利用农业废弃物固态发酵裂褶菌F17产锰过氧化物酶的基质优化(英文)

锰过氧化物酶(MnP)在环保领域有着广阔的应用前景。目前,利用廉价基质生产MnP,尤其是利用工农业废弃物生产MnP的研究受到了国内外学者的广泛关注。本实验利用响应面方法从几种不同的农业废弃物中筛选裂褶菌F17(Schizophyllum sp.F17)产MnP的固态发酵基质。结果表明,以0.52∶0.15∶0.33的比例组成的松木屑、稻草和黄豆粉的混合基质为发酵产MnP的最佳基质,发酵第6天MnP的活力最高,达到11.18 U/g。因此,利用农业废弃物固态发酵产锰过氧化物酶在减低酶的成本和环境污染物治理方面具有重要的意义。     

裂褶菌F17固态发酵产锰过氧化物酶及其发酵动力学模型的建立

白腐真菌分泌的锰过氧化物酶是木质素降解酶系统的主要组分,对木质素解聚,纸浆和染料的脱色均有重要作用.利用裂褶菌F17在自行设计的通气托盘式反应器中,以松木屑、稻草及黄豆粉为混合营养基质进行固态发酵生产锰过氧化物酶.在自制通气托盘式反应器中,裂褶菌F17能够产生锰过氧化物酶,发酵96 h时,最高酶活力达到13.51 U/...     

Rapid decolourization of azo dyes by a new isolated higher manganese peroxidase producer: Phanerochaete sp. HSD

In the present paper, a strain of higher MnP producer, Phanerochaete sp. HSD, was screened and the important medium components influencing MnP production were optimized using fractional factorial design and central composite experimental design; statistical analysis suggested diammonium tartrate and Mn²⁺ were the important factors and under the optimum conditions, MnP activity reached 2613 ± 22 U/l, accorded with the predicted value from response surface analysis. The feasibility of using this fungus to decolourize azo dyes was examined too. Results indicated that crude enzyme solution of it could decolourize three azo dyes efficiently and speedily: for 120 and 350 mg/l of Congo red, 95% decolourization rate was observed at the 5th and 8th hour; for 200, 350 and 600 mg/l methyl orange, 95% decolourization rate was obtained at the 5th, 6th and 9th hour; furthermore, the decolourization rates of 150 and 300 mg/l of Eriochrome black T were up to 97.1% and 91.4% at the 7th and 13th hour, respectively. In addition, MnP played a crucial role in the decolourization process.     

裂褶菌F17对刚果红的脱色及主要降解酶的研究

裂褶菌F17在限氮培养基中对偶氮染料刚果红表现出较高的脱色能力。最佳脱色条件为:温度28℃、初始pH值4.5以及菌体摇瓶培养3d加入染料,染料浓度以100mg/L为宜。诱导因子藜芦醇、吐温80、土豆汁和松木屑的加入明显提高了脱色率,而叠氮化钠和氰化钾的加入对脱色有显著的抑制作用。酶活检测表明,裂褶菌F17在刚果红脱色过程中主要产生MnP,LiP活力很小,未检测到Lac。此外,刚果红的脱色率与累积MnP酶活具有良好的线性关系,相关系数为0.973。推测裂褶菌F17对刚果红脱色的主要降解酶为MnP。     

裂褶菌F17锰过氧化物酶酶活力影响因素的响应面优化(英文)

锰过氧化物酶是真菌分泌的一种糖基化的含有血红素辅基的胞外蛋白,在染料降解和脱色过程中起着重要作用。本实验利用本实验室保存的的白腐真菌裂褶菌Schizophyllum sp.F17产锰过氧化物酶(MnP),研究MnP的酶学性质,并对酶活条件进行优化。实验通过超滤浓缩、DEAE-纤维素、DE52离子交换层析和Sephadex G-75凝胶过滤等步骤,分离纯化得到电泳纯的锰过氧化物酶。该酶蛋白含量为23μg/mL,分子量大小为49.2kDa,在0.1mmol/L H2O2中半衰期为5~6min。Mn2+、H2O2以及酶的用量可以影响MnP酶促反应的效率,在单因子分析法的基础上,通过全因子中心组合设计响应面分析表明:H2O2以及H2O2与酶用量之间的交互作用对酶促反应的作用是最显著的。在优化条件下,酶对偶氮染料金橙G、刚果红显示出较强的脱色能力。     

Oxidative degradation of azo dyes by manganese peroxidase under optimized conditions

