CONTINUOUS HARVEST OF CELLULOSIC FILAMENT DURING CULTIVATION OF ACETOBACTER XYLINUM Seiichi Tokura I, Hiroshi Tamura I, Mitsuo Takai *,Taiichi Higuchi and Hisashi Asano 'Faculty of Engineering, Kansai University and HRC, Yamate-cho, Suita, Osaka, 564-8680 Japan. 'Graduate School of Engineering, llokkaido University, Sapporo 065-0012. Japan. 'Graduate School of Environmental Earth Sci., Hokkaido University, Sapporo 065-0012 Japan.
ABSTRACT A shallow pan cultivation of Acetobactor xylinum has been studied using a continuous wind up roller to give swollen bacterial ccllulosc (BC) filament. The swollen filament was stretched with a twisting mode to rcmove water following the purifying procedures such as boiling in aqucous sodium dodecyl sulfate and in aqueous sodium hydroxidc, respectively. Modified BC filament containing residual N-acctylglucosamine (GlcNAc) was also obtaincd, when A. xylinum subcultured in GlcNAc medium was applied in a medium containing glucose and ammonium chloride. The BC and modified filaments showcd higher tensile strcngth than those of cotton and rayon, but poorer elongation and relatively lowcr Young' s modulus than those of cotton and rayon. Highcr orientation of molecule, as shown by X-ray diffraction pattern analysis, was achievcd by applying a heavier weight during drying procedure. The molecular weights of BC products were estimated viscometrically using coppcr-ammonium solvents.
INTRODUCTION It has been establishcd that A. xylinum produces bacterial cellulosc (BC) under static incubation in Hestrin-Schramm (HS) medium containing glucose as a carbon source 4. Because BC is a pure cellulose, it has attracted much attention in various manufacturing fields. In our previous studies on the preparation of BC, we had developed novel proccdures for producing molecular variants of BC. Incorporation of amino-sugar residues has been successfully attained by incubation of the bacteria that had been subcultured rcpcatedly in a medium containing N-acetylglucosamine (GlcNAc) and glucose (Glc) or only GlcNAc as carbon source 5. The subculture of bactcria was required in GlcNAc mcdium to form thc pcllicle containing GlcNAc. Sincc a similar dcgrcc of GlcNAc incorporation was found by employing media containing Glc and either galactosamine or glucosamine but not by mannosaminc, the activation of transaminase was assumed to be rout of metabolic cycle of thc bacteria 6. Undcr rotatory but not static conditions, a similar degree of GlcNAc incorporation was also observed when cultivation was carricd out with air bubbling in a medium containing Glc and ammonium chloride 6, Although much effort have bccn devoted to the preparation and utilization of BC and its analogs, these methods and agents have not yet bccn incorporatcd as an industrial resource,
4
New sources, structure and properties of cellulose
largely because of the high production costs involved. Given that complicated procedures are required for the preparation of fibers and films from these biosynthesized polysaccharides 7, a simple method, one that can serve to reduce these high production costs, has bccn required. We describe the results of our latest investigations to simplify the production processes, which were prompted by recent successes in regard to surface polymerization of nylon capable of giving supcrpolymer films or filaments directly from the interface of organic and aqucous phases *. We have designed shallow pans to increase the BC yield, to regulate the gel thickness during the incubation and to conserve the total amount of the medium used. The tensile strcngth of thc filamcnt was significantly greater than that of the ordinary cellulose fibers, and good orientation of molecules was revealed in the X-ray diffraction pattern.
EXPERIMENTAL Materials Chemicals wcre purchased from Wako Pure Chemicals Co. Ltd., (Osaka, Japan) and uscd without further purification. A wild type of A. xylinum, ATCC 10245 strain, was subcultured at 28 OC in Hestrin and Schramm (HS) medium containing Glc as a carbon source, and repeatedly transfcrrcd to the new culture medium every 3 days. Incubators A. Aerobic Rotatory Incubator A round vessel of 3.0L volume incubator with air supply was designed as shown in Fig. 1. The optimum rate of rotation was around 10 rpm when 2.5L of medium was applied
Heater
(Volume of medium is 2500 d)
Figure 1. Outline of aerobic rotatory incubator.
Continuous harvest of cellulosic filament 5 under our condition.
B. Shallow Pun Incubator with Wind up Roller Two culture pans (100 mm width x 400 mm lcngth x 7 mm depth and 200 mm width x 400 mm lcngth x 7 mm depth) were worked out by cutting stainless steel board. The inside shape of the pan was spccially designed to facilitate harvesting the thin gcl smoothly. The pan was cquippcd with a winding roller and a bath of 2% aqueous sodium dodccyl sulfate (SDS), as shown in Fig. 2. The whole apparatus was set in a sealed chamber in which the tempcrature was maintained at 28 "C and filtered air was passed through the incubator.
Figure 2. Outline of the culture pan for the direct filamcntation of BC a, sinker; b, roller; c, washing pan; d, wind up roller. Composition of mediums The compositions of medium are listed in Table 1 for BC production and Table 2 for the incorporation of GlcNAc residue into BC. Table 1. Composition of medium components Compounds of mcdium
Concentration (w/v%)
~~
Sugar Bacto peptone Yeast extract Disodium hydrogen phosphate Citric acid
2.0 0.5 0.5 0.27 0.115
Tablc 2. Composition of medium components for the incorporation of GlcNAc residue Compounds of medium Sugar Bacto peptone Yeast extract Disodium hydrogenphosphate Citric acid Ammonium salts or GlcNAc
Concentration (w/v%) 2.0
0.5 0.5 0.27 0.115
0.2
Culture mcdium and cultivation HS medium (150 ml or 300 ml) was added to the smallcr or larger culture pans,
6 New sources, structure and properties of cellulose
Figure 3. Pratical winding-up process of bacterial cellulose during incubation of A. xylinum in a shallow pan.
Figure 4. Pravtical incubation process in aerobic rotatory incubator by the time. respectively. The depth of the culture media in each pan was 3-4 mm. The media were inoculated with the subcultured A. xylinum under static conditions at 28 "C. After 2 days of incubation, the edge of the pellicle produced on the surface was picked up, passed through the SDS bath to denature the bactcrial cell wall, and set on the winding roller as shown in Fig. 3(a) and (b). The winding process was continued for a couple of weeks at the rate of 35-40m d h and 28 "C.During the incubation, the depth of the culture medium was maintained by the stepwise addition of HS medium every 8-12 h. 2.5L of HS medium was applied for aerobic rotatory incubator and incubated for 7-14days under conditions similar to those for shallow pan incubation, as shown in Fig. 4.
Purification of filament
Figure 5. Removal of water from fibrous gel by twisting by hand.
Continuous harvest of cellulosic filament 7 The wound filament was boilcd for 3 h in 2% SDS aqueous solution, washcd with distilled water, boiled again in the 4 % aqueous sodium hydroxide solution for 1.5 h. The wet filament was cxtcnsively rinsed with distillcd watcr and thcn air-dried at less than 60 "C under tension following a hand twist of the fibrous gel to exclud water as shown in Fig. 5.
Tensile strength The stress-strain diagrams of thc filaments were obtained using a Shimadzu Auto Graph AGS-5OOD apparatus at a guide distance of 25 mm,a chart speed of 100 mdrnin, and a load cell spced of 2 mm/rnin. The force at the brcaking point was taken as tensile stress, which was transferred to tensile strength and Young's modulus.
Wide-angle X-ray diffraction (WAXD) analysis WAXD patterns were rccorded by using a MAC M18XHF X-ray diffractometer. The X-rays werc gcncrated at 40 kV and 100 mA using nickel-filtered CuKa radiation. A vacuum camcra equipped with a 0.5 mm pin-holc collimator was used.
Scanning electron microscopy (SEM) SEM wits accomplished using an Akashi S-DS 130 microscope with gold-coated sample. Microsphercs wcrc sprinkled onto double-sidcd tape, sputter-coated with gold, and examined in the microscope at 10 kV. Estimation of molecular weight by viscometric measurement The molecular wcight of the BC produced was achieved according to the mcthod of Gralen and Ebcll in which BC was dissolved in coppcr-ammonium solution ( C U ;1.0 ~ k O.lg/L, ammonium; 210 k 5g/L and 1Og/L of sucrose as rcductant) under nitrogen atmosphere. Viscosity was measurcd in an Ubbelohde viscometer at 25°C g.
RESULTS AND DISCUSSION Design of the cultivation system An aerobic rotatory incubator was designed to prepare fibrous BC due to rotatory force to the alignmcnt of cellulose molecules and to increase the yield of BC (Fig. 2 of outline and Fig. 5 of practical proccss). A shallow pan was also dcviscd to make thinner BC gel suitable for dircct and continuous filamentation during the incubation of A. xylinum together with increase of yield. In a preliminary incubation under static conditions using a pan with 10 mm depth, thin BC gcl was obtained on the surface of thc culture medium, and gels werc strong and elastic enough to pick up and manipulate. On this basis, a direct filamentation system was dcsigncd as shown in Fig. 3, consisting of a shallow pan of 7 mm of dcpth (400 mm or length and 100 or 200 mm of width) with a winding up roller. One side of the pan was curved gently to permit harvesting of thc pellicle through a narrow mouth. The thin BC gel was directly passed through a bath containing aqueous SDS solution to rcducc the bacterial activity and thcn the filament was wound slowly on an attached roller. The shallow pan was also effective in conserving the total amount of culture mcdium used for the incubation.
8
New sources, structure and properties of cellulose
Direct filamentation Using the system described above, A. xylinum was incubated under static conditions at 28 "C in the shallow pan containing HS mcdium containing Glc and GlcNAc mixture or Glc mixcd with ammonium chloride as carbon source. Following static incubation for 2 or 3 days, thin BC gel was formed and harvesting was started on the roller system. Taking the growth of the BC gel into account, the optimum rate of wind up was found to be around 4Omn/hr for continuous filamentation. To maintain the depth of the culture medium, NS medium was supplied in increments during the incubation, without further addition of bacteria. From thc 100 mm pan, a filament of more than 5 m length was obtained by winding-up at a rate of 16 mm/h for 14 days. Fig. 3 shows the process of the direct filamentation. For purification, the BC filament thus obtained was succcssively treatcd with boiling 2% SDS solution and boiling 4 % sodium hydroxide solution. All filaments were subjected to air-drying under tension at less than 60 OC following the hand twist to remove water as shown in Fig. 5. Dependence of BC yields on the cultivation methods Yields of BC and of GlcNAc residues incorporated into BC are listed in Table 3. As seen in the Table, the shallow pan method improved the yield of BC remarkably in addition to the production of filament. The yield of BC was fairly constant on the aerobic rotatory incubator, whereas unstable yields were observed on static incubation. As volumes of medium are the final stage of cultivation, the increase of yield on shallow pan cultivation seems to be due to the freshness of medium by stepwise addition of medium. Sufficient air supply is also one of major factors in regulating yield, because a fresh surface is served every time on the shallow pan cultivation due to wind up thc product. X-ray diffraction patterns of filamcnts from three incubation methods are shown in Fig. 6. As seen, higher orientation is suggested for direct filamentation compared with those for the other two methods. The peak of (020) of a directly filarnented one, howcver, is slightly shifted to a lower value than those of static and aerobic rotatory methods probably due to an almost homogeneous crystalline structure. Table 3. Dependence of BC yield on the cultivation
Static culture Static culture Static culture Static culture Rotatory culture Filamentation Pilamentation Filamentation Filamentation
Mcdium (ml)
Days
Weight (g)
Yield (%)
15 30 50 300 2500 200 350 450 375
7 7 3 7 7 7 7 10 4
0.0026 0.0093 0.00 12 0.2200 1.3658 0.1493 0.3505 0.5001 0.2269
0.86 1.35 0.12 3.60 2.73 3.73 5.01 5.56 5.03
Physical properties of the filament The BC filament obtained by the direct filamentation was first examined by scanning elcctron microscopy (SEM) to confirm the success of the washing process. A SEM image
Continuous harvest of cellulosic filament 9
5
10
15
20
25
20 /degree
Figure 6. X-ray diffraction patterns of BCs produced by the three different cultivation methods. (Fig. 7) showed that the BC filament has good alignment of cellulose moieties and a slightly twisted fiber mode. The cut surface of filament, also shown in Fig. 7, shows the melting of the inner part of the filament. X-ray diffraction patterns of the filament were also examined as shown in Fig. 8. The filament from the shallow pan incubator shows a slightly higher orientation of molecules than that of filament prepared by aerobic rotatory incubation, whereas poor orientation was suggested for the filament from static cultivation.
Figure 7. Scanning electron microscopic pictures of the bacterial cellulose filament. surface view (left), cut surface view (right).
Figure 8. X-ray diffraction patterns of BCs incubated in different medium. Glc (left), Glc:GlcNAc = 7: 3(v/v) (center), Glc and NH4CI (right).
10 New sources, structure and properties of cellulose Thc tensile and the stress-strain properties for the filaments obtained by shallow pan incubation are listed in Table 4 as factors of medium composition.The stress-straindiagram shows that there is a little decrease of strength by the change of medium, though denier of filament becomes smaller probably due to thinner gel formation by poor yield especially in ammonium chloride HS medium, But stress of filament was inforced by the introduction of GlcNAc residue though there is almost similar level of ammonium chloride medium. Average values of the tensile strength and Young's module of GlcNAc incorporated filaments are 4.4 g/dcnier and 9.0 Gpa which are comparable to those of BC filamcnt, cotton and othcr fibers Thcsc tcnsilc properties of BC filaments are possibly improved by the finishing proccdurcs, bccausc ethylcnc glycol significantly changed these tensile properties by the addition at water rinsing process even if the content was small. Table 4. Tensile properties of BC filaments Sample
N1 N2 N3 N4 N5 NAc- 1 NAc-2 NAc-3 NAc-4 NH.Cl-1 NIIICl-2 NH.Cl-3 NH];Cl-4
Size (denier)
Elong (%)
Stress (GPa) (Average)
108.0 169.2 108.0 108.0 140.4 26.3 26.3 60.5 60.5 39.6 39.6 39.6 39.6
4.2 6.0 4.5 3.8 5.8 1.5 1.2 0.9 2.1 4.4 4.1 4.0 5.0
0.27 0.22 0.43 (0.33) 0.24 0.39 0.57 OS2 (0.48) 0.37 0.59 0.18 o-28 (0.33) 0.40 0.47
Strength Young's modulus (g/ denier) (GPd (Average) (Averagc) 3.9 5.6 3.6 (4.6) 3.0 6.1 7.4 5.9 (4.4) 1.3 4.6 2.4 2*8(4.0) 3.7 5.6
6.4 3.7 9.6 (6.7) 6.7 6.3 9.6 (9.0) 10.9 7.0 4.0 6*7 (7.0) 9.9 9.5
Pretreatment :treated with boiling 4% sodium dodecylsulfateaqueous solution for 2 hr and thcn 2% sodium hydroxide aqueous solution for 1.5 hr. Abbreviation : Elong ;Elongation ,Stress ;Tensile stress , Strength ;Tensile strength N: BC filaments cultured in HS medium containing Glc as carbon source. NAc: BC filaments containing N-acetylglucosamine residuces cultured in HS medium containing GlcNAc instead of Glc. NH,Cl: BC filaments containing N-acetylglucosamineresiduces cultured in HS medium containing Glc and 0.2% ammonium chloride.
Molecular weight of BC under static cultivation The molccular weight of BC produced under static conditions was estimated viscometrically by plotting reduced viscosity vs polymcr concentration and logarithm reduced viscosity vs polymer conccntration. Although molecular weight of 1 . 0 5lo6 ~ were given for both BCs of 1 day and 4 days incubations as shown in Fig. 9, this is only preliminary measurement.
11
Continuous harvest of cellulosic filament "4days" M w = 1.05 x lo6
"lday" M w = 1.07 x lo6
y = 5.9029 + 70.758 X R2 = 0.999
I 0
.
6
K =U.YVL
I
I
1 4
0.000
0.004 0.008 Concentration
0.012
y
P
= 6.9527 += 12.775 X
R2 = 0.955
1
0.00 0.02 0.04 0.06 0.08 0.10 C 12 Cvnccntration
Figure 9. Profiles of viscosity.
CONCLUSION Our design of a simple and direct filamentation system for the production of bacterial cellulose is, we believe, the first such procedure to be reported in the literature. The tensile strcngth of the filament was found to be significantly stronger than the ordinary cellulose fibers and a good orientation of molecules was shown both by the X-ray diffraction pattern and SEM obscrvation. Thcse simplificd methods for producing and harvesting BC, are cxpecting to lower the production cost of BC togcthcr with the basic research for the orientation of cellulose molecule.
ACKNOWLEDGEMENTS A part of this research was financially supported by the Kansai Univcrsity Special Research Fund. 1999.
REFERENCES 1. J. Brown, On an Acetic Fermant which forms Cellulose, J. Chern. Soc., 1886.49, 432-439. 2. M. Takai, Y. Tsuta, J. Hayashi & S. Watanabe, Biosynthesis of Cellulose by Acetobactcr Xylinum. 111. X-Ray Studies of Preferential Orientation of the Crystallites in a Bacterial Cellulose Membrane, Polym. J., 1975.7, 157-164. 3. M. Fujiwara, K. Fukushi. M. Fukaya, H. Okumura, Y. Kawamura, M. Takai & J. Hayashi, Construction of Shuttle Vectors Derived from Acetobacter xylinum for Cellulose Producing Bacterium Acetobacter xylinum, Biotech. Let., 1992, 14,539542. 4. S. Hestrin & M. Schramm, Synsethis of Cellulose by Acetobacter xylinum.2. Preparation of Freeze-dried Cells Capable of Polymerizing Glucose, Biochem. 1..
12 New sources, structure and properties of cellulose 1954,58,345-352. 5. R. Ogawa & S. Tokura, Preparation of bacterial cellulose containing NAcetylglucosamine residue, Carbohydy.Polymers, Carbohydr. Polymers, 1992, 19, 171-178. 6. A. Shirai, M. Takahashi, H. Kaneko, S.-I. Nishimura, M. Ogawa, N. Nishi & S. Tokura, Biosynsethis of a Novel Polysaccharide by Acetobaetcr xylinum, Int. J. Bioll. Macromol., 1994, 16,297-300. 7. H. Hibbert, Action of Bacteria and Enzymes of Carbohydrates and their Bearing on Plant Synsethis, Science, 1930,71,419-420. 8. E. E. Magat & R. D. Strachan, U.S. Patent, 1995,2,708,617. 9. N. Gralen & L. Ebell, ,J. Biol. Chem., 1924, GO, 257-266. 10. R. Meredith, A Comparison of the Tensile Elasticity of some Textile Fibers, J. Textile Inst., 1945,36, T 107-130. 11. L. Rebenfeld & W. P. Virgin, Relation bctwccn the X-Ray Angle of Cottons and Their Fibcr Mcchanical Properties, Textile Res. J., 1957,27,286-289.
