View drawing guides 1. Right-click on slide and select ’Grid and Guides...’ 2. Check ’Display drawing guides on screen’ 3. Select ’OK’ How Ethanol Production Technology Impacts DDGS Value in Animal Nutrition Don Cannon, Ph.D.; DuPont Industrial Biosciences Biorefineries Applied Innovation Center in Cedar Rapids, IA in collaboration with DuPont Danisco Animal Nutrition (Milan Hruby, Ph.D.) 18th Annual Distillers Grains Symposium, Dallas, TX; May 14, 2014Copyright © 2012 DuPont. All rights reserved. Phytic Acid Molecular Structures of Interest phyticphytateinositol acid Text starts with no bullet Click once on ’Increase Indent’ for bullet and click max. three times for more bulletdesign To get previous design back, click on ’Decrease Indent’ 3- + 6 PO4 Change Date and insert PresentationChange the Reference Title under >Insert >Headerfield under and ‘View’ Footer – and’Header write in and Fixed Footer’ Date andfield and field write‘Footer in ’ field>Apply ‘Footer’ to All DeactivateDeactivate by clicking by clicking off off thethe checkboxes checkbox Some Molecular Facts about Phytic Acid myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate phytate (ionic form); phytin (mineral complexed); IP6 Text starts with no bullet Click once on reactive acidic compound that chelates cations ’Increase Indent’ for bullet • more strongly associates with divalent over monovalent and click max. three times • 12 protonation sites; 6 pKa’s more acidic than the other 6 pKa’s for more bulletdesign 1 to 3 %w/w in seeds, cereals, and oilseeds used in animal feed To get previous design back, • different locations in different grains click on ’Decrease Indent’ • associated with protein in oilseeds and grain legumes • various amounts of endogenous phytase (little to none in corn, high variability in wheat) phytin accounts for up to 80% of all P in animal feed phytin must be degraded for sufficient P availability (and reduced environmental impact) availability of energy and amino acids is reduced when IP6 is present in ingredients and animal feeds Phytate alters the secretion of endogenous compounds (animals digestive enzymes, HCl, mucins, etc.) DDGS with a reduced phytate content may have a superior nutritional value compared Change Date and insert with conventional DDGS Presentation Title under >Insert >Header and Footer phytin levels in DDGS can vary greatly – phytase use in fuel ethanol process and write in Fixed Date field and field ‘Footer’ >Apply to All Deactivate by clicking off the checkboxes Phytate Content and Endogenous Phytase Enzyme Activity in Grains Grain/Cereal Phytate 1) Endogenous (% ds) Phytase 2) (units/kg) Corn 0.8 -1.2 ~20 Sorghum 0.8 -1.1 ~20 Wheat 0.25 -1.37 1193 Barley 0.38 -1.16 582 Rye 0.54 -1.46 5130 Triticale 0.50 -1.89 n.d. Reference: 1) Food Phytases,Edited by Reddy,N. R and Sathe,S. K (2002) CRC Press, New York 2) Ravidran V., W.L. Bryden and E. T. Kornegay. 1995. Phytates: Occurrence, bioavailability and implications in poultry nutrition. Poultry and Avian Biology Reviews. 6 (2): 125-143 DuPont Danisco Animal Nutrition Phytases granular phytase enzyme for reduced feed costs and a better environment Text starts with no bullet Click once on ’Increase Indent’ for bullet and click max. three times for more bulletdesign To get previous design back, click on ’Decrease Indent’ Change Date and insert Presentation Title under >Insert >Header and Footer and write in Fixed Date field and field ‘Footer’ >Apply to All Deactivate by clicking off the checkboxes Impact of Phytate on Fuel Ethanol Production http://en.wikipedia.org/wiki/File:Ethanol_plant.jpg (photo by Steven Vaughn), May 2011 Why Do We Care About Phytate in Fuel Ethanol Production? calcium is needed for direct protein contact a-amylase stability starch-protein can reduce stability of complexes are less enzymes soluble Starch-binding increases viscosity Zinc is an essential mineral for yeast 7 The Genencor Timeline for Phytase Application Paper presented at March: Product Launch World Ethanol 2004 & Text starts with no bullet GC 491 a phytase Ethanol Production Article Phytase in fuel ethanol Click once on production published in the ’Increase Indent’ for bullet product for ethanol Workshop Maximizing production by George Virgil International Sugar Journal Ethanol and click max. three times Production from for more bulletdesign Grains Product Launch Product Launch MAXALIQ One SPEZYME® RSL To get previous design back, click on ’Decrease Indent’ 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 April: Paper Product Launch presented at the DISTILLASE SSF+ 2001 Detmold Application of a blend for SSF which Phytases in Fuel Starch Conference