Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor Luana Oliveira Pitol a , Anelize Terezinha Jung Finkler a , Glauco Silva Dias b , Amanda Souza Machado c , Gisella Maria Zanin a , David Alexander Mitchell b , Nadia Krieger c, ⁎ a Departamento de Engenharia Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, Maringá 87020-900, Paraná, Brazil b Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil c Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil ARTICLE INFO Keywords: Biodiesel Lipases Rhizopus microsporus Solid-state fermentation Packed-bed bioreactor Scale-up ABSTRACT A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g -1 at 18 h in the laboratory bioreactor to 113 U g -1 at 20 h in the pilot bioreactor. However, the es- terification activity decreased by only 14%: from 12.1 U g -1 to 10.4 U g -1 . When the FS produced in the la- boratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h. 1. Introduction The most common process for the production of biodiesel involves the transesterification of fats and oils with short chain alcohols, espe- cially methanol, using a homogeneous alkaline catalyst. Biodiesel can also be produced using lipases to catalyze either transesterification or esterification reactions. This enzymatic route is more environmentally friendly than the alkaline catalysis route, as it avoids the generation of large volumes of alkaline wash water. Although biodiesel is produced using the enzymatic route at industrial scale in a few plants in the USA and China [1,2], the high cost of the lipases and the long reaction times prevent the widespread adoption of this technology [3]. Traditionally, lipases have been produced by submerged fermenta- tion (SmF) [4]. However, solid-state fermentation (SSF) represents an interesting alternative to reduce production costs. In fact, Castilho et al. [5] showed that the cost of producing lipases by SSF is about a third of the cost of producing them by SmF. This is because SSF allows the utilization of cheap agro-industrial byproducts as substrates and re- quires a lower total capital investment [5,6]. Over the past decade, our group has been working on a strategy that reduces the costs of producing lipases by SSF significantly in compar- ison to the process considered by Castilho et al. [5], who included the downstream steps of extraction and recovery. Our strategy involves simply air-drying the fermented solids at the end of the fermentation and then adding them directly to the reaction medium to catalyze es- terification and transesterification reactions [7–12]. This strategy also avoids the need for a separate immobilization step. It has recently been adopted by other groups [13,14]. Initially, we used fermented solids to catalyze biodiesel synthesis in media in which the substrates were dissolved in solvents such as n- hexane and n-heptane [7]. More recently, we have used solvent-free media [8–12]. The use of such media not only increases volumetric productivities but also avoids the need for solvent recovery and re- cycling. Our best results were obtained using fermented solids produced by growing Burkholderia cepacia LTEB11 (later reclassified as Bur- kholderia lata) on a mixture of sugarcane bagasse and sunflower seed meal. With this solid, we achieved a conversion of 95% in 46 h for transesterification of soybean oil with ethanol [8] and a conversion of http://dx.doi.org/10.1016/j.procbio.2017.07.019 Received 14 April 2017; Received in revised form 16 July 2017; Accepted 24 July 2017 ⁎ Corresponding author at: Departamento de Química, Universidade Federal do Paraná Cx. P. 19081 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil. E-mail address: nkrieger@ufpr.br (N. Krieger). Process Biochemistry xxx (xxxx) xxx–xxx 1359-5113/ © 2017 Elsevier Ltd. All rights reserved. Please cite this article as: Pitol, L.O., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.07.019