Oil Palm (Elaeis gcrineensis) Wastes as a Potential Source of Cellulose M o l d Azemi Mohd. Noor' and Harun Sari$ 'Biopolymer Research Group, School of Industrial Technology Universili Sains Malaysia, I 1800 Minden, Penang, Malaysia. 'Fibrotech Sdn. Bhd., No. I Lorong Terasek Kanan, Bangsar Baru, 59100 Kuala Lumpur, Mulaysia.
ABSTRACT Oil palms are an important cash crop in Malaysia and currently the total area under cultivation is approximately 2.5 million hectares. Oil palm cultivation generates a significant amount of lignocellulosic biomass dcrivcd from fronds, empty fruit bunches and trunks. About 36 million tons of these lignocellulosic wastes are generated annually and currently most of these wastes are either left in the plantations or burned illegally. Small amounts are being utilised for fibre production and energy generation. The biomass consists of about 40% cellulose, 40% hemicellulosc, 18% lignin and 2% extractives (sugars and phenolic substances etc.). Attempts are being made to fractionate, isolate and purify the cellulose fraction into microcrystalline cellulose. High pressure steam treatments followed by aqucous extraction and chlorite bleaching were employcd to fractionate, isolate and purify cellulosic materials derived from oil palm biomass. Highly crystalline cellulose derivatives resembling microcrystalline (MCC) with over 95% purity were successfully isolated. The properties are comparable to commercially available MCC and cfforts are being made to commercialise these products. INTRODUCTION The cultivation of oil palm (Elueis guineensis Jacq) is forecast to cover a total area of about 2.8 million hcctarcs in Malaysia by the year 20003. Besides palm oil, the by products of oil palm industry such as oil palm trunk (OPT), fronds, cmpty fruit bunches (EFB) and palm press fibre (PPF) contribute significant amounts of lignocellulosic biomass. Currcntly this biomass has yet to be fully utilised as a source of value added products especially chcmicals and its dcrivatives. Cellulose materials rcpresent about 70-80% (cellulose and hemicellulosc) of oil palm biomass and would certainly serve as raw materials for the production of chemical cellulose and its dcrivativcs3. This report describes the production of microcrystalline cellulose (MCC) through fractionation, isolation and purification of this oil palm biomass using high pressure treatments (steam explosion).
14 New sources, structure and properties of cellulose MATERIALS AND METHODS. Oil palm biomass, supplied by Sabutek (M) Sdn. Bhd. Malaysia, was subjected to high pressure steam treatmcnt and followed by aqueous extraction (Pig. 1). The steam exploded fibers were initially subjected to a watcr extraction process at room tempcrature then followed by alkaline treatment. The alkaline extracted fiber (AEF) fractions wcrc then bleached with 10% sodium chlorite and subsequently treated with alkali followcd by acid hydrolysis to produce MCC'. The composition of the water soluble constituents in the water extract liquor (WEL) and alkaline extract liquor (AEL) were analyscd for carbohydrate content and compared with the untreated lignocellulosic material (UT). The carbohydrate content was analysed using borate-anion cxchange chromatography (BAEC) tcchnique developcd by Simatupang4. The MCC was analysed for particle size distribution, degree of polymerisation (DP), viscosity and stability. The particle size distribution was analyscd using a coulter counter (Gulter Elcctronic, Herb, England), while viscosity measurcrnent and stability studies were carried out using Ubblohde capillary tube viscometer and cellulast enzyme (NOVO) digestion respectively.
RESULTS AND DISCUSSION As shown in Table 1, relative percentage of lignin contents after high pressure steam treatmcnt (ST%) varies according to the source of oil palm wastes and corrcspond with the amount of carbohydrate (exprcsscd in term of glucose)'. The relative percentage of lignin and cellulose (in term of glucose) dctected in high prcssure steam treated fibers somehow increascd; indicating only small amount being converted into water soluble fragments. Rhamnose, mannose, arabinose, galactosc and xylose wcre detected in all sources of oil palm wastes.
Table 1. Chemical analysis of various oil palm lignocellulosics before and after high pressure stcam trcatment and of water soluble parts Compound Palm Press Fiber Empty Bunches Oil Palm Trunk (PPF) (EFB) (OPT) U.T. S.T. W.S. U.T. S.T. W.S. U.T. S.T. W.S.
YO Klason lignin Rhamnose Mannose Arabinose Galactose Xylose Glucose 4 -m-MG
35.7
% 41.3
%
YO
1.0
23.3
% 33.3
% 3.3
1.43 0.38 0.17 1.09 0.40 0.18 1.50 2.05 3.78 0.89 1.03 0.79 1.5 1 3.68 1.40 5.40 0.78 1.97 0.74 0.23 2.30 23.02 13.75 44.68 21.45 15.05 25.45 24.22 37.29 2.48 40.22 43.47 1.93 0.51 0.40 0.74 0.35 U.T.= Untreated lignocellulosic material S.T.= High prcssure steam treated, defiberized and refined fibcrs W.S.= Watcr soluble parts Values are bascd on ovcn dried material
-
-
YO
YO
19.8
24.3
0.34 1.20 0.71 0.48 20.00 44.91 1.02
0.76
-
11.40 57.62 0.63
% 0.9
1.65 3.06 2.83 1.73 48.93 4.97
Oil palm wastes
15
Figure 1. Extraction and Purification of Oil Palm Wastes Lignocellulosic Matcrials
Oil Palm Lignocellulosic Materials
J Steam Explosion
J J
Steam Exploded Fibre (SEF) Hot Watcr Extraction
>
Watcr Extract Liqour (WEL)
Water Extracted Fiber (WEF)
J
Alkaline Extraction
Alkaline Extract Liquor (AEL)
J Acidification
4
Lignin
+
Alkaline Extracted Fiber (AEF) Bleaching
+ + 1
Alpha Ccllulose Mineral Acid Hydrolysis Washing & Pufification Microcrystalline Cellulosc
J
Product Characterization
16 New sources, structure and properties of cellulose
Micrograph 1. + Oil Palm Wastes MCC.
Micrograph 2. + Commcrcial MCC
Oil palm wastes
17
I Iowevcr upon high pressure steam treatment, arabinosc was completely solubiliscd as opposed to 4-rn-methyl-gluconocosylxylosc(4-m-MG). The presence of xylose in all water soluble fractions in increasing order is partly due to dcpolymerisation into water soluble low molecular wcight fragmcnts. The MCC samples obtaincd were evaluated for purity, molecular weight, DP, particle size distribution, degree of crystallinity and instrinsic viscosity (Table 2). The particle size distribution of oil palm waste MCC scemcd to be in thc larger range (3.5 30 pm) than the commercial MCC. However, the degree of polymerisation (DP) is lower than that of the commercial MCC. This is in line with the low instrinsic viscosity mcasurcd as compared to the commercial MCC which may contribute toward stability of colloidal suspension. The degree of crystallinity and purity are similar. As shown in micrograph 1 and 2, the properties of MCC derived from oil palm wastcs closely resemble commercially produced MCC. The DP is dcrivativc valuc of the intrinsic viscosity measurement. Table 2. Propertics of Oil Palm Wastcs MCC and Commercial MCC Properties Oil Palm Wastes MCC Commercial MCC I’article size distribution 3.64 pm-28.90 pm 3.12 pm-9.91pm DP 640 repeat unit 940 rcpcat unit Instrinsic Viscosity 3.36 4.96 Crystallinity Index 87.4 88.8 Purity > 95% > 95%
CONCLUSION Microcrystalline cellulose from oil palm wastes exhibited a closc rcscmblance to those produced from various sourccs of biomass and can be isolated through high pressure stcam treatmcnt.
REFERENCES 1. Mohd. Azcmi B.M.N., Sallch M., Wright R.S,and Glasscr W.G. (1998). Stcam Assisted Fractionation of Oil Palm Trunks Solids. Biomass and Bioenergy (In prcss - Elsevicr, Oxford UK). 2. Baltista 0. A., and Smith P A . (1962). Microcrystalline Cellulose in Industrial and Chemical Engineering. 54(9), pp 20-29. 3. H u s h M., IIassan A.H and Mohamad A.T. (1997). Availability and Potential Utilization of Oil Palm Trunks and Fronds upto Ycar 2000. PORIM Occasional I’apcr No. 20. pp 1 - 20 4. Simatupang, M.N. (1977). Ion exchange chromatography of some neutral monosaccharidcs and uronic acids. J. Chromatogr: 178: pp 588-591
ISOLATION AND CHARACTERISATION OF SAGO ( Metroxylon Sagu ) CELLULOSE Mohd. Zahid A., Mohd. Zulkali M.D. and Azemi B.M.N. Bio-polymer Research Group School of Industrial Technology, Universiti Sains Malaysia I I800 Minden, Penang, Maloysia
ABSTRACT Apart from starch, cellulosic materials constitute the major by products of sago starch extraction plants, notably from starch free fibrous materials and the barks. These potentially commcrcialisable cellulosic materials remain unexploited. This paper describes an attempt to isolate and purify the ccllulose fraction which forms about 40% of the total dry weight of the cellulosic materials from sago barks. Alkaline treatment and acidic precipitation of non-cellulosic materials namely lignin were employed to fractionate the cellulose component. Bleaching was accomplished by chlorite oxidation. Upon acid hydrolysis, a-cellulose was obtained and characterised for particle size distribution, cystallinity index, mean molecular weight, intrinsic viscosity, microscopic appearance and susceptibility to cellulase attack. The properties of thc sago dcrived cellulose were compared with that of commercial products.
INTRODUCTION East Malaysia (Sarawak) in particular is known for sago plantations. There are 32 sago mills supplied by 19,720 hectares of sago plantations4. These quality of the plantations are found in the area of Mukah, Igau and Oya-Dalat districts of Sibu Division and others in the Pusa-Saratok districts of Sri Aman Division6. Sibu contributes about 95% of thc starch exported from the State. A total of over 35,000 tons of sago starch and 4,180 tons of sago flour were exported in 1994 alone, procuring incomes of over RM22 million (USD 8 million) and RM2.4 million (USD 1 million) for the State, respectively’. The total annual export of premium quality sago flour to the world market was approximately 43,000 tons. It is predicted that a sufficient supply of harvestable palms would be availablc in four years’ time to support an annual production of 67,000 tons of sago starch. Besidcs starch, the sago industry generates a large variety of lignocellulosic biomass. These untapped potentially rich sourccs of cellulose and hemicellulose remain unexploited. In this paper attempts to isolatc, fractionate and characterise this cellulosic biomass in the form of microcrystallinecellulose (MCC) are reported.
20 New sources, structure and properties of cellulose MATERIALS AND METHODS
Sago palm barks were appropriately sized and subjected to high pressure steam treatments followcd by aqueous extraction. Thc methods used were similar to thosc employed by Azemi and Harun (199Q3. The MCC obtained was analysed for particle size distribution, crystallinity index, molecular weight, degree of polymerization (DP), viscosity, microscopic appearance and digestibility'. The particle size distribution was analyscd using a coulter counter (Coulter Electronic ,He&, England ). The crystallinity index and instrinsic viscosity were analysed using X-ray crystallography and Ubblohde capillary tube viscometer rcspectively2. Scanning electron micrograph (SEM, Leica Cambridge) was employed for microscopic appearance studies, while product digcstibility was carried out with cellulast enzyme (NOVO). RESULT AND DISCUSSION
The particle size distribution of the MCC obtained was similar to that of the commercial MCC, however, intrinsic viscosity and cellulase susceptibility varied (Table 1). The intrinsic viscosity of sago palm bark MCC was approximately one third that of commercial MCC. The crystallinity index was similar to that of commercial MCC, suggcsting the existcnce of amorphous regions in the sago bark MCC which was reflectcd in the cellulase susceptibility value of 1.5%. Howevcr, cellulase susceptibility value for commercial MCC was higher than that of sago bark MCC, suggesting more amorphous region. Micrographs 1 and 2 show the appearance of commercial MCC and sago bark MCC. They seemed to be similar in appearance*. This short study has elaborated the potential of the barks as a source of MCC, however ,further studies are required to fully exploit the wastes. Table 1: Properties of sago palm bark MCC Properties MCC of Sago Bark Commercial MCC Particle size distribution 2.18-8.72 pm 2.18-6.92 pm *DP 70 rcpeat unit 225 repeat unit Intrinsic viscosity 0.375 dL/g 1.179 dL/g Purity >95% >95% 36 000 kD 11 500 kD *Mean molecular weight 8 1.8% 83.4% Crystallinity Index Cellulase (cclluclast) susceptibility 1SO% 2.65% * = These value were derived from the intrinsic viscosity measurement CONCLUSION
,
The work has highlighted the potential of sago palm bark as a good and economically viable source of MCC.
Isolation and characterisation of sago cellulose 2 1 MICROSCOPIC APPEARANCE
- COMMERCIAL MCC
SAGO BARK MCC -
ACKNOWLEDGEMENT
This work was funded by C R A W Sarawak and Universiti Sains Malaysia
22 New sources, structure and properties of cellulose REFERENCES 1. Mohd. Azemi B.M.N., Salleh M., Wright R.S, and Glasser W.G. (1998). Steam assistcd Fractionation of oil palm trunks solids. Biomass and Bioenergy (in press), Elsevier, Oxford, UK. 2. Battista 0. A., and Smith P.A. (1962). Microcrystalline Cellulose in Industrial and Chemical Engineering. 54(9), 20-29. 3. Azemi B.M.N. and Harun S. (1998). Production and Characterization of Microsrytalline Cellulose from Stcam Exploded Oil Palm Fronds. M.Sc. Thesis USM. 4. Ark P.K. (1996). Inventory and Evaluation of Sago Palm (Metroxylon spp.) Distribution. In: 6Ih Sago Symposium: Sago, The Future Source of Food and Feed. Pckanbaru, Indonesia. 5 . Bujang K., K. Apun and M.A. Salleh (1996). A Study in the Production and Bioconversion of Sago Waste. In: 6Ih Sago Symposium: Sago, The Future Source of Food and Feed. Pekanbaru, Indonesia. 6. K.H. Ong (1976). Sago in Sarawak. In: 1'' Int. Sago Symposium: The Equatorial Swamp as a Natural Resource. Kuching, Sarawak. ,
A highly cellulosic exopolysaccharide produced from sugarcane molasses by a Zougfoea sp. M. Paterson-Beedle', L. L. Lloyd', J. F. Kennedy', F. A. D. Melo: & V. Medeiros2 'Birmingham Carbohydrate and Protein Technology Group, ChembiotechLaboratories, University of Birmingham Research Park, Birmingham, BIS ZSQ, UK 'EstaCSo Experimental de Cana-de-A qucar de Carpina- Universidade Federal Rural de Pernambuco, Carpina, Pernanibuco, CEP 55 810 000, Brazil
Brazilian Zoogloea sp. bacterium produced from sugarcane molasses a polymer substance containing glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0. l%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitoI(74.7%) and 2,3-di-Omethylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1+4)-8-D-glucopyranosyl units.
INTRODUCTION Cellulose is produced by Acetobacter xylinum and other, mainly Gram-negative, bacterial species (1,2), the cellulose being excreted into the medium, where it rapidly aggregates as microfibrils. Bacterial cellulose has long been used in foods in Asian countries (3,4) and its different properties from wood-derived cellulose opens new industrial applications. Bacterial cellulose possesses high crystallinity, high degree of polymerization, high tensile strength and tear resistance, and high hydrophilicity that distinguish it from other forms of cellulose (5). Various potential industrial applications for bacterial cellulose include acoustic diaphragms, artificial skin or wound healing, filter membranes, ultra strength paper and paper additives (5,6,7). In food processing, suspensions of disintegrated bacterial cellulose have been found useful as thickening and binding agents and as a dietary fibre supplement (5). Zoogloea ramigera produces zooglan, a polysaccharide composed of D-glucose, Dgalactose and pyruvic acid in an approximate ratio of 11:3:1.5 (8). It is a long chain polysaccharide consisting of mainly 1,Clinked glucose residues and 1,4- and 1,3- linked galactose residues with branches of glucose residues at the C-3 or C-6 positions of the galactose residues. .The pyruvic acid residues, the acidic component, are linked to the nonreducing end and/or 1,3-linked glucose residues through 4,6-ketal linkages (8). Here, we present recent studies on the composition and structure of an exopolysaccharide produced from sugarcane molasses by a Zoogloea sp. EXPERIMENTAL Materials
The following materials were obtained from sources indicated: sugarcane molasses ( M a c 2 0 Experimental de Cana-de-AGucar, Brazil); yeast extract, peptone and agar (Merck, USA); Celluclast 1.5 L (cellulase) (NOVONordisk, Denmark); cellulose powder (Sigma, UK); monosaccharide standards (myo-inositol, glucose, mannose, arabinose, xylose, ficose, galactose, ribose and glucuronolactone) (Sigma, UK); trifluoroacetic acid
24 New sources, structure and properties of cellulose (Janssen Chimica, UK); formic acid, diethylamine, l-methyl imidazole, dimethyl sulphoxide and acetic anhydride (BDH, UK); barium carbonate (Sigma, UK); sulphur dioxide (Fluka, UK), methyl iodide (Aldrich, UK). Other chemicals of analytical grade were obtained commercially. Microorganism An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated fiom an agro-industrial environment in the north-eastern region of Brazil. The identification of the microorganism was carried out at the Instituto de Antibibticos, Universidade Federal de Pernambuco, Brazil. The Zoogloea sp. was maintained as slant cultures at 4 "C. The yeast extract (5.0 peptone (3.0 culture medium consisted of glucose (20.0 agar (15.0 g/L) in deionised watcr and the pH was adjusted to 6.8 before sterilisation (120 "C, 20 rnin).
a),
a),
a),
Production of the exopolysaccharide sample
For the exopolysaccharideproduction, cells were transferred from agar slants to 100 mL of sterilised medium (120"C, 40 min), consisting of sugarcane molasses (15 "Brix, pH 5.0), in 250 mL Erlenmeyer flasks. Cultures were incubated at 30 "C for 7 days. The polysaccharide pellicles, a gel-like material, were formed at the air-liquid interface. The pellicles were washed in deionised water, sterilised (120"C, 40 min) and dried at 60°C on glass plates to form solid polysaccharide sheets. Determination of the water soluble material of the polysaccharide sheet
The polysaccharide sheet (21 1.04 mg) was cut into small particles (ca. 4 mm') and transferred to a centrifbge tube. Deionised water (6 mL) was added, the material was mixed for 1 min using a vortex mixer, left to stand for 15 min, mixed again for a further 1 min and centrihged at 8,OOOg for I5 rnin. The residue was washed with deionised water (6 mL) and centrihged at 8,000 g for 15 min. This procedure was repeated twice. The supernatants were combined, lyophilised and dried using an Abderhalden dryer at 39 "C. The residue was dried using an Abderhalden dryer (39 "C) and the dried weight of the residue determined. Mild acid hydrolysis of the polysaccharide smmple
The water insoluble residue (47.51 mg) was suspended in trifluoroacetic acid (2 M, 4 mL) in a round bottom flask, allowed to stand for 45 min and refluxed at 97 "C for 3 h. The hydrolysate was left at ambient temperature for 20 h. Trifluoroacetic acid was removed by drying the sample using a rotor evaporator. The hydrolysate was washed with deionised water (2 mL) which was also removed by evaporation. This procedure was repeated six times. The residue was transferred to a centrifbge tube and deionised water (1.3 mL) was added. The sample was centrihged at 8,000 g for 30 An. This procedure was repeated three times and thc supernatants were combined and stored at -20°C prior to anion exchange high performance liquid chromatography analysis. The acid insoluble residue was dried usirig an Abdcrhalden dryer (39 "C) and the dry weight determined.
