Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk

Su et al. BMC Biotechnology (2016) 16:81 DOI 10.1186/s12896-016-0311-8 RESEARCH ARTICLE Open Access Biodegradation of lignin and nicotine with white rot fungi for the delignification and detoxification of tobacco stalk Yulong Su1†, He Xian2†, Sujuan Shi1,3, Chengsheng Zhang1, S. M. Nuruzzaman Manik1, Jingjing Mao1, Ge Zhang1,3, Weihong Liao4, Qian Wang1* and Haobao Liu1* Abstract Background: Tobacco stalk is one kind of abundant crop residues in China. The high lignification of tobacco stalk increases its reusing cost and the existing of nicotine will cause serious pollution. The biodegradation of lignocellulosic biomass has been demonstrated to be an environmental and economical approach for the utilization of plant stalk. Meanwhile, many nicotine-degrading microorganisms were found in nature. However, microorganisms which could degraded both nicotine and lignin haven’t been reported. Therefore, it’s imperative to find some suitable microorganisms to break down lignin and simultaneously remove nicotine in tobacco stalk. Results: The nicotine in tobacco stalk could be degraded effectively by Trametes versicolor, Trametes hirsute and Phanerochaete chrysosporium. The nicotine content in tobacco stalk was lowered to below 500 mg/kg (a safe concentration to environment) after 10 days of fermentation with Phanerochaete chrysosporium and Trametes versicolor, and 15 days with Trametes hirsute. The degradation rate of lignin in the fermented tobacco stalk was 37.70, 51.56 and 53.75% with Trametes versicolor, Trametes hirsute and Phanerochaete chrysosporium, respectively. Meanwhile, 24.28% hemicellulose was degraded by Phanerochaete chrysosporium and 28.19% cellulose was removed by Trametes hirsute. Through the enzyme activity analysis, the main and highest ligninolytic enzymes produced by Phanerochaete chrysosporium, Trametes hirsute and Trametes versicolor were lignin peroxidase (88.62 U · L−1), manganese peroxidase (100.95 U · L−1) and laccase (745.65 U · L−1). Meanwhile, relatively high and stable cellulase activity was also detected during the fermentation with Phanerochaete chrysosporium, and the highest endoglucanase, exoglucanase and filter paper enzyme activities were 0.38 U · mL−1, 0.45 U · mL−1 and 0.35U · mL−1, respectively. Moreover, the products in the fermentation of tobacco stalk with P. chrysosporium were identified with GC-MS, besides the chemicals produced in the degradation of lignin and nicotine, some small molecular valuable chemicals and fatty acid were also detected. Conclusions: Our study developed a new method for the degradation and detoxification of tobacco stalk by fermentation with white rot fungi Phanerochaete chrysosporium and Trametes hirsute. The different oxidative enzymes and chemical products detected during the degradation indicated a possible pathway for the utilization of tobacco stalk. Keywords: Tobacco stalk, Nicotine degradation, Delignification, Phanerochaete chrysosporium, Lignocellulolytic enzymes * Correspondence: ; † Equal contributors 1 Key Laboratory of Tobacco Biology and Processing, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Su et al. BMC Biotechnology (2016) 16:81 Background Tobacco is an important cash crop in China and has been planted in many farming areas. It has been estimated that about 3.2 million tons of tobacco stalk were produced annually in China [1]. The reusing of tobacco stalk is costly due to its high lignification. Most of tobacco stalk is discarded in the field directly and the nicotine in tobacco stalk can easily permeate into the soil, affecting its ecological structure and also polluting the ground water. It is reported that the average nicotine content in tobacco stalk is up to 3800 mg/kg [2] and the nicotine-containing waste would be classified as “toxic and hazardous”, when the concentration of nicotine exceeds 500 mg/kg dry weight [3]. Therefore, it is imperative to remove nicotine from tobacco stalk so as to make better utilization of tobacco stalk. Significant progresses have been made in the utilization of tobacco stalk such as making fiberboard, tobacco sheet, acticarbon or extracting chemicals [4–7]. However, all of these methods were deficient due to the terrible pollution on the environment or the high cost, and it is necessary to explore new approaches to the safe and sustainable utilization of tobacco stalk. The biodegradation of lignocellulosic biomass has been demonstrated to be an environmental-friendly and economical way for the reusing of plant stalk [8]. It is already shown that fungi belonged to Moniliales Gliocephalias sp. [9] and Aspergillus sp. [10] could degrade lignin in tobacco stalk. However, the microbes that could be used for the biodegradation of tobacco stalk were still limited due to the high lignification and the existing of nicotine. Though many microorganisms have been demonstrated to be able to degradate lignin and nicotine separately [11, 12], microorganisms which could degradate the both haven’t been reported. This study aimed to screen anti-nicotine microorganisms for the degradation of tobacco stalk, and explore their degradation characteristics. The research will provide a better way for the innocent treatment of tobacco stalk. Results and discussion Effect of fungi fermentation on nicotine degradation in tobacco stalk The concentration of nicotine in tobacco stalk was 1900 mg/kg in this study (Fig. 1). Nicotine has been defined as one kind of the toxic and hazardous chemicals in tobacco wastes [13]. It can contaminate ground water when the concentration exceeds 500 mg/kg in dry weight. Therefore, it is necessary to prevent the contamination of nicotine during the biodegradation of tobacco stalk. Nicotine concentration analysis showed that all the fungi selected could efficiently degradate nicotine (Fig. 1). For P. chrysosporium and T.versicolor, the concentration Page 2 of 9 Fig. 1 The content of nicotine in tobacco stalk when fermentation with Phanerochaete chrysosporium, Trametes versicolor and Trametes hirsute in solid state. The culture temperature was 28 °C and the relative humidity was 80%, the nicotine content was detected every 5 days until 25 days after inoculation of nicotine in tobacco stalk remained lowe (...truncated)