Short Communications - Decolorization of Synozol Red 6HBN by Yeast, Candida tropicalis 4S, Isolated from Industrial Wastewater.
Azo dyes decolorizing yeast identified as Candida tropicalis 4S was isolated from wastewater treatment plant of textile and dyeing industry, Kot Lakhpat, Pakistan. The optimum temperature and pH for the growth of yeast were found to be 30oC and 7, respectively. C. tropicalis 4S was capable of decolorizing Synozol Red 6HBN up to 79%, 90% and 93% after incubation of 7, 14 and 21 days, respectively. Phyto- and microbial toxicity studies revealed that dye degraded products did not show any toxicity. C. tropicalis 4S can be exploited for bioremediation of wastewater containing azo dyes.
Keywords: Synozol Red 6HBN, Candida tropicalis, azoreductase, decolorization, degradation.
Dyes are colored substances that when applied to fibers give them a permanent color which is able to resist fading upon exposure to sweat, light, water and many chemicals, including oxidizing agents and microbial attack (Rai et al., 2005). It is estimated that 280,000 tons of textile dyes are discharged in such industrial effluents every year worldwide (Jin et al., 2007). With the increasing use of a wide variety of dyes, pollution by dye wastewater is becoming increasingly alarming. Among these azo dyes are important colorants and are characterized by the presence of one or more azo groups (N=N) and constitute the largest class of dyes having extensive applications in textiles, papers, leathers, gasoline, additives, foodstuffs and cosmetics (Chen et al., 2009).
The release of textile and dye-house effluent may cause abnormal coloration of the surface water and this creates the greatest immediate environmental concern with regard to water quality, and directly affects the aquatic flora and fauna. It has been found that purified forms of many azo dyes are directly mutagenic and carcinogenic (Chen, 2002).
In the last decade, several microorganisms have been investigated for decolorization of reactive dyes and its effectiveness depends on the adaptability and the activity of selected microorganisms (Aksu et al., 2007; Jadhav et al., 2007; Dave and Dave, 2009; Aftab et al., 2011; Ilyas and Rehman, 2013). Microbiological decolorization and degradation is an environmental- friendly and cost competitive alternative to the chemical decomposition process (Saratale et al., 2009b, 2010; Aftab et al., 2011).
Very little work has been done to explore the decolorization ability of yeast, and it has mainly been studied with regard to biosorption. Some ascomycetes yeast species, such as Candida tropicalis, Debaryomyces polymorphus, Candida zeylanoides (Yang et al., 2003) and Issatchenkia occidentalis (Ramalho et al., 2004), have been used to carry out putative enzymatic biodegradation and consequent decolorization of different azo dyes.
This study aims to investigate the potential of C. tropicalis 4S to decolorize an azo textile dye, Synozol Red 6HBN. The rate dependent environmental parameters such as temperature, pH and metal ions were studied. The phyto- and microbial toxicity of the products formed after degradation were also studied.
Materials and methods
Isolation and screening of yeast strains
Samples were collected from Kot Lakhpat industrial areas, Lahore. Isolation was done by spreading 100 L of wastewater sample on yeast extract peptone-dextrose (YEPD) agar plates. YEPD plates were prepared by dissolving 2g of peptone, 2g of dextrose and 1g of yeast extract and 2g of agar in 100 mL distilled water. In the preliminary study, Synozol Red 6HBN [CI Reactive Red 195; MF: C31H19ClN7Na5O19S6 (Fig.1)] was selected for determining the decolorization ability of the 34 yeast isolates. YEPD broth medium containing 20 g/mL of dye was used for screening of yeast incubated at 30oC.
DNA was isolated (Masneuf-pomarAde et al., 2007) and polymerase chain reaction (PCR) was performed by using two oligonucleotide yeast 18S rRNA primers, the forward primer (ITS5; 5-GGAAGTAAAAGTCGTAACAACG-3) and the reverse primer (ITS-4; 5-TCCTCCGCTTATTGATATGC-3) (Larena et al., 1999). The PCR consisted of 35 cycles, each of denaturation at 94oC for 4 min, annealing at 55oC for 2 min, and elongation at 72oC for 10 min. PCR reaction mixture contained 3 L of PCR buffer, 3 L of dNTPs, 2.5 L of each forward and reverse primer, 0.5 L of taq polymerase, 2.5 L of MgCl2, 5 L of nuclease free water and 6 L of genomic DNA.
