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Microbial Degradation and Decolorization of Reactive Dyes by Bacillus Spp. ETL-1979

Maulin P Shah*, Kavita A Patel, Sunu S Nair, A M Darji

Industrial Waste Water Research Laboratory, Applied & Environmental Microbiology Lab, Enviro Technology Limited (CETP), Gujarat, India

Abstract: A bacterial strain ETL-1979, identified as Bacillus spp. was isolated from activated sludge of textile wastewater treatment plant in Ankleshwar, Gujarat, India. Azo dyes constitute the largest and most versatile class of synthetic dyes. 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 Bacillus spp. ETL-1979, isolated from an activated sludge process of a textile company, for the sequential decolorization and detoxification of the azo dyes Reactive Yellow 107, Reactive Black 5, Reactive Red 198 and Direct Blue 71. 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: Bacillus, Reactive Yellow, Reactive Black, Reactive Red, decolorization

Received August 28, 2013; Revised November 01, 2013; Accepted January 22, 2014

Citation: Maulin P Shah, Kavita A Patel, Sunu S Nair, A M Darji, Microbial Degradation and Decolorization of Reactive Dyes by Bacillus Spp. ETL-1979. American Journal of Microbiological Research, 2 (1). 16-23. January 2014. doi:10.12691/ajmr-2-1-3

1. Introduction

Textile industry is one of the oldest industries in India with over 1000 industries. Taking into account the volume and composition of effluent, the textile wastewater is rated as the most polluting among all in the industrial sectors. In general, the wastewater from a typical textile industry is characterized by high values of BOD, COD, color and pH. It is a complex and highly variable mixture of many polluting substances ranging from inorganic compounds and elements to polymers and organic products. [43]. Reactive dyes, including many structurally different dyes, are extensively used in the textile industry because of their wide variety of color shades, high wet fastness profiles, ease of application, brilliant colors, and minimal energy consumption. The three most common groups are azo, anthroquinone and phthalocyanine [44,45]. The textile finishing generates a large amount of waste water containing dyes and represents one of the largest causes of water pollution [32]. Azo dyes have been used increasingly in industries because of their ease and cost effectiveness in synthesis compared to natural dyes. However, most azo dyes are toxic, carcinogenic and mutagenic [33,34]. Environmental pollution has been recognized as one of the major hazard of the modern world. Due to rapid industrialization, lot of chemicals including dyes manufactured and used in day to day life [36]. Dyes usually have a synthetic origin and complex aromatic molecular structures which make them more stable and more difficult to biodegrade [1]. Approximately 10,000 different dyes and pigments are used industrially and over 0.7 million tons of synthetic dyes are produced annually, worldwide. The three most common groups of dyes are azo, anthraquinone and phthalocyanine [5], most of which are toxic and carcinogenic. Disposal of these dyes into the environment causes serious damage, since they may significantly affect the photosynthetic activity of hydrophytes by reducing light penetration and also toxic to aquatic organisms due to their break down products [2,20]. One of the most pressing environmental problems related to dye effluents is the improper disposal of waste water from dyeing industry [23]. Traditional methods for the cleanup of azo dyes in the textile waste water usually involve the removal of unwanted materials through sedimentation, filtration and subsequent chemical treatments such as flocculation, neutralization and electro-dialysis before disposal. These processes may not guarantee the treatment of toxic dye in the effluent. Moreover, considering the volume of wastes released during the industrial production process these are often laborious and expensive [19]. Over the past decades, biological decolorization has been investigated as a method to transform, degrade or mineralize azo dyes [6]. A number of microorganisms have been found to be able to decolorize textile dyes including bacteria, fungi, and yeasts [37]. They have developed enzyme systems for the decolorization and mineralization of azo dyes under certain environmental conditions [38]. Several methods are used in the treatment of textile effluents to achieve decolorization but they have many disadvantages and limitations [15]. Biological processes provide alternative technologies that are more cost effective and environmentally friendly [3]. Many microorganisms belonging to the different taxonomic groups of bacteria have been reported for their ability to decolorize azo dyes [14]. Rapid industrialization has necessitated the manufacture and use of different chemicals in day to day life [44]. Reactive dyes, including many structurally different dyes, are extensively used in the textile industry because of their wide variety of color shades, high wet fastness profiles, ease of application, brilliant colors, and minimal energy consumption. The three most common groups are azo, anthroquinone and phthalocyanine [43]. The present study mainly focused on the degradation of four reactive azo dyes in a successive static/aerobic process using, exclusively, a Bacillus spp ETL-1979 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 metabolites 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 monitored during the biodegradation process. Biodegradation of the dyes was monitored for decolorization by UV–vis and degradation products were characterized using HPLC-MS. The effect of biotreatment on toxicity was evaluated using Daphnia magna the test organism.

