Viscosity reduction

Process Biochemistry 49 (2014) 1950–1957 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Viscosity reduction of cassava for very high gravity ethanol fermentation using cell wall degrading enzymes from Aspergillus aculeatus Aphisit Poonsrisawat a , Sittichoke Wanlapatit b , Atchara Paemanee c , Lily Eurwilaichitr a , Kuakoon Piyachomkwan b , Verawat Champreda a,∗ a Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Klong Luang, Pathum Thani 12120, Thailand b Cassava and Starch Technology Research Unit, National Center for Genetic Engineering and Biotechnology, Kasetsart University, Bang Khen, Bangkok 10900, Thailand c Genome Institute, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Klong Luang, Pathum Thani 12120, Thailand a r t i c l e i n f o Article history: Received 28 March 2014 Received in revised form July 2014 Accepted 17 July 2014 Available online August 2014 Keywords: Aspergillus aculeatus Bioethanol Cell wall degrading enzyme Very high gravity fermentation Viscosity a b s t r a c t Cassava is an important feedstock for bioethanol production; however, its use in very high gravity (VHG) fermentation is limited by the high viscosity of mash due to the presence of plant cell wall derived polysaccharides In this study, viscosity reduction of cassava root mash, chips, and pulp was achieved using a mixture of cell wall degrading enzymes prepared from solid state fermentation of Aspergillus aculeatus BCC17849 Proteomic analysis showed the mixture contained endo- and exo-acting cellulases, hemicellulases, and pectinases from various glycosyl hydrolase families Enzymatic pretreatment of cassava substrates with the enzyme mixture containing endoglucanase, FPase, xylanase, polygalacturonase, ␤-glucanase, and mannanase activities at 45 ◦ C, pH 5.0 for h reduced viscosity to the operating level of 300 g/L) to yield ethanol at high A Poonsrisawat et al / Process Biochemistry 49 (2014) 1950–1957 concentration in the fermentation product [3] This leads to an increase in fermenter throughput, saving in process water, reduced risk of bacterial contamination, and reduction in energy usage for downstream processing and effluent treatment cost VHG fermentation can hence lead to improvement of the overall process economics with no major alteration to the existing facilities [4] Currently, VHG fermentation has been mostly studied for ethanol production from cereal grains including wheat, corn, barley, rye, sorghum, and finger millet [3,5–10], while its application in root or tuber mash prepared from dicotyledonous plants is still limited This is mainly due to the highly viscous nature of root and tuber mashes caused by their high pectin content, which leads to entrapment of water in the cell wall matrix [11] This results in difficulties in mixing and solid–liquid separation leading to incomplete hydrolysis of starch to sugar, which reduces the overall fermentation efficiency [12,13] Enzymatic treatment with plant cell wall degrading enzymes has been used for viscosity reduction of mashes prepared from various sources, mostly from cereal grains Viscosity reduction results from the degradation of fibrous polysaccharides in the substrates In contrast with grain sources, enzymatic viscosity reduction for VHG fermentation has been less well studied for root and tuber mashes, with reports only for sweet potato [13], potato [14], and sugar beet [15], and to our knowledge, none on cassava feedstocks In this study, a mixture of enzymes with multiple cell wall degrading activities from an Aspergillus aculeatus strain was evaluated for viscosity reduction of fermentation mashes prepared from different cassava feedstocks for VHG fermentation The fungal enzyme mixture was characterized by proteomic analysis, which revealed multiple cell wall degrading enzymes with functions associated with viscosity reduction Materials and methods 2.1 Materials Cassava roots (variety Rayong at 10 months harvesting time) and cassava pulp were obtained from Chonchareon Ltd., Chonburi, Thailand Dried ground cassava chips were obtained from the Advance Flour Ltd., company, Chachoengsao, Thailand The cassava products were analyzed for moisture, protein, ash, and sand content using a standard method [16] Starch content was determined using the polarimetric method, according to the standard method of the Commission of the European Communities (1999) [17] and Thai Industrial Standard for Tapioca Flour/Starch [18] Neutral and acid detergent fiber (NDF and ADF) and lignin were analyzed using the standard AOAC method (1998) (Method 992.16 and Method 973.18) A aculeatus BCC17849 was obtained from the BIOTEC Culture Collection, Thailand (www.biotec.or.th/bcc) and maintained on potato dextrose agar (PDA) Polysaccharides used as substrates in enzymatic activity analysis were obtained from Sigma–Aldrich 2.2 “Multi-enzyme” production by solid state fermentation