Kinetic of Azo Dyes Decolourization by Enterobacteriaceae Species in the Intact Cell Assay System

The method “Intact cell assay” was adopted to assess the influencing factors on the process of decolourization ability of the enterobacteriaceaea specie isolates under predisposed environment. Taken into account several ingredient are added individually as well in combinations in the assay reaction. Values of dye decolourized in μg ml with Multi vitanmins solution (MVS) with glucose 42.53; MVS 36.00; Riboflavin (RF) 39.00; Yeast extract 36.00 and Bcomplex 23.00 from the initial dye of 46.00μg ml were observed , where as glucose, Ascorbic acid, Cysteine, Cetyl-trimethyl-ammonium bromide (CTAB), Sodium molybdate, Biotin, KNO3, NaNO2, folic acid and 1-amino-2naphtho-4-sulfonic acid (ANSA) does not shows influence on to the process, but, some of them showed inhibitory effect toward the decolourization. It was observed that the riboflavin addition at 19.95 n moles ml in the reaction mixture, rate of decolourization was suddenly change from 0.019 ∆A/minutes to 0.20 ∆A/minutes, which is extremely high by 10 fold fast and subsequently remains faster i.e 0.2 ∆A/minutes without further additional RF in the same assay mixture. Rate of decolourization with different concentrations of riboflavin i.e. 13.0 n moles ml to 59.9 n moles ml showed second order kinetic. This indicates that the minimum amount of RF is essential to trigger the process of decolourization by the intact cells under the assay condition. While five different azo dyes were subjected, showed diverse behavior on to the rate of decolourization. Results of entire study incite on role of riboflavin could to a certain extent act as a redox mediator in the reaction(s) process and electron mediator between intracellular pool to the dye available at periplasmic redox sink @ JASEM In recent years a large number of microorganisms have been isolated which are able to degrade compound which previously was considered to be non degradable. In nature there is a significant resource of biological agents, which can be nourished to be exploited and employed for biotechnological applications. Most microbial activities that can serve the basis of bioremediation do not function optimally under the process conditions. It is thus become necessary to isolate potent microbes and study their environmental requirements for their best expression and performance for degradation/ decolourization in the nature. Studies and explanation view of decolourization to degradation and ultimate mineralization varied due to diversity in the both. As allude in literatures, azo dye decolourization is a reductive process in which azo -N=Nbond (s) is converted to -HN-NHor 2 molecules of NH2 by the reductive enzymes present in specified bacterial strains. The products generated in process are two aromatic amines that do not absorb light in the visible spectrum and therefore azo dye reduction represents a decolourization process (Tan 2001) Several studies have published on the anaerobic decolourization of azo dyes by using intestinal anaerobic bacteria that subsequently found to be decolorized with various other anaerobic cultures (Walker and Ryan 1970, Allan and Raxon 1974, Chung et al. 1978, Brown and Laboureur 1983, Chung et al 1992, Carliell et al. 1994, Donlon et al. 1997, Razo-Flores et al. 1997, Beydilli et al. 1998). On the other hand azo dye reduction is a ubiquitous capacity of many microbes under anaerobic condition (Walker et al 1970, Rafii and Carliell et al. 1995, Donlon et al. 1997, Razo, Flores et al. 1997). An aerobic azo dye bioremediation also possible only by the bacterial consortium, but time required in days for decolorized 90 % out of 50 μg/ml concentration (Senan et al 2004), which not feasible at field level due to high volume of the effluent, Further in biodecolourization with anaerobic bacteria, according to Gingell and Walker (1971) and Haug et al. (1992) required electrons for the reductive cleavage of azo dyes which may derive from co-substrates. Nigam et al. (1996a) also reported the similar results thus Azo dye reduction by the microbes requires co-substrate as a source of electron equivalent. Various records on the types of co-substrates used to suit as electron donors were, glucose (Carliell et al. 1995), Nigam et al. (1996a), hydrolyzed starch (Willetts et al. 2000), topioca (Chinwekitvanich et al. 2000), yeast extract (Nigam et al. 1996a), a mixture of acetate, butyrate, propionate (Donlon et al. 1997, Van Der Zee et al. 2000) and the azo dye reduction product like 5aminosalicylic acid as well (Razo-Flores et al. 1997) are recorded. Anaerobic decolourization of azo dyes by bacteria, the reduction equivalents generated by the oxidation of auxiliary substrates, i.e. organic carbon complexes act as electron donor through NAD(P) reduce the azo bond via primarily assuming electron carriers (coenzymes) flavins nucleotides (FMN, FAD) or riboflavin as cofactors (Roxon et. al. 1967; Zimmermann et al. 1982, Rafii et at 1990, Chung and Stevens 1993). So incite that Kinetic of Azo Dyes Decolourization... SHAILESH B. GONDALIYA; SIDDHARTH J. PATHAK 46 reactions may be multi steps and complex to fulfill intracellular donor to xenobiotics (dyes) in the system The study of enzymes and enzyme-catalyzed reactions has contributed greatly to our understanding of microbial metabolism (Philips, 1999). It is always not possible to measure physiological or biochemical processes of bacteria by working with enzymatic reactions individually and to obtain molecular mechanisms. Philips (1999) again discusses the use of native intact cells to overcome the assays of individual enzymes due to the inability to make substances available to the enzyme in known and nonmilitant conditions. Several workers have recognized that causative agent(s) ought to be studied and also it is important, the native intact cell studies provide the confirmation of the existence of the causative agent(s) and also considerable mechanistic detail. This is also called in vivo method of quantitatively measurement of the process, which is not impossible, in vitro but necessitates expensive and cumbersome techniques. In this study decolourization rate was measured in different assay conditions created in the reaction mixture in vivo intact cell assay system to evaluate influencing parameter, subjective remarks that overall phenomenon are may operated by the reducing equivalents electron pool generated intracellular from anaerobic metabolism, then which are transfer from source to the azo dyes to reduce azo bonds via enzymatic system may at periplasmic place of the organism. Present study incite influences of supplementary substance and role of riboflavin on the kinetic of decolourization for establish a mechanism of the process for applications elaboration MATERIALS AND METHODS Dyes and chemicals: Four commercially available dyes, Reactive Magenta HB (C.I name Reactive Violate 13), Reactive Red PBX, Remazol Magenta HB, Sunzol Violet 5R were obtained from Saraf Chemical Ahemadabad, India and one laboratory dye Biebrich Scarlet was from BD chemical UK were selected for the studies purpose. stock solution of each dyes were prepared by dissolving 2.0g of each dye in 100 ml distilled water (20 mg ml), sterilized by autoclaving at 110 c for 30 minutes. Detailed structure of Reactive Violate 13 is Fig-A as a model structure available. All other chemical used are standard analytical grade produced from E Merck, BDH, S. D. fine chemical and Spectrochem, India