Textile azo dye degradation by Candida rugosa INCQS 71011 isolated from a non-impacted area in Semi-Arid Region of Brazilian Northeast

1 Fiocruz Oswaldo Cruz Foundation, National Institute for Health Quality Control, Avenida Brasil 4365, – Manguinhos, Rio de Janeiro, RJ 21040-900, Brazil. 2 Fiocruz Oswaldo Cruz Foundation, National Institute for Health Quality Control, Post-graduate Program in Health Surveillance, Avenida Brasil 4365, – Manguinhos, Rio de Janeiro, RJ 21040-900, Brazil. 3 Federal University of Minas Gerais, Microbiology Department, ICB, CP 486, Belo Horizonte, Minas Gerais, Brazil.


INTRODUCTION
Wastewaters from textile industries are a complex mixture of several pollutants such as heavy metals, pigments, and dyes (Coulibaly et al., 2003). The potential of pollution from textile dyes has been acknowledged primarily due to possible problems towards human and environmental health (Banat et al., 1996;Meehan et al., 2000;Stolz, 2001). Effluents discharged by textile industries, if not treated properly, can contain large quantities of hazardous chemical compounds (Chen and Hwang, 1997;Aksu and Dönmez, 2003). There is a necessity to develop better treatment technologies to remove color from industrial effluents that prove technically and economically satisfying as treatment technology. Therefore, several emerging approaches such as advanced oxidation process, membrane filtration, photocatalysis, and sonication, are being proposed and tested at different stages of commercialization (Anjaneyulu et al., 2005). A number of studies have focused on some microorganisms that are able to biodegrade and biosorb dyes in wastewaters. Some bacteria, fungi, and algae are capable of decolorizing a wide range of dyes (Fu and Viraraghavan, 2001). The treatment using fungi is a promising alternative to replace or complement conventional treatment processes due to the efficiency of dye decolorization by fungi, mainly because of the fungal enzymatic apparatus that enables the degradation of these compounds and for not producing material to be discarded. Additionally, fungal treatment has a good public acceptance (Fu and Viraraghavan, 2001). However, more studies on the ability of fungi to decolorize dyes used by the textile industry are needed. Little is known about the degradation of dyes and color removal by yeasts (Singh, 2006); nonetheless, most studies show positive results. Several genera of yeast have been studied such as Kluyveromyces, Schizosaccharomyces, Issatchenkia, Debaryomyces, Pseudozyma, and Saccharomyces (Aksu and Dönmez, 2003;Ramalho et al., 2004;Yang et al., 2003Yang et al., , 2005Yu and Wen, 2005;Kumari and Abraham, 2007). Candida species have been used in some works and good results were presented for removal of reactive dyes in concentrations ranging from 10 -700 mg l -1 , nevertheless, these studies showed adsorption, besides degradation (Gönen and Aksu, 2009;Ertugrul et al., 2009) and absorption (Dönmez, 2002;Aksu and Dönmez, 2003).
Compared with bacteria and filamentous fungi, the yeasts display attractive characteristics. Despite the fact that yeasts do not always grow as fast as bacteria, they grow faster than filamentous fungi and have the ability to resist to unfavorable environments (Pajot et al., 2007). Based on this context, the isolation of fungal species from an environment with potential bioremediation has been spreading (da Silva et al., 2003;Junghanns et al., 2008;Passarini et al., 2011) and different ecosystems have been exploited for this purpose.
Brazil has ecosystems and biomes that are not yet fully known, such as the semi-arid Caatinga, an area of uncertain rainfall situated in the northeastern state of Piauí. This biome represents a valuable biodiversity reservoir, in which there is an important reserve for the preservation of its specific environment, the Serra da Capivara National Park (Pessis, 1998;Gusmão and Maia, 2006).
From the surroundings of Serra da Capivara National Park, sediment samples of pristine water reservoirs where collected and mixed fungal cultures were selected for dye degradation. From the most efficient mixed fungal culture, Caldeirão Escuridão (CE), a yeast strain (CE-9) was isolated (Nascimento et al., 2011). Therefore, the aim of the present work was to identify by phenotypic and molecular methods the yeast CE-9 as well as to test its capacity to degrade three textile azo dyes.

Yeast isolation and preservation
The yeast strain CE-9 was isolated from a mixed fungal culture do Nascimento et al. 6637 collected from Caldeirão Escuridão (CE), a natural water reservoir situated in the surroundings of the Serra da Capivara National Park, at an altitude of 423 m (xUTM -769341, yUTM -9022784 (Nascimento et al., 2011 For routine work at laboratory scale, the selected yeasts were maintained on yeast extract-malt extract agar (YMA) slants at 4°C and subcultured at 15 day regular intervals.

