Decolorization and biodegradation of reactive sulfonated azo dyes by a newly isolated Brevibacterium sp. strain VN-15.pdf
RESEARCH Open Access Decolorization and biodegradation of reactive sulfonated azo dyes by a newly isolated Brevibacterium sp. strain VN-15 Elisangela Franciscon, Matthew James Grossman*, Jonas Augusto Rizzato Paschoal, Felix Guillermo Reyes Reyes and Lucia Regina Durrant Abstract Azo dyes constitute the largest and most versatile class of synthetic dyes used in the textile, pharmaceutical, food and cosmetics industries and represent major components in wastewater from these industrial dying processes. Biological decolorization of azo dyes occurs efficiently under low oxygen to anaerobic conditions. However, this process results in the formation of toxic and carcinogenic amines that are resistant to further detoxification under low oxygen conditions. Moreover, the ability to detoxify these amines under aerobic conditions is not a wide spread metabolic activity. In this study we describe the use of Brevibacterium sp. strain VN-15, isolated from an activated sludge process of a textile company, for the sequential decolorization and detoxification of the azo dyes Reactive Yellow 107 (RY107), Reactive Black 5 (RB5), Reactive Red 198 (RR198) and Direct Blue 71 (DB71). Tyrosinase activity was observed during the biotreatment process suggesting the role of this enzyme in the decolorization and degradation process, but no-activity was observed for laccase and peroxidase. Toxicity, measured using Daphnia magna, was completely eliminated. Keywords: Azo dyes, Textile wastewater, Decolorization, Biodegradation, Detoxification, Brevibacterium, Tyrosinase, Carcinogenic aromatic amine Background Azo dyes account for about one-half of all dyes pro- duced and are the most commonly used synthetic dyes in the textile, food, paper making, color paper printing, leather and cosmetic industries (Chang and Lin 2001). The textile industry accounts for two-thirds of the total dyestuff market and during the dyeing process approxi- mately 10% of the dyes used are released into the waste- water (Easton 1995). The amount of dye lost in industrial applications depends on the type of dye used and varies from 2% loss for basic dyes to about 50% loss for certain reactive sulfonated dyes when used with cellulosic fabrics due to the relatively low levels of dye fiber fixation (Shore 1995; McMullan et al. 2001; Pearce et al. 2003; Hai et al. 2007). The high color content of dye process wastewater makes the presence of these dyes obvious and inhibits photosynthetic aquatic plants and algae by absorption of light, and as a result dye decolorization has been a pri- mary goal of dye wastewater treatment processes (Banat et al. 1996). However, beyond color, the presence of these dyes in aqueous ecosystems presents serious envir- onmental and health concerns as a result of the toxicity of the free dyes themselves and their transformation into toxic, mutagenic and carcinogenic amines, primarily as result of anaerobic microbial reductive cleavage of the azo bond (Chung and Cerniglia 1992; Weisburger 2002; Asad et al. 2007). In addition to toxicity, textile dye was- tewaters have high TOC, high salt content and extremes in pH, with reactive dye baths having high pH and acid dye baths have low pH (Golob et al. 2005). Dye wastewaters are treated physically and chemically by flocculation, coagulation, adsorption, membrane fil- tration, precipitation, irradiation, ozonization and Fenton’s oxidation (Lodha and Choudhari 2007; Wong et al. 2007). * Correspondence: mjgrossman@biosagebiotech.com Department of Food Science, Food Engineering School, University of Campinas, (UNICAMP) Rua Monteiro Lobato 80, Cidade Universitária Zeferino Vaz, Campinas, SP 13083-862, Brazil a SpringerOpen Journal © 2012 Franciscon et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Franciscon et al. SpringerPlus 2012, 1:37 http://www.springerplus.com/content/1/1/37 Although effective in dye removal, particularly for non- ionic dyes, these methods are expensive, add operational complexity to the process and can generate large amounts of dye contaminated sludge that must be disposed of, all of which add significantly to process costs (Lee 2000). Due to the inherent drawbacks of physical, chemical and photochemical approaches to dye removal, the use of biological methods for the treatment of textile waste- waters has received attention as a more cost effective al- ternative (Olukanni Olukanni et al. 2006; Dos Santos et al. 2007). Anaerobic microbial wastewater treatment can be very effective in removing color, primarily by the activity of azo reductases that cleave the azo bond yield- ing the corresponding amines, which are frequently toxic, mutagenic and carcinogenic and resist further degradation under anaerobic conditions (Gottlieb et al. 2003; O’Neill et al. 2000; Pinheiro et al. 2004; Van der Zee et al. 2001; Van der Zee and Villaverde 2005). In this process the azo dye acts as the terminal electron