Enzymatic reduction of complex redox dyes using NADH-dependent reductase from Bacillus subtilis coupled with cofactor regeneration

Conventional vat dyeing involves chemical reduction of dyes into their water-soluble leuco form generating considerable amounts of toxic chemicals in effluents. In the present study, a new β-nicotinamide adenine dinucleotide disodium salt (NADH)-dependent reductase isolated from Bacillus subtilis was used to reduce the redox dyes CI Acid Blue 74, CI Natural Orange 6, and CI Vat Blue 1 into their water-soluble leuco form. Enzymatic reduction was optimized in relation to pH and temperature conditions. The reductase was able to reduce Acid Blue 74 and Natural Orange 6 in the presence of the stoichiometrically consumed cofactor NADH; meanwhile, Vat Blue 1 required the presence of mediator 1,8-dihydroxyanthraquinone. Oxygen from air was used to reoxidize the dyes into their initial forms. The enzymatic reduction of the dyes was studied and the kinetic constants determined, and these were compared to the chemically-reduced leuco form. The enzyme responsible for the reduction showed homology to a NADH-dependent reductase from B. subtilis based on results from the MS/MS peptide mass mapping of the tryptically digested protein. Additionally, the reduction of Acid Blue 74 to its leuco form by reductase from B. subtilis was confirmed using NADH regenerated by the oxidation of formic acid with formate dehydrogenase from Candida boidinii in the same solution.     

Implementation of a biotechnological process for vat dyeing with woad

The traditional process for vat dyeing with woad (Isatis tinctoria L.) basically relies on microbial reduction of indigo to its soluble form, leucoindigo, through a complex fermentative process. In the 19th century, cultivation of woad went into decline and use of synthetic indigo dye and chemical reduction agents was established, with a consequent negative impact on the environment due to the release of polluting wastewaters by the synthetic dyeing industry. Recently, the ever-growing demand for environmentally friendly dyeing technologies has led to renewed interest in ecological textile traditions. In this context, this study aims at developing an environmentally friendly biotechnological process for vat dyeing with woad to replace use of polluting chemical reduction agents. Two simple broth media, containing yeast extract or corn steep liquor (CSL), were comparatively evaluated for their capacity to sustain the growth and reducing activity of the strain Clostridium isatidis DSM 15098(T). Subsequently, the dyeing capacity of the CSL medium added with 140?g?L(-1) of woad powder, providing 2.4?g?L(-1) of indigo dye, was evaluated after fermentation in laboratory bioreactors under anaerobic or microaerophilic conditions. In all fermentations, a sufficiently negative oxidation/reduction potential for reduction of indigo was reached as early as 24?h and maintained up to the end of the monitoring period. However, clearly faster indigo dye reduction was seen in the broth cultures fermented under strict anaerobiosis, thus suggesting the suitability of the N(2) flushing strategy for enhancement of bacterial-driven indigo reduction.     

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.

     

我国蓝草的传统植物学知识研究

蓝草在我国历史上曾是重要的经济作物,蓝草制备的植物靛蓝具有抗菌消炎和抗紫外线等保健作用,然而在合成靛蓝商业化的冲击下,蓝草制靛技艺几乎消亡。近年来,伴随着人们对环保和生物多样性的关注,植物靛蓝需求量不断增加,对蓝草的研究也越来越受关注。该文在文献研究的基础上,简要介绍了我国民间利用的蓝草种类及分布,对我国蓝草制靛工艺的发展进行了梳理,重点阐述了制靛工艺原理和工艺传承现状,并对存在的问题进行了讨论。结果表明:文献记载的我国蓝草共10种及变种,分属于6科6属,现利用的蓝草仅5种;我国蓝草制靛工艺从浸揉染色法发展为固态发酵制蓝法和液态发酵制靛法,只有液态发酵制靛法沿用至今;制靛工艺是将蓝草中的前体物质转化为靛蓝并伴有靛玉红等副产物生成的过程,影响靛蓝纯度的因素包括蓝草的材料选择、温度、浸泡时间、pH值、溶氧量等。目前我国蓝草的研究已取得了一些成果,但在蓝草种类的古籍考证、蓝草种质资源的挖掘以及蓝草传统制靛工艺的还原和再现等方面仍有待于进一步研究。     