The application of enzyme-based systems in waste treatment is unusual, given that many drawbacks are derived from their use, including low efficiency, high costs and easy deactivation of the enzyme. The goal of this study is the development of a degradation system based on the use of the ligninolytic enzyme manganese peroxidase (MnP) for the degradation of azo dyes. The experimental work also includes the optimization of the process, with the objective of determining the influence of specific physicochemical factors, such as organic acids, H(2)O(2) addition, Mn(2+) concentration, pH, temperature, enzyme activity and dye concentration. A nearly total decolorization was possible at very low reaction times (10 min) and at high dye concentration (up to 1500 mg L(-)(1)). A specific oxidation capacity as high as 10 mg dye degraded per unit of MnP consumed was attained for a decolorization higher than 90%. Among all, the main factor affecting process efficiency was the strategy of H(2)O(2) addition. The continuous addition at a controlled flow permitted the progressive participation of H(2)O(2) in the catalytic cycle through a suitable regeneration of the oxidized form of the enzyme, which enhanced both the extent and the rate of decolorization. It was also found that, in this particular case, the presence of a chelating organic acid (e.g., malonic) was not required for an effective operation. Probably, Mn(3+) was chelated by the dye itself. The simplicity and high efficiency of the process open an interesting possibility of using of MnP for solving other environmental problems.     

Microbial decolorization of azo dyes by Proteus mirabilis

A bacterium identified as Proteus mirabilis was isolated from acclimated sludge from a dyeing wastewater treatment plant. This strain rapidly decolorized a deep red azo dye solution (RED RBN). Features of the decolorizing process related to biodegradation and biosorption were also studied. Although P. mirabilis displayed good growth in shake culture, color removal was best in anoxic static cultures. For color removal, the optimal pH and temperature were 6.5–7.5 and 30–35°C, respectively. The organism exhibited a remarkable color removal capability, even at a high concentration of azo dye. More than 95% of azo dye was reduced within 20 h at a dye concentration of 1.0 g L⁻¹. Decolorization appears to proceed primarily by enzymatic reduction associated with a minor portion, 13–17%, of biosorption to inactivated microbial cells. Received 06 January 1999/ Accepted in revised form 22 April 1999     

Decolorization of textile reactive dyes by the crude laccase produced from solid-state fermentation of agro-byproducts

Reactive dyes are one of the major sources of waste-water pollution. Efficient degradation of these dyes with enzymes produced from agricultural waste has attracted tremendous recent interests in both the scientific community and the general public. In this study, we took advantage of solid-state fermentation of four agro-byproducts (rape stem, wheat bran, peanut shell and rice hull) for producing laccase from the fungus Trametes sp. AH28-2. Higher laccase activities were obtained in multiple-substrates media than in single substrate media. The maximum yield of laccase (2.10 × 10⁶ U/kg) was obtained in the medium containing 60% rape stem, 20% peanut shell and 20% wheat bran, without the supplement of any toxic inducers. Our results further demonstrated that the textile reactive dyes Levafix Blue CA and Cibacron Blue FN-R (1.0 g/l) were completely decolorized by the crude laccase (5.0 U/l) obtained within 15 h in the absence of any mediator. Therefore, the agro-byproducts could be re-utilized to produce laccase for the decolorization of textile reactive dyes. Q.Y. Sun and Y.Z. Hong contributed equally to the study.     

Production of laccase and manganese peroxidase by Pleurotus pulmonarius in solid-state cultures and application in dye decolorization

The production of ligninolytic enzymes (laccase and Mn-dependent peroxidase) by the white-rot fungus Pleurotus pulmonarius (FR.) Quélet was studied in solid-state cultures using agricultural and food wastes as substrate. The highest activities of laccase were found in wheat bran (2,860?±?250 U/L), pineapple peel (2,450?±?230 U/L), and orange bagasse (2,100?±?270 U/L) cultures, all of them at an initial moisture level of 85 %. The highest activities of Mn peroxidase were obtained in pineapple peel cultures (2,200?±?205 U/L) at an initial moisture level of 75 %. In general, the condition of high initial moisture level (80–90 %) was the best condition for laccase activity, while the best condition for Mn peroxidase activity was cultivation at low initial moisture (50–70 %). Cultures containing high Mn peroxidase activities were more efficient in the decolorization of the industrial dyes remazol brilliant blue R (RBBR), Congo red, methylene blue, and ethyl violet than those containing high laccase activity. Also, crude enzymatic extracts with high Mn peroxidase activity were more efficient in the in vitro decolorization of methylene blue, ethyl violet, and Congo red. The dye RBBR was efficiently decolorized by both crude extracts, rich in Mn peroxidase activity or rich in laccase activity.     