Highly cellulosic exopolysaccharide 25 Strong acid hydrolysis of the polysaccharide sample
Trifluoroacetic acid (99%, 3 mL) was added to the dried acid insoluble residue (33.5 mg) and allowed to stand at ambient temperature (16 h) to swell. The sample was refluxed for 2 h, at 97 "C, diluted to 80% trifluoroacetic acid with deionised water and refluxed for 30 min. The sample was diluted again to 30% trifluoroacetic acid with deionised water and refluxed for a fbrther 4 h. The trifluoroacetic acid was removed and the sample worked up as for the mild acid hydrolysates. Monosaccharide analysis using anion exchange high performance liquid chromatograplry
The trifluoroacetic acid hydrolysates were neutralized with sodium hydroxide solution prior to the analysis. The analysis of monosaccharides of the hydrolysates was performed on a Dionex DX-500system consisting of a GP40 gradient pump and a Dionex ED40 electrochemical detector. The standard sequence of potentials for carbohydrate detection (50 mV for 200 ms; 750 mV for 200 ms and -150mV for 400 ms) was applied to the Au ED40 working electrode for pulsed amperometric detection. The columns used were a CarboPac PA1 analytical column (250 x 4.0 mm ID) and a CarboPac PA1 guard columns (50 x 4.0 mm ID). All eluents and reagents were prepared using water purified to 18.2 Mi2 using a UHQPS system (Elga). Eluents A, B and C were deionised water, sodium acetate (1 M) and sodium hydroxide (1 M), respectively. Sodium hydroxide (300 mM) at 0.5 a m i n was added as a post column reagent. After equilibrating the system with deionised water for 30 min a sample (50 pL) was injected. The sodium acetate concentration was then increased, linearly, to 200 mM over the following 10 min and held at this level for 10 min. Oligosaccharide analysis using anion exchange high performance liquid chromatography - method 1
The analysis of the oligosaccharide component of the water soluble extract and the mild trifluoroacetic acid hydrolysate was performed on a Waters 625-LC system fitted with a non-metallic flow path, a 464 pulsed amperometric detector (PAD) fitted with a gold working electrode and a base stable reference electrode and a Whisp 712 injector. Sodium hydroxide (300 mM), at a flow rate of 0.7 mWmin, was added to the eluent stream between the columns and detector. The PAD was operated in the cathodic mode with the following sequence of potentials: 50 mV for 200 ms; 800 mV for 200 ms; and 600 mV for 500 ms. The columns used were a CarboPac PA1 analytical column (250 x 4.0 mm ID) and a CarboPac PA1 guard column (50 x 4.0 mm ID). N1 eluents and reagents were prepared using water purified to 18.2 Mi2 quality using a UHQPS system (Elga). Eluents A, B, C, and D were sodium hydroxide (100 mM), sodium hydroxide (100 mM) containing sodium acetate (800 mM), sodium hydroxide (300 mM), and sodium hydroxide (500 mM), respectively. Eluent C was used as the post column reagent. After equilibrating the system with sodium hydroxide (100 mM) a sample (200 pL) was injected: The sodium acetate concentration was then increased, linearly, to 800 mM over the following 60 min and held at this level for 5 min.
26 New sources, structure and properties of cellulose Oligosaccharide analysis using anion exchange high performance liquid chromatography - method 2
The method used for the analysis of the oligosaccharide component of the strong trifluoroacetic acid hydrolysate and the enzyme hydrolysates was similar to method 1. The only difference was that the concentration of sodium acetate was increased, linearly, to 400 mh4 over 60 min. Methylation analysis
The water insoluble polysaccharide was methylated by three cycles according to method described by Isogai et al. (9).
Preparation of polysaccharide siispension The water insoluble polysaccharide (32.4 mg) was dispersed in dimethyl sulphoxide (2.84 mL). Concentrated sulphur dioxide-methyl sulphoxide solution (ca. 0.3 dmL) was prepared as follows: sulphur dioxide (15 g) was bubbled into methyl sulphoxide (50 mL). Sulphur dioxide-methyl sulphoxide solution (165 pL) was added to the sample. Then, diethylamine (61 pL) was added. The suspension was stirred at ambient temperature for 20.3 h. Complete dissolution was not obtained.
Methylation of polysuccharide Freshly powdered sodium hydroxide (389 mg) was added to the sample, at ambient temperature, and the mixture stirred for 1 h under helium. Methyl iodide (390 pL) was added dropwise, at ambient temperature, and the mixture was stirred for 1 h, and then kept at 40 "C for 0.5 h, at 50 "C for 0.5 h and at 60 "C for 1 h. The methylation procedure was repeated three times, starting from the preparation of polysaccharide suspension (from the addition of sulphur dioxide-methyl sulphoxide solution). When solidification occurred during stirring, methyl sulphoxide was added to the sample to regenerate the slurry (1 mL prior to the second methylation and 2 mL prior to third methylation). The methylated products were isolated by dialysis and subsequent lyophilised.
Formic acid and sulljhuric acid hydrorySis The dry methylated product (11.8 mg) was allowed to swell in formic acid solution (90%, 1.18 mL) overnight, at ambient temperature. The sample was then refluxed during 2 h, the formic acid evaporated, the residue washed once with deionised water (1 mL) and then evaporated. Sulphuric acid solution (0.125 M, 2.95 mL) was added to the sample and refluxed during 10 h, neutralised with barium carbonate (ca. 236 mg) and the insoluble barium sulphate removed by centrifhgation (7,500 rpm, 15 min). The partially methylated sugars present in the supernatant were then used for the preparation of alditol acetates.
Highly cellulosic exopolysaccharide 27 Preparation of alditol acetates
The resulting partially methylated aldoses were converted to partially methylated alditol acetates as described by Blakeney et al. (10). Ammonia (80 pL) was added to the hydrolysate (containing ca. 2 mg methylated monosaccharides in 0.5 mL). myo-Inositol solution (4 mg/mL, 100 pL) was added as an internal standard. Sodium borohydride (ca. 70 mg) was added and the sample was allowed to stand at 40 "C for 1.5 h. Excess borohydride was destroyed by addition of glacial acetic acid (0.2 mL). After cooling to ambient temperature, I-methyl imidazole (1.5 mL) was added and the sample shaken vigorously to ensure it was in solution. Acetic anhydride (5 mL) was added cautiously (in a bath containing a mixture of ice and cold water), causing an immediate rise in temperature. The sample was allowed to react for 20 min, with frequent gentle agitation. The excess anhydride was hydrolised by addition of deionised water (12 mL) and shaken to thoroughly mix the sample. It was cooled for 5 min in a bath containing a mixture of ice and water, then extracted twice with dichloromethane (1 mL). The dichloromethane (lower layer) was removed by dropping a pipette; the extracts from the sample were combined and submitted to gas liquid Chromatographyanalysis. Gas liquid chromatography nnalysis
Quantification of the partially methylated alditol acetates was carried out on a Carlo Erba GC 8000 series gas chromatograph fitted with a BPX 70 (70% biscyanopropylpolysilphenylene-siloxane, 25 m x 0.33 mm, 0.25 pm film thickness) capillary column (supplied by SGE, UK) and a flame ionisation detector. The column pressure was 150 kPa and the flow of helium was 2 mL/min. The initial temperature of the oven was 150 "C and was increased at a rate of 10 "Chin to 190 "C and maintained for 2 min. The temperature was then increased at a rate of 5 "Chin to a final temperature of 250 "C and maintained for 10 min. The injection temperature was 260 "C and the detector temperature was 300 "C. Gas cliromatography nnd mass spectrometry
Gas chromatography and mass spectrometry (GC/MS) analyses were carried out on a Prospec (VG Company) equipped with a BPX 70 (70% biscyanopropylpolysilphenylene-siloxane, 50 m x 0.33 mm, 0.25 pm film thickness) capillary column (supplied by SGE, UK). The initial temperature of the oven was 150 "C and was increased at a rate of 10 "Chin to 190 "C and maintained for 2 min. The temperature was then increased at a rate of 5 "Chin to a final temperature of 250 "C and maintained for 5 min. Enzymatic liydrolysis of the polysaccharide sample
Particles of the water insoluble fraction of the polysaccharide sheet (4.3 mg) were suspended in sodium acetate buffer (0.4 M, pH 5.0, containing Celluclast 1.5 L, 5 pL/mL, 860 to give a concentration of 5 mg sample/mL. Cellulose powder (3.5 mg) was also suspended in sodium acetate buffer (0.4 M, pH 5.0, containing Celluclast 1.5 L, 5 pWmL, 700 pL). The samples and control (sodium acetate buffer containing Celluclast 1.5 L) were mixed and incubated in a water bath at 50 "C for 68 h and then at 100 "C for 10 min to deactivate the enzyme. After centrihgation at 10,000 g for 30 min, the
a),
28
New sources, structure and properties of cellulose
supernatants were decanted and deionised water (same volume used for the enzymatic hydrolysis) was added to the residues. The samples were centrifbged at 10,000 g for 30 min. The supernatants were combined and stored at -20 "C prior to anion exchange high performance liquid chromatography analysis. RESULTS AND DISCUSSION
Composition analysis
The contents of the water soluble and insoluble components of the polysaccharide sheet were 12 and 88 % (w/w). respectively. The profile of the oligosaccharides present in the water soluble component, compared with an oligosaccharide fingerprint of a starch hydrolysate reference material, indicated the presence of more than one neutral monosaccharide and a number of disaccharides with different monosaccharide composition or linkage position or type. Polysaccharides are hydrolysed under acid conditions to their component monosaccharides by cleavage of the glycosidic linkage at the bond between the anomeric carbon atom and the glycosidic oxygen. Conditions chosen for acid hydrolysis of polysaccharides are always a compromise between release and destruction of the component monosaccharides. Depending on the type of structural information required, conditions can be selected to give optimum release for monosaccharides or to favour release of oligosaccharide fragments. In this study two acid hydrolysis conditions (mild and strong) were chosen to release the monosaccharides. The percentages of soluble and insoluble components obtained from the trifluoroacetic acid hydrolysis of the polysaccharide sheet, under mild conditions, were 15.8 and 84.2% (w/w), respectively. The percentages of soluble and insoluble components obtained from the trifluoroacetic acid hydrolysis (using strong conditions) of the acid insoluble fraction (obtained using mild conditions) of the polysaccharide sheet were 72.2 and 12.0% (w/w), respectively. The total percentage of soluble material obtained from the trifluoroacetic acid hydrolysis, using mild and strong conditions, was 88% (w/w) of the water insoluble fraction. The monosaccharide compositions of the soluble components obtained from the trifluoroacetic acid hydrolysis of the polysaccharide sheet, under mild and strong conditions, determined by anion exchange high performance liquid chromatography analysis, are shown in Table 1. Glucose accounted for 87.6% of the total monosaccharides present in the acid hydrolysates (mild and strong conditions). Galactose was present in a very small amount (0.1%). These results differ from those obtained in the literature for zooglan, which is composed of D-glucose, D-galactose and pyruvic acid in an approximate ratio of 11:3:1.5 (8). The content of xylose (8.6%) was higher than the other monosaccharides present in the hydrolysates. The percentage of monosaccharides present in the acid hydrolysates (strong and mild conditions), determined by anion exchange high performance liquid chromatography, is shown in Table 2. The total percentage of monosaccharides released by the acid hydrolysis was only 38.4% of the total acid soluble component. The oligosaccharide profile of the trifluoroacetic acid hydrolysate, under mild conditions, showed a main peak in the monosaccharide region and other small peaks in the region of di- and tetra-saccharides, It should be noted that the ratio of the two peaks in the monosaccharide component is significantly different for the trifluoroacetic acid hydrolysate produced using the mild conditions and the water soluble component. This ,
Highly cellulosic exopolysaccharide 29 would suggest that there are differences in the solubility of sample according to the monosaccharide composition. Table 1 Monosaccharide compositions of the soluble fractions produced by trifluoroacetic acid hydrolysis, under mild and strong conditions, determined by anion exchange high performance liquid chromatography
".Monosaccharide ............... Fucose
Mild conditions Strong conditions Total ............................e??.?..Y!!!~.......... ...Y!!?.. !.! ...... !............ !? S%?. I.Y!!.!) .....
Arabinose Rhamnose
Galactose Glucosc
Xylose Mannose Ribose
Glucuronic acid Total
.
0.0 I 1.91 0.22 1.58 55.41 29.7 1 1.96 2.37 6.83 100.00
0.01 0.26
0.03 89.88 7.06 0.74 1.63 0.39 100.00
0.01 0.37 0.01 0.13 87.57 8.58
0.82 1.68 0.83 100.00
Table 2 Percentnges of monosacchnridcs, present in the trifluoroacetic acid hydrolysates of the polysaccharide sheet, using mild and strong conditions, determined by anion exchange high performance liquid chroniatography
Treatment Mild
Monosaccharides (%, w/w)'
14.4 43.7 38.4 * Calculated in relation to the amount of material solubiliscd by thc acid treatmcnt Strnne Total
The trifluoroacetic acid hydrolysate, obtained using strong conditions, was analysed for oligosaccharide content at two different concentrations. The main peak was in the monosaccharide region and its elution position correlates with glucose, in the maltodextrin reference material. It should be noted that even with the higher column load the monosaccharide is a single peak unlike the doublet which was observed in the oligosaccharide profiles of the mild trifluoroacetic acid hydrolysate and of the water soluble component .of the polysaccharide sheet. However, the disaccharide peak in the hydrolysate does not correlate with the elution position of maltose, the disaccharide present in the maltodextrin reference material, which would indicate that although the monosaccharide residue is glucose the polysaccharide linkage is different. As expected, these results confirm that a higher amount of material was released from the polysaccharide sheet by the strong acid conditions compared with the mild conditions. Structure analysis
Methylation analysis was carried out in order to hrther study the polysaccharide sheet. Methylation of the polysaccharide sheet demonstrated the presence of the sugar residues shown in Table 3. The main components of the polysaccharide are (1-4) linked glucose (74.7%) and (1-4-6) linked glucose (17.7%). Other linkages detected are (1-3-4), (1-2-
30 New sources, structure and properties of cellulose 4). (1-3-4-6) and (1-2-4-6) linked branch points. A small amount (2.2%) of (1-4) linked pentose is also present. These results are in agreement with the compositional analysis of the polysaccharide sheet which showed mainly the presence of glucose and a small amount of xylose. Table 3 Partially methylated alditol acetates from the polysaccharidesheet obtained from Zoogloea sp. Peaks were assigned by comparison of the fragmentation patterns (GC-MS) with published data (11) % .............. Compound .................................................................... Typc . .....of .....l..i n k q ............................................................................ e Molar ratio
.2,3,4,6-tetra-O-methyIhcxitol 2,3,5-tri-O-methyl pentitol 2,3,6-tri-O-mcthyI hcxitol 2,6-di-O-methyl hexitol 3,6-di-O-methyl hexitol 2,3-di-O-mcthyl hcxitol 2-0-mcthyl hexitol 3-0-methyl hexitol .
Tcrminal hcxosc (1 - 4) pentose (1 - 4) hcxosc (1 3 - 4) hexose (1 - 2 4) hexose (1 - 4 - 6) hexosc (1 - 3 - 4 6) hexosc (1 2 4 - 6) hcxose
-
-
- -
-
2.0 6.2 208.7 1.1
1.o 49.6 3.6 7.2
0.73 2.23 74.69 0.4 1 0.36 17.74 1.28 2.56
Enzymatic hydrolysis
In order to hrther confirm the monosaccharide linkage position and type in the polysaccharide sheet, hydrolysis using an enzyme with a known specificity was used. The commercial enzyme preparation Celluclast 1.5 L hydrolyses cellulose, (1 +)-I3-Dglucopyranose residues in a polysaccharide. A sample of cellulose powder and of the polysaccharide sheet were incubated with the enzyme preparation Cellulcast 1.5 L and the hydrolysates were subjected to oligosaccharide analysis. The two oligosaccharide profiles were very similar. The monosaccharide peak elutes in the position identified as glucose from the analysis of the maltodextrin reference material. The smaller components, and possibly the dimer cellobiose, are common to both hydrolysates. From these analyses it can therefore be concluded that the polysaccharide sheet contains some (1+4)-fi-D-glucose residues which are hydrolysed to glucose by the enzyme. CONCLUSIONS
The contents of the water soluble and insoluble components of the polysaccharide sheet were 12 and 88%; respectively.The total percentage of soluble material obtained from trifluoroacetic acid hydrolysis of the water insoluble fraction of the polysaccharide sheet, using mild and strong conditions, was 88% (w/w). The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.I%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-mcthylhexitol (74.7%) and 2,3die-methylhexitol (1 7.7%). Enzyme hydrolysis of the polysaccharide with a cellulase (Celluclast 1.5 L) confirmed the presence of (1+4)-l3-D-glucopyranosyl units. ACKNOWLEDGMENTS
We thank Prof. J. Otamar, from the Instituto de Antibibticos, Universidade Federal de Pemambuco, Recife, Brazil, for his assistance in the identification of the Zoogloea sp.
Highly cellulosic exopolysaccharide 3 1 We also thank Mr. G. Burns, from the School of Chemistry, University of Birmingham, UK, for his assistance in the monosaccharide analysis.
