Screening and optimization of parameters affecting fungal pretreatment of oil palm empty fruit bunch (EFB) by experimental design

Agarat Kamcharoen 0 1 Verawat Champreda 0 1 Lily Eurwilaichitr 0 1 Piyarat Boonsawang 0 1 0 V. Champreda L. Eurwilaichitr Bioresources Technology Research Unit, Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park , Paholyothin Rd., Klong 1, Klong Luang, Pathumthani 12120, Thailand 1 A. Kamcharoen P. Boonsawang (&) Department of Industrial Biotechnology, Faculty of Agro- Industry, Prince of Songkla University , Hat Yai, Songkhla 90112, Thailand In the present study, various white-rot fungi were used for the pretreatment of oil palm empty fruit bunch (EFB) using solid-state cultivation. The results showed that Trametes versicolor TISTR 3224 gave the highest selectivity value (the ratio of lignin degradation to cellulose degradation) of 1.57. In comparison, Trametes sp. BCC 8729, Phanerochaete chrysosporium ATCC 24725, Marasmius sp. BCC 9542 and Xylaria sp. BCC 7749 gave selectivity of 0.60, 0.59, 0.30 and 0.06, respectively. Screening parameters for the fungal pretreatment of EFB using T. versicolor TISTR 3224 was studied by PlackettBurman design (PBD). It indicated that the moisture content and co-substrate gave a positive effect on the lignin degradation, while EFB concentration had a negative effect on cellulose degradation. The optimum conditions for lignin degradation obtained from Box-Behnken statistical experimental design (BBD) were 80 % moisture content, 2.29 % wheat flour and 23.3 % EFB. Under this condition, 15.6 % of delignification was obtained. After an enzymatic hydrolysis, the digestibility of fungal treated EFB under the optimum condition achieved 1.34-fold compared with untreated EFB. - Oil palm (Elaeis guineensis) is one of the most economical oil crops. The process of palm oil production has generated empty fruit bunches, fiber and palm shell as wastes [1]. With the increasing demand for energy, biofuel from renewable raw materials has been attractive because it is easily accessible, locally abundant and rich in lignocelluloses [2]. Recently, attempts have been made to apply EFB for bioethanol production [37]. Generally, bioethanol production from lignocellulosic materials employs three major steps: pretreatment for breakdown of lignin and opening up the crystalline structure of cellulosic materials; hydrolysis for fermentable sugar production; and bioconversion of fermentable sugar produced to bioethanol [8]. Although chemical and physicochemical pretreatments have been widely investigated, inhibitory compounds, e.g., furfural and hydroxymethyl furfural, are released and further affect the fermentation process [9, 10]. Therefore, biological pretreatment is interesting and has the additional advantages of simple technique and low pretreatment requirements resulting in low operating cost and environmentally friendly process [11]. White-rot fungi are important microorganisms involved in lignin degradation during pretreatment [1214]. Phanerochaete chrysosporium [1518] and Trametes versicolor [1922] have been reported for the pretreatment of lignocellulosic materials. Moreover, Xylariaceous fungi and Marasmius sp. have been reported as lignin-degrading microorganisms [13, 2325]. However, different white-rot fungi differed in their capabilities of cellulose and lignin degradation from one biomass to another [26]. Some fungi not only degrade lignin effectively, but also consume cellulose simultaneously leading to low available cellulose for bioethanol production. A common measure of delignification efficiency is the selectivity value (SV) of a fungal pretreatment, defined as the ratio of lignin degradation (LD) to cellulose degradation (CD) [18, 27, 28]. A low SV means a relatively high cellulose loss during fungal pretreatment. Thus, the SV of a fungal pretreatment is used to screen whiterot fungi for biological pretreatment [28]. The limitation of biological pretreatment is a lower reaction rate and requires longer pretreatment time than chemical pretreatment [14]. Although the strategies of strain improvement may help resolve some of the drawbacks, the technical process is quite challenging. Another approach to improve the efficiency of biological pretreatment is through the optimization of nutrient and environmental cultivation to reach maximum lignin degradation and minimum cellulose degradation. Shi et al. [16] found that the moisture content and culture time affected the fungal pretreatment of cotton stalk using P. chrysosporium. Alam et al. [29] indicated that the moisture content, inoculum size and wheat flour as co-substrate affected ligninase production during the fungal pretreatment of oil palm biomass. Levin et al. [30] reported that the balance of cellulose and ligninolytic enzyme production during fungal pretreatment depended on pH, peptone and copper. Although, there have been many researches that studied the factors affecting fungal pretreatment, those involved a onefactor-at-a-time experiment or examination of only a few factors. Moreover, the screening of significant factors affecting the fungal pretreatment of EFB has not been studied. In this research, the systematic evaluation of the optimization for the fungal pretreatment was investigated. Statistically designed experiments are a powerful tool to get more information about the system being studied with a minimum number of experiments [31]. The PlackettBurman design (PBD) has been frequently used for screening process variables that make the greatest impact on a process [32]. Response surface methodology (RSM) is a statistical and mathematical technique useful for developing, improving and optimizing the processes of an interest variable. RSM offers a large amount of information from a small number of experiments and reduces time [10, 33]. This research aims to identify the selective lignindegrading white-rot fungus with high lignin degradation and low cellulose loss. Also, the optimum condition was studied to improve the efficiency of fungal pretreatment. PBD was used for screening the significant factors for fungal pretreatment during solid-state cultivation. Box Behnken design was then applied to determine the optimum level of each of the significant factors for delignification with a high SV. EFB preparation EFBs were collected from Thai Tallow and Oil Co., Ltd., Thailand. The sample was dried and crushed into 510 mm fibrous length using a hammer mill, then ground to pass through a 1 mm screen (18 meshes) and kept for use in the whole experiment. The chemical composition of EFB, given on dry weight basis, was as follows: 37.6 % cellulose, 21.5 % hemicellulose and 19.0 % lignin [34]. Phanerochaete chrysosporium ATCC 24725 was obtained from the Faculty of Agro-industry, Prince of Songkla University. Xylaria sp. BCC 7749, Trametes sp. BCC 8729 and Marasmius sp. BCC 9542 were received from the BIOTEC culture collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Scien (...truncated)