Presentation Outline

Transcription

1 Successful Application of Dextranase in Sugar Beet Factories Eggleston, G. 1, A. Dilks 2, M. Blowers 2, and K. Winters 2

2 Presentation Outline Background information on commercial dextranases: Principle of dextranase action Problem associated with different activity units of commercial dextranases New ICUMSA method to measure dextranase activity at the factory Industrial conditions that affect the efficiency of dextranases Trials on the use of dextranase at Wissington British Sugar factory in the 2009/10 campaign Include effects on second carbonation filtration Cost evaluations ations of the dextranase trial Major conclusions

3 Background Information on Commercial Dextranases

4 Basic chemical structure of dextran ( -(1 6)- -D-glucan) (1 3) D glycoside branching is shown in the figure Glucose molecules are linked by (1 6)-D-glycoside bonds. Approximately 5% branching occurs in cane dextrans, mostly through (1 4), and (1 3), and to a lesser extent (1 2)-D-glycoside linkages.

5 Principle of Dextranase Action Dextranase hydrolyzes(1 (1 6) 6)-D-Glycoside Linkages in Random Endo Sites of fhmwd Dextran High MW dextran (>1000 KDa) H 2 O dextranase Med MW dextran (~ KDa) H 2 O dextranase Lower MW dextran (~ KDa) H 2 O dextranase Oligosaccharides (2 to 10 degrees of polymerization), i.e., isomaltotriose, isomaltose Reaction times are not represented H 2 O glucose* dextranase Dots and connecting lines represent chains of glucose molecules linked by (1 6) glycosidic bonds in the dextran molecule. From Eggleston et al (2005). Process. Biochem.

6 Current Problem At the present time, the activities or strengths of commercial dextranases as vendors use a multitude of methods with different units of activity to measure and quote the activity: For Example Current Different Units of Strength or Activity u/g Du/g U/mL Du/mL kdu-a/g The dextranase market is very dynamic activities and prices can change regularly l

7 A Large Range in Variation Exists in Activities of Commercial Dextranases Available in the U.S. to the Sugar Industry Commercial Dextranase Activity DU/mL Dextranase Classification A Concentrated B nd Non-Concentrated C Non-Concentrated D 5X 8000 Non-Concentrated D 3000 Non-Concentrated Eggleston Classification: Concentrated Dextranases 25,000-58,000 DU/mL Non-Concentrated Dextranases <25,000 DU/mL Eggleston et al (2007) ACS Book Chapter.

8 Activity per Unit Dollar Varies Enormously Among Commercial Dextranases Commercial Dextranase Dextranase Activity DU/mL/$ Classification A Concentrated B Non-Concentrated D 5X Non-Concentrated D Non-Concentrated t

9 Simple Titration Method to Measure Dextranase Activity at Sugar Beet and Sugarcane Factories Advantages of this method: burette 1. Simple 2. No need for sophisticated equipment 3. No need for standards and a standard curve Eggleston and Monge (2004). SPRI Proc.

10 Principle (a) K 3 Fe(CN) 6 + Reducing Sugar Na 2 CO 3 K 4 Fe(CN) 6 (b) 2K 3 Fe(CN) 6 + 2KI Ace c acid 2K 4 Fe(CN) 6 + I 2 (c) 2K 4 Fe(CN) + 3ZnSO 4 K 2 Zn[Fe(CN) ] K 2 SO 4 (d) I 2 + starch indicator starch I 2 complex (e) Starch I 2 complex + 2NaS 2 O 3 Na 2 S 4 O 6 + 2NaI Reaction End Color yellow orange orange dark blue white

11 Measuring Dextranase Activity Using Simple Equipment at the Factory Incubation at 37 o C Boiling Adding reagents Simple Titration