REFERENCES 1 I W Sutherland, Biotechnology of microbial exopolysaccharides, Cambridge, Cambridge University Press, 1990. 2 S Masaoka, T Ohe and N Sakota, ‘Production of cellulose from glucose by Acetobacter xylinirm’, Joiimal of Fermentation and Bioengineering, 1993 75( 1) 18-22. 3 A Okiyama, H Shirae, H Kano and S Yamanaka, ‘Bacterial cellulose I. Two-stage fermentation process for cellulose production by Acetobacter uceti’, Food Hydrocolloids, 1992 6(5) 471-477. 4 A Okiyama, M Motoki, and S Yamanaka, ‘Bacterial cellulose 11. Processing of the gelatinous cellulose for food materials’, Food Hydrocolloids, 1992 6(5) 479-487. 5 S Yamanaka and K Watanabe, ‘Applications of bacterial cellulose’. In R D Gilbert (Ed.), Cellitlosic Polymers, Blends and Composites (pp. 207-2 15). Munich, Hanser Publishers, 1994. 6 S Yamanaka, K Watanabe, N Kitamura, M Iguchi, S Mitsuhashi, Y Nishi, and M Uryu ‘The structure and mechanical properties of sheets prepared from bacterial cellulose’, Journal Material Science, 1989 24 3 141-3 145. 7 M Takai, ‘Bacterial cellulose’. In R D Gilbert (Ed.), Cellulosic Polymers, Blends and Composites (pp. 233-240). Munich, Hanser Publishers, 1994. 8 F Ikeda, H Shuto, T Saito, T Fukui, and K Tomita, ‘An extracellular polysaccharide produced by Zoogloea ramigera 115’, European Journal of Biochemistry, 1982 123 437-445. 9 A Isogai, A Ishizu, and J Nakano, ‘A new facile methylation method for cell-wall polysaccharides’. Carbohydrate Research, 1985 138 99-108. 10 A B Blakeney, P J Harris, R J Henry, and B A Stone, ‘A simple and rapid preparation of alditol acetates for monosaccharide analysis’, carbohydrate Research, 1983 113 291299. 1 1 P-E Jansson, L Kenne, H Liedgren, B Lindberg and J Lonngren, ‘A practical guide to the methylation analysis of carbohydrates’,Chemical Cornmimicatioris, 1976 8 1-74.
A highly cellulosic exopolysaccharide produced from sugarcane molasses by a Zougfoea sp. M. Paterson-Beedle', L. L. Lloyd', J. F. Kennedy', F. A. D. Melo: & V. Medeiros2 'Birmingham Carbohydrate and Protein Technology Group, ChembiotechLaboratories, University of Birmingham Research Park, Birmingham, BIS ZSQ, UK 'EstaCSo Experimental de Cana-de-A qucar de Carpina- Universidade Federal Rural de Pernambuco, Carpina, Pernanibuco, CEP 55 810 000, Brazil
Brazilian Zoogloea sp. bacterium produced from sugarcane molasses a polymer substance containing glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0. l%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitoI(74.7%) and 2,3-di-Omethylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1+4)-8-D-glucopyranosyl units.
INTRODUCTION Cellulose is produced by Acetobacter xylinum and other, mainly Gram-negative, bacterial species (1,2), the cellulose being excreted into the medium, where it rapidly aggregates as microfibrils. Bacterial cellulose has long been used in foods in Asian countries (3,4) and its different properties from wood-derived cellulose opens new industrial applications. Bacterial cellulose possesses high crystallinity, high degree of polymerization, high tensile strength and tear resistance, and high hydrophilicity that distinguish it from other forms of cellulose (5). Various potential industrial applications for bacterial cellulose include acoustic diaphragms, artificial skin or wound healing, filter membranes, ultra strength paper and paper additives (5,6,7). In food processing, suspensions of disintegrated bacterial cellulose have been found useful as thickening and binding agents and as a dietary fibre supplement (5). Zoogloea ramigera produces zooglan, a polysaccharide composed of D-glucose, Dgalactose and pyruvic acid in an approximate ratio of 11:3:1.5 (8). It is a long chain polysaccharide consisting of mainly 1,Clinked glucose residues and 1,4- and 1,3- linked galactose residues with branches of glucose residues at the C-3 or C-6 positions of the galactose residues. .The pyruvic acid residues, the acidic component, are linked to the nonreducing end and/or 1,3-linked glucose residues through 4,6-ketal linkages (8). Here, we present recent studies on the composition and structure of an exopolysaccharide produced from sugarcane molasses by a Zoogloea sp. EXPERIMENTAL Materials
The following materials were obtained from sources indicated: sugarcane molasses ( M a c 2 0 Experimental de Cana-de-AGucar, Brazil); yeast extract, peptone and agar (Merck, USA); Celluclast 1.5 L (cellulase) (NOVONordisk, Denmark); cellulose powder (Sigma, UK); monosaccharide standards (myo-inositol, glucose, mannose, arabinose, xylose, ficose, galactose, ribose and glucuronolactone) (Sigma, UK); trifluoroacetic acid
24 New sources, structure and properties of cellulose (Janssen Chimica, UK); formic acid, diethylamine, l-methyl imidazole, dimethyl sulphoxide and acetic anhydride (BDH, UK); barium carbonate (Sigma, UK); sulphur dioxide (Fluka, UK), methyl iodide (Aldrich, UK). Other chemicals of analytical grade were obtained commercially. Microorganism An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated fiom an agro-industrial environment in the north-eastern region of Brazil. The identification of the microorganism was carried out at the Instituto de Antibibticos, Universidade Federal de Pernambuco, Brazil. The Zoogloea sp. was maintained as slant cultures at 4 "C. The yeast extract (5.0 peptone (3.0 culture medium consisted of glucose (20.0 agar (15.0 g/L) in deionised watcr and the pH was adjusted to 6.8 before sterilisation (120 "C, 20 rnin).
a),
a),
a),
Production of the exopolysaccharide sample
For the exopolysaccharideproduction, cells were transferred from agar slants to 100 mL of sterilised medium (120"C, 40 min), consisting of sugarcane molasses (15 "Brix, pH 5.0), in 250 mL Erlenmeyer flasks. Cultures were incubated at 30 "C for 7 days. The polysaccharide pellicles, a gel-like material, were formed at the air-liquid interface. The pellicles were washed in deionised water, sterilised (120"C, 40 min) and dried at 60°C on glass plates to form solid polysaccharide sheets. Determination of the water soluble material of the polysaccharide sheet
The polysaccharide sheet (21 1.04 mg) was cut into small particles (ca. 4 mm') and transferred to a centrifbge tube. Deionised water (6 mL) was added, the material was mixed for 1 min using a vortex mixer, left to stand for 15 min, mixed again for a further 1 min and centrihged at 8,OOOg for I5 rnin. The residue was washed with deionised water (6 mL) and centrihged at 8,000 g for 15 min. This procedure was repeated twice. The supernatants were combined, lyophilised and dried using an Abderhalden dryer at 39 "C. The residue was dried using an Abderhalden dryer (39 "C) and the dried weight of the residue determined. Mild acid hydrolysis of the polysaccharide smmple
The water insoluble residue (47.51 mg) was suspended in trifluoroacetic acid (2 M, 4 mL) in a round bottom flask, allowed to stand for 45 min and refluxed at 97 "C for 3 h. The hydrolysate was left at ambient temperature for 20 h. Trifluoroacetic acid was removed by drying the sample using a rotor evaporator. The hydrolysate was washed with deionised water (2 mL) which was also removed by evaporation. This procedure was repeated six times. The residue was transferred to a centrifbge tube and deionised water (1.3 mL) was added. The sample was centrihged at 8,000 g for 30 An. This procedure was repeated three times and thc supernatants were combined and stored at -20°C prior to anion exchange high performance liquid chromatography analysis. The acid insoluble residue was dried usirig an Abdcrhalden dryer (39 "C) and the dry weight determined.
Highly cellulosic exopolysaccharide 25 Strong acid hydrolysis of the polysaccharide sample
Trifluoroacetic acid (99%, 3 mL) was added to the dried acid insoluble residue (33.5 mg) and allowed to stand at ambient temperature (16 h) to swell. The sample was refluxed for 2 h, at 97 "C, diluted to 80% trifluoroacetic acid with deionised water and refluxed for 30 min. The sample was diluted again to 30% trifluoroacetic acid with deionised water and refluxed for a fbrther 4 h. The trifluoroacetic acid was removed and the sample worked up as for the mild acid hydrolysates. Monosaccharide analysis using anion exchange high performance liquid chromatograplry
The trifluoroacetic acid hydrolysates were neutralized with sodium hydroxide solution prior to the analysis. The analysis of monosaccharides of the hydrolysates was performed on a Dionex DX-500system consisting of a GP40 gradient pump and a Dionex ED40 electrochemical detector. The standard sequence of potentials for carbohydrate detection (50 mV for 200 ms; 750 mV for 200 ms and -150mV for 400 ms) was applied to the Au ED40 working electrode for pulsed amperometric detection. The columns used were a CarboPac PA1 analytical column (250 x 4.0 mm ID) and a CarboPac PA1 guard columns (50 x 4.0 mm ID). All eluents and reagents were prepared using water purified to 18.2 Mi2 using a UHQPS system (Elga). Eluents A, B and C were deionised water, sodium acetate (1 M) and sodium hydroxide (1 M), respectively. Sodium hydroxide (300 mM) at 0.5 a m i n was added as a post column reagent. After equilibrating the system with deionised water for 30 min a sample (50 pL) was injected. The sodium acetate concentration was then increased, linearly, to 200 mM over the following 10 min and held at this level for 10 min. Oligosaccharide analysis using anion exchange high performance liquid chromatography - method 1
The analysis of the oligosaccharide component of the water soluble extract and the mild trifluoroacetic acid hydrolysate was performed on a Waters 625-LC system fitted with a non-metallic flow path, a 464 pulsed amperometric detector (PAD) fitted with a gold working electrode and a base stable reference electrode and a Whisp 712 injector. Sodium hydroxide (300 mM), at a flow rate of 0.7 mWmin, was added to the eluent stream between the columns and detector. The PAD was operated in the cathodic mode with the following sequence of potentials: 50 mV for 200 ms; 800 mV for 200 ms; and 600 mV for 500 ms. The columns used were a CarboPac PA1 analytical column (250 x 4.0 mm ID) and a CarboPac PA1 guard column (50 x 4.0 mm ID). N1 eluents and reagents were prepared using water purified to 18.2 Mi2 quality using a UHQPS system (Elga). Eluents A, B, C, and D were sodium hydroxide (100 mM), sodium hydroxide (100 mM) containing sodium acetate (800 mM), sodium hydroxide (300 mM), and sodium hydroxide (500 mM), respectively. Eluent C was used as the post column reagent. After equilibrating the system with sodium hydroxide (100 mM) a sample (200 pL) was injected: The sodium acetate concentration was then increased, linearly, to 800 mM over the following 60 min and held at this level for 5 min.
26 New sources, structure and properties of cellulose Oligosaccharide analysis using anion exchange high performance liquid chromatography - method 2
The method used for the analysis of the oligosaccharide component of the strong trifluoroacetic acid hydrolysate and the enzyme hydrolysates was similar to method 1. The only difference was that the concentration of sodium acetate was increased, linearly, to 400 mh4 over 60 min. Methylation analysis
The water insoluble polysaccharide was methylated by three cycles according to method described by Isogai et al. (9).
Preparation of polysaccharide siispension The water insoluble polysaccharide (32.4 mg) was dispersed in dimethyl sulphoxide (2.84 mL). Concentrated sulphur dioxide-methyl sulphoxide solution (ca. 0.3 dmL) was prepared as follows: sulphur dioxide (15 g) was bubbled into methyl sulphoxide (50 mL). Sulphur dioxide-methyl sulphoxide solution (165 pL) was added to the sample. Then, diethylamine (61 pL) was added. The suspension was stirred at ambient temperature for 20.3 h. Complete dissolution was not obtained.
Methylation of polysuccharide Freshly powdered sodium hydroxide (389 mg) was added to the sample, at ambient temperature, and the mixture stirred for 1 h under helium. Methyl iodide (390 pL) was added dropwise, at ambient temperature, and the mixture was stirred for 1 h, and then kept at 40 "C for 0.5 h, at 50 "C for 0.5 h and at 60 "C for 1 h. The methylation procedure was repeated three times, starting from the preparation of polysaccharide suspension (from the addition of sulphur dioxide-methyl sulphoxide solution). When solidification occurred during stirring, methyl sulphoxide was added to the sample to regenerate the slurry (1 mL prior to the second methylation and 2 mL prior to third methylation). The methylated products were isolated by dialysis and subsequent lyophilised.
Formic acid and sulljhuric acid hydrorySis The dry methylated product (11.8 mg) was allowed to swell in formic acid solution (90%, 1.18 mL) overnight, at ambient temperature. The sample was then refluxed during 2 h, the formic acid evaporated, the residue washed once with deionised water (1 mL) and then evaporated. Sulphuric acid solution (0.125 M, 2.95 mL) was added to the sample and refluxed during 10 h, neutralised with barium carbonate (ca. 236 mg) and the insoluble barium sulphate removed by centrifhgation (7,500 rpm, 15 min). The partially methylated sugars present in the supernatant were then used for the preparation of alditol acetates.
Highly cellulosic exopolysaccharide 27 Preparation of alditol acetates
The resulting partially methylated aldoses were converted to partially methylated alditol acetates as described by Blakeney et al. (10). Ammonia (80 pL) was added to the hydrolysate (containing ca. 2 mg methylated monosaccharides in 0.5 mL). myo-Inositol solution (4 mg/mL, 100 pL) was added as an internal standard. Sodium borohydride (ca. 70 mg) was added and the sample was allowed to stand at 40 "C for 1.5 h. Excess borohydride was destroyed by addition of glacial acetic acid (0.2 mL). After cooling to ambient temperature, I-methyl imidazole (1.5 mL) was added and the sample shaken vigorously to ensure it was in solution. Acetic anhydride (5 mL) was added cautiously (in a bath containing a mixture of ice and cold water), causing an immediate rise in temperature. The sample was allowed to react for 20 min, with frequent gentle agitation. The excess anhydride was hydrolised by addition of deionised water (12 mL) and shaken to thoroughly mix the sample. It was cooled for 5 min in a bath containing a mixture of ice and water, then extracted twice with dichloromethane (1 mL). The dichloromethane (lower layer) was removed by dropping a pipette; the extracts from the sample were combined and submitted to gas liquid Chromatographyanalysis. Gas liquid chromatography nnalysis
Quantification of the partially methylated alditol acetates was carried out on a Carlo Erba GC 8000 series gas chromatograph fitted with a BPX 70 (70% biscyanopropylpolysilphenylene-siloxane, 25 m x 0.33 mm, 0.25 pm film thickness) capillary column (supplied by SGE, UK) and a flame ionisation detector. The column pressure was 150 kPa and the flow of helium was 2 mL/min. The initial temperature of the oven was 150 "C and was increased at a rate of 10 "Chin to 190 "C and maintained for 2 min. The temperature was then increased at a rate of 5 "Chin to a final temperature of 250 "C and maintained for 10 min. The injection temperature was 260 "C and the detector temperature was 300 "C. Gas cliromatography nnd mass spectrometry
Gas chromatography and mass spectrometry (GC/MS) analyses were carried out on a Prospec (VG Company) equipped with a BPX 70 (70% biscyanopropylpolysilphenylene-siloxane, 50 m x 0.33 mm, 0.25 pm film thickness) capillary column (supplied by SGE, UK). The initial temperature of the oven was 150 "C and was increased at a rate of 10 "Chin to 190 "C and maintained for 2 min. The temperature was then increased at a rate of 5 "Chin to a final temperature of 250 "C and maintained for 5 min. Enzymatic liydrolysis of the polysaccharide sample
Particles of the water insoluble fraction of the polysaccharide sheet (4.3 mg) were suspended in sodium acetate buffer (0.4 M, pH 5.0, containing Celluclast 1.5 L, 5 pL/mL, 860 to give a concentration of 5 mg sample/mL. Cellulose powder (3.5 mg) was also suspended in sodium acetate buffer (0.4 M, pH 5.0, containing Celluclast 1.5 L, 5 pWmL, 700 pL). The samples and control (sodium acetate buffer containing Celluclast 1.5 L) were mixed and incubated in a water bath at 50 "C for 68 h and then at 100 "C for 10 min to deactivate the enzyme. After centrihgation at 10,000 g for 30 min, the
a),
28
New sources, structure and properties of cellulose
supernatants were decanted and deionised water (same volume used for the enzymatic hydrolysis) was added to the residues. The samples were centrifbged at 10,000 g for 30 min. The supernatants were combined and stored at -20 "C prior to anion exchange high performance liquid chromatography analysis. RESULTS AND DISCUSSION
Composition analysis
The contents of the water soluble and insoluble components of the polysaccharide sheet were 12 and 88 % (w/w). respectively. The profile of the oligosaccharides present in the water soluble component, compared with an oligosaccharide fingerprint of a starch hydrolysate reference material, indicated the presence of more than one neutral monosaccharide and a number of disaccharides with different monosaccharide composition or linkage position or type. Polysaccharides are hydrolysed under acid conditions to their component monosaccharides by cleavage of the glycosidic linkage at the bond between the anomeric carbon atom and the glycosidic oxygen. Conditions chosen for acid hydrolysis of polysaccharides are always a compromise between release and destruction of the component monosaccharides. Depending on the type of structural information required, conditions can be selected to give optimum release for monosaccharides or to favour release of oligosaccharide fragments. In this study two acid hydrolysis conditions (mild and strong) were chosen to release the monosaccharides. The percentages of soluble and insoluble components obtained from the trifluoroacetic acid hydrolysis of the polysaccharide sheet, under mild conditions, were 15.8 and 84.2% (w/w), respectively. The percentages of soluble and insoluble components obtained from the trifluoroacetic acid hydrolysis (using strong conditions) of the acid insoluble fraction (obtained using mild conditions) of the polysaccharide sheet were 72.2 and 12.0% (w/w), respectively. The total percentage of soluble material obtained from the trifluoroacetic acid hydrolysis, using mild and strong conditions, was 88% (w/w) of the water insoluble fraction. The monosaccharide compositions of the soluble components obtained from the trifluoroacetic acid hydrolysis of the polysaccharide sheet, under mild and strong conditions, determined by anion exchange high performance liquid chromatography analysis, are shown in Table 1. Glucose accounted for 87.6% of the total monosaccharides present in the acid hydrolysates (mild and strong conditions). Galactose was present in a very small amount (0.1%). These results differ from those obtained in the literature for zooglan, which is composed of D-glucose, D-galactose and pyruvic acid in an approximate ratio of 11:3:1.5 (8). The content of xylose (8.6%) was higher than the other monosaccharides present in the hydrolysates. The percentage of monosaccharides present in the acid hydrolysates (strong and mild conditions), determined by anion exchange high performance liquid chromatography, is shown in Table 2. The total percentage of monosaccharides released by the acid hydrolysis was only 38.4% of the total acid soluble component. The oligosaccharide profile of the trifluoroacetic acid hydrolysate, under mild conditions, showed a main peak in the monosaccharide region and other small peaks in the region of di- and tetra-saccharides, It should be noted that the ratio of the two peaks in the monosaccharide component is significantly different for the trifluoroacetic acid hydrolysate produced using the mild conditions and the water soluble component. This ,
Highly cellulosic exopolysaccharide 29 would suggest that there are differences in the solubility of sample according to the monosaccharide composition. Table 1 Monosaccharide compositions of the soluble fractions produced by trifluoroacetic acid hydrolysis, under mild and strong conditions, determined by anion exchange high performance liquid chromatography
".Monosaccharide ............... Fucose
Mild conditions Strong conditions Total ............................e??.?..Y!!!~.......... ...Y!!?.. !.! ...... !............ !? S%?. I.Y!!.!) .....