White-rot fungus <Emphasis Type="Italic">Ganoderma</Emphasis> sp.En3 had a strong ability to decolorize and tolerate the anthraquinone,indigo and triphenylmethane dye with high concentrations

The ability of the white-rot fungus Ganoderma sp.En3 to decolorize different kinds of dyes widely applied in the textile and dyeing industry, including the anthraquinone dye Remazol Brilliant Blue R (RBBR), indigo dye indigo carmine and triphenylmethane dye methyl green, was evaluated in this study. Ganoderma sp.En3 had a strong capability of decolorizing high concentrations of RBBR, indigo carmine and methyl green. Obvious reduction of Chemical Oxygen Demand was observed after decolorization of different dyes. Ganoderma sp.En3 had a strong ability to tolerate RBBR, indigo carmine and methyl green with high concentrations. High concentrations of RBBR, indigo carmine and methyl green could also be efficiently decolorized by the crude enzyme of Ganoderma sp.En3. Different redox mediators such as syringaldehyde, acetosyringone and acetovanillone could enhance the decolorization capability for higher concentration of indigo carmine and methyl green. Different metal ions had little effect on the ability of the crude enzyme to decolorize indigo carmine and methyl green. Our study suggested that Ganoderma sp.En3 had a strong capability for decolorizing and tolerating high concentrations of different types of dyes such as RBBR, indigo carmine and methyl green.     

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.     

Bacterial Indigo Reduction

We have examined bacterial indigo reduction to provide a basis for the development of a sustainable alternative to the present chemical methods used to reduce indigo for denim dyeing. Indigo was reduced by Clostridium isatidis, but not by the related Clostridium aurantibutyricum, Clostridium celatum nor Clostridium papyrosolvens. However C. papyrosolvens could, like C. isatidis, reduce the soluble dye, indigo carmine. Of the bacteria examined only the supernatant from C. isatidis cultures decreased indigo particle size. An anthraquinone-rich madder root extract, the soluble anthraquinone-2,6-disulfonic acid and humic acid all stimulated the reduction of indigo by C. isatidis, without affecting the redox potential of the cultures. C. isatidis cultures generated redox potentials from ?476 to ?602 mV vs SCE, which were about 100 mV more negative than those of the other bacteria examined. The mechanism of bacterial indigo reduction remains unknown, but the unique features of the indigo-reducing C. isatidis indicate possible mechanisms.     

Bacterial Indigo Reduction

We have examined bacterial indigo reduction to provide a basis for the development of a sustainable alternative to the present chemical methods used to reduce indigo for denim dyeing. Indigo was reduced by Clostridium isatidis, but not by the related Clostridium aurantibutyricum, Clostridium celatum nor Clostridium papyrosolvens. However C. papyrosolvens could, like C. isatidis, reduce the soluble dye, indigo carmine. Of the bacteria examined only the supernatant from C. isatidis cultures decreased indigo particle size. An anthraquinone-rich madder root extract, the soluble anthraquinone-2,6-disulfonic acid and humic acid all stimulated the reduction of indigo by C. isatidis, without affecting the redox potential of the cultures. C. isatidis cultures generated redox potentials from -476 to -602 mV vs SCE, which were about 100 mV more negative than those of the other bacteria examined. The mechanism of bacterial indigo reduction remains unknown, but the unique features of the indigo-reducing C. isatidis indicate possible mechanisms.     