12 Accuracy Spect trophotometr ric method DU/g Thousa ands Comparison of Two Different Dextranase Activity Methods Simple Titration Method DU/mL Thousands R-square = 1 # pts = 4 y = x The excellent correlation between the two methods, confirms the accuracy of the simple titration method. ICUMSA Method GS7-8 (2010): Standard Measurement of the Activity ( ) y of Dextranases at Sugarcane or Sugar Beet Factories using a Simple Titration Method Tentative Status

13 Also Need an ICUMSA Dextranase Method Because the Activity of Commercial Dextranases Changes on Storage U/mL Conc. dextranase activity D Thousands concentrated non concentrated Non-con nc dextranase activity DU/ml Thousands Simulated Factory Storage Stored at room temperature in shaded place (~25 o C) over 90 day processing season Storage time (days) 50 6 Conc. de extranase activity DU U/mL Thousands concentrated Refrigerated Temperature 20 (4 o C) non concentrated Non-conc dextranase activity DU/mL Thousands St ti (d ) 2

14 Industrial Conditions That Affect The Efficiency of Dextranases

15 Effect of Temperature 140 (isomaltotriose peak ht) Rel. Dextranase Activity ( Thou usands Conc 4.6X dil Non-conc. A Non-Conc. B Pure Dextran T ppm 50ppm dextranase ph min Temperature oc isomaltotriose peak ht) Rel. Dextranase Activity ( ands Thousa Juice Addition Evaporator Syrup Addition Dextran in Juice 3177ppm/Brix 100ppm dextranase Conc. 4.6X dil Non-conc. A ph Non-conc. B 25 min Temperature oc

16 Effect of Brix (% Dissolved Solids) Rel. Dextran nase Activity (isomaltot triose peak ht) Tho usands Juices Juice application Evaporator application Evaporator Syrups Brix 1000ppm Dextran 100 ppm dextranase ph min 120 o F Conc 4.6X dil Non conc. A Non conc B ph optimum is between 5 and 6

17 Effect of Dextran Concentrations on Dextranase Action 100 % dextran breakdow wn Antibody dextran (ppm/brix) It is easier to breakdown large amounts of dextran than smaller amounts with dextranase

18 Contact Between Dextran (Substrate) and Concentrated Dextranase (Enzyme) Need Factory Working Solutions Dextran Low Contact Conc dextranase No dilution High Contact :1 dilution Low Contact 1:4 dilution Same ppm of dextranase to juice

19 Studies into the Use of Dextranase at Wissington Factory During the 2009/10 Campaign

20 Problem During the 2009/10 beet campaign the UK experienced a v. cold winter with freezing temperatures for 15 days during January 2010, which was followed by gradual warming conducive to Leuconostoc mesenteriodes growth and dextran formation The large crop and long campaign lengths (up to 180 days) also led to difficult beet processing conditions later in the campaign Problems Caused by Dextran in Factory Higher 2 nd carbonation filtration pressures which significantly slow down beet processing rates High mol. wt. dextran detrimentally affects the crystallization of calcium carbonate - thus small calcium carbonate particles are formed which detrimentally impact 2 nd carbonation filtration Increased viscosity by high concentrations of dextran can cause sugar crystallization problems, but this issue is more prevalent in the cane industry

21 Simple Scheme Showing Dextranase Addition Point Raw Juice to Carbonation Heaters Cossettes RT Diffuser R t = 18 min Cold Raw Juice o C Mingler Hot Raw Juice (71 o C) Dextranase addition Concentrated dextranase added as a working solution (1:4 in tap water)