acceptor in anaerobic respiratory oxidation of carbon sources and other electron donors (Carliell et al. 1995; Ryan et al. 2010). In contrast, aerobic biological methods, such as acti- vated sludge processes, are largely ineffective in the treatment of textile wastewaters as a stand alone process, resulting in little or no color removal from azo dyes, with dye removal primarily occurring through ad- sorption to the sludge (Nigam et al. 1996; Brik et al. 2006; Singh et al. 2007). Bacteria capable of aerobic decolorization and mineralization of dyes, specially sul- fonated azo dyes, have proven difficult to isolate and the bacteria need to be specially adapted (McMullan et al. 2001; Pearce et al. 2003). However, both mixed and pure cultures of bacteria have been shown to be able to aerobically degrade and detoxify aromatic amines pro- duced by anaerobic decolorization of azo dyes (Khalid et al. 2009). The first azo reductases identified from anaerobic microorganisms were found to be oxygen sensitive, how- ever, recent work described an Enterococcus gallinarum isolate, obtained from the effluent of a textile industry wastewater treatment plant, capable of decolorizing azo dye DB38 by azoreductase enzyme action under aerobic conditions (Amit et al. 2008). In another example, an azoreductase gene from Bacillus latrosporus RRK1, pro- ducing an azoreductase able to decolorize several azo dyes under aerobic conditions, was cloned and expressed in E. coli, which was then able to decolorize Remazol Red, and a level of 0.8 mg L-1 of dissolved oxygen was required (Sandhya et al. 2008). A number of studies on the degradation of azo dyes by bacteria and fungi have indicated the involvement of extra- cellular oxidative enzymes such as, tyrosinase, lignin and manganese peroxidases and laccase (Fu and Viraraghavan 2001; Shanmugam et al. 2005; Zille et al. 2005; Ulson et al. Ulson de Souza et al. 2007; Kaushik and Malik 2009; Joshi et al. 2010; Kurade et al. 2011). Phenol oxidases, which can be divided into tyrosinases and laccases, are oxidore- ductases that can catalyze the oxidation of phenolic and other aromatic compounds without the use of cofactors. In cultures which have shown the activity of these enzymes during dye degradation it has been observed that the dye structures can be cleaved symmetrically and asym- metrically (Duran et al. 2002; Dawkar et al. 2008; Dhanve et al. 2008). Due to the recalcitrance of azo dyes to strictly aerobic conditions and the production and accumulation of en- vironmentally deleterious amines under anaerobic condi- tions a combination of anaerobic decolorization followed by aerobic degradation of the amines to non-toxic pro- ducts is considered most viable as a biological treatment scheme (Seshadri et al. 1994; Kudlich et al. 1996; Supaka et al. 2004). Moreover, the high salt content and extremes in pH associated with textile dye wastewaters have been shown to inhibit azo dye degradation by was- tewater microbial communities indicating that tolerance to these conditions is also an important parameter to consider for efficient biotransformation of azo dye waste streams (Manu and Chauhari 2003; Sen and Demirer 2003; Asad et al. 2007). However, to achieve this, suit- able strains of microorganisms must be identified and characterized to identify the best conditions for effective biological treatment of azo dyes. Previous studies have shown that strains of Brevibacter- ium are able to degrade aromatic compounds including polynuclear aromatics, dibenzofurans and nitroaromatic compounds (Strubel et al. 1991; Stefan et al. 1994; Jain et al. 2005; Ningthoujam 2005; Onraedt et al. 2005). How- ever, there is only one report of azo dye decolorization by Brevibacterium (Ng et al. 2010). In this case a Brevibacter- ium casei, isolated from sewage sludge from a dyeing factory, was shown to reduce Cr(VI) in the presence of azo dye Acid Orange 7 (AO7), possibly with the dye acting as the electron donor. Further degradation of the decolorized dye was not reported. The present study was focused on the degradation of four reactive sulfonated azo dyes in a successive static/ aerobic process using, exclusively, a Brevibacterium sp. isolated from a textile dye wastewater treatment plant. Dye decolorization was carried out under static conditions until no color was observed. The medium was then aerated by stirring to promote further degradation of the metabo- lites formed by cleavage of the azo bond into non-toxic metabolites. Tyrosinase, laccase and peroxidase enzyme activities as well as total organic carbon (TOC) were moni- tored during the biodegradation process. Biodegradation of the dyes was monitored for decolorization by UV–vis and degradation products were characterized using Franciscon et al. SpringerPlus 2012, 1:37 Page 2 of 10 http://www.springerplus.com/content/1/1/37 measuring the increase in absorbance at 280 nm. One unit of tyrosinase activity was equal to a Δ280nm of 0.001 per min at pH 7.0 at 25°C in a 1.0 mL reaction mix containing 