Arabinose Rhamnose
Galactose Glucosc
Xylose Mannose Ribose
Glucuronic acid Total
.
0.0 I 1.91 0.22 1.58 55.41 29.7 1 1.96 2.37 6.83 100.00
0.01 0.26
0.03 89.88 7.06 0.74 1.63 0.39 100.00
0.01 0.37 0.01 0.13 87.57 8.58
0.82 1.68 0.83 100.00
Table 2 Percentnges of monosacchnridcs, present in the trifluoroacetic acid hydrolysates of the polysaccharide sheet, using mild and strong conditions, determined by anion exchange high performance liquid chroniatography
Treatment Mild
Monosaccharides (%, w/w)'
14.4 43.7 38.4 * Calculated in relation to the amount of material solubiliscd by thc acid treatmcnt Strnne Total
The trifluoroacetic acid hydrolysate, obtained using strong conditions, was analysed for oligosaccharide content at two different concentrations. The main peak was in the monosaccharide region and its elution position correlates with glucose, in the maltodextrin reference material. It should be noted that even with the higher column load the monosaccharide is a single peak unlike the doublet which was observed in the oligosaccharide profiles of the mild trifluoroacetic acid hydrolysate and of the water soluble component .of the polysaccharide sheet. However, the disaccharide peak in the hydrolysate does not correlate with the elution position of maltose, the disaccharide present in the maltodextrin reference material, which would indicate that although the monosaccharide residue is glucose the polysaccharide linkage is different. As expected, these results confirm that a higher amount of material was released from the polysaccharide sheet by the strong acid conditions compared with the mild conditions. Structure analysis
Methylation analysis was carried out in order to hrther study the polysaccharide sheet. Methylation of the polysaccharide sheet demonstrated the presence of the sugar residues shown in Table 3. The main components of the polysaccharide are (1-4) linked glucose (74.7%) and (1-4-6) linked glucose (17.7%). Other linkages detected are (1-3-4), (1-2-
30 New sources, structure and properties of cellulose 4). (1-3-4-6) and (1-2-4-6) linked branch points. A small amount (2.2%) of (1-4) linked pentose is also present. These results are in agreement with the compositional analysis of the polysaccharide sheet which showed mainly the presence of glucose and a small amount of xylose. Table 3 Partially methylated alditol acetates from the polysaccharidesheet obtained from Zoogloea sp. Peaks were assigned by comparison of the fragmentation patterns (GC-MS) with published data (11) % .............. Compound .................................................................... Typc . .....of .....l..i n k q ............................................................................ e Molar ratio
.2,3,4,6-tetra-O-methyIhcxitol 2,3,5-tri-O-methyl pentitol 2,3,6-tri-O-mcthyI hcxitol 2,6-di-O-methyl hexitol 3,6-di-O-methyl hexitol 2,3-di-O-mcthyl hcxitol 2-0-mcthyl hexitol 3-0-methyl hexitol .
Tcrminal hcxosc (1 - 4) pentose (1 - 4) hcxosc (1 3 - 4) hexose (1 - 2 4) hexose (1 - 4 - 6) hexosc (1 - 3 - 4 6) hexosc (1 2 4 - 6) hcxose
-
-
- -
-
2.0 6.2 208.7 1.1
1.o 49.6 3.6 7.2
0.73 2.23 74.69 0.4 1 0.36 17.74 1.28 2.56
Enzymatic hydrolysis
In order to hrther confirm the monosaccharide linkage position and type in the polysaccharide sheet, hydrolysis using an enzyme with a known specificity was used. The commercial enzyme preparation Celluclast 1.5 L hydrolyses cellulose, (1 +)-I3-Dglucopyranose residues in a polysaccharide. A sample of cellulose powder and of the polysaccharide sheet were incubated with the enzyme preparation Cellulcast 1.5 L and the hydrolysates were subjected to oligosaccharide analysis. The two oligosaccharide profiles were very similar. The monosaccharide peak elutes in the position identified as glucose from the analysis of the maltodextrin reference material. The smaller components, and possibly the dimer cellobiose, are common to both hydrolysates. From these analyses it can therefore be concluded that the polysaccharide sheet contains some (1+4)-fi-D-glucose residues which are hydrolysed to glucose by the enzyme. CONCLUSIONS
The contents of the water soluble and insoluble components of the polysaccharide sheet were 12 and 88%; respectively.The total percentage of soluble material obtained from trifluoroacetic acid hydrolysis of the water insoluble fraction of the polysaccharide sheet, using mild and strong conditions, was 88% (w/w). The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.I%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-mcthylhexitol (74.7%) and 2,3die-methylhexitol (1 7.7%). Enzyme hydrolysis of the polysaccharide with a cellulase (Celluclast 1.5 L) confirmed the presence of (1+4)-l3-D-glucopyranosyl units. ACKNOWLEDGMENTS
We thank Prof. J. Otamar, from the Instituto de Antibibticos, Universidade Federal de Pemambuco, Recife, Brazil, for his assistance in the identification of the Zoogloea sp.
Highly cellulosic exopolysaccharide 3 1 We also thank Mr. G. Burns, from the School of Chemistry, University of Birmingham, UK, for his assistance in the monosaccharide analysis.
REFERENCES 1 I W Sutherland, Biotechnology of microbial exopolysaccharides, Cambridge, Cambridge University Press, 1990. 2 S Masaoka, T Ohe and N Sakota, ‘Production of cellulose from glucose by Acetobacter xylinirm’, Joiimal of Fermentation and Bioengineering, 1993 75( 1) 18-22. 3 A Okiyama, H Shirae, H Kano and S Yamanaka, ‘Bacterial cellulose I. Two-stage fermentation process for cellulose production by Acetobacter uceti’, Food Hydrocolloids, 1992 6(5) 471-477. 4 A Okiyama, M Motoki, and S Yamanaka, ‘Bacterial cellulose 11. Processing of the gelatinous cellulose for food materials’, Food Hydrocolloids, 1992 6(5) 479-487. 5 S Yamanaka and K Watanabe, ‘Applications of bacterial cellulose’. In R D Gilbert (Ed.), Cellitlosic Polymers, Blends and Composites (pp. 207-2 15). Munich, Hanser Publishers, 1994. 6 S Yamanaka, K Watanabe, N Kitamura, M Iguchi, S Mitsuhashi, Y Nishi, and M Uryu ‘The structure and mechanical properties of sheets prepared from bacterial cellulose’, Journal Material Science, 1989 24 3 141-3 145. 7 M Takai, ‘Bacterial cellulose’. In R D Gilbert (Ed.), Cellulosic Polymers, Blends and Composites (pp. 233-240). Munich, Hanser Publishers, 1994. 8 F Ikeda, H Shuto, T Saito, T Fukui, and K Tomita, ‘An extracellular polysaccharide produced by Zoogloea ramigera 115’, European Journal of Biochemistry, 1982 123 437-445. 9 A Isogai, A Ishizu, and J Nakano, ‘A new facile methylation method for cell-wall polysaccharides’. Carbohydrate Research, 1985 138 99-108. 10 A B Blakeney, P J Harris, R J Henry, and B A Stone, ‘A simple and rapid preparation of alditol acetates for monosaccharide analysis’, carbohydrate Research, 1983 113 291299. 1 1 P-E Jansson, L Kenne, H Liedgren, B Lindberg and J Lonngren, ‘A practical guide to the methylation analysis of carbohydrates’,Chemical Cornmimicatioris, 1976 8 1-74.
THE SUPRAMOLECULAR STRUCTURE OF CELLULOSE 11. STUDIES WITH %-CP/MAS-NMR AND CHEMOMETRICS Helcna Lennholm Dep. of Pulp and Paper Chemistry and Technology KTH, SE-100 44 Stockholm, Sweden
INTRODUCTION Native cellulose I can be turned into cellulose I1 by mercerisation or regeneration. When a sample is mercerised it is swelled in strong alkali solution, and then washcd with water. Regeneration is achieved when the cellulose sample is dissolvcd, and then precipitated in water. Merccrisation is thus a solid state reaction, but regeneration is a reaction from solution state to solid state. Differences in origin of a ccllulose sample can thus be expccted to remain more after a mercerisation than after regencration. 13~-cross-po~arisation magic angle spinning nuclear magnetic resonance (CP/MASNMR)-spcctra of cellulose contain information regarding the amounts and structures of the different cellulose polymorphs and of unordered cellulose [l]. NMR-spectra of lignocellulosic samples, however, contain broad and overlapping peaks. This problem can be 0,vcrcomeby using chemometrics or spectral fitting. NMR-spectra of cellulose I have recently been thoroughly assigncd, using spectral fitting [2]. In earlier work [3] we used chemometrics to evaluate NMR-spectra of cellulose I and 11. In this work we wanted to study more thoroughly which factors determine the production of cellulose I1 from cellulose 1. We chose the starting cellulose materials to represent variations in the proportion of cellulose Ia and Ip, microfibril size and state of order. We used algal ccllulose, bacterial cellulosc, cotton linters and birch kraft pulp. Further, we varied the state of order of the starting materials by ball milling. Thc starting materials and ball-milled starting materials were all mercerised and regenerated. We recordcd 13C-CPAVIAS-NMR-spectraon all samples and evaluated the spectra with chemometrics. EXPERIMENTAL Starting materials
The cotton linters and bleached birch kraft pulp wcre commercially available samples. Bacterial cellulose was generated from commercial Nata de COCO,by boiling in 1 % NaOH for 2 days, then washing with deionised water. Algal cellulose was generated from Cfadophoru from the Baltic: Thc Cladophora was treated with 0.25 M HC1 for 3 days, then washed with deionised watcr. It was then treated with 0.05 g NaC102 and 0.015 ml acetic aced per gram Cfudophorufor 18 h. The chlorite treatment was repeatcd 3 times. After washing with deionised water the algal cellulose was treatcd with 0.1 M NaOH for 2 h, and washed. Mercerisation The samples were immersed in 16 % NaOH and kept at -18 "C for at least 18 h. The mcrcerised samples were then washed with watcr.
34 New sources, structure and properties of cellulose Regeneration A total of 5 g of each sample was dissolved in 50 ml tetrabutylammonium hydroxide (40% in water) and 50 ml dimethylsulphoxide and stirred for 2 h. The solution was then poured into water with some drops of acetic acid during agitation. The regenerate was then washed with water. Milling The samples were milled in a vibratory ball-mill, for 10 min.
NMR-spectroscopy Thc 13C-CJ?/MASNMR spectra were recorded on wet samples on a Bruker AMX-300 instrumcnt at ambient temperature. The spectromctcr operated at 75.47 MHz using a double air-bearing probe and ZrO2 rotors. Spinning rate was 5 kHz, contact time was 0.8 ms, acquisition time was 37 ms, sweep width was 368 ppm and delay betwcen pulses was 2.5 s. For each spectrum, 2000-15000 transients were accumulated with 2048 data points and zero fillcd to 4096 data points. The spectra were referenced to carbonyl in external glycine (6= 176.03 ppm). Chernometrics Principal component analysis (PCA) was carricd out using the SIMCA 7.0 s o b a r c .
RESULTS AND DISCUSSION Fig. 1 shows reference NMR-spcctra of cellulose Ia, cellulose IP, cellulose I1 and unordered cellulosc [3].
110
105
c z , 3,5
c4
GI
100
95
90
85
80
75
70
C6
65
60
55
Figure I I3C-CP/MAS-NMR-spectra representing different cellulose polyinorphs [3].
The NMR-spectra of the starting materials are shown in Fig. 2. Assignments are according to [4]. The spectra of algal and bacterial cellulose show typical appearance of cellulose Ia-pcaks (cf. Fig. 1). The spectrum of cotton show a more typical appcarance of the cellulose IP-peaks (cf. Fig. 1). In the spectra of birch pulp we see thc fcatures of
Supramolecular structure of cellulose I1 35
Cotton linters
110
105
100
95
85
90
70
75
80
65
60
55
50
Figure 2 13C-CP/MAS-NMR-spectra of the starting materials.
Merc linters
A
Merc bacterial
A Alg
A
A -_____
Merc alg Merc birch
A
Bacterial
'TGGrs
A
A
Birch
Figure 3 PCA scores-plot of the NMR-spectra of the starting materials and the mercerised samples.
a more disordered sample (cf. Fig. 1). The spectra of algal and bacterial cellulose reprcscnt samples with high proportion of cellulose Ia,whereas the spectra of cotton and birch represcnt samples with low proportion of cellulose Ia.If we study the peaks originating from thc surfaces o f the microfibrils (80-85ppm, cf. [2]), we furthcr sce that
36
New sources, structure and properties of cellulose
the algal cellulose and cotton have large microfibrils (small surface peaks), whereas bacterial cellulose and birch have small microfibrils (large surface peaks). Fig. 3 shows a principal component analysis (PCA) scores-plot where we can sce the NMR-spectra of the starting materials and the merccrised samples. There is a grouping of the starting materials to the right and mercerised samples to the left. We thus have the merccrisation along principal component 1 (PC1). The birch sample is, however, very close to the merceriscd algal and birch sample. The question is then if the mcrceriscd algal and birch sample still contain some cellulosc I?
Bacterial cellulose Cotton linters
110
105
I00
95
90
85
80
75
70
65
Figure 4 NMR-spectra of the mercerised samples.
Reg bacterial
A
A
Birch .Linters
Keg birch
A Reg linters
A
A
A Bacterial
60
55
50
Supramolecular structure of cellulose I1 37 The NMR-spectra of the mercerised samples are shown in Fig. 4. Indccd the mcrccrised algal and birch samplcs have NMR-signals corresponding to ccllulose 11, but also some features of ccllulose I (cf. Fig. 1). We further see that the merccrised bacterial and cotton samples show very broad patterns, corresponding to totally unordcrcd cellulose. These samples obviously did not turn into ccllulose I1 upon merccrisation. In Fig. 5 the PCA scores-plot of the NMR-spectra of the starting materials and the rcgeneratcd samples is shown. Here we see a clear grouping of starting materials (to the right) and regeneratcd samples (to the left) along PC1. The corresponding NMR-spectra of the rcgenerated samples are shown in Fig. 6 . These spectra are not as similar as the spectra of the merceriscd samples (Fig. 4). Upon a dissolution and regeneration we expected the samples to lose information about the cellulose structure of the starting materials, but from Fig. 6 we see that this is not the case. The samples with small microfibrils (bacterial and birch) thus seem to yicld regenerated samplcs with low amounts of cellulose 11, compared to the cotton sample (with large microfibrils).
Bacterial cellulose
Cotton linters
110
105
100
95
90
85
80
75
70
65
60
55
50
Figure 6 NMR-spectra of the regenerated samples.
Fig. 7 shows NMR-spcctra of some of the merceriscd and regencratcd milled samples. Here thc pattern is obvious; all the samples are now cellulose I1 (cf. Fig. 1). The disordering or lowering of molecular weight the milling introduced rcsulted in a thorough formation of ccllulose I1 upon mcrccrisation and regeneration, cf. Figs 4 and 6.
38 New sources, structure and properties of cellulose
110
105
I00
95
90
85
80
75
70
65
60
55
50
Figure 7 Some NMR-spectra of the regenerated and mercerised milled samples. CONCLUSIONS
In the study which factors determine the gcneration of cellulosc I1 from cellulose I we have indications that thc origin of the cellulose I-matcrials affected the result of both merccrisation and regeneration. We have so far not obscrved any systematic effect of the proportion cellulose Ia and Ip, neither upon merccrisation nor regeneration. Wc have indications that the microfibril size affect the regeneration of ccllulose I to cellulose 11. Ball milling of the starting materials resulted in increascd formation of cellulose 11, both during mercerisation and regeneration. ACKNOWLEDGEMENTS
Johan Fransson for the skilful expcrimental work. Dr. Tomas Larsson and Professor Tommy Ivcrscn for fruitful discussions. Carl Tryggers Stiftelse for financial support. RFFERENCES 1 A Isogai, M Usuda, T Kato, T Uryu and R H Atalla, Macromolecules, 1989 22 3168. 2 H Lcnnholm and T Iversen, Holzforschung, 1995 49(2) 119-126. 3 P T Larsson, K Wickholm and T Iversen, Carbohydrate Research, 1997 302 19-25. 4 D L Vandcrhart and R €1 Atalla, Macromolecules, 1984 17 1465-1472.
EFFECTS OF PULPING ON CRYSTALLINITY OF CELLULOSE STUDIED BY SOLID STATE NMR T. Liitiii', S. L. Maunu' and B. Hortling'
I Laboratory of Polymer Chemistty, P.O. Box 55, FIN-00014 Universi& of Helsinki, Finland 2 The Finnish Pulp and Paper Research Institute, Paper Science Centre,P.O. Box 70, FIN02151 E S ~ Q O FinIand ,
Solid state NMR spectroscopy has been used in this work to investigate crystallinity of cellulose in spruce wood before and after kraft pulping. Effects of bleaching and refining in water and in weak alkali have also been studied. It has been noticed that the I, crystalline form of cellulose predominates over the Ip form in native spruce and vice versa in all the pulps studied. In pulping part of the cellulose 1, is converted to the more stable Ip form mainly by heat. Cellulose I1 is also formed during pulping and the content of form I1 seems to be higher on the fiber surface. Any measurable changes in the degree of crystallinity or in the relative proportions of different crystalline forms of cellulose could not be seen in pulps after refining or TCF-bleaching. However, it was noticed that the degree of crystallinity was considerably lower in the fines than in the bulk fiber.