A review on applications of liposomes in textile processing

This review discusses the properties of liposomes and their role in the textile process, including textile preparation and dyeing. Liposomes have a surface activity effect due to a hydrophilic head group and hydrophobic hydrocarbon tail. Its preparations do not tend to foam, which advantageously distinguishes them from other textile auxiliaries. According to the carrier role of liposomes, they can be used in several textile processes such as textile finishing and dyeing with several types of dyes and fibers. Each application is discussed in this review paper. Several types of dyes are encapsulated by liposomes in the dyeing process and their presence indicates that they have retardant and leveling effects according to their gradual release of dyes. In addition, the presence of liposomes in the textile process can improve the mechanical properties of textile products, resulting in better wash fastness properties and leveling effect and handle properties. The best character of liposomes is a reduction in temperature of process resulting to save energy and they are environment degradable materials.     

Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.     

Reuse of reactive dyes for dyeing of jute fabric

The aim of the work was to find out suitable method of dyeing so that costly reactive dye can be reused without draining them. The bleached jute fabric was dyed with four different class of reactive dyes namely, cold brand, hot brand, vinyl sulphone and high exhaustion (HE) brand. It is found that the two-step two-bath method of reactive dyeing, where exhaustion and fixation step is separated, is most ideal for reuse of dye bath. Separate original samples produced K/S value same as that of original sample and the K/S value of separate reuse sample varied from 50% to 80% of the original sample depending on the class of dye. In case of same bath method, colour yield of original reuse samples varies from only 10% to maximum 30% of the original samples depending on the class of dyes. Reuse of reactive dyes following separate bath method is particularly suitable for higher depth of shade (4% and above). This process not only utilises costly reactive dyes to the maximum extent but it also produces low water pollution as the effluent contain minimum amount of dye. So the process is economic and eco-friendly as well.     

Polysaccharide conjugated laccase for the dye decolorization and reusability of effluent in textile industry

Nine different polysaccharides were screened for conjugation with laccase and evaluated for pH and thermal stability. All the polysaccharides decreased the thermal and pH stability of laccase at 50 °C and 60 °C, where conjugation with gum Arabic showing the most pronounced effect. Thermal instability of gum Arabic conjugated laccase was affirmed by differential scanning calorimeter while the structural changes in the conjugated laccase responsible for thermal instability was analysed by fluorescence spectrophotometer. The gum Arabic conjugated laccase showed an unusually high tolerance to sodium chloride, thermal instability and lower stability in alkaline conditions. Gum Arabic conjugated laccase was found to decolorize Remazol brilliant blue R in the textile effluent at a slower rate without any microbial growth which was unlike that observed in effluent treated with free laccase. Further, effluent treated with conjugated laccase enabled its reuse as liquor for the dyeing to get desired shade.     

Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres

New cellulases from the fungi Acrophialophora nainiana and Penicillium echinulatum were used in the finishing of knitted cotton fabrics (biopolishing) and compared with the well established enzymes from Trichoderma reesei. Both cellulases reduced the pilling tendency with a lower weight loss than T. reesei cellulases. Cellulases from P. echinulatum were also studied in stonewashing of denim fabrics to obtain the fashionable aged look in indigo dyed jeans ware and were found to remove more colour from denim fabrics and produce less indigo dye redeposition (back-staining) than commercial acid or neutral cellulases under the test conditions. Efficiency was found to be influenced by pH during textile processing and the substrate used for the production of cellulases. Cellulases produced by P. echinulatum grown on cellulose showed better stonewashing results (higher colour removal and less back-staining) than cellulases produced on sugar cane bagasse. The substrate used during enzyme production of P. echinulatum cellulases seems to have a significant influence on cellulose composition, which affects textile processing results.     