Yeast identification
The isolate CE-9 was characterized by standard methods (Yarrow, 1998) and the identification followed the keys of Kurtzman and Fell (1998). Concurrently, the characterization by the Vitek ® 2 using the Yeast Biochemical Card (YBC) (bioMérieux, Durham, NC) was conducted. Genomic DNA of CE-9 and of the reference strain C. rugosa ATCC 10571 was prepared after 2 days of incubation on YMA using the methodology described by de Barros Lopes et al. (1998). The D1/D2 variable domains of the large-subunit rDNA were amplified by polymerase chain reaction (PCR) using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') according to Lachance et al. (1999). Amplification was performed as follows: 95°C for 2 min, followed by 35 cycles of 95°C for 15 s, 54°C for 25 s, and 72°C for 20 s, and a final extension at 72°C for 10 min. The amplified DNA was concentrated and cleaned (Kit Wizard Plus SV Minipreps DNA Purification System-Promega, USA). Sequencing was conducted using an ET dynamic terminator kit in a MegaBACE 1000/ automated 96 capillary DNA sequencer (GE Healthcare, Buckinghamshire, UK). The quality of each electropherogram was evaluated using Phred-Phrap software and consensus sequences were obtained by alignment of both sequence strands using CAP3 software available on the Eletropherogram quality analysis web page (http://asparagin.cenargen.embrapa.br/phph). The nucleotide sequences and other related sequences were aligned using the CLUSTALW software package (EMBL-EBI) (http://www.ebi.ac.uk/clustalw/). Phylogenetic relationships were estimated using the MEGA program Version 4.0 (Tamura et al., 2007). The phylogenetic trees were constructed using the neighbor joining (NJ) algorithm with bootstrap values calculated from 1,000 replicate runs. The Maximum Composite Likelihood model was used to estimate evolutionary distance.

Yeast growth
CE-9 was grown on potato dextrose agar (PDA) with 200 mg l -1 of each of the three dyes separately and incubated for 48 h. Aliquots from these cultures were transferred to sterile purified water and the turbidity was adjusted to 2 McFarland standard (6 x 10 8 CFU ml -1 ). From this suspension, 2 ml were used to inoculate 100 ml of potato dextrose broth ( rotary shaker (New Brunswich, Innova 4335, USA) at 140 rpm. After this incubation period, 100 mg l -1 of the dyes were added separately in all flasks. The cultures were kept incubating under the same conditions during 7 days. Control experiments were conducted by means of incubating PDB medium without yeast and with the same concentrations of the dye tested as well as with PDB without either dye or yeast.

Evaluation of dye decolorization
The evaluation of dye decolorization by CE-9 was conducted during the 7-day incubation at the following times: 24, 48, 72, 96, 120, 144 and 168 h. After centrifugation at 10,000 rpm for 10 min of 2 ml aliquots of the cultures, 0.5 ml of supernatant was diluted 10 times in purified water in order to be analyzed using spectrophotometer UV/VIS (Shimadzu -UV-1601, Japan). The culture medium with dye and without inoculum was used as a negative control whereas medium without either dye or inoculum was used as blank. The spectra were scanned between 200 to 700 nm of absorbance (Máximo et al., 2003) in order to analyze dye disappearance. Decolorization activity (%) was calculated for the two flasks (duplicate) of each culture according to the formula below, considering the maximum wavelength of 520 nm for RR198, 548 nm for RR141, and 608 nm for RB214.

Compound
Result Assimilation of carbon compounds Glucose

Phenotypic characterization
The macro-morphological observations on YMA medium after 2 days at 25°C of the isolate CE-9 demonstrated a butyrous texture, colored white to cream. From the micromorphological analysis, cells were elongate, single and in pairs and pseudo-hyphae were produced. These are morphological characteristics typical of C. rugosa. In Table 1, the physiological results obtained from CE-9 were present. The yeast was negative for urea hydrolysis and formation of extracellular amyloid compounds. It did not ferment carbohydrates, it did not grow in the absence of vitamins, it was sensitive to cycloheximide, it did not assimilate the NaNO 2 and KNO 3 as nitrogen source, but assimilated the following carbon compounds: glucose, Dgalactose, D-xylose, glycerol, D-mannitol, D-glucitol, lactic acid, and succinic acid. The other carbon compounds used in the test were not assimilated by the yeast. Hence, the isolate was identified biochemically and physiologically as C. rugosa. The automated method of identification using Vitek ® 2 was also used for the biochemical characterization whereas the YBC was applied to confirm the conventional method. As the result, the identification was 97% of probability for C. rugosa. The D1/D2 sequence of strain C. rugosa ATCC 10571 has 527 nucleotides, where 24 and 19 correspond to the nucleotide primers NL1 and NL4, respectively. Thus, the sequence to be obtained from isolate CE-9 should be 484 nucleotides.
Forward and reverse strands of D1/D2 domain of isolate CE-9 were sequenced using the primers NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL-4 (5'-GGTCCGTGTTTCAAGACGG-3'). Consensus sequence had 447 nucleotides and was aligned with the sequences of C. rugosa ATCC 10571. Manual editing of the sequence was performed based on the chromatograms obtained and no differences between the sequences were observed. When performing blast, it had 99% of identity with only one Gap of difference. Therefore, these results confirm the phenotypic characterization, leading to the identification of the isolate CE-9 as the species C. rugosa.