INTRODUCTION Cellulose is a scmicrystalline biopolymcr with ordered crystalline and disordered amorphous regions. The crystalline cellulose may crystallize in several different polymorphs. Cellulose I is synthesized natively and exists in two crystalline forms, cellulose I, and Ip.' Cellulose I, is metastable and can be converted to the more stable form Ip by heat?. It is assumed that I, and Ip cellulose have different conformations and that they differ also in hydrogen bonding.'. The difference between cellulose I and I1 is that cellulose chains in cellulose I are thought to be parallel, whereas in cellulose I1 they are probably antiparalle1.d Cellulose I1 is formed when the lattice of cellulose I is destroyed. Cellulose I can be converted to cellulose I1 by alkali treatment called mercerisation or by regeneration of dissolved cellulose. NMR spectroscopy in solid state enables us to investigate wood and pulp fibres in their original state. Thus the physical structure of the samples rcmains unchanged and ordinary "C CPMAS measurements can bc uscd to determine the degree of crystallinity of cellulose. The relative proportions of I,, Ip and I1 crystalline forms of cellulose can be obtained by deconvolution after resolution enhancement of NMR data. Solid statc NMR spectroscopy has been used in this work to investigate the crystallinity of cellulose in spruce wood bcfore and after kraft pulping. Effccts of TCFbleaching and refining in water and in weak alkali have been studicd. We have also separated thc fincs after refining and compared the crystallinity of cellulose between the
'
'
40 New sources, structure and properties of cellulose fines and the bulk fiber to find out whether the crystallinity of cellulose on the fiber surface differs from the bulk fiber.
EXPERIMENTAL Materials
We have studied Wiley-milled (40 mesh) spruce wood (Picea abies) and conventional haft pulp made of the same spruce. The pulp has been refined with a Voith Sulzcr laboratory refiner in water in two different ways (Kraftl, Kraft2) and also in 0.01 M NaOH solution (Kraft3). The first fraction of the pulp has been refined in water so that the fibers are ground more on the surface. The second refining in water has more cutting effect on the fibers. Refining in weak alkali has been made like the refining of Kraftl in water. The refining processes have been explained in more detail elsewhere5and Kraftl, Kraft2 and Kraft3 were referred to as Sal, Sa2 and Sa3, respectively. After refining the fines (Finel, Fine2, Fine3) have been separated from the bulk fibers (Kraftl-F, Kraft2F, Kraft3-F). The effects of TCF-bleaching have been studied after oxygen (0)stage and after oxygen, peroxide, ozone and peroxide treatments (OPZP). Methods
All the ordinary I3C CPMAS NMR measurements have been done with a Varian m"YINOVA 300 NMR spectrometer operating at 75.5 MHz for carbons. The spinning speed was 5000 Hz,contact time 1 ms, acquisition time 20 ms and delay between pulses 2 s for all samples. The time of accumulation has been 20-26 hours for pulps and 2.5 days for spruce wood. The measurements have been long in order to get good signal-tonoise ratio, because the amount of noice increases during resolution enhancement. All samples have been moistened with de-ionised water (-50 w-% H,O), because it is known that water improves resolution and signal-to-noise-ratio in the spectra of cellulose.6 All the spectra have been referenced using the C1 signal as an internal reference (6 105 ppm). The degrce of crystallinity of cellulose (CrI) has been defined from the areas of the crystalline (86-92 ppm) and amorphous (79-86 ppm) C4 signals by deconvolution using Lorenzian line shape.' To be able to see the different crystalline forms of cellulose, the spectra must be resolution enhanced. The resolution enhancement has been done using negative line broadening (lb) together with Gaussian hnction (go. Values of those parameters have been selected experimentally. After the resolution enhancement the relative proportions of different polymorphs have been defined from the deconvolution of the crystalline C4 signal as represented in the figurc 1.b. The signals of the polymorphs arc ovcrlapping doublets and thus the most intense peak at 88.9 ppm is believed to arise from the contribution of all three polymorphs. The fractions of Lorcnzian or Gaussian line shape in deconvolutions have been chosen so that the sum of the areas of the peaks at 89.7 ppm, 88.1 ppm and 87.5 ppm is closest to the area of the peak at 88.9 ppm.
Crystallinity of cellulose 41
110
loo
90
80
70
60
50
PPm
Figure I . a) The deconvolution of KrafiOPZP spectrum for determination of the degree of crystallinity of the cellulose. b) The deconvolution of crystulline C4 signal afier resolution enhancement for the determination of relative proportions of different crystalline forms of cellulose.
RESULTS AND DISCUSSION The crystallinity index of cellulose in spruce wood could not be determined exactly, becausc there are many signals of lignin and hemicelluloses at the same range as the amorphous C4 transition used for determination of the CrI. The CrI values determined for the pulps are not any absolute values of crystallinity either due to solubilisation of unordered materials during kraft pulping* and because pulp contains also some lignin and hemicelluloses. Thcir cffcct on crystallinity index of pulps is anyway thought to be very small and in this work we have used CrI as an approximate value for comparison between pulps. Any measurable changes in the degree of crystallinity could not be seen in pulps after refining or TCF-bleaching. It was noticcd, however, that the degree of crystallinity was considerably lower in the fines than in the bulk fibcr. In all pulps studied the crystallinity iiidcx is approximately 50%, whereas in the fines it is 37-40%. The content of lignin was noticcd to be higher in fines than in the pulps. That may affect little on CrI, but does not explain totally the lower amount of the crystalline cellulose in fines. Mechanical forces may break crystallites during refining and thus lower the CrI in fines. It is also possible that degree of crystallinity is lower on the fiber surface than inside the fiber. Thc "C CPMAS measurements proved that the I, crystalline form of cellulose predominates over the Ipin native spruce. The amount of the cellulosc I, is almost three times the amount of cellulose Ipin spruce wood. In the kraft pulps studicd the amount of cellulose Ip is twice the amount of cellulosc I,. Ccllulose I, is known to be
42 New sources, structure and properties of cellulose metastable and it can be converted to the more stable Ip form by heat. Conversion of the 1, form to the I p form has been found to require 260-280 "C temperatures in highly crystalline algal and bacterial celluloses? Although the temperature is only 170 OC in haft pulping the NaOH media probably accelerates the transformation of cellulose I, into cellulose Ip?. Samples of lower crystallinity, like wood in this case, have also been suggested to convert more easily? According to the NMR spectra small amount of cellulose I1 was also formed during haft pulping. 100% 80% 60%
mCellulose Ib
40%
20% 0% Kraft Pulp
Spruce Wood
Figure 2. Relative proportions of polymofls of the crystalline cellulose in spruce wood and in b a j l pulp.
The relative proportions of the different cellulose polymorphs and proportions of amorphous cellulose in refined and TCF-blcached pulps are represented in figures 3 and 4. Only marginal changes in the amounts of the crystalline forms of cellulose are observed during refining or bleaching. The proportions of cellulose I,, Ip and I1 of all the cellulose in pulps are 11-16%, 28-32% and 5-7%, respectively. In fines the proportions of cellulose I, and Ip are 9-1 1% and 20-23%, respectively, because of the lower CrI. The ratio I& is anyway 0.4 0.5 like in all the other pulps. The relative proportion of cellulose I1 is slightly higher in Finel and Fine3 than in the other samples studied. The ratio 1/11 is 4 in Finel and Fine3, whercas in all the other pulps it is 6-9. In Kraft3-F 1/11 ratio is as large as 11. Finel and Fine3 probably represent the surface of the fiber more than Fine2, because the first and third refining should be more surface active? In the second refining the fibers are more cut. Besides the lower CrI and the higher cellulose I1 content, no significant differences between the bulk fiber and fiber surfaces could be seen.
-
x)O%
90% 00% 70%
60% 50% 40%
30% 20%
UCellulose II .Cellulose Ib pCellulose la
lo% 0%
Figure 3. Relative proportions of different celhlose polymorphs and amorphous cellulose (Am4 in the refinedpulps andfines.
Crystallinity of cellulose 43 .DO% 90%
80% 70%
60% 50% 40%
cellulose II mCellulose Ib
30% 20%
10% 0%
Figure 4. ReIative proporlions of different cellulose polymorphs and amorphous cellulose (Aml) in TCF-bleachedpubs.
CONCLUSIONS
Part of the ccllulose I, is converted to cellulose Ip during haft pulping. This is mainly caused by high tcmperature (170 "C), but the cooking liqour and the lower crystallinity of cellulose in wood probably accelerate the transformation, too. Cellulose I1 is formed also during pulping and the content of cellulose I1 seems to be higher on the fiber surface thhn inside the fiber. The crystallinity index of the surface material of the fiber was detected to be lower than in the bulk fiber. The refining or TCF-bleaching were not observed to cause any signinficant changes in the degree of crystallinity or in the relative proportions of different crystalline forms of cellulose. ACKNOWLEDGEMENT
We are indcbtcd to the Technology Development Ccntrc of Finland (TEKES) for financial support. REFERENCES
1 2 3 4 5
6 7 8
VandcrIIart, D. L. and Atalla, R. H. Macromolecules 17(1984) 1465. Debzi, E. M., Chanzy, H., Sugiyama, J., Tekely, P. and Excoffier, G. Macromolecules 24 (1991) 6816. Sugiyama, J., Pcrsson, J. and Chanzy, H. Macromolecules 24 (1991) 2461. Sjostrom, B. Wood Chemistry, Fundamentals and Applications, Academic Press, Inc. 1981, New York, p.53-55. Hortling, U., Jousimaa, T. ,Hyviirinen, 13.-K. and Holopainen K., Cellucon '98, In this volume. Willis, J. M. and Herring, F. G. Macromolecules 20 (1987) 1554. Tecaiir, I .0 k
!
6060
8
$
301 30 A
0
0
30
A 60
90
0 1
i
0
30
60
90
Time (mln)
Figure 1. Change in spccific viscosity (q) of CM-celluloses as a function of time by cndoglucanase (S 1 1 subunit) of C.thermocellum cellulosome. A, CM-cclluloses with high (0)and medium (A) viscosities; B, CM-celluloscs with DS 0.78 (O), 0.93 (A), 1.3 (H), 1.7 (0) and 2.1 (+). Each point is the mean of two assays. 600
-
400
-
200
-
B
Kelalionship between change in viscosity and the release of reducing sugars from CM-celluloses by endoglucanases The plot of change in spccific viscosity versus rcducing sugar released is used to distinguish whcther an endoglucanase is a more or lcss randomly acting enzyme (7,8). The endoglucanase (Sl I subunit) of C. thermocellum reduced the specific viscosity of high and mcdium viscosity CM-celluloscs from 138 to 40 cp and 28 to 5 cp, respectively, while releasing almost the same amount of reducing sugar (-160 pg) from both substrates (Fig. 3 A, lines I and 2). However, the determination of change in specific viscosity as a percentage of the initial specific viscosity revealcd that the enzyme reduced the specific viscosity of both CM-celluloses by 80% of their original. This suggested that the major endoglucanase of C. thermocellum cellulosomc attacked both CM-celluloses with equal efficicncy. Nevcrthelcss, the rapid decreasc in the specific viscosity of high viscosity CM-cellulose suggcstcd that the enzyme is a more randomly acting endoglucanase. Similar rcsults wcrc obtained with the cndoglucanase from T,aurantiacus.
86 Application of enzymes to pulp, fibres and cellulose Thc change in specific viscosity and rcducing sugars released by endoglucanases from C. therrnocellurn cellulosome and T. uuruntiacus were inversely proportional to the degree of substitution of CM-celluloses. Thus, with the increase in degree of substitution from C.78 to 2.1, the release of reducing sugars and change in specific viscosity of CM-celluloses by the major endoglucanase from C. therrnocellum cellulosome dccrcased rapidly and reached almost to zero (Fig. 3 A and B). These results demonstrated that the degree of substitution rather than viscosity influences the action of both endoglucanases on CM-cellulose. 150
1
A
3 120 .-2 In g .-
90
u)
Reducing sugar (pg glucose equivalent)
Figure 3. Relationship between the change in specific viscosity (71) and the release of reducing sugars from CM-celluloscs of varying viscosity and DS by the action of endoglucanase (SIIsubunit) of Cthermocellum cellulosome. A, B, symbols 0 and A correspond to CM-celluloses with high and medium viscosities, while symbols 0, A, V and correspond to CM-celluloses with DS 0.78, 0.93, 1.3, 1.7 and 2.1, respectively. Each point is the mean of two assays.
.,
+
IIPLC analysis of hydrolysis products released from CM-celluloses by endoglucanase from T. aurantiacits The enzyme released mainly cellobiose and cellotriose from all CM-cclluloses testcd. Also, the endoglucanase released noticeable amounts of cellotetraose and ccllopentaose from all CM-celluloses during the initial stages of the reaction, and thcir conccntration dccreascd with the increase in incubation time. Nevertheless, the conccntration of all cello-oligosaccharides released from CM-celluloses decreased with their increase in viscosity and degree of substitution. In fact, the level of cellobiose and cellotriose rcleascd from low viscosity CM-cellulose by T. auruntaicus endoglucanase was 2 and 8 folds higher respectively, than that rcleascd from CMcellulosc with high viscosity and dcgrcc of substitution 1.7. Also, thc lcvcl of glucose rcleascd from all CM-celluloses by this endoglucanase was negligible.
CONCLUSIONS 1. Degrce of substitution rathcr than viscosity of CM-ccllulosc influcnccs thc action of major endoglucanases from C. thermocellum and T. uurantiacus. 2. Both endoglucanascs showed similar modcs of action on CM-celluloscs and appcarcd to bc randomly acting cndoglucanascs.
Thermophilic bacterial and fungal endoglucanases
87
3. Endoglucanase from T. aurantiacus released mainly ccllobiose and cellotriose from all CM-celluloses tested, but the level of cello-oligosaccharides released, decrcascd with increase in viscosity and degree of substitution. References 1. D E Coffey, D A Bell and A Henderson, ‘Cellulose and cellulose derivatives’. In: Food polysaccharides and their applications, cd. A M Stephen, 1995 123-153, Marcel Dckker Inc. 2. C H N Sieger, A G M Kroon, J G Batelaan, and G G van Ginkel, ‘Biodegradation of carboxymethyl celluloses by Agrohucterium CM-1’. Carhohydr Poly, 1995 27 137143. 3. D V Garcia-Martinez, A Shinmyo, A Madia, and A L Dcmain, ‘Studies on cellulase production by Clostridium thermocellum’, Eur J Appl Microhiol Biotechriol, 1980 9 189 - 197. 4. M Mandcls and D Sternberg, ‘Recent advance in cellulase technology’, (1976) J Ferment Technol, 1976 54 267-286. 5. M Somogyi. ‘Notes on sugar determination’,J Biol Chem, 1952 195 19-23. 6. T M Wood and K M Bhat, ‘Measurement of ccllulasc activities’, In: Methods Enzymol, eds. W A Wood & S T Kcllogg, 1988 vol. 160 87-112, Academic Press, London. 7. T M’ Wood and S I McCrae, ‘The cellulase of T. koningii: purification and properties of some endoglucanase components with special reference to thcir action on cellulose when acting alone and in syncrgism with the cellobiohydrolasc’, Biochem J, 1978 171 61-72. ’ 8. K M Bhat, S I McCrae and T M Wood, ‘The endo-( 1->4)-P-D-glucanase system of P. pinoplzilum cellulase: isolation, purification and characterisation of five major endoglucanase components’, Carhohydr Res, 1989 190 279-297.
T l l E EFFECT OF ANTIIKAQUINONE ON WOOD CAKSO HYD RATES I)U R 1NG A L KA L1NE PULPI NG IN AQUEOUS ORGANIC SOLVENTS M.F.Kiryushina, M.I.Ermakova, A.S.Olefirenko, E.-M.Bennacer,T.G.Fcdulina, A.B.Nikandrov, M.Ya.Zarubin St.-Pelersburg Academy of Forestry, St.-Petersburg, Russia ABSTRACT The protcctive effect of anthraquinone (AQ) on wood carbohydrates during alkaline pulping of spruce sawdust (25% NaOH, 17OoC, ratio solid-liquid 1:100, 1% AQ) and the samples of kraft pulp in water and aqueous organic solvents: water-EtOH (1: I), water-MeOH (1 :I), water-dioxan (7:3), water-DMSO (7:3), (NaOH 1.25 mol/l, 17OoC,lh, ratio solid-liquid 1:20, AQ 0.016 mol/l) has been investigated. The stabilizing effect of AQ on carbohydrate redox end groups in the above mentioned media is shown on cellobiose an example. The lower content of aliphatic acids in the black liquor and the highcr yield of the wood residue (at the same degree of delignification) after cooking of spruce sawdust and kraft pulp with the addition of AQ was established. It allows confirmation that the prevention of "pccling" reactions by oxidation of aldehyde groups up to carboxyl ones occurs in the presence of AQ more extensively than in its absence. At the same time the decrease of the degree of polymerisation (DP) in nondissolved samples of krafi pulp after treatment indicates that AQ promotes the alkaline solvolysis at arbitrary sites of carbohydrate chains in all investigatcd media and that this effect is grcatcr in the solvents with the highest basicity. INTRODUCTION The use of AQ in alkaline wood delignification has attracted the attention of rcscarchcrs after the publications of Holton, who has offered AQ as a catalyst accelerating the delignification and promoting the increase of the pulp yield ( I ,2). The last fact is provided by the stabilizing of the wood carbohydrates in the presence of AQ (3-5). A large interest to organosolve pulping including thc alkaline ones is connected with the increase of the delignification rate. However, at the same time the selectivity of the process decreases because of a high degrcc of carbohydrate destruction (6). Thc aim of this research was to study the protectivc cffcct of AQ with regard to the wood carbohydrates at the heating in aqueous organic solvents with alkali. RESULTS AND DISCIISSION The samples of spruce sawdust (lignin content 27.6 Ya) were treated by aqueous sodium hydroxide (25%) at 17OoCwith and without AQ (1%) for 0-6 h at a ratio solidliquid of 1 :100. The yield, the amount of dissolved carbohydrates, the lignin content in the wood residue and thc amount of nonvolatile acids in the black liquor were dctcrmined. It was found that the wood residuc yields at the same lignin contcnt (16.5%) are 65.2% (with AQ) against 56.7% (without AQ). Simultaneously, in black
92 Pulp production and processing liquor AQ provides the smaller contcnt of nonvolatilc aliphatic acids (Fig.1). This is confirmcd by the published data on stabilization of carbohydrates in aqueous alkali. It was interesting to ascertain whether this is the case for alkaline pulping with organic solvents. Samples of kraft pulp from pinc wood (lignin content 6.3%, DP 1700) were treated in alkaline solutions (NaOII 1.25 mol/l) of aqueous organic solvents containing AQ (0.016 mol/l) and without AQ for Ih at 170OC for a solid-liquid ratio of 1:20. The yield of nondissolved wood residue and DP were determined (see Table 1). Table 1. The pulp yield and degree of polymerization after alkaline treatment of pine kraft pulp samples in water-organic solvent systems with and without anthraquinone. I
Water-organic solvent systcm
Degree of polymerization, DP with AQ without AQ 840 960 410 460 460 500 400 460 220 220
water watcr-methanol(1:1) watcr-ethanol(1: 1) water-dioxan (7:3) water-DMSO (7:3)
.
