ISOLATION AND CHARACTERISATION OF MICROBIAL STRAIN AZO29 CAPABLE OF AZO DYE DECOLOURIZATION

M. Grekova-Vasileva1, I. Popov2, D. Vassilev2, Y. Topalova1 1 Sofia University St. Kliment Ohridski, Faculty of Biology, Department of General and Applied Hydrobiology, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria 2 AgroBioInstitute, Department of Bioinformatics, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria Correspondence to: Martina Grekova-Vasileva E-mail: marty_grekova@abv.bg

ABSTRACT
Activated sludge samples collected from wastewater treatment plant (WWTP) in textile factory Giorgetti Bulgaria AD were exploited for isolation of dye decolorizing bacteria. A microbial strain AZO29 was selected based on its efficiency, showing maximum and faster decolourization of textile dye Amaranth. Identification of isolate by 16SrRNA technique revealed that the microorganism were from family Pseudomonadaceae of Gammaproteobacteria (99% confidence), the closest matches found after BLAST alignment were 16S sequences of Pseudomonas strains (97% similarity). Bacterial isolate AZO29 performed 100% decolourization after 24 hours of cultivation at the presence of 700 mg.l-1 Amaranth and degrade up to 1400 mg.l-1 of the azo dye for 72 hours in liquid medium. The decolourization rate (rama) increased with respect of increase of Amaranth concentration from 0.165 to 0.265 mM. Over 90% conversion can be achieved by AZO29 for a range of azo dye concentration up to 0.265 mM. Amaranth, microbial Keywords: phylogenetic characterization decolourization, breakdown products under anoxic conditions, it is repeatedly suggested to combine the anaerobic reduction with subsequent aerobic treatment of textile wastewater (5). Hence, interest is now focused on the bacteria, which can perform high rate decolourization under anoxic conditions and provide detoxification of aromatic products under aerobic environment. Most probably these microorganisms will be with facultative metabolism and capable of utilizing wide range of aromatic substrates. This paper describes the isolation and characterization of a bacterial culture AZO29, which is capable to decolourize azo dyes Amaranth and Acid Black 1. We operated an anoxic batch bioreactors with conventional activated sludge that effectively decolourized azo dyes and obtained enrichment bacterial culture, which was adapted to rising concentrations of azo dyes. An attempt to cultivate and enrich the most effective decolourizers enabled the successful isolation and identification of a member of this consortia; this member belongs to a family Pseudomonadaceae and is closely related to Pseudomonas marginalis (97% identity).

Introduction
Sulfonated azo dyes are the most numerous of the manufactured synthetic azo dyes. The electron withdrawing character of azo bond makes azo compounds problematic for oxidative strategies of microbial degradation (1). The presence of SO3H groups as substituents results in poor or incomplete biodegradation of sulfunated azo dyes, which are frequently found chemically unchanged in effluents of wastewater treatment plants (4). Under anaerobic and anoxic conditions various bacterial strains can reduce azo dyes, but this unspecific reduction in the biological systems usually proceeds rather slowly (4). To establish a biological wastewater treatment of sulfonated azo dyes is essential to discover the microorganisms that carry the sulfonated azo dye degrading enzymes (6). The anaerobic degradation of azo dyes results in accumulation of aromatic amines, which are potentially carcinogenic and mutagenic compounds. Only aerobic microorganisms may further oxidize the reduced products via deamination or hydroxylation reactions (2). To overcome the problem of the recalcitrance of azo dye
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Materials and methods

