Microbial Decolorization Of Dyes And Its Mechanism

Dyes have been widely utilized in many industries, such as textile, plastics, paper, food,cosmetic and pharmaceuticals. However,10%-20%of dyes are discharged into theenvironment during the process of their production and application. The discharged dyescreate a bulk of colored wastewater and cause serious pollution problems. Effluent containingdyes is highly colored and therefore leads to the reduction of dissolved oxygen concentration.Moreover, these dyes are resistant to degrade and are toxic and potentially carcinogenic owingto their complex aromatic structure. Thus, treatment of effluent containing dyes is a difficultand significant issue, especially the decolorization of dyes. Recently, many researches focusedon the decolorization of dyes from the effluent through microbial approach which includedbacterial decolorization, and white-rot fungal decolorization, etc. Moreover, the white-rotfungal decolorization can be classified into two processes, in vivo and in vitro. In the processof in vitro decolorization, enzymes produced in fungal cultivation were extracted and thenwere utilized to decolorize dyes under suitable conditions. In the process of in vivodecolorization, dyes were firstly mixed with substrates and then were decolorized bycultivation of fungi in the substrates. During the cultivation, the fungal growth, enzymessecretion and dyes decolorization were synchronous.On the basis of careful investigations and detailed surveys of literature, five differentdyes (Congo red, Methyl orange, Aniline blue, Indigo carmine, Azure B) and three strains(Phanerochaete chrysosporium, Trametes sp. SYBC-L4, Bacillus sp. MZS10.) are employedin this thesis for research on dyes decolorization via in vitro decolorization, a novel processincorporating adsorption and in vivo decolorization, and bacterial decolorization, respectively.Moreover, the decolorization metabolites were identified by various analytical techniquesincluding ultraviolet-visible spectrophotometry, ultra high performance liquidchromatography-mass spectrometry, gas chromatography-mass spectrometry, Fouriertransform infrared spectrometry and scanning electron microscopy. And the mechanism ofdecolorization was proposed on the basis of the decolorization metabolites analyses. Theseresults are references for research on technique of dyes decolorization.1. In vitro decolorization. Bioconversion of lignocellulosic wastes to added-valueenzymes for dyes decolorization has economic and ecological benefits. Manganese peroxidase(MnP) was produced by Phanerochaete chrysosporium in solid cassava residue substratefermentation, and then crude MnP extracted from the fermentation was employed for in vitrodecolorization of Indigo carmine and Methyl orange, respectively.(1) Inducers of MnPproduction were screened and optimized by one-at-a-time experiment and Box-Behnkendesign. Maximum MnP activity of186.38nkat·gds-1after6days of fermentation wasachieved with the supplement of79.5mM·kg-1acetic acid,3.21mL·kg-1soybean oil and28.5g·kg-1alkaline lignin. Then, in vitro decolorization of Indigo carmine by the crude MnP wascarried out and attained by the decolorization ratio of90.18%after6h incubation.Furthermore, an oxidative mechanism of Indigo carmine decolorization with MnP wasproposed on the basis of isatin identification by analyses of high performance liquid chromatography and gas chromatography-mass spectrometry.(2) Mathematical model andtechnical condition of methyl orange decolorization with MnP were investigated by artificialneural network and genetic algorithms. The error, correlation coefficient determination, rootmean square error and absolute average deviation of the established model through artificialneural network were0.0009,0.9971,1.21and6.82, respectively. Moreover, the optimumcondition of decolorization through genetic algorithms was found to be MnP amount of0.6mL (0.7U·mL-1), Mn2+concentration of4mM and H2O2concentration of0.49mM, for themaximum decolorization of90.74%. Results suggest the coupling of artificial neural networkand genetic algorithms is an effective technique for the investigation of methyl orangedecolorization with manganese peroxidase.2. In vivo decolorization and adsorption using lignocellulosic biomass. In vitrodecolorization is a feasible approach for removal dyes from effluent. However, this approachis inconvenient and uneconomical. Therefore, In vivo decolorization and adsorption usinglignocellulosic biomass were further investigated.