Pulp yield, % with AQ 89.6 78.9 82.4 85.0
67.8
without AQ 79.9 66.6 69.8 71.6 59.1
It is shown that in all tested systems: water-EtOH (l:l), water-McOH (I:]), waterdioxan (7:3), water-DMSO (7:3) as well as during pulping in aqueous alkali without solvent, AQ prevents the dissolution of carbohydrates. The yield of the fibrous product after heating with AQ is approximately 10-14% higher than without it (scc Fig. 1). AQ promotes the delay of "peeling" rcactions. This is confirmed by the data on cellobiose cleavage by aqueous NaOH (OSmol/l) in all the above mentioned media during diffcrent times (SCC Fig. 2) in the prcscncc of AQ the proccss occurs slowly. In the
-
0
100
200
300 400 Time, min
-
Figure 1. Dissolution of carbohydrates (% to dry wood weight) 1,2 and change of nonvolatile acids content 3,4. 12- without AQ; 2,4 with AQ.
-
-
Effect of anthraquinone on wood carbohydrates 93 %
100
90 80 70 60 50 40
30 20 10
0 20
0 '
40
60 Time, min
-
-
Figure 2. Degree of cellobiose cleavage (YO):1,2 in water; 3,4 in water:MeOH; 5,6 in water:dioxan; 7,8 in water:DMSO. 1,3,5,7 without AQ; 2,4,6,8 with AQ.
-
-
-
-
absence of organic solvents and heating for 40 min , we observed a stabilization of the glucosidic bond to cleavage. The "peeling" reaction stops and it results in an increase of thc fibrous product yield. Concerning DP, we observed an opposite picture. On the presence of AQ, the DP of the fibrous residue is lower than without it (see Table 1). In water without solvent, AQ decreases the DP more than in water-solvent systems. However, in systems with high basicity as water-DMSO (7:3),the solvent effect covers that of AQ (scc Table 1). The alkaline solvolysis rate of glucosidic bonds in these mcdia is very high (7). The bond cleavage in arbitrary sites of carbohydrates results in DP reduction. Apparently, AQ promotes this process as it can oxidize primary and secondary alcoholic groups (8). AQ oxidizes them to carbonyl groups neighboring with the glucosidic groups and creates the additional possibility for the cleavage of carbohydrates chains through the carbonylic mechanism. This promotes the decrease of the product degree of polymerisation. CONCLUSION The addition of AQ at alkaline pulping in water and in aqueous organic solvents results in a reduction of the "peeling" reactions of the carbohydrate end groups, but docs not prevcnt and even promotes the cleavage of glucosidic bonds in arbitrary sites of the carbohydrate chains. In solutions with high basicity the effect of AQ is masked by the effect of the medium.
94
Pulp production and processing
REFERENCES
H H Holton and F L Chapman, ‘Kraft-pulping with anthraquinone’, TAPPI Journal, 1977 60(11) 121-125.
H IIolton, ‘Better cooking with anthraquinone’, Pulp and Paper Internat, 1978 20(9) 49-52.
L Lowcndahl and 0 Samuelson, ‘Carbohydrate stabilization during krall cooking with addition of anthraquinone’, Svenskpapperstidn, 1977 SO( 17) 549-551. L Lowendahl and 0 Samuelson, ‘Carbohydrate stabilization with anthraquinone during pulping’, Polymer Bull., 1978 l(3) 205-210. L Lowendahl and 0 Samuelson, ‘Carbohydrate stabilization during soda pulping with anthraquinone’, TAPPI Journal, 1978 61(2) 19-21. M F Kiryushina, M I Ermakova, E M Bennacer, A S Olefirenko and M Ya Zarubin, ‘Ccllulose destruction in solutions with high basicity’, Wood Chemisrry (in Russian), 1991 (1) 38-42. E M Bennacer, M F Kiryushina and M Ya Zarubin, ‘Effect of organic solvents on cleavage of beta-alkyl-O-arylic bond kinetics’, 5”’ ISWPC, Ralleigh, 1989. A F Wallis and R H Wearne, ‘Oxidation of monohydric alcochols with anthraquinone and its derivatives under soda pulping conditions’, Journal Wood Cliem. and Teclzol., 1987 7(4) 5 13-525.
TWO PHASE EQUILIBRIA OF METAL IONS IN PULPING UNIT OPERATIONS: FROM IMPREGNATION TO OXYGEN BLEACHING J. Karhu, P. Snickars, L. IIarju and A. Ivaska Laboratory of Analytical Chemistry, Process Chemisty Group, Abo Akademi University, Biskopsgatan 8, FIN-20500 Turku-Abo, Finland
ABSTRACT Two phase equilibria of metal ions at diffcrent steps of the pulping process were studied. Pulp samples were taken from different process steps in a modern Finnish kraft pulp mill, that produces batch cooked kraft pulp from a mixture of coniferous trecs. The concentrations of Na, K, Ca, Mg, Zn, Fe, Mn, Al, Ba and Si both in the liquid and the fiber phases were dctermincd mainly using ICP-MS and DCP-AES. Also wood chips were analyzed for metal ions. Conditional distribution coefficients wcre determined for sevcral mctal ions in different pulp samples. The metal ions can be arranged in affinity orders at the different steps of the pulping process. INTRODUCTION The interest in the ion exchange propcrtics of kraft pulps has strongly grown during the 1990's as totally chlorine free (TCF) bleaching processes have become more common in pulping industry. Some cations, especially transition metal ions such as manganese, iron, cobalt, copper, nickel and chromium have disturbing effect on the TCF process Other metal ions, such as magncsium and calcium have protecting effect on the fibers in alkaline bleaching with oxygen-based chemicals High concentrations of Na and the non-process elements (NPE) K, C1, Al, Si, Ca, P and Mg can be found in spent process liquors. NPEs can cause problems in the recovery processes. Effluents containing nitrogen, phosphorus and heavy metal ions are detrimental to the environmcnt 3*4. Both desorption of metal ions from pulp and wood chips and sorption of metal ions on pulp fibers have reccived increased interest when the demand to close pulping processes has increased. Furthermore, in certain process steps where thc pH is low thc metal ions may desorb from the fibers and precipitate in the following process steps whcre pH is increased. Thc dcposits are one of the biggest obstacles when closing the processes. Most of the metal ions present in the pulping processcs originate from the wood used as raw material. Some of the metal ions e.g. sodium and calcium come mainly from the chemicals used in the mill. On the other hand also the raw water used and corrosion of equipment increase the metal ion content in the process solutions, The main mechanism for sorption of metal ions to wood and pulp is assumed to be cation exchange 5-8. The main binding sites are the carboxylic groups. Their number in pulps is usually rather low, ca 40 mmol/kg in unbleached and oxygen blcached softwood kraft pulp '. Other potential sites for ion exchange are phenol groups and lignosulfonic groups in sulphite pulps lo. Grubhofcr ' I gives a thorough description of ion exchange resins based on native cellulose.
'.
96 Pulp production and processing Metal ions bound to the functional groups in wood and pulp can be desorbed by sequestration with strong chelating agents like EDTA and DTPA or by stripping with acidic solutions. Both desorption of metal ions from wood chips or pulp and the sorption of metal ions on pulp fibers need to be understood when the water circulation in pulping processes becomes more closed.
EXPERIMENTAL Sampling Samples were taken from a Finnish kraft pulp mill working in .the batch mode. A mixture of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) was used as raw material. The actual mixture at the time of sampling consisted of approx. 25 % of pine chips, 35 % of spruce chips and 40 % of softwood chips from saw mills. The sampling sites at the mill are shown in Figure 1. The process delays were considered in sampling in order to have the same original raw material. The washers mentioned in the figure are wash presses. Pulp samples from two batch cooks were studied. However, it was possible to get only one sample from the cooked unwashed pulp. This sample was also diluted to some degree with tap water in the sampling device. The metal concentrations of this sample are therefore not presented in this work.
Wood chips
I
Impregnation
I
Batch cook Pulp suspension out
Wash liquor Pulp suspension in Pulp out
*I
2nd postox washer Pulp suspension in Pulp out
Figure 1. Sampling sites at the pulp mill.
Filtrate out I
I
Wash liquor to the 2nd shower Filtrate out
Two phase equilibria of metal ions 97 Metal concentrations were determined both in the dried fibres (or chips) and in the liquors. The pulp samples were taken as such or filtered if the sample consistency was lower than ca. 20 %. The liquor samples were taken in plastic bottles and filled to maximum volume. The fiber and liquor samples were stored in the freezer (-18 "C) or in the refrigerator (5 "C).
Sample preparation The wood chip samples and the fiber samples were dried at 105 OC in order to determine the metal ion concentrations on dry wood (d.w.) or dry pulp (d.p.) basis. The samples were ground to a suitable particle size using a Cyclo-Tec (Tecator Inc.) cutter mill. The solid samples (500 mg) were digested in a mixture of HNO3 (5 ml) and H202 (2 ml) by the microwave oven technique before the analytical determinations 12. Impregnation experiments were made in the laboratory with the same chips and liquors that were used in the mill.
Instrumental techniques Concentrations of Na, K, Ca, Mg, Zn, Fe, Mn, Al, Ba and Si in the sample solutions were mainly determined using inductively coupled plasma mass spectrometry (ICP-MS) and direct current plasma atomic emission spectroscopy (DCP-AES). For ICP-MS a Perkin-Elmer Elan 6000 (PE Sciex, Toronto, Canada) instrument and for DCP-AES a Spectraspan IIIB instrument (Spectrametrics Inc., Andover, MA) were used for the elemental analyses. The concentrations of some elements were determined both by ICPMS and DCP-AES for the control of the reliability of the used method. A more detailed description of the methods that can be used for determination of metal ions in wood related materials and pulping solutions has been given by Ivaska and Harju If.
RESULTS AND DISCUSSION Metal ion content in wood chips, pulps and process solutions The metal ion concentrations in the dried wood chip and pulp samples are presented in Table 1. The numbers 1 and 2 after the different process steps in the tables represent samples taken from two different batches in the same point in the process. The fiber mat after filtration including the solution in it is regarded as the solid phase l4,I5. Main cations in wood chips are Ca, K, Mg and Na, and their concentrations exceed 100 mgkg dry wood (d.w.). Other metal ions included in the table are in the concentration range 10 - 50 mgkg d.w. The Si concentration in the impregnated chips varied strongly and therefore two values are shown in the table. More information about the metal ion content in wood can be found in the literature 16-18. As could be expected, the concentration of the main component sodium increases clearly in the impregnation stage - from slightly over 0.1 to over 6 g k g dry pulp (d.p.). The measured sodium concentration is at its highest, ca 77 g/kg d.p., in pulp that is pumped to the second brown stock pulp washer. In this washer the sodium concentration decreases down to ca 35 g/kg d.p. Pulp that goes to the second washer after the two oxygen bleaching stages has a lower sodium concentration (ca 3.2 g k g d.p.). This value remains constant in the washing operations. A similar concentration pattern as for sodium can be found for potassium. The increase in potassium Concentration in unbleached pulp is about 60-fold, if compared with the content in wood chips. The magnesium concentration in pulp- is about twice the
98 Pulp production and processing concentration in the wood raw material. The calcium concentration increases from ca 600 mgkg in wood chips to 1200 - 1500 mgkg in unbleached pulp. For the other metal ions the variations are relatively small and generally a decrease in the metal concentration in pulp can be observed during the pulping process studied. The metal ion concentrations determined in process solutions are given in Table 2. The pH values of the different steps are also included. The highest sodium concentration, ca 40 g/l, was found in the impregnation liquors and it is the highest of all the ions in the process solution studied in this work. The sodium concentration, however, decreases radically down to the level of 1 g/1 in the solution after the first washing of the oxygen-bleached pulp. Similar trends can also be seen for potassium. A distinct decrease in concentrations of the other metal ions can be observed for process solutions at the same process step. This is due to addition of cleaner water to this process stage.
Table 1. Metal contents ( m a g ) in dry wood chips and pulps from different steps in the pulping process. b.d.1. = below detection limit. "a1 138 102 6440 6130 78300 75500 36800 32600 3480 2950 3530 3790
[KI 123 125 1030 990 8770 6430 4220 3710 114 112 61.1 71.9
597 572 661 579 1340 1490 1220 1330 1100 1150 1020 1120
[Mgl 108 131 69.3 75.0 256 224 163 168 255 25 1 215 223
[Znl 15.3 3.71 6.58 5.96 21.6 17.0 29.4 16.7 17.2 15.7 17.6 23.7 -
Wood chip mixture, 1 Wood chip mixture, 2 Chips from lab. impregnation, 1
[Fel [Mnl [All 10.7 53.9 9.62 3.74 58.3 b.d.1. 1.10 80.3 28.1
Pal 11.1 8.56 6.64
Chips from lab. impregnation, 2
1.15
61.9
24.3
4.27
Pulp to 2ndpreox washer, 1 Pulp to 2ndpreox washer, 2 Pulp from 2ndpreox washer, 1 Pulp from 2ndpreox washer, 2 Pulp to 2ndpostox washer, 1 Pulp to 2"dpostox washer, 2 Pulp from 2ndpostox washer, 1 Pulp from 2nd postox washer, 2
24.1 20.5 43.7 19.4 39.7 28.2 27.2 20.6
46.1 41.9 36.1 36.3 23.2 21.6 20.7 20.8
35.6 29.5 29.6 22.2 26.6 21.7 23.3 20.9
7.15 8.10 6.43 7.67 9.49 9.84 9.25 9.46
Pi1 23.3 2.04 8391 66.1 245 I 144 243 206 116 119 177 179 126 137
Wood chip mixture, 1 Wood chip mixture, 2 Chips from lab. impregnation, 1 Chips from lab. impregnation, 2 Pulp to 2ndpreox washer, 1 Pulp to 2ndpreox washer, 2 Pulp from 2ndpreox washer, 1 Pulp from 2ndpreox washer, 2 Pulp to 2ndpostox washer, 1 Pulp to 2ndpostox washer, 2 Pulp from 2ndpostox washer, 1 Pulp from 2ndpostox washer, 2
[Gal
Table 1 continues.
Liquor to impregnation, 1 Liquor to impregnation, 2 Liquor from lab. impregnation, 1 Liquor from lab. impregnation, 2 Liquor from lab. impregnation without wood chips, 1 Liquor from lab. impregnation without wood chips, 2 Solution in pulp suspension to 2ndpreox washer, 1 Solution in pulp suspension to 2ndpreox washer, 2 Washing filtrate to Znd preox washer, 1 Washing filtrate to 2ndpreox washer, 2 Filtrate from 2"dpreox washer, 1 Filtrate from 2ndpreox washer, 2 Solution in pulp suspension to 2"dpostox washer, 1 Solution in pulp suspension to 2"dpostox washer, 2 Last washing filtrate to 2ndpostox washer, 1 Last washing fiItrate to Znd postox washer, 2 Filtrate from Znd postox washer, 1 Filtrate from 2ndpostox washer, 2
13.3 13.2 12.9 12.9 13.3 13.4 12.9 12.9 10.3 10.4 12.8 12.9 9.7 9.7 10.6 10.8 9.7 9.7
PH "a1 41800 42500 30100 32900 40700 38100 18200 18100 0600 0600 6000 8200 1110 1140 1040 1050 1290 1290
Table 2. Metal ion concentrations (mgn) in different process solutions. [KI 6080 6330 4330 4880 5750 5400 1720 2210 1160 1120 2110 2270 47.3 50.5 22.0 20.1 28.6 29.2 [Ca] 46.2 54.3 115 122 85.5 106 46.6 42.4 24.4 41.4 32.0 46.9 4.55 4.54 4.67 2.93 4.95 5.42
[Mg] 42.7 40.6 40.6 38.1 43.5 37.1 37.7 48.0 46.7 47.9 27.2 55.3 13.6 13.1 3.87 3.53 14.1 13.4
0.03 0.27 0.28
[Zn] 1.17 2.91 5.94 4.40 31.4 32.9 2.06 1.91 4.13 4.00 3.16 4.13 0.33 0.33 0.03
[Fel 4.79 19.2 4.75 10.9 23.9 33.9 2.95 2.48 3.78 1.84 2.27 3.74 0.19 0.19 0.08 0.07 0.21 0.36
[Mnl 13.7 15.1 8.67 17.8 12.5 12.8 6.74 6.40 4.21 7.03 4.12 2.83 0.81 0.81 0.06 0.07 0.73 0.72
[All 23.1 27.8 13.4 23.3 43.3 32.8 4.46 5.13 6.38 4.35 4.72 6.59 0.49 0.48 0.10 0.08 0.47 0.42
[Bal 1.64 1.24 2.93 1.99 3.76 3.09 1.24 1.27 1.48 1.36 1.37 1.42 0.24 0.26 0.41 0.40 0.30 0.30
13.7 13.7
10.1
[Sil 125 149 102 152 126 127 36.4 47.4 24.1 25.0 33.5 46.8 13.7 13.4 10.0
5
5-
EL
9
B
a
g-
5
f.
(D
g
"3
4
3
100 Pulp production and processing Determination of conditional distribution coefficients, DM Distribution of a metal ion, M”’, between the solid and the aqueous phase can be described by the conditional distribution coefficient, DM’. It is used in this work as defined by Ringbom l9 according to the following equation:
DM =[M’],/[M’] where [M’], is the total analytical concentration of the metal ion in the solid phase and is expressed as molkg d.p. The total concentration of the species containing the metal ion in the aqueous phase, [M’]. is given as mol/l. The distribution coefficient defined in equation [l]is a kind of conditional constant, which strongly depends on pH and other experimental parameters in the two-phase system. Table 3 gives the results of the dctermination of the conditional distribution coefficients, DM’,for several metal ions in different steps of the pulping process in the particular mill studied. The pH values of the solution phase are also included. DM’ values are also given for impregnated wood chips, but they cannot directly be compared to the DM’values reported for pulp samples. The values of DM’are low indicating a relatively weak binding of metal ions to pulps. Large variations of DM’can be observed for most metal ions at different stages of the process. Generally the value of DM’ is increasing towards the end of the pulping process. This is mainly explained by the decrease in pH during the process from pH ca 13 to 10. At pH around 13 there is a strong interference of hydroxide ions on the reactions of transition metal ions, resulting in decrease in the value of the conditional distribution coefficient. Also precipitation of metal hydroxides, silicates and other salts can take place. The alkali metal ions are an exception. These metal ions form very weak hydroxo complexes at very alkaline conditions. In spite of the great variations of the concentrations of e.g. Na and K in the two-phase system the conditional distribution constants are surprisingly constant. The conditional distribution coefficients can also be used for comparison of the binding strength of different metal ions to pulp in a batch with constant pH and other chemical conditions, i.e. in different steps of the pulping process. Due to the small number of functional groups in the pulp, all metal ions probably have the same stoichiometry for the ion exchange reaction. This holds at least for cations of thc samc valency. The following affinity order for different metal ions can be found for the two oxygen bleached pulp samples that were taken from pulp pumped to the 2”dpostox washer: C a > Fe > Al, Z n > B a > Mn > Mg > Si > N a> K Conditional distribution coefficients can be used for theoretical studies of metal ion desorption. Metal ions can be dcsorbed from pulps by addition of chelating agents. The effect of these agents on the sorption processes can easily be considered by so-called side-reaction coefficients (or-coefficients) introduced by Ringbom 19. A complete desorption (99.9 %) of a metal ion is achieved if DM’< 10” V/m
131
where V is the volume of the aqucous phase and m the mass of the solid phase.