In vivo and in vitro decolorization of synthetic dyes by laccase from solid state fermentation with Trametes sp. SYBC-L4

Synthetic decolorization of dyes through solid cassava residue substrate fermentation with Trametes sp. SYBC-L4 via in vivo and in vitro processes was investigated in this study. Effects of pH and mediator (1-hydroxybenzotriazole, HBT) concentration on dyes decolorization were evaluated. In vitro, decolorization ratios of dyes differed considerably in pH and increased with the increasing of HBT concentration. Crude laccase (50 U/L) derived from Trametes sp. SYBC-L4 decolorized 67.91 ± 1.25 % Congo red (100 mg/L), 94.58 ± 1.05 % aniline blue (100 mg/L) and 99.02 ± 0.54 % indigo carmine (100 mg/L) with 2.5 mM HBT at pH 4.5 in 36 h of incubation. In vivo, decolorization ratios of dyes were not enhanced by usage of the mediator. After 10 days of fermentation, decolorization ratio of Congo red (1,000 mg/kg), aniline blue (1,000 mg/kg) and indigo carmine (1,000 mg/kg) was 57.82 ± 0.84, 92.53 ± 1.12 and 97.26 ± 1.92 % without the usage of mediator at pH 4.5, respectively. Moreover, there was no obvious difference between the in vivo decolorization of aniline blue and indigo carmine in the pH range of 3.0–9.0. Results showed that Trametes sp. SYBC-L4 had great potential to be used for dyes decolorization via in vivo and in vitro processes. Moreover, in terms of pH range and mediator, in vivo decolorization with Trametes sp. SYBC-L4 was more advantageous since laccase mediator was needless and the applicable range of pH was broader.     

Decolorization and biosorption for Congo red by system rice hull- Schizophyllum sp. F17 under solid-state condition in a continuous flow packed-bed bioreactor

Synthetic dyes are important chemical pollutants from various industries. This work developed an efficient and relatively simple continuous decolorization system rice hull-Schizophyllum sp. F17 under solid-state condition in a packed-bed bioreactor, for decolorizing Congo red. In the decolorization system, two decolorization mechanisms exist, one is decolorization by Schizophyllum sp. F17, the other is biosorption by rice hull. The decolorization efficiency was greatly affected by dye concentration and hydraulic retention time (HRT), which were quantificationally analyzed and optimized through response surface methodology (RSM). A 2(2) full factorial central composite design (CCD) was performed, and three second order polynomial models were generated to describe the effects of dye concentration and HRT on total decolorization (R2=0.902), decolorization by Schizophyllum sp. F17 (R2=0.866) and biosorption by rice hull (R2=0.890). Response surface contour plots were constructed to show the individual and cumulative effects of dye concentration and HRT, and the optimum values. A maximum total decolorization 89.71% and maximum decolorization by Schizophyllum sp. F17 60.44% was achieved at dye concentration 142.63mg/L, HRT 41h, and dye concentration 110.7mg/L, HRT 29.4h, respectively. Meanwhile, the role of manganese peroxidase (MnP) in the decolorizaion process was investigated. This study proved the feasibility of continuous mode for decolorizing synthetic dyes by white-rot fungi in solid-state fermentation bioreactors.     

红酵母固态发酵类胡萝卜素产物稳定性的研究

探讨了红酵母固态发酵类胡萝卜素产物的干燥及储存方法;比较了真空干燥和鼓风干燥2种方法对固态发酵产物稳定性的影响;考察了光照、空气、储藏温度对固态发酵产物稳定性的影响。结果表明:在相同温度下,真空干燥(5.67±0.58)h比鼓风干燥所需时间短(9.33±0.58)h、类胡萝卜素损失小,效果也较好;光照和空气对其储藏过程中色素稳定性影响很大,30 d后类胡萝卜素损失了53%;培养物烘干后用透明塑料袋真空避光储藏效果显著,6个月后类胡萝卜素只损失19%,不抽真空损失了46%;温度对培养产物中色素稳定性影响不显著;红酵母菌株固态发酵培养产物应选择真空避光室温储藏。     

Decolorization of triphenylmethane and azo dyes by Citrobacter sp.