2. Materials & Methods

2.1. Chemicals and Culture Medium

The azo dyes Reactive Yellow 107, Reactive Red 198, Reactive Black 5, and Direct Blue 71 were kindly provided as a gift. All other reagents were analytical grade and purchased from Sigma and used without further purification. Mineral salts medium (MM), pH 7 was prepared as previously described (Franciscon et al. 2009a, 2009b). To evaluate the effect of different carbon sources on dye decolorization MM was supplemented with the indicated amounts of glucose, sodium pyruvate and/or yeast extract, and 100 mg/L of dye. The highest degree and rate of decolorization occurred using MM supplemented with 3g/l glucose and 1g/l pyruvate, and this medium was used for all subsequent biodegradation experiments and was designated MM rich mineral medium (MMR). Strain isolation and characterization Bacillus spp. ETL-1979 was isolated from activated sludge obtained from the Textile Company, Ankleshwar, India. Serial dilutions (10⁻¹ to 10⁻⁶) of the sludge were inoculated by the spread plate technique onto Nutrient Agar plates containing azo dyes (100 mg L⁻¹) and incubated under low oxygen conditions. Bacillus spp. ETL-1979 was chosen for further evaluation based the production of a large decolorization zone in the azo dye containing plates. The strain was maintained on slants of Nutrient Agar. The identification of Bacillus spp. ETL-1979 was based on standard morphological and biochemical methods as described by Benson (1985), and 16S rRNA gene sequence analysis. Genomic DNA was obtained according to Ausubel et al. (1989). The 16S rRNA gene was amplified by PCR using the bacteria specific primers, 27f and 1401r [29].

2.2. Identification

Screening of the strains for dye decolorization was performed by enrichment culture technique using NM9 and DNM9 media as described by Hong et al. After purification by successive single colony isolation on a DNM9 agar plate, strain ETL-1982 was identified by carbon source utilization patterns using Biolog GN2 microplate (Biolog, USA) and the analysis of 16S rDNA sequences. For the 16S rDNA sequence analysis, bacterial genomic DNA was extracted and purified using a Wizard Genomic DNA Prep. Kit (Promega Corp., Madison). Two primers annealing to the 5` and 3` end of the 16S rRNA gene were 5`-GAGTTTGATCCTGGCTCAG-3` (positions 9 to 27 (Escherichia coli 16S rDNA numbering)) and 5`-AGAAAGGAGG TGATCCAGCC-3` (positions 1542 to 1525 (E. coli 16S rDNA numbering)), respectively. Polymerase Chain Reaction (PCR) was performed as follows: pre-denaturation at 95°C for 5 min, 30 cycles at 95°C for 40 s, 55°C for 40 s and 72°C for 2 min. The PCR product was subcloned into pGEM-T easy vector (Promega, Madison, USA) and its nucleotide sequence was determined by Banglore Genei Ltd. (Banglore, India). The partial rDNA sequences were analyzed using a BLAST search algorithm to estimate the degree of similarity to other rDNA sequences obtained from the NCBI/GenBank. Phylogenetic trees were constructed by the ClustalX program [48]. Physiological characteristics were determined according to the procedures outlined in Bergey`s Manual of Determinative Bacteriology [46].