22 Prior to First Dextranase Trial First Trial 2nd Carb Filtration (Pre Dextranase 12h0m0s Chemically cleaned (1.5% HCl) 28/02/ :00:00 01/03/ :00: factory throughput 000 ** #1 (A) pi0206d.pv 01/03/ :00: BAR (Raw) 2FILT F2 PRESS RESV PRES ** #2 (A) pi0106d.pv 01/03/ :00: BAR (Raw) 2FILT F1 PRESS RESV PRES ** #3 (A) pi0406d.pv 01/03/ :00: BAR (Raw) 2FILT F4 PRESS RESV PRES ** #4 (A) pi0506d.pv 01/03/ :00: BAR (Raw) 2FILT F5 PRESS RESV PRES ** #5 (A) li5085d.pv 01/03/ :00: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK ** #6 (A) TOTSLICE.PV 01/03/ :00: TON/HR Min) TOTAL BEET SLICED ** #7 (A) FI6014C.PV 01/03/ :00: M3/HR (Raw) SODA TO 2ND CARB FLOW ~30 cleanings per day of carbonation filters During the First Dextranase Trial nd Carb Filtration (During Dextranase 12h0m0s 3 ppm concentrated (52000 Du/ml) dextranase A 02/03/ :00:00 03/03/ :00:00 No change in factory throughput because of existing limits on the diffuser ~ 8 cleanings per day ** #1 (A) pi0206d.pv 03/03/ :00: BAR (Raw) 2FILT F2 PRESS RESV PRES ** #2 (A) pi0106d.pv 03/03/ :00: BAR (Raw) 2FILT F1 PRESS RESV PRES ** #3 (A) pi0406d.pv 03/03/ :27:00 BAR (Raw) 2FILT F4 PRESS RESV PRES ** #4 (A) pi0506d.pv 03/03/ :00: BAR (Raw) 2FILT F5 PRESS RESV PRES ** #5 (A) li5085d.pv 03/03/ :00: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK ** #6 (A) TOTSLICEPV 03/03/ :00: TON/HR Min) TOTAL BEET SLICED

23 The First Trial finished on 5 March 2010 after which the 2 nd carbonation filter pressures increased resulting in more acid washing and PCC reactor blockage 2nd Carb Filtration (Post Dextranase 12h0m0s 05/03/ :00:00 06/03/ :00: ** #1 (A) pi0206d.pv 06/03/ :00: BAR (Raw) 2FILT F2 PRESS RESV PRES ** #2 (A) pi0106d.pv 06/03/ :00: BAR (Raw) 2FILT F1 PRESS RESV PRES ** #3 (A) pi0406d.pv 06/03/ :00: BAR (Raw) 2FILT F4 PRESS RESV PRES ** #4 (A) pi0506d.pv 06/03/ :00: BAR (Raw) 2FILT F5 PRESS RESV PRES ** #5 (A) li5085d.pv 06/03/ :00: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK ** #6 (A) TOTSLICE.PV 06/03/ :00: TON/HR Min) TOTAL BEET SLICED ** #7 (A) FI6014C.PV 06/03/ :00: M3/HR (Raw) SODA TO 2ND CARB FLOW Second Trial The Second Trial re-started with another dextranase B (54302 DU/ml) at 1 ppm, but the filtration conditions continued to be poor with some periods of markedly lower throughputs being experienced: During the Second Dextranase Trial 2nd Carb Filtration ( Dextranase 12h0m0s 1 ppm concentrated (54302 DU/ml) dextranase B lower /03/ :00:00 06/03/ :00:00 throughput period **#1 (A) pi0206d.pv 05/03/ :56: BAR (Raw) 2FILT F2 PRESS RESV PRES **#2 (A) pi0106d.pv 05/03/ :56: BAR (Raw) 2FILT F1 PRESS RESV PRES **#3 (A) pi0406d.pv 05/03/ :56: BAR (Raw) 2FILT F4 PRESS RESV PRES **#4 (A) pi0506d.pv 05/03/ :56: BAR (Raw) 2FILT F5 PRESS RESV PRES **#5 (A) li5085d.pv 05/03/ :56: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK **#6 (A) TOTSLICE.PV 05/03/ :56: TON/HR Min) TOTAL BEET SLICED **#7 (A) FI6014C.PV 05/03/ :56: M3/HR Min) SODA TO 2ND CARB FLOW

