental results well. The approaches of experiments and mathematical modeling in this study can be applied to design a full-scale fluidized bed process to treat reactive dye in textile wastewater.     

Metabolomic differential display analysis of the white-rot basidiomycete Phanerochaete chrysosporium grown under air and 100% oxygen

When the cultural atmosphere of the white-rot basidiomycete, Phanerochaete chrysosporium, was changed from air to 100% oxygen, the lyophilized mycelial weight increased and thickening of extracellular glucan layer was observed in 2-3 days. To better understand the oxygen-stress responsive mechanism of P. chrysosporium, the metabolomic differential display analysis was performed using metabolites isolated from fungal cells grown under either air or 100% O(2) atmosphere. In the GC-MS total ion chromatogram of methanol-extracts from fungal cells, at least 183 peaks were detected and 53 compounds were identified. Among them, veratryl alcohol (VA), threonate, and erythronate were identified as oxygen-stress responsive metabolites. The intracellular concentration of VA increased dramatically within 1 h after an oxygen purge, indicating that VA production is sensitive to the oxygen stress in P. chrysosporium.     

Biodegradation of triphenylmethane dye Malachite Green by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis>

Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Sphingomonas paucimobilis, was isolated from the soil sample collected from contaminated sites of textile industry located in KsarHellal, Tunisia, and it was able to decolorize Malachite Green (MG) dye (50 mg/l) within 4 h under shaking condition (pH 9 and temperature 25°C). The effect of inoculum size, dye concentration, temperature and initial pH of the solution were studied. The results obtained from the batch experiments revealed the ability of the tested bacteria to remove dye. UV–Vis spectroscopy and FTIR analysis of samples before and after decolorization confirmed the ability of the tested strain to decolorize MG. In addition, the phytotoxicity study revealed the degradation of MG into non-toxic product by S. paucimobilis.     

Comparison of the performance of one stage and two stage sequential anaerobic-aerobic biological processes for the treatment of reactive-azo-dye-containing synthetic wastewaters

In the present study the performance of anaerobic-aerobic one and two stage processes for the biological treatment of synthetic wastewaters containing Reactive Black 5 (RB5) were studied and compared with each other. In both processes the majority of colour removal by biodegradation occurred under anaerobic environment. The colour change under aerobic conditions was correlated with extent of anaerobic decolourisation in the preceding phase/stage of the process. Partial mineralisation of the anaerobic dye metabolites, roughly to the same extent, was achieved aerobically in both one stage and two stage processes. The majority of COD was removed in the anaerobic stage for two stage processes and aerobic stage in one stage processes. In one stage processes, the exposure of anaerobic sludge to alternating anaerobic-aerobic environment decreased anaerobic decolourisation efficiency and COD removal; when employing activated sludge, the same exposure enhanced anaerobic substrate utilisation whereas the effect on the anaerobic decolourisation efficiency depended on RB5 concentration. The comparative performance of one and two stage processes in terms of overall dye decolourisation depended on RB5 concentration. Both types of processes brought about similar overall COD removal. Increase in RB5 concentration, in the range studied, resulted in decrease in overall COD removal for both processes.     

Biodecolorization of textile azo dyes by isolated yeast from activated sludge: Issatchenkia orientalis JKS6

An ascomycetous yeast strain isolated from activated sludge could decolorize Reactive Black 5 azo dye at 200 mg l^?1 up to 90 % within 12–18 h under agitated condition. Yeast decolorization ability was investigated at different RB5 concentrations and, at higher dye concentration, 500 mg l^?1, the decolorization was found to be 98 % after 36 h incubation time. Extensive decolorization (95–99 %) was obtained in presence of five other azo dyes, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12, and Direct Black 22, by isolated yeast. HPLC analysis, UV–vis spectra and colorless biomass obtained after complete decolorization showed that the decolorization occured through a biodegradation mechanism. Decolorization was occurred during the exponential growth phase which is associated to primary metabolism. Laccase production by the yeast cells was not detected. The isolated yeast was characterized according to phenotypical and molecular procedures and was closely related (99 % identity) to Issatchenkia orientalis.     

Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol

After analysis of batch culture and identification of the ways for prolongation of citric acid active synthesis by yeast, repeat-batch (RB) cultivation was suggested. Yarrowia lipolytica strain RB cultivation was studied and optimal conditions for cultivation selected. It was shown that when applying RB cultivation, better results were obtained than for batch cultivation. The activity of the culture remained stable after cultivation for more than 700 h. Comparative analysis of enzyme activities confirmed the regularity of the effect described, as the activity of practically of all the enzymes participating in ethanol oxidation and citric acid biosynthesis remained stable over time during RB cultivation. Advantages of RB cultivation for the production of citric acid by yeast are discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 July 1999     

Ammonium Excretion by an l-Methionine-dl-Sulfoximine-Resistant Mutant of the Rice Field Cyanobacterium Anabaena siamensis

An ammonium-excreting mutant (SS1) of the rice field nitrogen-fixing cyanobacterium Anabaena siamensis was isolated after ethyl methanesulfonate mutagenesis by selection on 500 muM l-methionine-dl-sulfoximine. SS1 grew in the presence and absence of (l)-methionine-dl-sulfoximine at a rate comparable to that of the wild-type strain, with a doubling time of 5.6 h. The rate of ammonium release by SS1 depended on cell density; it peaked at the 12th hour of growth with 8.7 mumol mg of chlorophyll h (at a chlorophyll concentration of 5 mug ml) and slowed down to almost nil at the fourth day of growth. A similar pattern of release by immobilized SS1 was observed between 12 to 20 h after loading alginate beads in packed-bed reactors at the rate of 11.6 mumol mg of chlorophyll h. The rate was later reduced significantly due to the fast growth of SS1 on the substrate. Prolonged release of ammonium at the peak level was achieved only by maintaining SS1 under continuous cultivation at low chlorophyll levels (5 to 7 mug ml). Under these conditions, nitrogen fixation in the mutant was 30% higher than that in its parent and glutamine synthetase activity was less by 50%. Immunoblot analysis revealed that SS1 and its parent have similar quantities of glutamine synthetase protein under ammonium excretion conditions. In addition, a protein with a molecular weight of about 30,000 seems to have been lost, as seen by electrophoretic separation of total proteins from SS1.     

Effect of nitrogen source concentration on decolouration rates of laboratory dyes by immobilized cells of two bacterial species

The aim of this study was to assess the effect of sodium nitrate concentration on the decolouration of laboratory dyes (bromothymol blue, crystal violet, eosin blue, eosin yellow and methylene blue), by alginate immobilized cells of Pseudomonas aeruginosa and Bacillus subtilis. The sodium nitrate concentrations used in the study were 5, 10, 15 and 20 g/L. A control setup that contained no sodium nitrate was also studied. During incubation, aliquot samples were withdrawn from each flask every 24 for 144 h duration for the estimation of decolouration rate of the dyes, using standard procedures. The results revealed remarkable decolouration of the bromothymol blue and crystal violet in presence of the P. aeruginosa occurring at sodium nitrate concentrations of 10 and 15 g/L, respectively. In the case of media that was inoculated with the B. subtilis cells, although no remarkable decolouration of the bromothymol blue and crystal violet was observed throughout the period of incubation, highest decolouration were observed at sodium nitrate concentration of 5 and 10 g/L, respectively. For the eosin blue and methylene dyes, no remarkable decolouration were observed in presence of the test bacterial species at the respective sodium nitrate concentrations. Highest decolouration of the eosin yellow was however observed in media with sodium nitrate concentration of 5 g/L. The results of this study could be applied in scale up studies and continuous process, for implementation in biological decolouration of dye effluents.     

Passing of fluorescein derivatives into the hyphae of<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Fluorescein derivatives added into the growth medium were decolorized during submerged cultivation of Phanerochaete chrysosporium. The highest decrease of absorbance A450 was observed in the growth phase regardless of the presence of inducers Tween 80 or poly(ethylene glycol) (PEG). Fluorescein linked to PEG was prepared and, after addition to cultures, shown to stimulate the production of lignin peroxidase. Passing of fluorescing substances into hyphae (observed by confocal microscopy) showed that they were concentrated on some structures inside hyphae.     