Screening of azo-degrading bacteria. Isolation and

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cultivation of microbial culture:The activated sludge, which was obtained from denitrifying reactor of wastewater treatment plant (WWTP) in textile factory Giorgetti Bulgaria AD Elin Pelin, was used as a source of highperformance bacterial decolourizer AZO29. The enrichment bacterial culture from the sludge samples was acclimated for 1 month in the presence of azo dye Amaranth and then served as a stock culture. The bacterial isolating procedures were carried out in a screening medium containing the following components (g per liter): peptone (1,5), yeast extract (0,75), NaCl (0,75), 50 mg Amaranth (7). Single strains were further isolated from the stable cultures with agar plates of the same enrichment medium (2% agar). The colonies around which clean zones expanded quickly were collected for further experiments. The single strains were cultivated in 500 ml flasks containing 150 ml growth medium at 20oC in a rotary shaker for 24 hours. Equal volume of mineral solution containing (g per liter): NaH2PO4 (3,5), K2HPO4 (5), (NH4)2SO4 (2,5), MgSO4.7H2O (0,3), FeSO4 (0,05mg), CuSO4 (0,01mg), ZnSO4 (0,005mg), CoCl2 (0,005mg), MnCl2 (0,005mg), CaCl2 (0,005mg), Na2MoO4 (0,005mg) and nutrient solution containing (g per liter): peptone (10), yeast extract (5), NaCl (5) was mixed to make the growth medium. The isolates were tested for their color removal ability at 35 oC for 72 hours, under static anoxic conditions and different concentrations of Amaranth ranging from 100 to 1400 mg.l-1. One isolate performed 100% decolourization after 72 hours of cultivation at the presence of 1400 mg.l-1 of azo dye and was selected for further investigations. 16 rDNA sequencing and phylogenetic analysis: For sequencing partial 16s rDNA from bacterial isolate, the combination of primers F968 and R1378 was used. PCR products were purified with High-Pure PCR product purification kit (Boehringer Mannheim, Almere, NL) and directly sequenced. The sequencing was performed in Department of Microbial Ecology, University of Groningen, NL. The partial 16S rDNA sequences (length about 500bp 16S sequence of good quality) obtained from isolate AZO29 was compared against those available in the database using standard BLAST-N provided on the NCBI server (www.ncbi.nlm.nih.gov/BLAST) and the Ribosomal Database Project (http://rdp.cme.msu.edu/). The alignment of the sequences was done using the CLUSTALW multiple sequence alignment software (www.ebi.ac.uk/Tools/clustalw2) using the neighbour joining algorithm. A phylogenetic tree was constructed from the alignment by average distance of the percentage identity, using Jalview. A second clustering
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was done with the MEGA 4 software, with the same algorithm. After 1000 iterations of bootstrapping the AZO29 strain clustered in the Pseudomonas group with a bootstrap value of 95 (95% repeatability of the clustering). Reduction of Amaranth by bacterial strain AZO29: The stock culture was growth aerobically for 24 hours at 20oC in a rotary shaker. The aerobically growth culture was transferred to plastic tubes (15 ml). The azo dye was added at different concentrations and the tubes were completely filled with the bacterial culture sealed with screw caps and incubated at 35 o C. The incubation time depended on test conditions and purposes of the experiments and varied from 5 to 24 hours. All assays were conducted in triplicates with non-inoculated controls. Analyses: The azo dye used in this study was Amaranth (C.I. No. 16185), which was obtained from Fluka. After centrifugation at 10 000 g for 15 min, the supernatants were decanted and used for determination of dye concentration. The concentration of Amaranth in samples was determined by measuring the absorbance of the supernatant of the sample at 520 nm with UV-VIS spectrophotometer (Ultrospec 3000, Pharmacia Biotech Ltd). The samples were diluted to less than 0.8 absorbance units. The dye concentration was determined from absorbance calibration curve of standard solutions. Scanning electron microscopy: A 0.2-ml bacterial suspension was filtered through a 0.2 m isopore membrane filter. Bacteria were rinsed with sterile distilled water for 30 s, and then fixed for SEM by the following series of treatments: 2.5% gluteraldehyde (30 min); 0.15 M phosphatebuffer (3 x 15 min); 50% ethanol (1 x 15 min); 70 ethanol (1 x 15 min); 90% ethanol (1 x 15 min) and 100% ethanol (3 x 15 min). Specimens were dried and mounted on aluminum stubs and gold-coated for 5 min in a JEOL-JFC-1200 and examined using the JEOL-JSM-5510 scanning electron microscope. Azoreductase assay: The biomass for enzyme assays was harvested by centrifugation at 10, 000 x g for 10 min at 4 oC. The pellet was washed twice with 0.003mM Ka-phosphate buffer pH 7,5 and resuspended in 0.033 mM phosphate buffer pH 7,5 (0,15 g wet weight per ml). The cell extracts were prepared by sonic disruption at 20 kHz at 4 oC. The unbroken cells and cell debris was removed by centrifugation at 13, 000 x g for 30 min at 4 oC and the supernatant was used as a intracellular crude enzyme source. One unit of azoreductase activity was defined as the amount of enzyme required to
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reduce 1 M of Amaranth per min per mg of protein. The reaction mixture contained at a total volume of 3 ml, 100 mM Ka-phosphate buffer (pH 7,5), Amaranth at different concentrations (0,02 - 0,15 mM), NADH (1 mM) and cell extract (0,8 0,9 mg of protein). The stock solutions were made anaerobically by repeated gassing with N2. The reaction was performed in gastight cuvettes and the decrease in concentration of Amaranth was determined spectrophotometrically at 520 nm, the initial velocity was calculated by using molar extinction coefficient of 20.76 mM-1 cm-1. Determination of protein content: The protein content of cell extract was determined by the method of Lowry, using bovine serum albumin as a standard (3). The protein content of whole cells was determined by the modification of Lowry method.