2.3. Dye Decolorization

Decolorization experiments for each dye were performed by growing Bacillus spp. ETL-1979 in 500 mL Erlenmeyer flasks containing 350 mL of sterile MM supplemented with 100 mg L⁻¹ of dyes plus the indicated carbon source. Cultures were first incubated under static conditions to provide conditions of oxygen limitation, at 30°C, for the indicated times, until decolorization was complete. All cultures were then aerated by stirring for 140 h without any further supplementations to the medium. Dye decolorization was evaluated by measuring the change in absorbance between 200 and 800 nm with a Shimadzu 1800 UV-visible spectrophotometer in the static and aerobic stages. Preparation of enzyme extract Bacillus spp. ETL-1979 was grown in 500 mL Erlenmeyer flasks containing 350 mL of sterile MMR, containing 100 mg L⁻¹ dyes and inoculated with a 3% by volume culture of Bacillus spp. ETL-1979 previously grown for 24 h in MMR without dyes. Samples were harvested and the cells were sonicated with 5 seconds pulses separated by 2 minutes intervals repeated 6 times. The sonicated cells were then centrifuged at 18,000 x g for 20 min, to remove cell debris, and the supernatant was used as the source of enzyme. Determination of enzyme activity Tyrosinase enzyme activity was assayed as described by Kamahldin and Eng (2003). Tyrosinase activity was determined using 0.1 mL of 1mM tyrosine solution (1 M phosphate buffer at pH 7) as substrate, 0.6 mL of the enzyme preparation and 0.3 mL of distilled water in a final volume of 1 mL. The oxidation of tyrosine to dihydroxyphenylalanine was monitored spectrophotometrically by 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 solution in ethanol (due to its limited solubility in aqueous solutions) as substrate, 0.2 mL of 0.05 M citrate phosphate at pH 5, 0.6 mL of enzyme preparation and 0.1 mL of distilled water in a final volume of 1 mL [47]. The oxidation of syringaldazine was monitored spectrophotometrically at 525 nm. Peroxidase activity 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 produced by Bacillus spp. ETL-1979 in MMR were analyzed by HPLC-MS. Culture samples were centrifuged (18,000 × g for 20 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 quadrupole (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 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 biodegradation 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), desolvation 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), collision 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 [16,17].

2.4. 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 [16,17].

3. Results

3.1. Characterization & Isolation

Isolate ETL-1979 was initially isolated from activated sludge from a textile plant wastewater treatment facility based on its ability to produce large zones of decolorization around colonies grown on nutrient agar containing 100 L⁻¹ of various azo dyes. The 16S rRNA gene sequence of the ETL-1979 strain was determined and compared with 16S rRNA gene sequences in the Genbank nucleotide databases. The ETL-1979 strain was phylogenetically positioned in the genus Bacillus (Figure 3). The nucleotide alignment of the partial 16S rRNA gene sequence of this strain had identity values of 98 to 99%, with different Bacillus strains.

3.2. Decolorization Assays

The ability of Bacillus spp. ETL-1979 to decolorize four azo dyes Reactive Yellow 107, Reactive Red 198 , Reactive Black 5 , and Direct Blue 71 was evaluated in a static/ agitated sequential batch process as described in methods. Bacillus spp. ETL-1979 could decolorize the dyes efficiently only when the medium was supplemented with carbon sources (data not shown). The medium that promoted the most rapid and efficient decolorization (>95%) under static conditions for all azo dyes was MM containing 3g L⁻¹glucose and 1g L⁻¹ sodium pyruvate (MMR) and this medium was used for all subsequent experiments for determining enzyme activities, degradation products and toxicity reduction. When sodium pyruvate was substituted by yeast extract (1g L⁻¹) a similar rate of decolorization was observed. When glucose alone was added at a concentration of 1 or 3 g L⁻¹ the rate of dye decolorization was reduced resulting in only 30 and 50% color removal after 168 h, respectively (data not shown). In MMR the monoazo dyes (RY107 and RR198) were decolorized to their maximum extent after 96 and 120 h respectively, while the more complex diazo RB5 and triazo DB71 dyes required 144 to 168 h, respectively, for maximum decolorization (Table 1). Subsequent aeration via stirring for a period of 168 h resulted in greater than 99% decolorization of all dyes being. (Table 1).

3.3. Enzyme Activities Determination

The activities of the oxidoreductase enzymes (peroxidase, laccase and tyrosinase) were measured during the decolorization process in MMR. Static conditions were maintained until complete decolorization occurred as indicated in Table 1. This was followed by an aeration phase for and additional 168h. In the static and stirring conditions laccase and peroxidase activities were very low and likely did not play much of a role in dye decolorization/degradation. However, tyrosinase activity was observed both under static and aerated conditions (Figure 1). Under static conditions the activity increased until decolorization was complete and then decreased over of time for all dyes. Following initiation of aerated conditions the tyrosinase activity increased again for all dye cultures.

Table 1. Azo dye decolorization by Bacillus spp. ETL-1979 under static and aerobic conditions in the presence of 100 mg L⁻¹ of aze dyes and 3 g L⁻¹ glucose and 1g L⁻¹ pyruvate

Figure 1. Time course of tyrosinase activity in Bacillus spp. ETL-1979 cultures during biodegradation of azo dyes

Figure 2. UV-vis spectra of the azo dyes – Before (straight line) and after microaerophilic (dashed line) and aerobic (dotted line) treatments – A: RY 107; B: RR198; C: RB5; D: DB71

3.4. UV–Vis Characterization

As shown in Figure 2(a-d), virtually complete decolorization of all four azo dyes occurred under static conditions as shown by the disappearance of, the absorbance peaks in the visible region (390 to 750 nm). In the UV spectra of samples taken during static conditions, the absorbance observed in the 280-350 nm absorption profiles of all of the dyes diminished and were replaced by a new peak at 260 nm, which then either increased or diminished, depending on the dye, under aerated conditions.