Ganoderma lucidum U-281漆酶催化偶氮染料活性黑5脱色

漆酶在纺织染料脱色及印染废水处理领域有着广阔的应用前景。活性黑5是纺织印染中应用广泛的偶氮类活性染料,结构复杂,生物降解性低。以灵芝菌Ganoderma lucidum U-281所产漆酶对活性黑5进行氧化脱色,采用单因素逐一优化方法得到了U-281漆酶催化活性黑5脱色的工艺参数：染料初始浓度25mg/L、漆酶用量2.0U/mL、铜离子添加量40mmol/L、pH 6.0、40℃。在优化条件下,4h可使RB5脱色62.34%,24h可完成90%以上的脱色效果。     

Reactive blue 19 decolouration by laccase immobilized on silica beads

Laccase (31.5 U of activity/g or 4.39 μg of protein/m²) from Trametes versicolor was immobilized on controlled-porosity-carrier silica beads and evaluated for the decolouration of Reactive blue 19, an anthraquinone dye. Although there was an initial, rapid adsorption of the dye to the packed bed in a recirculating reactor, about 97.5% of Reactive blue 19 removal was due to enzymatic degradation. The free enzyme lost 52% of its activity in 48 h. However, the activity of the immobilized laccase was unchanged after 4 months of storage in phosphate buffer under ambient conditions followed by three successive decolourations over 120 h. Treating the laccase immobilized beads with ethanolamine reduced dye adsorption by 40%.     

Azo dyes decolorization under high alkalinity and salinity conditions by Halomonas sp. in batch and packed bed reactor

Biodecolorization and biodegradation of azo dyes are a challenge due to their recalcitrance and the characteristics of textile effluents. This study presents the use of Halomonas sp. in the decolorization of azo dyes Reactive Black 5 (RB5), Remazol Brilliant Violet 5R (RV5), and Reactive Orange 16 (RO16) under high alkalinity and salinity conditions. Firstly, the effect of air supply, pH, salinity and dye concentration was evaluated. Halomonas sp. was able to remove above 84% of all dyes in a wide range of pH (6–11) and salt concentrations (2–10%). The decolorization efficiency of RB5, RV5, and RO16 was found to be ≥ 90% after 24, 13 and 3 h, respectively, at 50 mg L⁻¹ of dyes. The process was monitored by HPLC-DAD, finding a reduction of dyes along the time. Further, Halomonas sp. was immobilized in volcanic rocks and used in a packed bed reactor for 72 days, achieving a removal rate of 3.48, 5.73, and 8.52 mg L⁻¹ h⁻¹, for RB5, RV5 and RO16, respectively, at 11.8 h. The study has confirmed the potential of Halomonas sp. to decolorize azo dyes under high salinity and alkalinity conditions and opened a scope for future research in the treatment of textile effluents.

     

Stainless steel sponge: a novel carrier for the immobilisation of the white-rot fungus Trametes hirsuta for decolourization of textile dyes

Alginate beads, polyurethane foam, nylon sponge and stainless steel sponge were tested as carrier materials for the white-rot fungus Trametes hirsuta for laccase production under submerged fermentation conditions. Stainless steel sponge was the best carrier material leading to the highest laccase activities of up to 800 U/l after 8 days of cultivation. These values are higher than those reported to date operating with inert supports and without inducer addition. In a 1-l bioreactor containing T. hirsuta immobilised on stainless steel sponge laccase activities of about 2200 U/l were obtained when the culture medium was supplemented with 1 mM copper sulphate. There were no operational problems with this system during culturing time. The textile dye Indigo Carmine was almost totally degraded in 3 days by T. hirsuta grown in this bioreactor, while Lanaset Marine was degraded in two successive batches, reaching in the first batch a decolourization percentage of about 82% in 15 h and in the second one by 71% in 28 h. Results obtained after inhibition of growth of T. hirsuta by antibiotics indicated that dye decolourization could not exclusively be attributed to laccase activity.     

Differential Expression of Antioxidant Enzymes During Degradation of Azo Dye Reactive black 8 in Hairy roots of Physalis minima L.