The PCR products was extracted by the thermo scientific gene JET gel extaction kit method. The sequencing was carried out by Genetic analysis system model CEQ-800 (Beckman) Coulter Inc. Fullerton, CA, USA. Nucleotide sequence similarities were determined using BLAST (NCBI database; http://www.ncbi.nlm.nih.gov/BLAST).
Microorganism and maintenance conditions
The yeast culture was maintained on YEPD agar plates at 4oC. The yeast culture was also maintained in salt medium containing 10 g/L glucose, 1 g/L (NH4)2SO4, 0.15g/L KH2PO4, 0.1g/L K2HPO4, 0.1g/L MgSO47H2O, 0.026g/L FeSO4 and 0.086g/L CaCl2. The pH of the medium was adjusted to 7.0-7.2 and was sterilized at 121oC for 20 min. The 20 g/mL of Synozol Red 6HBN was added to the cell culture on 2nd day of incubation. After 7 days of incubation cells were harvested by centrifugation (6000 g, 10 min), washed twice with sodium phosphate buffer (50 mM, pH 7.0) and lysed by sonication for 2-3 times at 4oC for 15 s with a 60 s interval. The lysate was centrifuged at 4000rpm for 10 min.
For the determination of optimum temperature of the yeast YEPD broth medium was inoculated with fresh yeast culture and kept at 20, 30, 37, 50 and 70oC under shaking conditions. For determining optimum pH 100 mL YEPD broth medium with pH 5, 6, 7, 8, and 9 was inoculated with fresh yeast culture and incubated at 30oC for 24 h. Growth was monitored by taking the optical density at 600 nm.
Effect of dye on growth
For analyzing the effect of dye on the growth of yeast isolate YEPD broth medium (100 mL) was prepared and inoculated with yeast culture in 250 mL of Erlenmeyer flask and marked as control. Another flask containing 100 mL of salt medium was inoculated with culture and 20 g/mL of Synozol Red was also added in it and marked as treated. The flasks were incubated at 30oC in shaker (120 rpm) and 3 mL sample was withdrawn after regular time interval of 4, 8, 12, 16, 20, 24, 28 and 32 h. The absorbance was taken at 600nm.
Enzyme activity assay
Yeast cells were harvested by centrifugation at 4000 rpm for 10 min and an appropriate amount of lysis buffer (SDS and mercaptoethanol: 1:1 Check spelling) was added to the cell pellet and then sonicated at 4oC for 15 s with a 60 s interval 2-3 times and centrifuged at 4000 rpm for 10 min. The lysate or sonicated suspension was used for determining and analyzing the activity of crude enzyme. Azo reductase activity was determined by initiating the reaction by addition of 200 l of 100 M NADH in enzyme reaction mixture comprising 200 l of 25M Synozol Red 6HBN, 600 L of crude enzyme and 1 ml of 25mM tris-HCl buffer with pH 7.4. The reaction was incubated at 30oC for 30 min and relative activity of the enzyme was measured by taking absorbance at 436 nm using UV-VIS spectrophotometer (UV-4000, Germany). One unit of azoreductase can be defined as the amount of enzyme required to decolorize 1 mol of Synozol Red 6HBN per min.
Effects of temperature, pH and metalions
The activity of azoreductase enzyme was checked by incubating the enzyme reaction at temperature (30-90oC) by standard enzyme assay. The optimum pH of the crude enzyme was investigated over a pH range of 4.0-9.0. The enzyme was kept at 30oC for 30 min in various buffers (50 mM) and the residual azoreductase activity was determined under standard assay conditions. The buffer systems used comprised of 50 mM sodium acetate buffer pH (5.0-6.0), 50 mM sodium phosphate buffer pH (7.0-8.0) and 50 mM Tris-HCl buffer pH (9.0). For each pH a reaction mixture without enzyme (control) was prepared under the same condition and was used to measure the possible changes in absorbance. The effect of metal ions (FeCl3, CoCl2, NaCl, CuSO4, ZnCl2, MgCl2 and MnCl2; 1 mM each separately) on the azoreductase activity was also investigated.