1mM tyrosine solution. Laccase activity was assessed using 0.1 mL of 0.5 mM syringaldazine so- lution in ethanol (due to its limited solubility in aqueous solutions) as substrate, 0.2 mL of 0.05 M citrate phos- phate at pH 5, 0.6 mL of enzyme preparation and 0.1 mL of distilled water in a final volume of 1 mL (Szklarz et al. 1989). The oxidation of syringaldazine was moni- tored spectrophotometrically at 525 nm. Peroxidase ac- tivity was monitored using the same substrate used for laccase with 0.1 mL of 2mM hydrogen peroxide solution instead of distilled water. All enzyme assays were run in triplicate. High performance liquid chromatography mass spectrometry analysis (HPLC-MS) The biodegradation products of azo dye RR198 pro- duced by Brevibacterium sp. strain VN-15 in MMR were analyzed by HPLC-MS. Culture samples were centri- fuged (20,000 × g for 15 min) and filtered through a 0.25 μm pore size filter. Aliquots of 25 μL were injected into a HPLC-MS system consisting of an HPLC system (Waters, USA) coupled to a mass spectrometer with hybrid quadru- pole (Q) and time-of-flight (ToF) mass analyzers from Micromass (Waters, USA), with an electrospray source interface (LC-ESI-MS-MS). Instrument control and data processing were carried out by Masslynx 4.0 software. The mobile phase components used were degassed in an ultrasonic bath (Model USC 700, Unique Thorton, Brazil) before use in the LC system. A Varian reverse phase C18 HPLC column (150 × 2.1 mm, 5 μm particle size) was used to separate the biodeg- radation products. The column temperature was set at 25°C. The mobile phase was composed of water and methanol, using gradient elution. The gradient elution profile, using a flow rate of 0.2 ml min-1, consisted of (in percent by volume; duration (min)) water (100; 30), water:methanol (50:50; 3), ending with methanol (100; 2). The quadrupole analyzer was programmed to select ions with m/z in the range from 50 to 1200 u. The ionization conditions selected were: cone gas flow (150 L h-1), deso- lvation gas flow (350 L h-1), polarity (ESI+), capillary energy (2900 V), sample cone energy (30 V), extraction cone energy (2.0 V), desolvation temperature (350°C), source temperature (120°C), ionization energy (2.0 V), col- lision energy (4 V), and multi-channel plate detector energy (2700 V). Tentative identification of the metabolites from azo dye biodegradation was obtained by comparing the acquired mass spectra to spectra in the MS Database using the Cambridge SoftChem Office 2008 program. TOC measurement The change in organic carbon of the biotreated azo dye cultures was monitored by measuring the Total Organic Carbon (TOC) using a TOC analyzer (Shimadzu 5000A) as previously described in (Franciscon et al. 2009a, 2009b). Toxicity test Daphnia magna is a commonly used bioindicator test aquatic organism in acute and chronic toxicity studies of chemical compounds present in aquatic ecosystems (USEPA 1985). The acute toxicity tests using D. magna were carried as previously described (Franciscon et al. 2009a, 2009b). Competing interests The authors declare they have no competing interests in relation to this article. Authors’ contributions EF participated in the design of the study, performed the microbiology, 16S rRNA gene sequencing, toxicology and enzymology work and participated in the preparation of the manuscript. MJG evaluated the data and prepared the manuscript. JARP and FGRR performed the HPLC-MS analysis. 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Title: Decolorization and biodegradation of reactive sulfonated azo dyes by a newly isolated Brevibacterium sp. ​ strain VN-15
Authors: Elisangela Franciscon, Matthew James Grossman, Jonas Augusto Rizzato Paschoal, Felix Guillermo Reyes Reyes, Lucia Regina Durrant ​
Publisher: SpringerPlus
Date:  October 23, 2012 ​
Abstract: azo dyes constitute the largest and most versatile class of synthetic dyes used in the textile, pharmaceutical, food, and cosmetics industries and represent major components in wastewater from these industrial dyeing processes. ​ Biological decolorization of azo dyes occurs efficiently under low oxygen to anaerobic conditions. ​ However, this process results in the formation of toxic and carcinogenic amines that are resistant to further detoxification under low oxygen conditions. ​ Moreover, the ability to detoxify these amines under aerobic conditions is not a widespread metabolic activity. ​ In this study, we describe the use of Brevibacterium sp. ​ strain VN-15, isolated from an activated sludge process of a textile company, for the sequential decolorization and detoxification of the azo dyes Reactive Yellow 107 (RY107), Reactive Black 5 (RB5), Reactive Red 198 (RR198), and Direct Blue 71 (DB71). ​ Tyrosinase activity was observed during the biotreatment process, suggesting the role of this enzyme in the decolorization and degradation process, but no activity was observed for laccase and peroxidase. ​ Toxicity, measured using Daphnia magna, was completely eliminated. ​