A aculeatus grown on rice grains was inoculated in liquid spore production medium (2% peptone, 2% yeast extract, 1% KH2 PO4 , and 1% glucose) at the ratio of g rice: mL medium The culture was incubated at 30 ◦ C for d The fungal spores were resuspended in 0.1% Tween 80 and the suspension was used as the starter culture The starter was inoculated into the solid-state production medium (20 kg on dried weight basis) containing 3.5:1.5 (w/w) of dried cassava pulp: soybean meal, supplemented with 20 g/kg yeast extract, 20 g/kg peptone, 250 mL/kg salt solution (40 g/L KH2 PO4 , 90 g/L Na2 HPO4 , and g/L KCl), and 10 mL/kg trace element solution (14.3 g/L ZnSO4 ·H2 O, 0.5 g/L NiCl2 ·6H2 O, 2.5 g/L CuSO4 ·5H2 O, 1951 and 13.8 g/L FeSO4 ·H2 O) and the moisture was adjusted to 70% The culture was grown at 30 ◦ C for d in a tray reactor system The enzyme was extracted by adding 50 mM sodium acetate buffer, pH 5.5 containing 0.1% Tween 80 to the solid state culture with the ratio of 1:4 (solid:liquid) The suspension was mixed and incubated at ◦ C overnight The crude enzyme mixture was clarified by centrifugation and filtered sequentially through 0.45 and 0.2 ␮m nitrocellulose membranes This mixture of enzymes with multiple activities, referred to hereafter as “multi-enzyme” was then concentrated 15-fold on a Minimate tangential flow filtration system using a 30 kDa MWCO membrane (Pall, Port Washington, NY) The enzyme was stored at ◦ C and used for subsequent studies 2.3 Enzyme activity assay Polysaccharide degrading activities were analyzed based on the amount of liberated reducing sugars using the 3,5-dinitrosalisylic acid (DNS) method [19] One-hundred microliter reaction mixtures contained the appropriate dilution of enzyme in 50 mM sodium acetate buffer, pH 5.0 and 1% of the corresponding substrate: carboxymethyl cellulose for CMCase activity, birchwood xylan for hemicellulase activity, pectin from citrus for pectinase activity, ␤-glucan for ␤-glucanase activity, and locust bean gum for mannanase activity The reaction was incubated at 45 ◦ C for 30 Cellulase activity, expressed as filter paper unit (FPU), was analyzed in a mL reaction using Whatman number filter paper (size cm × cm) as the substrate and incubated at 45 ◦ C for 60 The amount of reducing sugars was determined from the absorbance measurement at 540 nm and interpolated from a standard curve of the corresponding sugar ␤-Glucosidase and ␤-xylosidase activities were determined using p-nitrophenyl-␤d-glucopyranoside (PNPG) and p-nitrohenyl-␤-d-xylopyranoside (PNPX) as the substrate [20] One international unit (IU) was defined as the amount of enzyme which produced ␮mol of reducing sugar or p-nitrophenolate in under the assay conditions 2.4 Proteomic analysis The multi-enzyme preparation was applied to a 10% SDS-PAGE gel and separated using a MiniProtean II cell (Biorad, Hercules, CA) The protein bands were visualized using Coomassie blue R-250 staining and were manually excised into four fractions according to their apparent molecular weights (up to 116.0 kDa) The polypeptides in gel were then digested with trypsin (Ettan Spot Handling Workstation User Manual 18-1153-55 Edition AC, GE Healthcare Biosciences, Uppsala, Sweden) The tryptic peptides were resuspended with 0.1% formic acid and analyzed on a SYNAPT HDMS mass spectrometer (Waters, Milford, MA) according to Wongwilaiwalin et al [21] All MS/MS spectra were searched using the Mascot® search engine (Matrix Science, Boston, MA) against the NCBI-nr database using the following criteria: enzyme trypsin, static modification of cysteine (+57.05130 Da), and differential modification of methionine (+15.99940) The search results were filtered by cross-correlation versus charge state (+1 ≥ 1.5, +2 ≥ 2.0, +3 ≥ 2.5) and protein probability (minimum 1.00E-3) The candidate protein queries were mapped to the UniProt Knowledge base (UniProtKB) [22] 2.5 Enzymatic viscosity reduction of cassava substrates The effects of the multi-enzyme on viscosity reduction of cassava substrates were studied under conditions for VHG fermentation Cassava roots (unpeeled) were chopped and homogenized using a blender, and the solid content adjusted to 32% (w/w) on dried weight basis with tap water Ground cassava chips were 1952 A Poonsrisawat et al / Process Biochemistry 49 (2014) 1950–1957 used for preparation of slurry at 32% (w/w) solid content Fresh cassava pulp was adjusted to 10% (w/w) solid content The substrates were adjusted to pH 5.0 and treated with 0.05–2.5% (w/w) of the multi-enzyme at 45 ◦ C for h with 160 rpm mixing Samples were collected after 0, 30, 60, and 120 of enzyme treatment for viscosity analysis using a Rapid Visco Analyzer (RVA 4, Newport Scientific, Australia) at 32 ◦ C with an agitation speed of 160 rpm for Reactions with no-enzyme were used as controls The experiments were done in duplicate The target operational viscosity is set at