For decolorization study, a loop full culture of yeast isolate from YEPD plate was used to inoculate 250-mL Erlenmeyer flask containing 100 mL salt medium under optimal growth conditions (pH 7.0; 30oC). The flask was kept at 30oC with shaking (130 rpm) for 2 days. The 48 h grown culture was incubated with Synozol Red 6HBN (20 g/mL) for 7, 14 and 21 days under aerobic condition. The decolorization process was monitored by taking 5 mL sample at regular intervals and O.D was measured spectrophotometrically at 436 nm. Control had no inoculated yeast culture except dye. Experiment was run in triplicate. The percentage decolorization was calculated (Saratale et al., 2009a) as follows:
Microbial and phytotoxicity
Toxicity effects were studied using dye degraded metabolites of 7 days old filtrate of yeast isolate. LB plates were inoculated with Bacillus cereus and Azotobacter and incubated at 37oC for 24 h. The yeast culture supernatant was then poured into the wells of the plate. The plates were incubated at 37oC for 24 h. Control contained distilled water in place of yeast culture supernatant. The effect was measured in terms of a zone of inhibition (diameter; cm) after 24 h of incubation at 37oC. To know the phytotoxic effect of Synozol Red decolorized products Vigna radiata was used. The pot containing four seeds of V. radiata was watered by 10 mL of yeast decolorized water per day after centrifugation at 4000 rpm for 10 min. Control pot was watered with distilled water at the same time. Growth was monitored after 7 days of incubation under 1:1 light and dark period (Ilyas and Rehman, 2013).
Results and discussion
Growth characteristics of C. tropicalis
The nucleotide sequences coding for the 18S rRNA gene (GenBank database under accession number JN009853) after BLAST query revealed that this gene is 100% homologous to Candida tropicalis.
The isolate showed maximum growth in YEPD medium (Control). Yeast isolate also showed good growth in dye containing salt medium but it was less as compared to control (data not shown). The optimum temperature and pH of the yeast was found to be 30oC and 7, respectively.
Effects of temperature, pH and metal ions on enzyme activity
It was determined that C. tropicalis 4S showed maximum azo reductase activity at 30oC (108%) and pH 7 (Fig. 2A). The decline at higher temperatures can be attributed to the loss of cell viability or the denaturation of azo reductase enzyme (Chang et al., 2001a; Saratale et al., 2009c).
The effects of pH (Fig. 2B) may be correlated to the transport of dye molecules across the cell membrane, which is considered as the rate limiting step for the decolorization (Chang et al., 2001b).
The enzyme activity increased 37% in the presence of K+ (Fig. 2C). The presence of heavy metal ions in the industrial effluents is a major obstacle which could potentially affect the activity of the reductase enzymes.
The color removal was 79% after 7 days, 90% after 14 days and 93% after 21 days. Some studies also show that yeast species act as a promising dye adsorbent and are able to uptake higher dye concentration (Safari'kova' et al., 2005), while Galactomyces geotrichum MTCC can decolorize triphenylmethane, azo and reactive high exhaust textile dyes (Jadhav et al., 2008a). Another detailed study was conducted on the decolorization of Navy Blue HER by using Trichosporon beigelii NCIM-3326, with the enzymatic mechanism and toxicity of the degradation products also reported (Saratale et al., 2009a).
In general, during azo dye degradation initial reductive cleavage of the azo bonds takes place, with the help of azoreductase enzymes which results into the production of colorless solutions containing potentially hazardous-aromatic amines (Van der Zee and Villaverde, 2005) which are further degraded aerobically or anaerobically (Joshi et al., 2008).
Microbial and phytotoxicity
Microbial toxicity and phytotoxicity results revealed no germination inhibition of plant by Synozol Red 6HBN metabolites. The growth of V. radiata was as good as its growth was observed in distilled water. This indicates that the yeast decolorized/degraded dye supernatant was safe for irrigation.
In the present study C. tropicalis 4S exhibited maximum ability to decolorize Synozol Red 6HBN upto 93%. It was observed that biodegradation efficiency is strongly affected by pH, temperature and type of metal ions used. Thermal stability of azo reductase was found to be 30oC at pH 7. Biodegraded products of Synozol Red 6HBN by C. tropicalis 4S have shown that these metabolites are safe and non-toxic for microbial flora and agricultural crops; thus this treated wastewater can be at-least used for irrigation purposes. Therefore the nature of the metabolites and their biodegradability require further investigation for complete mineralization of these compounds by the yeast strain.
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|Author:||Ilyas, Sidra; Bukhari, Dilara A.; Rehman, A.|
|Publication:||Pakistan Journal of Zoology|
|Date:||Aug 31, 2015|
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