Reduction in Acute Ecotoxicity of Paper Mill Effluent by Sequential Application of Xylanase and Laccase Saurabh Sudha Dhiman 1,2. , Gaurav Garg 3,4. , Jitender Sharma 3 , Vipin C. Kalia 5 *, Yun Chan Kang 1 *, Jung- Kul Lee 1,2 * 1 Department of Chemical Engineering, Konkuk University, Gwangjin–Gu, Seoul, Republic of Korea, 2 Institute of SK–KU Biomaterials, Konkuk University, Gwangjin–Gu, Seoul, Republic of Korea, 3 Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India, 4 Department of Biotechnology, Maharishi Markandeshwar University, Mullana-Ambala, Haryana, India, 5 Microbial Biotechnology and Genomics, CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Delhi, India Abstract In order to reduce the ecotoxicity of paper mill, four different enzymatic pretreatment strategies were investigated in comparison to conventional chemical based processes. In strategy I, xylanase-aided pretreatment of pulp was carried out, and in strategy II, xylanase and laccase-mediator systems were used sequentially. Moreover, to compare the efficiency of Bacillus stearothermophilus xylanase and Ceriporiopsis subvermispora laccase in the reduction of ecotoxicity and pollution, parallel strategies (III and IV) were implemented using commercial enzymes. Conventional C D E OP D 1 D 2 (C D, Cl 2 with ClO 2 ;E OP, H 2 O 2 extraction; D 1 and D 2, ClO 2 ) and X/XLC D E OP D 1 D 2 (X, xylanase; L, laccase) sequences were employed with non- enzymatic and enzymatic strategies, respectively. Acute toxicity was determined by the extent of inhibition of bioluminescence of Vibrio fischeri with different dilutions of the effluent. Two-fold increase was observed in EC 50 values for strategy I compared to the control process. On the other hand, sequential application of commercial enzymes resulted in higher acute toxicity compared to lab enzymes. In comparison to the control process, strategy II was the most efficient and successfully reduced 60.1 and 25.8% of biological oxygen demand (BOD) and color of effluents, respectively. We report for the first time the comparative analysis of the ecotoxicity of industrial effluents. Citation: Dhiman SS, Garg G, Sharma J, Kalia VC, Kang YC, et al. (2014) Reduction in Acute Ecotoxicity of Paper Mill Effluent by Sequential Application of Xylanase and Laccase. PLoS ONE 9(7): e102581. doi:10.1371/journal.pone.0102581 Editor: Ligia O. Martins, Universidade Nova de Lisboa, Portugal Received March 23, 2014; Accepted June 20, 2014; Published July 24, 2014 Copyright: ß 2014 Dhiman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscript and supporting information files. Funding: This work was supported by the Energy Efficiency & Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea (201320200000420). This research was also supported by a grant from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031955) funded by the Ministry of Science, ICT and Future Planning, Republic of Korea. This work was supported by 2013 KU Brain Pool fellowship of Konkuk University. The authors also acknowledge the support provided by Kurukshetra University, BILT and RCDP, India. VCK wish to thank CSIR-WUM (ESC0108) Government of India for providing necessary funds and facilities. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: vckalia@igib.res.in (VCK); yckang@konkuk.ac.kr (YCK); jkrhee@konkuk.ac.kr (JKL) . These authors contributed equally to this work. Introduction In the last two decades, use of enzymes, especially hemicellu- lases, has revolutionized the pulp and paper industry and provided a glimpse of hope that application of enzymes at various levels can reduce the industrial pollution and effluent’s toxicity. However, the current scenario continues to be challenging because of the high pollution load released by the pulp and paper industries, which are still using chlorine-based bleaching sequences [1]. The conventional bleaching methodology is chemical dependent and energy intensive; therefore, a novel enzymatic treatment with safe level of discharge needs to be developed [2]. Hydrolytic enzymes along with a laccase mediator system (LMS) have been more beneficial in reducing the pollution load of industries compared to other strategies [3]. An N-hydroxy-based synthetic mediator was predominantly used for this purpose [4]. However, toxicity and cost are two of the major hurdles, which hamper the industrial applications of these synthetic mediators. Therefore, the applica- tion of natural mediators in LMS is one of the alternatives to overcome these disadvantages, even though their application may cause grafting onto the pulp, an increase in kappa number, and a reduction in brightness of the pulp. Since chemical-intensive conventional strategies and enzymatic processes utilizing synthetic mediators release high levels of toxic compounds into water bodies, the entire processing of the effluents should be character- ized to analyze their ecotoxicity and other hazardous properties [5]. It has been observed that pretreatment with xylanase alone cannot reduce the pollution load of pulp and paper industry significantly. Hence, it is believed that a cocktail of two or more enzymes could reduce the release of hazardous materials to safer levels [4]. Although enzymes are effective at the pretreatment level, effluents from the entire process should be analyzed to study the enzymatic after-effects. Few reports deal with the character- ization of effluents from enzyme-aided bleaching processes, but without evaluating the interaction of technical parameters at the PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e102581