Amaranth for 24 hours in liquid medium (table 1). TABLE 1. Decolourization (%) of different initial concentrations of Amaranth by AZO29. Decolourization (%) Initial concentration After 24h After 48h After 72h of of of azo dye of -1 (mg.l ) cultivation cultivation cultivation 300 100 400 100 500 100 600 100 700 100 800 85 100 900 79 100 1000 76 100 1200 65 84 100 1400 58 76 100 The equilibrium conversion (Rama) and specific decolourization rate (rama) at the initial stage of Amaranth biodegradation were assessed over period of 5 hours (fig. 2). A positive correlation between the two parameters was established, as their values increased in a linear fashion. The azoreductase activity was determined during 5-h period and observed values were in the range of 4.28 4.52 M.min1 .mgP-1. In the course of the experiment azoreductase showed stable activity which resulted in high decolourization potential of the microbial culture. The effect of dye concentration on the equilibrium conversion (Rama) and specific decolourization rate (rama) of Amaranth by AZO29 is shown in fig. 3. The rama value tended to increase as the concentration of Amaranth was increased from 0.165 to 0.264 mM; the rama value reached plateau as the dye concentration was higher than 0.264 mM. In contrast to rama value, the equilibrium conversion was not enhanced by the increase of dye concentration, as the Rama value decreased between 8 - 22% (from 100% to 78%) with respect to an increase of dye concentration from 0.198 to 0.330 mM. This means that over 90% conversion can be achieved by AZO29 for a range of azo dye concentration up to 0.265 mM.

Results and Discussion

Isolation and identification of bacterial culture:The most active microbial strains from the mixed population of activated sludge were selected based on their ability to form a clear halo on agar plate containing the azo dye Amaranth. The colonies around which clean zones expanded quickly were collected for further experiments. Among eight isolates, one was selected based on its colour removal ability. The selected strain performed 100% decolourization after 72 hours of cultivation at the presence of 1400 mg.l-1 of azo dye and was further identified by 16S rRNA sequencing. After the initial alignment at NCBI and RDP, the relevant sequences were downloaded and phylogenetic analysis was done (fig. 1). In the RDP analysis the isolate aligned with the family Pseudomonadaceae of Gammaproteobacteria with 99% confidence. The closest matches found for AZO 29 after BLAST alignment are 16S sequences of Pseudomonas strains (97% similarity). After multiple sequence alignment of results from BLAST search in the referent sequence database (RefSeq) of NCBI and the creation of phylogenetic tree (fig. 1) the partial sequence of AZO29 clustered with g. Pseudomonas. Decolourization of Amaranth by microbial culture AZO29: The influence of initial substrate concentration on the decolourization activity of microbial strain AZO29 was investigated. The loading test revealed that AZO29 is highly efficient decolourizer, capable to degrade up to 700 mg.l-1

