Effects of activation on cellulose dissolution Enzymatic treatments for improving cellulose dissolution Patrick Navard

Transcription

1 Effects of activation on cellulose dissolution Enzymatic treatments for improving cellulose dissolution Patrick Navard MINES Paristech Centre de Mise en Forme des Matériaux - CEMEF Sophia Antipolis - France 1

2 Outline I Dissolution and activation II Dissolving cellulose in NaOH-water III Effects of enzymatic treatments IV Effects of pectinase treatments V Conclusions 2

3 I Dissolution and activation 3

4 P Navard 4

5 Diluted Concentrated f< f* one chain in 4/3p(<S 2 > 0 ) 3/2 Semi-diluted f > f** = (m l V p )/l 3 f* < f < f** = (m l V p )/l 3 P Navard 5

6 Free energy: H: enthalpy S: entropy Solution thermodynamics G = H TS DG m = G 12 (G 1 + G 2 ) G 12 : free energy of the solution G 1 : free energy of solvent G 12 : free energy of solute

7 Polymers Calculation of DS m The idea of Flory and independently Huggins is to cut the polymer chain into pieces that have the same volume as solvent molecule. f i : volume fraction of component i N i number of molecules of component i DS m = -k ( N 1 lnf 1 + N 2 lnf 2 ) Or With r i : number of segments of component i r i = 1 for solvent and r i >> 1 for polymer 7

8 DS m = -k ( N 1 lnf 1 + N 2 lnf 2 ) or D S m f f 1 f 2 f r r 1 2 A[ ln ln ] 1 2 With r i : number of segments of component i r i = 1 for solvent and r i >> 1 for polymer D S m >> DS m r r r >> r

9 D S m >> DS m r r r >> r Mixing two simple components Mixing one simple component and a polymer Difficult to mix solvent and polymers 9

10 Dissolution Can you cite entropic factors which will help dissolution? Positive contributions of entropic factors: - entropy of mixing (translational entropy): influence of molar mass - entropy of conformation mobility (rigidity) - if applicable, entropy gain due to counter ions What about kinetics??

11 Dissolution and kinetics Physical processes linked to kinetics A B C D E B- solid phase goes above Tg C- point of chain disentanglement D- chains can move to the solvent phase

12 Dissolution and kinetics Cellulose should be easy to dissolve: Not too rigid backbone, less rigid than derivatives that are easily soluble: Polymer Cellulose Cellulose Cellulose Cellulose acetate Hydroxyethyl cellulose Solvent cadoxen FeTNa NMMO acetone water q/nm Large amount of OH groups, able to bind to other compounds or to be dissociated Amphiphilic molecule Why is it so difficult to dissolve cellulose?

13 Cellulose dissolution: difficultés Cellulose as a polymer: should follow thermodynamics and kinetics laws Cellulose as a material extracted from Nature: more complicated than a simple polymer

14 Cellulose dissolution Some solvent families which are or could be industrially used: Amine oxides Phosphoric acids Ionic liquids NaOH-based solvents Let s compare with polyethylene Aromatic hydrocarbons (toluene or xylene) Chlorinated solvents ( trichloroethane or trichlorobenzene)

15 Cellulose dissolution In many cases, there is a difficulty for solving all the cellulose fractions. This is particularly sensitive when willing to use NaOHbased solvents. The same difficulty, usely called accessibility, is true when willing to transform cellulose into glucose. Cellulose-based materials are thus «activated» in order to help dissolution or degradation.

