450 INVOLVEMENT OF EXTRACELLULAR FUNGAL ENZYMES IN BIOREMEDIATION OF TEXTILE EFFLUENT Rina D. Koyani and Kishore S. Rajput* Address(es): Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India-390002. *Corresponding author: ks.rajput15@yahoo.com ABSTRACT Keywords: Irpex lacteus, dye decolourisation, dye degradation, solid state fermentation, ligninolytic enzymes INTRODUCTION The huge manufacturing of the dyes due to their massive applications in textile industries has abundantly enhanced the effluent disposal into the environment. As a technological and scientific development in dye technology, they are synthesized as chemically and photolytically more stable and therefore persist in natural environment (Rieger et al., 2002). Consequently leads to worsening of the environment which is inevitably linked with overall quality of life. To mitigate these xenobiotic pollutants, their complete mineralization or transformation into the degradable forms is the only imperative solution on it. Although, many physicochemical techniques are available for efficient mineralization of these dyes, they are very expensive and commercially unattractive. However, biological treatment or biodegradation is an environment friendly and cost-effective alternative to these technologies (Gueu et al., 2007). The Basidiomycetes fungi have the ability of metabolizing lignin. To meet the challenge of lignin degradation, white rot fungi produce one or more of the three principal extracellular enzymes i.e. lignin peroxidises (LiP, EC 1.11.1.14), manganese peroxidase (MnP, EC 1.11.1.13) and laccases (EC 1.10.3.2) (Hatakka, 1994; Asgher et al., 2004). Identification of these enzymes generated one of the credible evolutions for the degradation of aromatic xenobiotics and/or environmental pollutants. Perusal of literature demonstrates the potential of white rot fungi to degrade pollutants by producing extracellular ligninolytic enzymes (Valentin et al., 2007; Wen et al., 2010) and most of them have been focused on dye decolourisation and degradation (Enayatzamira et al., 2009; Champagne et al., 2010). The initial recognition of the white rot fungi for their decolourizing competency lays the foundation for their application in dye degradation. Therefore, the present study paved the way from dye decolourisation to degradation using the potential of white rot fungus Irpex lacteus. From extremely diverse range of the textile dyes, most unanimously used reactive dyes (Reactive yellow FG and Reactive orange 2R) have been opted in the present study. The key role of ligninolytic enzymes yielded by Irpex lacteus under solid state fermentation has been emphasized for their ability to degrade the dyes. As the complete purification of enzyme is costly, crude and partially purified enzyme was preferred for their application in enzyme assay and degradation. Influence of different physico-chemical parameters such as inoculum size, effect of pH, temperature, incubation and reaction time, supplementation of carbon/nitrogen sources, and metal ions on enzyme productivity was also investigated. The intermediates formed during degradation of the dyes were analysed by FTIR (Fourier Transform Infrared Spectroscopy). The main objectives of the present study were: i) to evaluate the potential of Irpex lacteus in decolourisation of textile dyes i.e. Reactive yellow FG and Reactive orange 2R; ii) whether the dyes are decolourising or undergoing structural alterations due to enzyme action? and iii) rectification of reactive dyes degradation using ligninolytic enzymes through FTIR. MATERIALS AND METHODS Isolation and screening of the fungi Thirty five strains of wood rot fungi collected from the different forests of Gujarat State (India) were isolated and plated on optimised malt extract agar (MEA) medium. The purified cultures were subjected to Bavendamm’s test (Bavendam, 1928) for the screening of white rot fungi. Among six screened white rot fungi, KSR-70 was considered for the present studies. For molecular identification fungal DNA was extracted as per Möller et al., (1992) and extracted DNA was subjected to Polymerase chain reaction followed by sequencing, BLAST and its submission to the NCBI with Accession No. KJ670229. Chemicals and dyes DMAB (3-dimethyl amino benzoic acid), MBTH (3-methyl-2- benzothioazolinone hydrazone hydro chloride), H2O2 and Manganese Sulphate (MnSO4) were procured from National chemicals Ltd., (Vadodara, India). The textile dyes used in the present study were kindly provided by dying, printing and processing houses (Gujarat, India). Chemicals required for the biochemical studies were purchased from Qualigens Fine chemicals (Mumbai, India). All the other chemicals used were commercially available products of analytical grade. The plentiful use and reckless discharge of textile effluent to the nature witnessed the rising of water and soil pollution. Biological remediation of these compounds is the most desirable technique to overcome the elevated environmental pollution. Present study evaluates the efficiency of a wild strain of Irpex lacteus in decolourisation and degradation of Reactive yellow FG and Reactive orange 2R. Media supplemented with different carbon/nitrogen sources and inoculum size play important role in enhancing the ability in which dextrose and aspargine boosted the process while inoculum size one-three (10 mm diameter) were more significant with solid and liquid decolourisation respectively. The ligninolytic enzyme production under Solid State Fermentation (SSF) was carried out using different lignocellulosic substrates. Among different substrates wheat straw produced highest amount (560.6 IU/ml) of manganese peroxidase. Optimization of particle size and time of incubation were also assorted to define the efficient enzyme activity; where one mm particle size and 6 th day of incubation period were the most felicitous. The influence of physico-chemical factors like pH, temperature, reaction time and metal ions were assessed with respect to enzyme activity. The partial purification of crude enzymes was achieved at different percent saturations, where 40% saturated fraction yielded maximum (560.6 IU/ml) MnP activity. Molecular weight of the partially purified enzyme was 58.3 kDa. The degradation of dyes was confirmed with shift of the dominant peaks found on the FTIR graphs. ARTICLE INFO Received 26. 6. 2014 Revised 21. 4. 2015 Accepted 17. 12. 2015 Published 1. 4. 2016 Regular article doi: 10.15414/jmbfs.2016.5.5.450-455