Available online at www.sciencedirect.com Enzyme and Microbial Technology 43 (2008) 124–129 Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer’s spent grain Solange I. Mussatto ∗ , Marcela Fernandes, Adriane M.F. Milagres, Inˆ es C. Roberto Department of Biotechnology, Engineering College of Lorena, University of S˜ ao Paulo, Estrada Municipal do Campinho s/no., Cep: 12602-810 Lorena/SP, Brazil Received 2 July 2007; received in revised form 5 October 2007; accepted 12 November 2007 Abstract Enzymatic hydrolysis of brewer’s spent grain in three different forms: original (untreated), pretreated by dilute acid (cellulignin), and pretreated by a sequence of dilute acid and dilute alkali (cellulose pulp), was studied to verify the effect of hemicellulose and lignin on cellulose conversion into glucose. The hydrolysis was carried out using a commercial cellulase concentrate (Celluclast 1.5 L) in an enzyme/substrate ratio of 45 FPU/g, 2% (w/v) substrate concentration, 45 ◦ C for 96 h. According to the results, the cellulose hydrolysis was affected by the presence of hemicellulose and/or lignin in the sample. The cellulose conversion ratio (defined as glucose yield + cellobiose yield) from cellulignin was 3.5-times higher than that from untreated sample, whereas from cellulose pulp such value was 4-times higher, correspondent to 91.8% (glucose yield of 85.6%). This best result was probably due to the strong modification in the material structure caused by the hemicellulose and lignin removal from the sample. As a consequence, the cellulose fibers were separated being more susceptible to the enzymatic attack. It was concluded that the lower the hemicellulose and lignin contents in the sample, the higher the efficiency of cellulose hydrolysis. © 2007 Elsevier Inc. All rights reserved. Keywords: Brewer’s spent grain; Enzymatic hydrolysis; Cellulase; Cellulose; Hemicellulose; Lignin 1. Introduction Agricultural, agro-industrial and forestry lignocellulosic residues have potential as cheap and renewable feedstocks for large-scale production of fuels and chemicals. Currently, biopro- cessing of lignocellulosics is focused on enzymatic hydrolysis of the cellulose fraction to glucose, followed by fermentation to fuel-grade ethanol. However, enzymatic hydrolysis of cellu- losic materials to produce fermentable sugars has also enormous potential in meeting global food and energy demand via biolog- ical route [1]. Enzymatic hydrolysis of cellulose is a reaction carried out by cellulase enzymes, which are highly specific [2]. Cellulases are usually a mixture of several enzymes, among which at least three major groups are involved in the hydrolysis process of cellulose: (1) -1-4-endoglucanase (EC 3.2.1.4.), which attacks regions of ∗ Corresponding author. Universidade do Minho, Departamento de Engen- haria Biol´ ogica, Campus de Gualtar, 4710-057 Braga, Portugal. Tel.: +351 253 605 413; fax: +351 253 678 986. E-mail addresses: solange@deb.uminho.pt, solangemussatto@hotmail.com (S.I. Mussatto). low crystallinity in the cellulose fiber creating free chain ends; (2) -1-4-exoglucanase or cellobiohydrolase (EC 3.2.1.91.), which degrades the molecule further by removing cellobiose units from the free chain ends; (3) -glucosidase or cellobiase (EC 3.2.1.21.), which hydrolyzes cellobiose to produce glucose [3–5]. Because enzymatic hydrolysis is a heterogeneous reac- tion and requires direct physical contact between enzyme and substrate, the enzymes must diffuse from the bulk aqueous solu- tion to the particle surface, diffuse through physical barriers such as lignin, adsorb on the substrate surface, and then catalyze the hydrolysis [5,6]. Consequently, these reactions are complex and can be affected by the physicochemical properties of the sub- strate such as crystallinity, degree of polymerization, surface area, and lignin and hemicellulose contents [6–10]. In lignocellulosic materials cellulose is physically associated with hemicellulose, and physically and chemically associated with lignin. The presence of these two fractions is reported to make the access of cellulase enzymes to cellulose difficult, thus reducing the efficiency of the hydrolysis. The effect of hemicel- lulose and lignin matrix on enzymatic hydrolysis of cellulose has been investigated for several lignocellulosic materials including dairy manure [11], corn stover [12], sugarcane bagasse [13], and 0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.11.006