16 Cellulose activation Can you cite activation methods? Mechanical activation Steam explosion Ball milling Chemical activation Treatment with various chemicals to remove hemicellulose or lignin Enzymatic treatments. 16

17 Cellulose activation Activation of cellulose to improve solubility of pulps in NaOHwater Increasing the number of accessible paths inside the cell wall structure able to bring ions close to cellulose chains? But cellulose crystals are dissolving, and they are very dense. Opening the structure which could be blocked by hemicellulose or lignin? But these compounds are soluble in these solvents?.all these activation methods are strongly decreasing molar mass. This is not the only parameter able to explain activation effects. 17

18 II - Dissolving cellulose in NaOH-water 18

19 Cellulose dissolution: NaOH/ water 1850: John Mercer found that the already-known interactions between cellulose and soda could be used for producing a beautiful fibre. Addition of ZnO or compounds like urea are improving mercerisation. The first to find out that cellulose can be soluble in NaOH-water is G. Davidson (British cotton) in Cellulose must be treated, of low DP. Crystalline parts are less soluble. Following Mercer, he found that the addition of compounds like ZnO is strongly improving dissolution. 19

20 Cellulose dissolution: NaOH/ water Sa, % -5 C 4 C 15 C 30 C Asahi Chemicals in Japan in the 1980 s Steam exploded cellulose was readily soluble in NaOH-water (Yamashiki et al. 1988) Ca, wt% 13 (O3-H O5 ) H-bonds must be weakened to favor dissolution. Crystallinity or accessibility are not the only factors limiting solubility. Solutions are molecularly dispersed. Theta solvent at 40 C. Cellulose is a semi-rigid chain having a partially free draining behaviour. The unperturbed dimensions decreased with temperature increase. If the proper intermolecular hydrogen bonds have been broken, the factor that controls dissolution is the structure of the alkali ions. 20

21 Cellulose dissolution: NaOH/ water Isogai and Atalla (1998) Good dissolution in 8.5% NaOH is frozen at -20 C before thawing at room temperature while adding water to reach 5% NaOH in the solvent. Additives Laszkiewicz and Cuculo (1993) addition of thiourea and urea improves the solubility of a cellulose in NaOH-water. Biocelsol (2000) enzymatic activations and dissolved in NaOH-water with the addition of ZnO. Starting in 2000, the group of L. Zhang studied extensively the dissolution of cellulose in NaOH-water using urea, thiourea with or without ZnO. Cemef since 2001 (Understanding solution properties with and without additives. Preparation of various products). 21

22 Cellulose dissolution: NaOH/ water Potentially interesting technology Advantages Probably low cost of operation if dissolution can be made around room temperature. Use of NaOH well mastered in paper mills. Environmental benefits!! Disadvantages Difficulty to stabilize the solution (need of additives that are increasing cost and are difficult to be recycled in the process). Difficulties to solubilize large amounts of cellulose of high DP. Potential development in other than in textile fields Beads Aerogels 22

23 III- Effects of enzymatic treatments 23

24 III Enzymatic treatments Structural changes and solubility after the enzymatic treatment of wood cellulose fibres. Wart layer tertiary wall S2 wall S1 wall Removal of the external walls Celluclast 1,5L mixture of cellobiohydrolase and endo-glucanase Primary wall Econase HC 400 endo-1.4- beta-xylanase N. Le MOIGNE, K. JARDEBY et P. NAVARD Carbohydrate Polymers, 79, (2010) 24

25 III Enzymatic treatments Insoluble material fraction after solubilization in NaOH 8%-water at -6 C versus peeling time Borregaard dissolving sulphite Norway spruce pulps Super Aceta SA [h]= 880 ml/g VHV-S [h]=1430 ml /g Fast increase and large of solubility during the first 5 minutes of treatment 25

26 III Enzymatic treatments Amount of insoluble fractions versus intrinsic viscosity and crystallinity Linear decrease of the amount of insoluble fraction in function of the intrinsic viscosity and the crystallinity. Is the increase of solubility only due to thermodynamics and crystallinity?!! 26

27 Minimum insoluble amount % Intrinsic viscosity ml / g III Enzymatic treatments ,8 75, ,1 44, VHV-S A 1430 ml/g Enz. Peeling VHV-S A 880 ml/g (Yield 94%) SA B 880 ml/g Enz. Peeling SA B 670 ml/g (Yield 90%)

28 Minimum insoluble amount % Intrinsic viscosity ml / g III Enzymatic treatments ,8 75, ,1 44, VHV-S A 1430 ml/g Enz. Peeling VHV-S A 880 ml/g (Yield 94%) SA B 880 ml/g Enz. Peeling SA B 670 ml/g (Yield 90%) Conclusions : What counts is the structure of the pulp, not its molar mass. Can we increase solubility while not touching molar mass?