Decolorization
C. rugosa INCQS 71011 decolorized 78.46% of RR198 at 100 mg l -1 dye concentration after 24 h of incubation and reached 100% decolorization of this same dye after 48 h of incubation (Table 2), respectively. Regarding RR141 dye, decolorization reached 75.60% after 24 h of incubation, and complete decolorization was achieved within 144 h of incubation (Table 2). In both cases, there was no adsorption by the biomass but degradation, as demonstrated by the results of spectrophotometric analyses that showed the disappearance of the characteristic peak of those two dyes (Figures 2a and 2b). The other dye tested, RB214, was less efficiently decolorized by C. rugosa INCQS 71011. In the first 24h incubation, decolorization achieved 50%. From 48 to 144 h, decolorization increased progressively, however slowly; and at the end of the incubation period (168 h), the decolorization rate was 76.05% (Table 2). The adsorption to the biomass just occurred with this dye, which decreased up to the end of the incubation period (data not shown), while the characteristic peak of this dye was transformed into a new peak ( Figure 2c).

DISCUSSION
In some investigations, positive results for textile dye decolorization were obtained using fungi isolated from non-impacted areas (Pajot et al., 2011;Martorell et al., 2012), as similarly obtained in the present study. The ascomycetous yeast C. rugosa INCQS 71011 showed a very good result mainly in the decolorization of RR198, a monoazo, decolorizing 100% of the dye at a concentration of 100 mg l -1 after 48 h, while within 24 h the decolorization was 75.60%. Regarding RR141, the results were also satisfactory 75.20% was decolorized after 24 h incubation and the complete decolorization was achieved within 144 h incubation. For RB214, however, the decolorization took longer, 76.05% was decolorized at the end of the incubation period (168 h) ( Table 2). The decolorization of a number of simple azo dyes derived from 2-naphtholaminoazobenzene and p-N,Ndimethylaminoazobenzene in concentrations of 10-50 mg l -1 were tested in liquid aerated batch cultures using a strain of the yeast Candida zeylanoides UM2, by which method a 90% adsorption was obtained from the best result after seven days of incubation (Martins et al., 1999). This same strain was used by Ramalho et al. (2005) which, after changing some parameters, such as medium, for example, C. zeylanoides UM2 showed 100% efficiency of decolorization without traces of absorption after 60 h.
Dye degradation occurs when the biomass remains with its original color and the medium becomes decolorized (Chen et al., 2003;Yu and Wen, 2005). It can be monitored during cultivation of the fungus in liquid medium through the ratio between two characteristic wavelengths of the dye. Adsorption occurs when the characteristic peak of absorption of the dye remains constant during the incubation period, while degradation occurs when this peak disappears completely or a new peak appears (Glenn and Gold, 1983). In the present study, C. rugosa 71011 succeeded in promoting the disappearance of the characteristic peaks of absorption of visible light from both dyes, namely RR198 (520 and 370 nm) and RR141 (548 and 522 nm) (Figures 2a and  2b), with no dye adsorption by the biomass, thus typifying this fungal activity as the degradation of these dyes.
Degradation was also determined by Yang et al. (2003) when using Candida tropicalis Y2-0814 and Debaryomyces polymorphus Y1-0813 to decolorize 100 mg l -1 of seven synthetic dyes. Within 16-48 h of incubition the color disappeared completely without residual color on the biomass in the case of only one of these dyes, the Reactive Black 5. The metabolic profile species-specific, such as enzymatic activity (Ramalho et al., 2005;Martorell et al., 2012), media culture (Ramalho et al., 2002;Kaushik and Malik, 2010) and the structure complexity of the dye (Fu and Viraraghavan, 2001;Bergsten-Torralba et al., 2009) can greatly influence the final response of fungal degradation. In the present work, C. rugosa INCQS 71011 was much more efficient degrading RR198, a single azo class dye, than it was towards RR 141, a double azo class dye, which took longer to be degraded and towards RB 241, another double azo class dye, which was not completely degraded under the conditions established in this study.
The present results demonstrated the efficiency of C. rugosa INCQS 71011 to degrade textile azo dyes and indicates its potential as a biological treatment agent of textile effluent, despite its origin, a non-impacted area in Semi-Arid Northeastern Brazil. To our knowledge this is the first report of a strain of C. rugosa with the ability to degrade textile azo dyes. Further studies on the optimization conditions for this yeast to degrade azo dyes, nonetheless, are needed.

ACKNOWLEDGMENTS
The present work was supported by a grant from Fiocruz/CNPq (PAPES V -403647/2008-8). The authors are indebted to DyStar and Clariant industries (SP, Brazil) for donating the dyes used in the present investigation.