A Citrobacter sp., isolated from soil at an effluent treatment plant of a textile and dyeing industry, decolorized several recalcitrant dyes except Bromophenol Blue. More than 90% of Crystal Violet and Methyl Red at 100 M were reduced within 1 h. Gentian Violet, Malachite Green and Brilliant Green lost over 80% of their colors in the same condition, but the percentage decolorization of Basic Fuchsin and Congo Red were less than the others, 66 and 26%, respectively. Decolorization of Congo Red was mainly due to adsorption to cells. Color removal was optimal at pH 7–9 and 35–40 °C. Decolorization of dyes was also observed with extracellular culture filtrate, indicating the color removal by enzymatic biodegradation.     

Dye decolorization by manganese peroxidase in an enzymatic membrane bioreactor

In the present work an enzymatic membrane reactor (EMR) for the oxidation of azo dyes by manganese peroxidase (MnP) has been developed. The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane. The membrane allowed for most of the enzymatic activity to be recovered while both the parent dye and the degradation products could pass through. Different operational strategies (batch, fed-batch, and continuous) and parameters such as enzyme activity, H(2)O(2) feeding rate, hydraulic retention time (in continuous operation), and dye loading rate were studied. At best conditions, a continuous operation with a dye decolorization higher than 85% and minimal enzymatic deactivation was feasible for 18 days, attaining an efficiency of 42.5 mg Orange II oxidized/MnP unit consumed.     

Evidence on manganese peroxidase and tyrosinase expression during decolorization of textile industry dyes by Trichosporon akiyoshidainum

Textile dyes are engineered to be resistant to environmental conditions. During recent years the treatment of textile dye effluents has been the focus of significant research because of the potentially low cost of the process. Mechanisms of biological textile dye decolorization depend greatly on the chemical structure of the dye and the microorganisms used. While basidiomycetous filamentous fungi are well recognized for dye decolorization through ligninolytic enzymes, reports on textile dye decolorization mechanisms of basidiomycetous yeasts have been scarce. Decolorization of several textile dyes by Trichosporon akiyoshidainum occurs during the first 12 h of cultivation. This fast decolorization process could not be solely related to siderophore production or dye sorption to biomass; it was shown to be a co-metabolic process. T. akiyoshidainum could use glucose, sucrose, and maltose as alternative carbon sources, and urea as an alternative nitrogen source with similar decolorization rates. The activity of two enzymes, manganese peroxidase and tyrosinase, were induced by the presence of dyes in the culture media, pointing to their potential role during the decolorization process. Manganese peroxidase titers reached 666 U l⁻¹ to 10538 U l⁻¹, while tyrosinase titers ranged between 84 U l⁻¹ and 786 U l⁻¹, depending on the dye tested. The present work provides a useful background to propose new eco-friendly alternatives for wastewater treatment in textile dying industries.     

Accelerated decolorization of structurally different azo dyes by newly isolated bacterial strains

Wastewater effluents from the textile and other dye-stuff industries contain significant amounts of synthetic dyes that require treatment to prevent groundwater contamination. In research aimed at biotechnology for treatment of azo dyes, this study examined 288 strains of azo-dye degrading bacteria to identify efficient strains and determine incubation times required for decolorization. Initial enrichment cultures were carried out using a mixture of four structurally different dyes (Acid Red 88, Reactive Black 5, Direct Red 81, and Disperse Orange 3) as the sole source of C and N to isolate the bacteria from soil, activated sludge, and natural asphalt. Six strains were selected for further study based on their prolific growth and ability to rapidly decolorize the dyes individually or in mixtures. Treatment times required by the most efficient strain, AS96 (Shewanella putrefaciens) were as short as 4 h for complete decolorization of 100 mg l⁻¹ of AR-88 and DR-81 dyes under static conditions, and 6 and 8 h, respectively, for complete decolorization of RB-5 and DO-3. To our knowledge, these bacterial strains are the most efficient azo-dye degrading bacteria that have been described and may have practical application for biological treatment of dye-polluted wastewater streams.     

Microbial decolorization of azo dyes and dye industry effluent by Fomes lividus

The white rot fungus, Fomes lividus, was isolated from the logs of Shorea robusta in the Western Ghats region of Tamil Nadu, India. The fungus was tested for decolorization of azo dyes such as orange G (50 M) congo red (50 M) amido black 10B (25 M) and also for colour removal from dye industry effluents. The results revealed that the fungus could remove only 30.8% of orange G in the synthetic solution, whereas congo red and amido black 10B were removed by 74.0 and 98.9% respectively. A dye industry effluent was treated by the fungus in batch and continuous mode. In batch mode treatment, a maximum decolorization of 84.4% was achieved on day 4, and in continuous mode a maximum decolorization of 37.5% was obtained on day 5. The colour removal by the basidiomycete fungus might be due to adsorption of the dyes to the mycelial surface and metabolic breakdown. These results suggested that the batch mode treatment of Fomes lividus is one of the most efficient ways for colour removal in dye industry effluents.     