29 IV- Effects of pectinase treatments 29

30 IV Pectinases There is a small amount of pectin(s) in wood cell walls : 2-3% of wood, mainly concentrated in primary cell wall. Pulping treatments remove most of the pectins. Remaining fractions may cause detrimental effects in paper making (difficulties in de-watering). In the present case, there is NO remaining pectin. Objective of the work To explore the effect of pectinase on the ability to improve dissolution of a large range of pulps which are without pectins. 30

31 IV Pectinases Pulp Protocol Extracts Enzymatic incubation Washing / Filtration Treated pulp Enzyme

32 IV Pectinases Three enzymes: Biopectinase CCM, Pektinase L40 and Cellulase (EG) Method Yield Carbohydrate composition Molar mass Fiber dimension Crystallinity Viscosity Microscopy Pulp Extraction Control Nitren Enzyme Extract Extracted pulp Carbohydrate composition Molar mass Yield Carbohydrate composition Molar mass Fiber dimension Crystallinity Viscosity Microscopy Yield Insolubles Carbohydrate composition Molar mass Crystallinity Microscopic Analysis Mechanisms Kinetics NMMO 80% NaOH 8% Dissolution NaOH 8% Fiber dimension Microscopy Yield Solubles Carbohydrate composition Molar mass 32

33 IV Pectinases PULP 1% 1% cellulose pulp in in NaOH 8% - water Centrifugation Soluble - 6 C, 2 hours mixing stirring at 1000 rpm 8 min, 9050 g Insoluble Precipitation in methanol Gravimetric measurements Freeze drying Wash with methanol and water

34 Enzymatic activity units U/ml IV Pectinases Evaluation of the enzymatic activities towards cellulose and pectins for the three enzymes. Biopectinase CCM * Pektinase L40 Cellulase (EG) Provider Kerry Bio-Science, Ireland ASA, Germany Megazyme, Ireland Origin Aspergillus niger Aspergillus niger Talaromyces emersonii Optimum temperature (ºC) Optimum ph Endopectinase Pectinesterase Cellobiohydrolase (C1-Cellulase) Endoglucanase (CMCase) * Product no longer marketed CCM has a rather high endoglucanase activity L40 has no endoglucanase activity EG EG has no endopectinase activity 34

35 IV Pectinases Total activity units Enzyme dosage and the incubation time affect the treatment, Total activity units: parameter taking in consideration the initial activity units (units per gram of pulp) and the incubation time (in hours): Total activity units = initial activity units incubation time (hour) grams of pulp U g h 35

36 IV Pectinases Time and concentration conditions used for keeping a constant total pectinase activity for the L40 and CCM Sample Endopectinase activity units U/g (pulp) Endoglucanase activity units U/g (pulp) Total endopectinase activity (U*h/g pulp) * Total endoglucanase activity (U*h/g pulp) * Time (hours) Cuen viscosity (ml/g) NaOH dissolution yield (%) CCM_T CCM_T CCM_T CCM_T L40_T L40_T L40_T

37 Cuen viscosity (ml/g) Activity units (U/g) Dissolution yield in NaOH (%) Time (h) IV Pectinases Dissolution yield (%) Cuen viscosity (ml/g) Endopectinase activity U/g Endoglucanase activity U/g Time (h) 37

38 Cuen viscosity (ml/g) Dissolution yield in NaOH (%) Softwood Pre-hydrolysis Kraft pulp (Buckeye) Total enzymatic activity (U*h/g pulp) EP - EG Dissolution yield (%) Cuen viscosity (ml/g)

39 Cuen viscosity (ml/g) Dissolution yield in NaOH (%) Softwood Pre-hydrolysis Kraft pulp (Buckeye) Total enzymatic activity (U*h/g pulp) EP - 0 EG Dissolution yield (%) Cuen viscosity (ml/g)