3.5. HPLC-MS Analyses of RR198 Dye Biodegradation Products

The biodegradation products of the RR198 dye produced under static and aerated conditions were analyzed by HPLC-MS for tentative identification of the unknown compounds. Out of forty possible compounds identified as metabolites from RR198 dye biodegradation only three had good matches to structures clearly consistent with the structure of RR198 (data not shown). These tentatively identified degradation products indicate that RR198 was cleaved at the azo bond as well as the amine bond between the naphthalene group and triazenic ring and at one of the sulfonate bonds producing the identified compounds 4-chloro-N-o-tolyl-1,3,5-triazin-2-amine; sodium 4-aminonaphthalene-2-sulfonate and 3,6-dimethyl-7-(o-tolyldiazenyl) naphthalen-1-amine.

Figure 3. Phylogram (neighbor-joining method) showing genetic relationship between strain ETL-1979 and other microorganisms based on the 16S rRNA gene sequence analysis

3.6. Toxicity Test and TOC Reduction

The TOC of the medium was 2000 mg L-1 with the portion attributable to the azo dyes equal to 60 mg L⁻¹. After decolorization (data not shown) under microaerophilic (static) conditions the TOC was reduced from between 65% to 82% for all dyes, and after susequent aeration, for an additional 168 h, the TOC was further reduced (Table 2). The extent of the reduction in TOC demonstrates that the carbon sources were being consumed during the dye degradation process in both the static and aerobic phases. The results for Daphnia magna toxicity tests are presented as the percentage of death in the presence of samples taken from the static and stirring treatments compared to controls composed of the dye culture medium without bacterial treatment. The tests were carried out in a 1:4 dilution of the original supernatant concentration because 100% mortality occurred in the undiluted and 1:2 diluted dye media. The uninoculated controls showed mortality between 40 to 47% at a dilution of 1:4. Samples taken from static cultures had mortality values much lower for all the dyes (≤ 13%), and samples from stirred cultures had no detectable toxicity for any dyes.

Table 2. Mortality of Daphnia magna exposed to a 1:4 dilution of culture supernatants containing azo dyes and the % TOC removal after incubation with Bacillus spp. ETL-1979 under static and aerobic conditions