Decolorization of azo dyes by Shewanella sp. under saline conditions

Wastewaters from textile processing and dye-stuff manufacture industries contain substantial amounts of salts in addition to azo dye residues. To examine salinity effects on dye-degrading bacteria, a study was carried out with four azo dyes in the presence of varying concentrations of NaCl (0-100 g l(-1)) with a previously isolated bacterium, Shewanella putrefaciens strain AS96. Under static, low oxygen conditions, the bacterium decolorized 100 mg dye l(-1) at salt concentrations up to 60 g NaCl l(-1). There was an inverse relationship between the velocity of the decolorization reaction and salt concentration over the range between 5 and 60 g NaCl l(-1) and at dye concentrations between 100 and 500 mg l(-1). The addition of either glucose (C source) or NH(4)NO(3) (N source) to the medium strongly inhibited the decolorization process, while yeast extract (4 g l(-1)) and Ca(H(2)PO(4))(2).H(2)O (1 g l(-1)) both enhanced decolorization rates. High-performance liquid chromatography analysis demonstrated the presence of 1-amino-2-naphthol, sulfanilic acid and nitroaniline as the major metabolic products of the azo dyes, which could be further degraded by a shift to aerobic conditions. These findings show that Shewanella could be effective for the treatment of dye-containing industrial effluents containing high concentrations of salt.     

假单胞菌S—42对偶氮染料的脱色和降解代谢

Pseudomonas S-42 was capable of decolorizing azo dyes such as Diamira Brilliant Orange RR(DBO-RR), Direct Brown M (DBM), Eriochrome Brown R(EBR) and so on. The cell suspension, cell-free extract and purified enzyme of Pseud. S-42 could decolorize azo dyes under similar conditions: the optimum pH and temperature laid 7.0 and 37 degrees C respectively. The efficiencies of decolorizing of DBO-RR, DBM, EBR by intact cells stood more than 90%. When the cell concentration was 15 mg(wet)/ml and the reaction time was 5 hours, the decolorizing activity for above three azo dyes by intact cells were 1.75, 2.4, 0.95 micrograms dye/mg cell, respectively. Cell-free extract and purified enzyme could well express the decolorizing activity only under the anaerobic condition and added NADH. Purified enzyme belongs to azoreductase, its molecular weight is about 34,000-2000 daltons, and its Vmax and Km for DBO-RR are 13 mumol.mg protein-1.min-1 and 54 mumol/L. The results of the detection of the biodegrading products of DBO-RR by spectrophotometric and NaNO2 reactional methods showed that the biodegradation of azo dyes was initiated by the reduction cleavage of azo bonds. It was hypothesized that biodegrading metabolism pathway of DBO-RR by Pseudomonas S-42.     

氧气对混合菌群脱色降解偶氮染料效果的影响

【背景】偶氮染料及其中间产物具有一定的环境毒性,利用混合菌群降解偶氮染料是一种环境友好型方法,但降解过程中氧气的存在起到至关重要的作用,可以促进或抑制偶氮染料的微生物降解作用。【目的】探讨氧气对偶氮染料微生物脱色液的影响,分析氧气对混合菌群脱色降解偶氮染料效果的影响。【方法】利用混合菌群DDMY1在3种培养条件(好氧、厌氧、兼氧)下,对7种偶氮染料进行脱色降解,探讨偶氮染料脱色液对氧气的响应情况,利用紫外可见分光光度法(ultraviolet visible spectrophotometry,UV-vis)和傅里叶变换红外光谱法(Fourier transform infrared spectroscopy,FTIR)对脱色产物进行分析。【结果】在兼氧和厌氧条件下反应48 h后的染料脱色液,与氧气充分接触后,部分偶氮染料微生物脱色液发生较为明显的复色现象,如活性黑5、直接黑38;UV-vis分析结果表明,这种复色现象是由于脱色液与氧气接触之后产生新物质所致;FTIR分析结果表明,混合菌群对发生复色反应的偶氮染料仍然具有一定脱色降解效果,但是脱色尚不够完全。【结论】兼氧和厌氧条件下,氧气对部分偶氮染料微生物脱色液具有较为明显的影响,从而影响混合菌群对偶氮染料的整体脱色效果,这可为今后研究偶氮染料彻底生物降解提供理论基础。