角质酶/角蛋白酶一浴法处理对羊毛性能的影响

采用T.fusca产角质酶以及Bacillus subtilis产角蛋白酶一浴法的方式处理羊毛,通过毡缩率、断裂强力、碱溶解度、上染速率、K/S值和接触角等指标考察了该处理对羊毛的改性效果,并运用XPS、氨基酸分析和SEM考察了其对羊毛结构与性质的影响。实验结果表明：经一浴法处理后,羊毛织物的毡缩率下降明显,达到机可洗要求;断裂强力下降较少,碱溶解度增加较少,上染速率提高,K/S值增加;XPS分析表明,经处理后羊毛纤维表面的元素含量变化较大;氨基酸分析表明,经处理后羊毛纤维中的胱氨酸质量分数有所降低;SEM显示,羊毛鳞片层大部分被剥除,综上可以说明角质酶/角蛋白酶的一浴法处理对羊毛具有明显的改性作用。     

麻栎壳斗染料在羊毛染色中的应用

本文研究了提取自麻栎壳斗的植物染料(麻栎染料)的耐酸、碱稳定性,染浴pH值及铝、铁离子等环保型媒染剂对其染毛织物效果的影响,并且探究了其染色动力学.研究表明,麻栎染料在强酸性染浴(pH=3)中对羊毛织物直接性好,染色后毛织物得棕色,也可采用铝离子、铁离子对直接染色后的毛织物进行后媒染,以得到不同色相的毛织物,尤其是铁后...     

Cultivation and use of isatis tinctoria L. (Brassicaceae) in Southern Italy

Isatis tinctoria L. (Brassicaceae), commonly known as wood, is a biennial species with erect stem, hastate leaves, and yellow flowers clustered in racemes. Fruits are pendulous siliques. This species, probably indigenous of southeastern Asia, was used for the extraction of a dyeing agent called “indigo.” Wood was introduced in ancient times in Italy and the first records of its cultivation date back to the Roman period. For many centuries, wood cultivation remained stable, but grew dramatically in the eighteenth century. In that century, the Societá Economiche established by Bourbons encouraged the cultivation of it in Southern Italy. Near Caserta, in Campania region (Italy), a factory for the extraction of dyeing agents was established and the dye was used in textile production in San Leucio (Caserta). The cultivation of I. tinctoria is abandoned today, although this species grows spontaneously as a weed in Italy. The authors discuss the history of wood and some ancient extractive and dyeing methods.     

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.     

Performance of combined process of anoxic baffled reactor-biological contact oxidation treating printing and dyeing wastewater

A study of the treatment of printing and dyeing wastewater was carried out using the combined process of anoxic baffled reactor-biological contact oxidation. The results showed the pH ascended continuously and the oxidation-reduction potential dropped gradually from compartment 1-6 in ABR. When hydraulic retention time was 12h, color removal efficiency was 92% and the color of effluent of ABR could satisfy the professional emission standard (grade-1) of textile and dyeing industry of China. The total COD removal efficiency of the combined process was 86.6% and the COD of effluent could satisfy the professional emission standard (grade-2) of textile and dyeing industry of China.     

Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.     

Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases

The potential of three oxidoreductases, a laccase preparation of Pleurotus sajor-caju PS-2001, horseradish peroxidase (HRP) and a microbial peroxidase (MP) was evaluated for the decolorization of disperse textile dyes (CI Disperse Red 343, CI Disperse Red 167 and CI Disperse Blue 148) used in polyester dyeing. Decolorization was studied in aqueous solutions varying in dye concentration, pH, temperature, enzyme concentration and the addition of mediators HBT and syringaldazine. The best conditions found for Disperse Red 343 with laccase, HRP and MP were: 15 mg L^?1 dye concentration, 50°C, pH 3.0 for laccase and pH 5.0 for peroxidases. Without mediator, the highest decolorizaton results (38.5% and 58.6%) were achieved with the highest tested concentrations of laccase (10 U mL^?1) and HRP (89.7 U mL^?1), respectively, but no significant difference in decolorization was found for the tested MP concentrations (29.9–89.7 U mL^‐1). HBT or syringaldazine increased decolorization with peroxidases significantly, but no effect was observed for the laccase. Decolorization of Disperse Red 167 (up to 15%) and Disperse Blue 148 (up to 25%) was much lower than of Disperse Red 343. With respect to enzyme concentration, the use of mediator and under the selected test conditions the laccase of P. sajor-caju PS-2001 turned out to be more efficient in disperse dye decolorization, than peroxidases HRP and MP.