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Fig. 1. Average distance phylogenetic tree based on 16S rRNA sequences from activated sludge-derived Pseudomonas isolate. The cutoff value is selected in order to show the separate clustering of the g. Pseudomonas and Xanthomonas. The partial sequence of AZO 29 falls in the Pseudomonas group. The sequences used for the phylogenetic analysis were restricted to the BLAST results belonging to the family Pseudomonadaceae.

The observed correlation can be described by MichaelisMenten kinetics (rama = rama,max[Amaranth]/(Km + [Amaranth])), rama,max denotes maximum specific -1 -1 decolorization rate (M.min .mg of protein ), Km is Michaelis constant (mM) and [Amaranth] represents the concentration of Amaranth (mM). The kinetics constants estimated from experimental data (fig. 3) are 0.863 M.min1 .mg-1 for rama,max and 0.424mM for Km.
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The cell morphology of strain AZO29 under SEM is presented at fig. 4. Microscopic observation revealed that cells exhibited a typical morphology of pseudomonades. It had rod-shaped vegetative cells (1.0 to 1.2 by 2.0 to 4.0 m) and formed short chains (Fig. 4).

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of unstable biodegradation performance in the real technical

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Fig. 2. Time course of equilibrium conversion (Rama) and specific decolorization rate (rama) of azo dye Amaranth by AZO29. Initial dye concentration 0,165 mM.

Concentration of Amaranth (mM)

Fig. 3. Dependence of equilibrium conversion (Rama) and specific decolorization rate (rama) of AZO29 on the substrate (dye) concentration (incubation time 24 hours).

dumped into the aquatic ecosystem.applications and facilitate the removal of the azo compounds. Our further investigations will be focused on the degradation of aromatic amines formed by azo dye reduction and the potential of bacterial culture AZO29 to provide complete detoxification of these by-products. Acknowledgements This work was financed by the National Scientific Fund of the Ministry of Education and Science (Republic of Bulgaria) Bioremediation Technologies for Detoxification of Water and Sediments, Polluted by Textile Industry 2005-2008.

REFERENCES
1. Field, J.A., Brady, J. (2003) Wat. Sci. Thechnol., 48, 187-193. 2. Levine WG. (1991) Drug Metab Res, 23: 253-309. 3. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. (1951) Journal of Biological Chemistry, 193, 265-75. 4. Rau, J., Knackmuss, H.-J., Stolz A. (2002) Environ. Sci & Technol., 36, 1497-1504. 5. Stolz A. (2001) Appl. and Environ. Microbiology, 56, 69-80. 6. Vijaykumara M.H., Parag A. Vaishampayanb, Yogesh S. Shoucheb, T.B. Karegoudara (2007) Enz. and Microb. Technol., 40 (2), 204-211. 7. Yu, J., Wang, X., Yue, P.L. (2001) Water Research, 35, 3579-3586.

Fig. 4. Scanning electron micrographs of 24-h culture from bacterial isolate AZO29.

Conclusion
This preliminary characterization of the isolate regarding its morphological, biochemical characters and decolourization activity as well as the molecular identity gives useful information with regard to the further application of strain AZO29 for various purposes. The property of the isolate for azo dye decolourization would help for solving the problems

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Specific decolourization rate(rama, M.min-1.mg-1)

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0.40