24 Second Trial Trend of second carbonatation filtration and throughput conditions during the Second Trial showing effect of PCC reactor blockage. 2nd Carb Filtration ( Dextranase 12h0m0s 1 ppm concentrated (54302 DU/ml) dextranase B 06/03/ :00:00 06/03/ :00: ** #1 (A) pi0206d.pv 06/03/ :00: BAR (Raw) 2FILT F2 PRESS RESV PRES ** #2 (A) pi0106d.pv 06/03/ :00: BAR (Raw) 2FILT F1 PRESS RESV PRES ** #3 (A) pi0406d.pv 06/03/ :00: BAR (Raw) 2FILT F4 PRESS RESV PRES ** #4 (A) pi0506d.pv 06/03/ :00: BAR (Raw) 2FILT F5 PRESS RESV PRES ** #5 (A) li5085d.pv 06/03/ :00: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK ** #6 (A) TOTSLICE.PV 06/03/ :00: TON/HR Min) TOTAL BEET SLICED ** #7 (A) FI6014C.PV 06/03/ :00: M3/HR Min) SODA TO 2ND CARB FLOW

25 Second Trial Dextranase B addition rate was increased to 2.1 ppm and conditions improved: 2nd Carb Filtration (Dextranase 12h0m0s 2.1 ppm concentrated (54302 DU/ml) dextranase B 09/03/ :00:00*** 09/03/ :00:00*** ** #1 (A) pi0206d.pv 09/03/ :00: BAR (Raw) 2FILT F2 PRESS RESV PRES ** #2 (A) pi0106d.pv 09/03/ :00: BAR (Raw) 2FILT F1 PRESS RESV PRES ** #3 (A) pi0406d.pv 09/03/ :00: BAR (Raw) 2FILT F4 PRESS RESV PRES ** #4 (A) pi0506d.pv 09/03/ :00: BAR (Raw) 2FILT F5 PRESS RESV PRES ** #5 (A) li5085d.pv 09/03/ :00: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK ** #6 (A) TOTSLICE.PV 09/03/ :00: TON/HR Min) TOTAL BEET SLICED ** #7 (A) FI6014C.PV 09/03/ :00: M3/HR Min) SODA TO 2ND CARB FLOW Improved conditions allowed the increase in alkali addition to the 2 nd carbonation vessel to aid limesalts control Sodium carbonate addition increased 4-fold without any detrimental impact on 2 nd carbonation filtration and allowed a reduction in filtered 2 nd carbonation limesalts from ~0.01g to 0.086gCaO/100Brix

26 End of Campaign During the remainder of the 2009/10 0 campaign (after dextranase ase trial) 2 nd carbonation filtration continued to impact on factory throughput. This continued to lead to increased chemical cleaning of filters and higher 2 nd carbonation limesalts due to the limited addition of alkali to the process. Trend of second carbonation filtration and throughput conditions after the end of the second dextranase trial 2nd Carb Filtration ( Dextranase 12h0m0s 10/03/ :00:00 11/03/ :00: High rate of cleaning ** #1 (A) pi0206d.pv 10/03/ :22: BAR (Raw) 2FILT F2 PRESS RESV PRES ** #2 (A) pi0106d.pv 10/03/ :22:35 BAR (Raw) 2FILT F1 PRESS RESV PRES ** #3 (A) pi0406d.pv pv 10/03/ :22: BAR (Raw) 2FILT F4 PRESSRESVPRES RESV ** #4 (A) pi0506d.pv 10/03/ :22: BAR (Raw) 2FILT F5 PRESS RESV PRES ** #5 (A) li5085d.pv 10/03/ :22: PERCENT (Raw) GAUDFRIN ACID DRAIN TANK ** #6 (A) TOTSLICE.PV 10/03/ :22: TON/HR Min) TOTAL BEET SLICED ** #7 (A) FI6014C.PV 10/03/ :22: M3/HR Min) SODA TO 2ND CARB FLOW Higher limesalts