contains phytase Ethanol by Gerhard Production Konieczny Janda Change Date and insert Presentation Title under >Insert >Header and Footer and write in Fixed Date field and field ‘Footer’ >Apply to All Fuel Ethanol Workshop Deactivate by clicking presentation given by Dr. Jay Shetty off the checkboxes Engineered Phytases with Enhanced Thermostability 9 Where Phytase is Applied in Fuel Ethanol Production Today Text starts with no bullet Click once on ’Increase Indent’ for bullet and click max. three times for more bulletdesign To get previous design back, click on ’Decrease Indent’ In Liquefaction To enhance the pH and temperature stability of alpha amylases Liquefaction at pH 5.0 to 5.3 reduces the amount of H2SO4 and anhydrous ammonia needed • Lower total cost of liquefaction • Decreasing the sulfur present in wet distillers grains and DDGS In Fermentation Change Date and insert To generate inositol*, an important yeast nutrient Presentation Title under >Insert >Header and Footer To maximize the level of free phosphate in residuals (DDGS) and write in Fixed Date field and field ‘Footer’ >Apply to All Reference: * Determining the effects of inositol supplementation and the opi1 mutation on ethanol tolerance of Saccharomyces cerevisiae Erin L. Krause, Deactivate by clicking Manuel J. Villa-García, Susan A. Henry, Larry P. Walker. Industrial Biotechnology. October 2007, 3(3): 260-268. doi:10.1089/ind.2007.3.260. off the checkboxes Dry Grind Liquefaction Has Some Issues Backset, Water Liquefaction has typically required two pH Plant Backset and Corn adjustments A 4.3 5.4 • From 5.1 to 5.8 B 4.2 5.7 • Back to the chosen fermentation pH (5.8 to C 4.3 5.1 4.5) It requires tight pH and temperature control D 3.4 5.6 • A pH variance of 0.2 can hurt performance E 3.7 5.2 • A temperature variance of +2° can hurt F 4.2 4.8 performance G 4.3 5.1 Adds significant sulfur to the system and eventually to DDGS (0.08 to 0.12% dmb) H 3.7 4.7 At many plants, two enzyme doses are I 4.3 5.1 necessary – adding complexity J 4.5 5.0 Poor liquefaction reduces ethanol yields and K 3.6 4.9 plant performance L 3.4 5.1 M 3.8 4.7 Average 4.0 ± 0.4 5.1 ± 0.3 Recommended Conditions for Liquefaction Conventional a-amylase use: Solids 30 to 36% wt/wt pH Range 5.6 to 6.0 Temperature Range 170 to 190° F Liquefaction Time 90 to 140 minutes Recommended Dose: 0.020 to 0.024 %wt/wt as-is grain SPEZYME® RSL (a-amylase + phytase) use: Solids 30 to 36% wt/wt pH Range 5.0 to 6.0 Temperature Range 170 to 195° F Liquefaction Time 90 to 140 minutes Recommended Dose: 0.020 to 0.024 %wt/wt as-is grain 12 Ion Chromatography for Inositol Phosphates 15 various phytase dosings in slurry 14 no phytase 1x phytase Text starts with no bullet 13 Click once on 12 3x phytase ’Increase Indent’ for bullet Dionex OmniPac PAX-100 column 11 chloride used in 0.20M NaOH gradient (2.5% IPA and click max. three times 10 isocratic) for more bulletdesign sample prep 5%-25% in 8 min 9 To get previous design back, 25%-70% in 7 min 8 click on ’Decrease Indent’ mV regen and equil steps 7 IP6 suppressed conductivity detection 6 phosphate, nitrate and supernatant dilution to 0.5M HCl 5 sulfate IP4 4 3 IP2 IP3 2 1 0 IP5 Change Date and insert 0 2 4 6 8 10 12 14 16 18 20 Presentation Title under >Insert >Header and Footer Minutes and write in Fixed Date field and field ‘Footer’ >Apply to All liquefaction supernatant: 0.25 %w/v supernatant, (31.6 DS% equates to 0.8 %w/w corn as is basis) Deactivate by clicking off the checkboxes The Analytical Method: sample preparation multiple chemical equilibriums to consider: Ka’s...Kchel’s...Ksp’s 12- 12- 2+ IP6H12 ↔ IP6 ; IP6 + M ↔ IP6M6; IP6 s ↔ IP6 aq Text starts with no bullet Click once on ’Increase Indent’ for bullet and click max. three times for more bulletdesign To get previous design back, click on ’Decrease Indent’ 1%1% IP6 IP6 in in 1%1% IP6 IP6 in in 1%1% IP6 IP6 in in 1%1% IP6 IP6 in in 2+ 2+ Change Date and insert HHO2O + HHOO + 200200 ppm ppm Mg Mg2+ 200200 ppm ppm Mg Mg2+ Presentation Title under 2 22 >Insert >Header and Footer 1M HCl 1M NaOH + 1M HCl + 1M NaOH and write in Fixed Date field and field ‘Footer’ >Apply to All Deactivate by clicking off the checkboxes Phytate is Soluble in Acidic Environments (i.e. phytate is NOT soluble in many conditions) Applegate, T.J. and Angel, R.; “Phytase: Basics of Enzyme Function,” Purdue Univ. Extension, Farm Animal Management @ Purdue, 2004, Doc. AS-560-W. 15 Phytase Use at Low pH Reduces the Antinutrient Effects of Phytate Text starts with no bullet Phytate is more soluble at low pH and Click once on ’Increase Indent’ for bullet more susceptible to phytase attack and click max.