4. Discussion

Based on the biochemical tests performed in this study, strain ETL-1979 was identified as Bacillus due to the differentiating characteristic in the oxidase test (Table 1). However, several reclassifications have been made and the genus has been restricted to those bacteria with a close resemblance to the type species B. linens based on 16S rRNA gene sequence analysis and DNA-DNA hybridization experiments; twelve species are currently classified in this genus [13]. Bacillus spp. ETL-1979 completely decolorized four azo dyes (Reactive Yellow 107, Reactive Red 198, Reactive Black 5 and Direct Blue 71) in a static/agitated sequential process only in the presence of a carbon source (glucose, pyruvate or yeast extract). In these experiments MMR, containing 3 gL⁻¹ glucose and 1 gL⁻¹ pyruvate as carbon sources, produced the best results. However, yeast extract, which has been the most commonly used a nutrient additive for dye bio-decolorization processes [40] could be substituted for pyruvate with similar results. Previous studies have shown that pyruvate is able to enhance the degradation of aromatic compounds [8]. Azo bond reduction of the azo dye amaranth by Shewanella decolorationis S12 was most effective using lactate or formate as electron donors, while pyruvate also increased the reduction of amaranth but to a lesser extent [21]. The chemical structures of the dyes also influence the decolorization rates [39]. Dyes with simple structures and low molecular weights usually exhibit higher rates of color removal, whereas color removal is less efficient with highly substituted and higher molecular weight dyes. Consistent with these observations, Bacillus spp. ETL-1979 required 96 h to decolorize the mono azo dye RY107 (the least substituted and least structurally complex dye used in this study) in comparison to 120 h for the highly substituted mono azo dye RR198, and 144 h for the diazo dye RB5 and 168 h for the triazo DB71. Induction of tyrosinase activity for all dyes under static conditions for up to 96-120 h suggests this enzyme was involved in the decolorization process. In addition, under aerated conditions, the activity also increased and remained increased for the entire 168 h aeration period suggesting that tyrosinase could be involved in further biodegradation of the decolorized azo dye metabolites as well. In contrast, significant peroxidase or laccase activity was not detected. In recent studies, induction of the oxidative enzymes lignin peroxidase, laccase and tyrosinase was observed during the decolorization of sulfonated azo dyes by the bacteria P. desmolyticum and an Exiguobacterium sp. [10,25,26,]. While in another report on dye decolorization by Comamonas sp. UVS, the induction of laccase and lignin peroxidase was observed but tyrosinase activity was not [50]. Phenol oxidases, which can be divided into tyrosinases and laccases, are oxidoreductases that can catalyze the oxidation of phenolic and other aromatic compounds without the use of cofactors [12,31]. Tyrosinases use molecular oxygen to catalyze two different enzymatic reactions: (I) the ortho-hydroxylation of monophenols to o-diphenols (monophenolase, cresolase activity) and (II) the oxidation of o-diphenols to o-quinones (diphenolase, catecholase activity) [49,30]. However, aromatic amines and o-aminophenols have also been recognized as tyrosinase substrates that undergo similar ortho-hydroxylation and oxidation reactions [9,18]. In addition, oxidation products of tyrosinase can be further converted into heavier oligomeric species [11]. Under static conditions the UV–Vis spectroscopy demonstrated a decrease in absorbance between 280–350 nm (consistent with substituted benzene and naphthalene compounds) was observed for all dyes, with the corresponding formation of a new peak at 260 nm. The decrease in the absorbance of the peak between 280–350 nm and the formation of a new peaks at 260 nm suggest that there were changes to substituents on the aromatic groups, consistent with azo bond cleavage and other transformational changes to the aromatic structure. Aromatic amine spectra show extensive structure in the 260–300 nm range, and removal of absorption in the visible range and the production of absorption peaks around 260 nm has been observed with azo dyes chemically reduced with sodium sulfide and after their biological reduction with activated sludge under anaerobic conditions. The peaks at 260 nm can be associated with the presence of oxidized aromatics such as phenolic and naphthoquinone compounds [35,22]. In addition, the absorbance peak at 260 nm can be attributed to the absorption by diketones [28]. These compounds may be generated during bacterial degradation after cleavage of aromatic rings under aerobic conditions. After subsequent aeration the absorbance in the 260 region increased for the mono azo dye RY107 and for the highly substituted mono azo dye RR198, while the absorbance in this region decreased for the diazo dye RB5 and 168 h for the triazo DB71. The reason for this difference may be due to differences in the rates of transformation of the decolorized products during this phase. The increase in absorbance in the 260 nm region for RY107 and RR198 may then be attributable to a greater rate of additional transformation to intermediate products with absorbance in this region, resulting in their accumulation at greater rate than that for the other dyes. The biodegradation products of the RR198 dye were analyzed by HPLC-MS for tentative identification of the unknown metabolites. Between possible compounds of the biodegradation RR198 azo dyes, three had reasonable matches in relation to metabolites presents in the sample: 4-chloro-N-o-tolyl-1,3,5-triazin-2-amine; sodium 4-aminonaphthalene-2-sulfonate and 3,6-dimethyl-7-(o-tolyldiazenyl) naphthalen-1-amine. After aerobic conditions the concentration of these metabolites was reduced indicating they were degraded further. The formation of these products would require the cleavage of azo bonds and sulfonate bonds. Cleavage of both the azo bond and the sulfonate bond was observed with the degradation of the diazo dye Reactive blue 172 by Exiguobacterium sp. RD3 (isolated from the dyestuff contaminated soil), which also expressed lignin peroxidase and laccase activity during the degradation process [10]. In addition to decolorization and degradation of the azo dyes, Bacillus spp. ETL-1979 also dramatically reduced the toxicity of the dye solutions after the static phase of incubation. Moreover, subsequent aeration of the static cultures further reduced toxicity below detectable levels. The present study demonstrates that Bacillus spp. ETL-1979 has the potential to decolorize and degrade toxic azo dyes to nontoxic products. The detection of tyrosinase activity throughout static and stirred stages indicates tyrosinase was involved in the biodegradation of the azo dyes. The addition of carbon substrates in the form of glucose, pyruvate or yeast exact was required for efficient decolorization indicating the dyes may be being used as terminal electron acceptors during their degradation.

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