(1) To highlight the comparison of in vitrodecolorization and in vivo decolorization, the laccase-producing fungal, Trametes sp.SYBC-L4was employed to decolorize Congo red, Aniline blue and Indigo carmine,respectively. Effects of pH and mediator (1-hydroxybenzotriazole, HBT) concentration ondyes decolorization were evaluated. In vitro, Crude laccase (50U·L-1) derived from Trametessp. SYBC-L4decolorized67.91%Congo red (100mg/L),94.58%Aniline blue (100mg·L-1)and99.02%Indigo carmine (100mg·L-1) with2.5mM HBT at pH4.5in36h of incubation.In vivo, decolorization ratio of Congo red (1000mg/kg), Aniline blue (1000mg·kg-1) andIndigo carmine (1000mg·kg-1) was57.82%,92.53%and97.26%without the usage ofmediator at pH4.5, respectively. Results showed that Trametes sp. SYBC-L4had greatpotential to be used for dyes decolorization via in vivo and in vitro processes. Moreover, interms of pH range and mediator, in vivo decolorization with Trametes sp. SYBC-L4was moreadvantageous since laccase mediator was needless and the applicable range of pH was broader.(2) Dyes adsorption onto lignocellulosic biomass and strains are tolerant on dyes arenecessary for the application of in vivo decolorization. Therefore, a novel processincorporating adsorption using cassava residue and in vivo decolorization with Trametes sp.SYBC-L4was investigated. Analyses of Fourier transform infrared spectroscopy andscanning electron microscopy indicated that the carbonyl (C=O), hydroxyl (-OH) and amino(-NH) groups in cassava residue were the potential adsorption sites for interaction with Congored. Cassava residue could adsorb Congo red effectively, and the Langmuir andpseudo-second order models were found to fit well with the data of equilibrium adsorptionand kinetics adsorption, respectively. The maximum adsorptive capacity was59.2mg·g-1,calculated by the Langmuir model. Then, Congo red-loaded cassava residue was furtherdecolorized via an in vivo process by Trametes sp. SYBC-L4. Decolorization of81.6%wasachieved under the conditions of pH5.5, temperature30℃, and moisture content of60%after16days of cultivation.3. Bacterial decolorization. White-rot fungal decolorization involved in vitrodecolorization and in vivo decolorization is a feasible approach for dyes removal from effluent.However, this approach is low efficiency owing to the long term of fungal growth and metabolism. Therefore, isolation and application of bacteria for dyes decolorization werefurther investigated.(1) A newly isolated Bacillus sp. MZS10strain was employed todecolorize Azure B dye. Based on the optimization of cultivation medium using one-at-a-timeexperiment, the decolorization reached93.55%(40mg·L-1) after14h of cultivation in a5-Lstirred-tank fermenter. The decolorization metabolites were identified with ultra performanceliquid chromatography-mass spectroscopy. A mechanism for decolorization of Azure B wasproposed as follows: the C=N in Azure B was initially reduced to-NH, and then the-NHfurther combined with-OH derived from glucose to form a stable and colorless compoundthrough a dehydration reaction. Moreover, the phytotoxicity was evaluated for both Azure Band its related derivatives produced by Bacillus sp. MZS10decolorization, indicating that thedecolorization metabolites were less toxic than the original dye.(2) Decolorization of Indigocarmine dye with strain Bacillus sp. MZS10was further evaluated. The decolorizationreached87.31%(100mg·L-1) after15h of cultivation in a5-L stirred-tank fermenter. In theprocess of decolorization, the intermediate metabolites derived from Indigo carminedecolorization were analyzed by UV-visible spectroscopy, mass spectroscopy, and Fouriertransform infrared spectroscopy, respectively. Based on the intermediate metabolites analyses,a novel pathway for decolorization of Indigo carmine was suggested as follows: bonds ofC=C and C=O in Indigo carmine were reduced in the process of decolorization, and anindoline derivative, indoline sulfonic acid was produced by biotransformation of Indigocarmine.