27 Effect of Dextranase on 2 nd Carbonation Particle Size

28 Continuous Precipitated Calcium Carbonate (PCC) Reactors Used during the 2009/10 campaign to aid 2 nd carbonation filtration How Does It Work? By adding additional PCC to the second carbonation vessel, smaller calcium carbonate crystals formed within the gassing vessel agglomerate -this results in a lowering of the very small crystals fines in the 2 nd carbonation juice leading to improved filterability References Burrough and Wones (2003). The Effect of frost damaged beet and other factors on Dorr 2 nd Carbonation juice particle size, Proc. of European Society for Sugar Technology (ESST), pp Struijs, Jaspers, van Dijk (2009). Methods used in the Netherlands to limit frost j, p, j ( ) damage and to process frost-deteriorated beets,proc. of European Society for Sugar Technology (ESST), pp 33-38

29 % F r e q u e n c y fines 2nd Carbonation Juice with and without PCC addition and Dextranase PCC only no PCC, no Dextranase Dextranase and PCC Dextranase only Particle size µm Typical 2 nd carbonation particle size distribution, with no PCC and no dextranase, with PCC only, with dextranase only and with PCC and dextranase.

30 14 12 % 10 F r e q u e n c y fines 2nd Carbonation Juice with and without PCC addition and Dextranase PCC only no PCC, no Dextranase Dextranase and PCC Dextranase only Particle size µm Modal Particle size/ µm % < 3µm No PCC and no dextranase With PCC addition only With Dextranase only With PCC and Dextranase improved factory operations p y p more stable beet-end flows smoothing of vapor demands a reduction in the amount of recycle to raw juice from filter cleaning

31 Dextran Levels In Juice Streams British Sugar haze dextran method at absorbance 720 nm Dextran levels in 2 nd carbonation juice of 60 ppm lead to filtration problems (information supplied by Nordic Sugar)

32 Dextran Levels in Juice Streams ppm Dextranase A Dextranase on 1 ppm Dextranase B 140 Dextranase addition stopped Dextran/ pp pm Trial start 120 Dextranase back on at 9:15am 100 Dextranase 80 Ran Out 60 Addition increased to 2.1 ppm Dextranase Ran Out filtration problems Time

33 Cost Evaluation of the Dextranase Trial

34 Breakdown of Cost Evaluation of the Dextranase Trial based on 3 ppm of Concentrated (52000 DU/ml) Dextranase A addition as much more cost effective (activity it per unit $ higher) h Cost of conc. Dextranase % Reduction Trial Saving $/day Cost of acid washing filters 73% CaO to process* 11% Anthracite 9% Throughput h costs 84% (LOP) Total Savings 3,180 * Lime required to aid filtration Other Financial Benefit: Reduction in water to ponds and shorter campaign lengths

35 Major Conclusions Commmercial dextranases occur in concentrated and nonconcentrated forms; activities, and activites per unit $, vary widely A new ICUMSA tentative method is now available to uniformally and easily measure the activity of dextranases at the factory to (1) economically compare activites of different commercial dextranases, (2) monitor the changing activities of dextranases on storage, and (3) measure activity of delivered batches 2 nd Carbonation filtration significantly improved by adding dextranase in a number of ways: Frequency of 2 nd carbonation filter chemical cleaning reduced by 73% Reduced chemical usage Reduction in the volume of water discharged to the effluent treatment plant with the used acid by 418 m 3 /day Adding dextranase significantly improved beet throughput $3180/day saved by using concentrated DU/ml dextranase (3 ki l ti )

36 Acknowledgements British Sugar Operations Services Science Department Wissington Factory Laboratory Team Geoff Parkin Garry Bowler Bob Howe Barbara Muir Jan-Maarten de Bruijn Arend Wittenberg (Suiker Unie) Jan Struijs (Suiker Unie) Eldwin St. Cyr (USDA)