Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Application of a combined fungal and diluted acid pretreatment on olive tree biomass José Carlos Martínez-Patiño a , Thelmo A. Lu-Chau b, ⁎ , Beatriz Gullón b , Encarnación Ruiz a , Inmaculada Romero a , Eulogio Castro a , Juan M. Lema b a Department of Chemical, Environmental and Materials Engineering, Universidad de Jaén, Campus Las Lagunillas, 23071, Jaén, Spain b Department of Chemical Engineering, Institute of Technology, Universidade de Santiago de Compostela, 15782, Santiago de Compostela, Spain ARTICLE INFO Keywords: Olive tree biomass (OTB) Fungal pretreatment Combined diluted acid pretreatment White-rot fungi Bioethanol ABSTRACT A biological pretreatment of olive tree biomass (OTB) was carried out. First, seven white-rot fungi (WRF) were screened on solid-state fermentations by analyzing the substrate composition, ligninolytic enzymes production and enzymatic hydrolysis yields at three different pretreatment times (15, 30 and 45 days). Glucose released by enzymatic hydrolysis of OTB pretreated with Irpex lacteus for 45 days doubled that obtained with the control (non-inoculated). In addition, to enhance this yield, the combination of fungal pretreatment with a chemical pretreatment was studied. It was also found that the order of the pretreatment combination has a relevant effect on the glucose yield. Thereby, the best option determined, fungal pretreatment with I. lacteus after 28 days of culture followed by diluted acid pretreatment (2% w/v H 2 SO 4 , 130 °C and 90 min), enhanced 34% the enzymatic hydrolysis yield compared with the acid pretreatment alone. Applying the best pretreatment combination, the overall sugar yield of the whole process (sequential pretreatment plus enzymatic hydrolysis) was 51% of the theoretical yield. 1. Introduction Olive tree biomass (OTB) is a lignocellulosic residue generated yearly as a consequence of the pruning carried out to remove old branches and prepare the tree for the next crop. With an estimation of 1.5 tons per ha per year (Ruiz et al., 2017), more than 3 million tons OTB are generated yearly in Spain. Most part of OTB is eliminated by burning or gridding and spreading across the fields with environmental risks without generating any added value (Martínez-Patiño et al., 2017). OTB composition and its availability allow to propose this bio- mass as a raw material to produce second-generation bioethanol and other high added-value products, developing a multiproduct industry (Romero-García et al., 2014; Ruiz et al., 2017). Lignocellulosic biomass has a very complex structure that hinders the accessibility of enzymes to cellulose during enzymatic hydrolysis. For this reason, pretreatment is an essential step in the biorefinery process (Sindhu et al., 2016). Different pretreatments such as liquid hot water (Cara et al., 2007; Requejo et al., 2012), steam explosion (Cara et al., 2008), phosphoric acid (Martínez-Patiño et al., 2015), inorganic salts (López-Linares et al., 2013) and organosolv pretreatment (Díaz et al., 2011; Toledano et al., 2011) have been applied to OTB. However, these pretreatments require high energy demand and high capital cost. Moreover, they often generate toxic compounds and cause corrosion problems, which make the process commercially uncompetitive with a negative impact on the environment (Alvira et al., 2010). To overcome these inconveniences, over the last years the biological pretreatment has gained great attention by researchers (Deswal et al., 2014). The potential of biological pretreatment is explained by the ability of certain microorganisms to degrade lignin from lignocellulosic biomass. White-rot fungi (WRF), a small group of basidiomycetes, are the most effective microorganisms in breaking down and mineralizing lignin due to the extracellular secretion of oxidative enzymes, namely manganese peroxidase (MnP), versatile peroxidase (VP), lignin perox- idase (LiP) and lacasse (Lac). Fungal pretreatment avoids the use of chemicals and the formation of inhibitory compounds. Besides, other advantages of the biological pretreatments are their low capital cost and energy requirement. On the contrary, the main disadvantages are long pretreatment times, carbohydrates loss and low hydrolysis rates compared with other pretreatments (García-Torreiro et al., 2016; Martín-Sampedro et al., 2015). A strategy to overcome the drawbacks of biological pretreatment can be the combination of fungal pretreatment with another physical or chemical pretreatment such as diluted acid (Gui et al., 2013; Ma et al., 2010), alkali (Zhong et al., 2011), steam explosion (Taniguchi et al., https://doi.org/10.1016/j.indcrop.2018.04.078 Received 13 February 2018; Received in revised form 25 April 2018; Accepted 26 April 2018 ⁎ Corresponding author. E-mail address: thelmo.lu@usc.es (T.A. Lu-Chau). Industrial Crops & Products 121 (2018) 10–17 0926-6690/ © 2018 Elsevier B.V. All rights reserved. T