The enzymes involved in the protection of plant metabolism in presence of azo dye was characterized by studying activities of the role of antioxidant enzymes in the hairy roots (HRs) of Physalis minima L. during degradation of an azo dye, Reactive Black 8 (RB8). When the HRs were exposed to RB8 (30 mg L^?1), a nine fold increase in SOD activity was observed after 24 h, while 22 and 50 fold increase in activity was observed for POX and APX respectively after 72 h, whereas there was no significant change in activity of CAT. The activation of different antioxidant enzymes at different time intervals under dye stress suggests the synchronized functioning of antioxidant machinery to protect the HRs from oxidative damage. FTIR analysis confirmed the degradation of dye and the non-toxic nature of metabolites formed after dye degradation was confirmed by phytotoxicity study.     

Decolorization of azo dyes and simulated dye bath wastewater using acclimatized microbial consortium--biostimulation and halo tolerance

Anaerobic acclimatization of activated sludge from a textile effluent treatment plant to high concentration of RB5 could effectively decolorize RB5 dye solution. The strains viz. Pseudomonas aeruginosa and Bacillus circulans and other unidentified laboratory isolates (NAD1 and NAD6) were predominantly present in the microbial consortium. The conditions for efficient decolorization, biostimulation to increase effectiveness of microbial consortium, its tolerance to high salt concentration and non-specific ability towards decolorization of eight azo dyes, are reported. The optimum inoculums concentration for maximum decolorization were found to be 1-5 ml of 1800+/-50 mg l(-1) MLSS and 37 degrees C, respectively. The decolorization efficiency was 70-90% during 48 h. The biomass showed efficient decolorization even in the presence of 10% NaCl, as tested with RB5. In the presence of flavin adenine dinucleotide (FAD) more than 99% decolorization occurred in 8h. The decolorization of RB5 was traced to extracellular enzymes. The effectiveness of acclimatized biomass under optimized conditions towards decolorization of two types of synthetic dye bath wastewaters that were prepared using chosen azo dyes is reported.     

黄孢原毛平革菌对RBBR脱色降解及其降解机制

为研究白腐真菌对蒽醌染料的生物降解机制,以白腐真菌黄孢原毛平革菌为脱色降解菌株,分析了蒽醌染料活性艳蓝KN-R(RBBR)的浓度、金属离子及脱色参数对染料脱色的影响;采用紫外-可见光谱、红外光谱、气相色谱-质谱(GC-MS)分析和植物种子毒性实验进行降解产物分析,以揭示RBBR可能的降解路径及其产物的毒性结果表明:在p...     

Decolorization of textile dye by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from industrial effluents

The aim of the present work was to observe microbial decolorization and biodegradation of the Direct Violet 51 azo dye by Candida albicans isolated from industrial effluents and study the metabolites formed after degradation. C. albicans was used in the removal of the dye in order to further biosorption and biodegradation at different pH values in aqueous solutions. A comparative study of biodegradation analysis was carried out using UV–vis and FTIR spectroscopy, which revealed significant changes in peak positions when compared to the dye spectrum. Theses changes in dye structure appeared after 72 h at pH 2.50; after 240 h at pH 4.50; and after 280 h at pH 6.50, indicating the different by-products formed during the biodegradation process. Hence, the yeast C. albicans was able to remove the color substance, demonstrating a potential enzymatic capacity to modify the chemical structure of pigments found in industrial effluents.     

Starch-based plastic polymer degradation by the white rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse pith: enzyme production

In this study, starch metabolites and enzymes were determined during starch-based plastic polymer biodegradation by the white rot fungus Phanerochaete chrysosporium, grown in sugarcane bagasse pith in tubular reactors. Various metabolites, amylase, ligninase and cellulase production were measured during P. chrysosporium growth on sugarcane bagasse pith with added glucose and starch polymer. On-line respirometric analyses followed during 32 days confirmed the P. chrysosporium capability of growing on sugarcane bagasse pith with starch polymer degradation. Enzyme activity during secondary metabolism increased, and a 70% and 74% starch degradation was reached with and without glucose addition, generating low molecular weight metabolites (e.g.) dextrin, maltotriose, maltose and glucose that were detected by high performance liquid chromatography.