40 Cuen viscosity (ml/g) Dissolution yield in NaOH (%) Softwood Pre-hydrolysis Kraft pulp (Buckeye) Total enzymatic activity (U*h/g pulp) EP EG Dissolution yield (%) Cuen viscosity (ml/g)

41 Cuen viscosity (ml/g) Dissolution yield in NaOH (%) Softwood Pre-hydrolysis Kraft pulp (Buckeye) Total enzymatic activity (U*h/g pulp) EP EG Dissolution yield (%) Cuen viscosity (ml/g)

42 Cuen viscosity (ml/g) Dissolution yield in NaOH (%) Softwood Pre-hydrolysis Kraft pulp (Buckeye) Total enzymatic activity (U*h/g pulp) EP EG Dissolution yield (%) Cuen viscosity (ml/g)

43 Cuen viscosity (ml/g) Dissolution yield in NaOH (%) IV Pectinases Softwood Pre-hydrolysis Kraft pulp (Buckeye) Pectinase has an influence on the accessibility of the pulp fibers Endoglucanase cannot reproduce the results achieved by the mixture of pectinase + endoglucanase The mixture of the two enzymes is the more efficient. Dissolution yield (%) Cuen viscosity (ml/g) 43

44 IV Pectinases Be-S: Beech bleached sulphite pulp (Lenzing) S-PhK: Softwood Pre-hydrolysis Kraft pulp (Buckeye) NS-S: Northern softwood sulphite pulp (Tembec) SS-S: Southern softwood sulphite pulp (Tembec) Eu-Vis: Eucalyptus viscose grade (Sappi) Eu-Kp: Eucalyptus bleached kraft pulp (Sappi) 44

45 IV Pectinases Effect on fiber structure and composition Intrinsic viscosity / molar mass distribution Beech bleached sulphite pulp (Lenzing) 0.8 Cuen viscosity (ml/g) Be-S Be-S_Blk Be-S_CCM Be-S_L40 differencial weight fraction (1/log(g/mol)) Be-S Be-S_Blk Be-S_CCM Be-S_L40 1e+4 1e+5 1e+6 molar mass (g/mol)

46 IV Pectinases

47 IV Pectinases

48 IV Pectinases Effect on fiber surface Northern softwood sulphite pulp (Tembec) NS-S_CCM NS-S NS-S_L40 39

49 IV Pectinases Optical and electron microscopy observations Observations of fiber surfaces by FEG-SEM endopectinase treatments: similar to the initial pulp (presence of micro fibrils) endoglucanase: no more microfibrils L40 EG First conclusions: Pectinase itself is efficient for improving dissolution in cold NaOH without decreasing significantly molar mass for the S-PhK pulp. The main increase on dissolution yield of CCM is due to the pectinase while the decrease of viscosity is due to the endoglucanase. 49

50 IV Pectinases Importance of primary wall for cellulose dissolution. What is the relation with pectin?

51 Dissolution yield in NaOH (%) IV Pectinases 100 Reaching 100% dissolution??? S-PhK Eu-KP 40 Eu-Vis Linéaire (S-PhK) R² = 0, Linéaire (Eu-KP) R² = 0,9589 Linéaire (Eu-Vis) R² = 0, Cuen viscosity (ml/g) 51

52 IV Pectinases Conclusions Endopectinase improves dissolution in NaOH-water even if there are no detectable pectin present. Enzyme are changing the internal structure of pulps. This is the most important factor helping dissolution, together with DP. To understand the nature and origin of these subtile changes in the internal structure would enormously increase the potential of cellulose to perform chemistry, dissolution or transformation into glucose. 52

53 Acknowledgements Coworkers Bodo Saake and Jürgen Puls (Hamburg) Students Nicolas Le Moigne, Nuno dos Santos Sponsors Lenzing Sappi Spontex Tembec Viskase Dow Wolff Boregaard 53

54 Thank you for your attention 54