Two phase equilibria of metal ions
101
Table 3. Log D' for the diffcrent samplcs. pH Chips from lab. impregnation, 1 Chips from lab. impregnation, 2 Pulp to preox washer, 1 Pulp to 2"d prcox washer, 2 pulp from 2"d preox washcr, 1 Pulp from Pd preox washer, 2 Pulp to 2ndpostox washer, 1 Pulp to 2ndpostox washer, 2 Pulp from postox washer, 1 Pulp from 2"d postox washer, 2
Na
K
Ca
Mg
Zn
Fe
Mn
A1
Ba
Si
12.9 -0.67 -0.63 0.76 0.23 0.04 -0.64 0.97 0.32 0.36 0.921 -0.19 12.9 -0.73 -0.64 0.68 0.29 0.13 -0.98 0.54 0.02 0.33 0.211 -0.02 12.9 0.63 0.71 1.46 0.83 1.02 0.91 0.84 0.90 0.76 0.82 12.9 0.62 0.46 1.55 0.67 0.95 0.92 0.82 0.76 0.80 0.64 12.8 0.36 0.30 1.58 0.78 0.97 1.28 0.94 0.80 0.67 0.54 12.9 0.25 0.21 1.45 0.48 0.61 0.71 1.11 0.53 0.73 0.41 9.7
0.50 0.38 2.38 1.27 1.72 2.32 1.46 1.73 1.60 1.11
9.7
0.41 0.35 2.40
9.7
0.44 0.33 2.31 1.18 1.81 2.11 1.45 1.70 1.49 0.96
9.7
0.47 0.39 2.32
1.28 1.68 2.17
1.43 1.66 1.58 1.13
1.22 1.93 1.76 1.46 1.70. 1.50 1.00
CONCLUSIONS The results of the present work givc detailed information on the metal ion profilcs in a kraft pulp mill and show how the metal ion concentrations change in different process steps. Impregnated chips, batch-cooked pulps, washed unbleached pulps and oxygenbleached pulps were studied. Also the incoming wood materials were analyzed. Conditional distribution coefficients wcre determined for several metal ions for pulp samples from different steps in the pulping process. One main factor affecting the values of DM and thus the binding strcngth of metal ions to pulps is pH. The main pH governed reactions arc formation of metal hydroxo complexes in the aqueous phase and protonation of the functional groups in the fiber phase. The metal ions can be arrangcd in the order of affinity for the different pulp batches studied. Conditional distribution coefficients can be useful for theoretical optimization of the desorption of metal ions from pulp with complexing agents. Howevcr, the ion exchange reactions in pulps are very complicated if compared for instance with synthetic cation exchangers. Further research is still nceded for a better undcrstanding of these processes.
ACKNOWLEDGEMENTS The financial support of The Finnish Technology Development Centre (TEKES), Ahlstrom Machinery Corporation, Enso Group and Metsii-Rauma Oy is gratefully acknowledged. We also thank Tech. Lic. Paul Ek for performing the ICP-MS analyses.
102 Pulp production and processing REFERENCES 1. 2. Yuan, M. D’Entremont, Y. Ni and A. R. P. van Heinigen, ‘The role of transition metal ions during peracetic bleaching of chemical pulps’, Pulp & Paper Canada, 1997,98(1 l), T408-T413. 2. 0. Dahl, J. Niinimai, T. Tirri, A.-S. Jabkelainen and H. Kuopanportti, ‘Bleaching softwood kraft pulp: The role of certain common chemical elements in the peracetic acid stage’, Tappi Pulping Conference, San Francisco, California, Tappi Press, Atlanta, 1997, pp. 1061-1067. 3. P. Ulmgren, “on-process elements in a bleached kraft pulp mill with a high degree of system closure - state of the art’, Nordic Pulp Paper Research Journal, 1997, 12(1), 32-41. 4. T. Krantz, ‘Kretsloppsanpassning i massabruket - var stir man?’, Svensk Papperstidning/Nordisk Cellulosa 1998, 101( l), 32-33 (in Swedish) 5. E. Sjostrom, J. Jansson, P. Haglund and B. Enstrom, ‘The acidic groups in wood and pulp as measured by ion exchange’, J. Polym. Sci, 1965, C1 1,221-241. 6. P. S. Bryant and L. L. Edwards, ‘Cation exchange of metals on kraft pulp’, J. Pulp Pap Sci, 1996,22(l), J37-542. 7. G. Eriksson and U. G r h , ‘Pulp washing: sorption equilibria of metal ions on kraft pulps’, Nord Pulp Pap Res J., 1996, 11(3), 164-176. 8. S. M. Abubakr, B. F. Hrutfiord, T. W. Reichert and W. T. McKean, ‘Retention mechanism of metal ions in recycled and never-dried pulps’, Tappi J., 1997, 80(2), 143-148. 9. J. Karhu, P. Forslund, L. Harju and A. Ivaska, ‘Characterization of carboxyl and phenol groups in kraft pulps at different temperatures’, paper in this Proceedings, 1999. 10. T. Lindstrom, ‘Chemical factors affecting the behaviour of fibres during papermaking’, Nordic Pulp Pap. Res. J., 1992,7(4), 181-192. 1 1. N. Grubhofer, Cellulose ion exchangers, in Zon Exchangers, Ed. K . Dorfner, Walter de Gruyter, New York, 1991, pp. 443-460. 12. Anonymous, Milestone application notes for microwave digestions, Milestone Application Lab, Pergamon, Sorisole, 1995. 13. A. Ivaska and L. Harju, Analysis of inorganic constituents, Analytical Methods in Wood Chemistry, Pulping and Papemaking, Eds E. Sjostrom and R. Altn, SpringerVerlag, Berlin, 1998, pp. 287-304. 14. P. S. Bryant, Transition metal measurement and control in closed kraft mills with hyrogen peroxide bleach lines, Doctoral dissertation, Ann Arbor, Michigan, University of Michigan, 1993. 15. R. Jkvinen and 0. Valttilii, ‘A practical method for studying NPEs in a kraft mill’, 1998 International Chemical Recovery Conference, Tampa, Florida, Tappi Press, Atlanta, 1998, Vol. I , 107-1 16. 16. L. Harju, K.-E. Saarela, S.-J. Heselius, F. J. Hernberg and A. Lindroos, ‘Analysis of trace elements in trunk wood by thick-target PIXE using dry ashing for preconcentration’, Fresenius J. Anal. Chem., 1997,358(4), 523-528. 17. P. Koch, ‘Utilization of hardwoods growing on southern pine sites’, US Dep. Agric. For. Serv., 1985, 1,438-445. 18. P. Koch, ‘Lodgepole pine in North America’, Forest Product Society, Madison, Wisconsin, 1996, Vol. 2,621-638. 19. A. Ringbom, Complexation in Analytical Chemistry, Wiley, New York, 1963.
CATALYSIS OF OXYGEN-ACETONE DELIGNIFICATION Ivan Deineko and Inna Deineko StPetersburg Forestry Academy, Institutsky per.5, 194021,St.Petersburg, Russia, E-ma*
[email protected] ABSTRACT A catalyst for oxygen-acetonedeligniflcationhas been suggested. The research has been carried out on spruce sawdust. Use of the catalyst in wood oxidation by oxygen in 60 % acetone allows transfer to the solution of about 90 % lignin during the reaction time of 24 hours at the reaction temperature of 120°C. Kinetics of delignification and influence of the catalyst concentration (0.2-2.0 g/l, liquid-to-wood ratio 25, P0(Oz) 0.7 MPa) on dissolution of wood components have been investigated. The rate of delignification in the catalytic process (120°C) is an order of magnitude higher than the rate of lignin dissolution in the process carried out in the absence of the catalyst.
INTRODUCTION The process of oxygen-organosolvdelignificationof wood started to be developed in StPetersburg Forestry Academy about ten years ago. The oxygen-organosolv process (oxysolvolysis) consists of processing wood raw material by oxygen in water-organic solutions under high temperature (140-160°C). The detailed researches have shown that this process has essential advantages compared to traditional and newly developed pulping processes /I/. The advantage of oxysolvolysis in comparison with other organosolv processes is the possibility to obtain pulp fiom both hardwood and softwood. In comparison with oxygen-alkaline pulping the offered process does not require application of alkalis and allows use of wood chips as raw material. However, oxygenorganosolv pulping is relatively low quality of cellulose resulted. The rather low mechanical properties of the fibrous material are apparently associated with a strong destroying action of oxygen on cellulose at high temperatures. A possible way to decrease the role of undesirable reactions resulting in cellulose destruction is to use selective catalysts of delignikation allowing one to carry out the process under softer conditions. An effective catalyst for oxygen-organosolvpulping has been recently offered /2/, and results here indicate there is an opportunity to develop a catalytic pulping process. In the present work, the results of preliminary researches on the use of new catalystsof oxysolvolysis are given.
EXPERIMENTAL Spruce sawdust (0.25-0.50 mm) was used as substrate. The sawdust contained extractives 1.1% (diethyloxide) and Klasson lignin 28.1%. Oxidation of wood by oxygen was conducted in a 1 L rocking autoclave (50 min-'). Sawdust (10 g o.d.), a catalyst and 250 ml of 60% (vol.) acetone were placed in the autoclave and then oxygen (0.7 m a ) was introduced into the autoclave. The time to temperature (1 18'C) was 60 min. After
104 Pulp production and processing the oxidation, the wood residue was separated from the solution on a filter, washed, oven-dried (1 03'C) and weighed. Klasson lignin was again determined.
RESULTS AND DISCUSSION To study the action of catalysts, the reaction conditions were chosen so that the degree of deligniiication in the absence of a catalyst was insignificant. The mixture of two inorganic compounds A and B were used as catalysts. Results given in Table 1 indicate that the compounds A and 13 showed a certain catalytic activity. Table 1. The influence of catalysts on the degree of dissolution of wood componcnts during oxysolvolysis (1 18"C, 2 h) Catalyst
Concentration of catalyst, mM/L No 0 A 8.0 B 8.0 8.0 A( 1)B(2) A(5)B(2)* 17.0 * Temperature was 121'C
Residue yield,
Lignin content,
% 94.0 89.1 81.2 73.3 53.4
% 27.4 27.0 18.0 17.6 5.6
Dissolved substances, % from initial Lignin Carbohydrates 8.3 3.7 14.4 8.1 48.0 6.1 54.1 14.7 28.8 89.4
Probably, that delignification process with catalysts is followed according to the next scheme:
+ +
Lo + Ox, L'o +Redl L'o + O2 products Red1 + 0 x 2 0 x 1+ Red2 Red2 + 0 2 90x2 where LOand L'o are, respectively, native and oxidised lignins Oxl and Red, are, respectively, oxidised and reduced forms of compound A 0x2 and Red2 are, respectively, oxidised and reduced forms of compound B. The results testify that the efficiency of the chosen catalysts differ appreciably. In the presence of compound A, the rates of transformation of both lignin and carbohydrates grow. Nevertheless, the rates of their dissolution increase insigniticantly. The compound B strongly increases the rate of lignin oxidation not rendering essential influence on the dissolution of carbohydrates. The greatest effect is reached with joint introduction of these two compounds (AF3). As the data obtained show, the catalytic activity of the AB system is observed at various mass proportions of the compounds used. An increase in the concentration of the catalytic system up to 17 mM/L allows removal of almost 90 % of lignin. The rate of dissolution of wood components largely depends on the conccntration of the catalyst in a solution. The influence of the concentration of the catalytic system on the dissolution of lignin and carbohydrates was investigated with an equal molar ratio of both substances in a mixture. Data given in Table 2 show that
Catalysis of oxygen-acetone delignification 105 acceleration of the oxidising processes is observed even with addition of small amounts of the catalyst to the solution. Table 2. The influence of the AB (1:l) concentration on sawdust dissolution (1 18'C, 2 h) Concentr ation of
Residue yield, %
Lignin content, %
94.0 88.6 85.1 83.0 82.0 78.0 76.2 72.6
27.4 25.2 24.2 23.0 24.2 22.7 21.3 18.1
AB,
mM/L 0 3.2 4.0 5.0 6.0 8.0 10.0 12.0
Figure 1 shows that the dependence of the amount of the dissolved wood components on the catalyst concentration in solution is linear and can be expressed by the following equation:
Y=P+QX where Y is the mass portion of the substrate (lignin, carbohydrates) dissolved in the presence of the catalyst, P is the mass portion of the substrate dissolved in a noncatalytic process, X is the concentration of the catalyst, Q is a constant.
50 40
30 20
10
0
I
I
I
I
I
I
2
4
6
8
1 10
12 mM/L
Figure 1. The dependence of the degree of dissolution on the concentration of AB (1:l) in solution (1 1 8"C,2 h). 1 - Lignin, 2 Carbohydrate
-
106 Pulp production and processing Factor Q in the given equation reflects the catalytic activity of the catalytic system used and can serve as a criterion of the efficiency of the catalysts. In the system investigated, it is equal to 0.06 LImM for lignin dissolution and 0.005 L/mM for dissolution of carbohydrates. These data testifL that the catalyst demonstrates considerably large catalytic activity in lignin substrate reactions as compared with carbohydrate substrate ones. The kinetics of the process have also been investigated and experimental data reflecting dependence of the delignification degree fiom the reaction time are given in Table 3. Table 3. The influence of oxysolvolysistime on sawdust dissolution (1 1S0C,2 h) in the presence of Al3 (SmM, 1 :1) Oxysolvolysis time
Residue yield
Lignin content
YO
%
91.4 81.9 78.0 66.5 55.6
26.1 24.2 22.7 15.8 6.9
m i n '
0 30 60 120 240
For kinetic curve construction, the results of cxperiments conducted under isothermal conditions were used. Therefore, the contents of wood components determined in the wood residues after achievement of the final temperature (oxysolvolysis time 0) have been taken as their initial contents in the substrate. Figure 2 shows that the delignification process is satisfactorily dcscribcd by a first order mathematical equation. The reaction rate constant of the catalytic process (1.3-104s-') conducted at the catalyst concentration of 8 mM/L is more than five times higher than the reaction rate constant of non-catalysed process (2.4.10-5s-'). The process rate can be increased in magnitude by an order and more (see Table 1) with an increase in the catalyst concentration.
I
0
100
200 Time. min
300
-In YNo 2.5 1
100 200 Time. rnin
300
Figure 2. Kinetics of dissolution of lignin (1) and carbohydrates (2)during oxysolvolysis (1 1S'C, AB 8mM/L. 1:1): a - kinetic curve, b - semilogarithmic anamomhoses
Catalysis of oxygen-acetone delignification 107 The kinetic data also show that the rate of the carbohydrate dissolution expressed through the rate constant of a first order reaction (3.10”s-’) is much lower than the rate of lignin dissolution, and an increase in the rate of carbohydrate destruction with introduction of the catalyst into solution is rather insignificant. The results indicate the possibility of obtaining pulp by the oxysolvolysis method with use of the catalytic system suggested. The purpose of research in the near future will be to check this conclusion.
REFERENCES 1. Deineko I. Oxysolvolysis of lignocellulosic materials. Fourth Brazilian Symposium on the Chemistry ofLignins and other Wood Components. 1995, V.5,5-10. 2. Evtuguin D.V., Net0 C.P., Marques V.M. Delignification by oxygen in the presence of polyoxometalates: mcchanism proposal and possible application. 9Ih International Symposium on Wood and Pulp Chemistry. Poster Presentation. 1997,25-1 - 25-4.
CHARGED GROUPS IN WOOD AND MECHANICAL PULPS Bjame Holmbom, Andrey V. Pranovich, Anna Sundberg and Johanna Buchert a b o Akademi University, Laboratory of Forest Products Chemistry, FIN-20500 Turkulabo, Finland
ABSTRACT Analysis by acid methanolysis and gas chromatography revealed that galacturonic acid, which is the main sugar unit in pectin, was the most abundant uronic acid in Nordic spruce, pine, birch and aspen wood, as well as in mechanical pulps. However, since most of the galacturonic acids are methylesterified in native wood, the 4-0-methyl glucuronic acid in xylan is the main charged group in wood and in mechanical pulps. Mechanical pulp fines were found to contain much more galacturonic acid, rhamnose, aiabinose and galactose, and slightly more xylose and methylglucuronic acid, than the long fibre fractions. Alkaline treatment of mechanical pulp more than doubled the fibre charge, and alkaline peroxide bleaching produced still more new acid groups. Charged groups were formed in the fibres with the same kinetics as the release of methanol and acetic acid. It was concluded that the new acid groups originate mainly from demethylation of pectin, and in case of peroxide bleaching also from oxidation of lignin. INTRODUCTION Charged groups are key functional groups in papermaking fibres. They are important both for the various chemical interactions with fibres in papermaking, and for the properties of the paper. Many paper chemicals are cationic and are sorbed to the fibres through interactions with the anionic groups, and the performance of e.g. retention aids and wet-end sizes is strongly affected by the amount of charged groups. Anionic groups interact with metal cations by ion exchange both in pulping, bleaching and papermaking processes, leading to fibre swelling and softening, and consequently strongly affecting many fibre properties (1). Table 1 lists the various charged groups that can occur in wood and mechanical pulps. Table 1. Charged groups in wood and mechanical pulps. Chemical group Carboxyl, uronic acid oxidised lignin fatty and resin acids Phenolic Hydroxyl Sulphonic acid Protein: both carboxyl and amino groups
Structure R-CO2H Ar-OH R-OH R-SO3H R-CO2H R-NH2
Acid constant, PKA 3.5 - 4 ca. 5 5.5-6.4 ca. 10 >12 ca. 1
110 Pulp production and processing Most charged groups in wood and mechanical pulps are carboxyl groups of uronic acids which are units of certain hemicelluloses and pectins (2, 3). Extractives such as fatty and resin acids also contain carboxylic acids. Carboxyl groups in uronic acids have acid constants of 3.5-4,whereas the carboxyl groups in oxidised lignin and in fatty and resin acids have constants in the range 5-6.5 (4,5). Alkaline treatment of mechanical pulps, as in peroxide bleaching, leads to the formation of new carboxyl groups (2,6). We have recently presented evidence that the new carboxyl groups are formed mainly by demethylation of galacturonic acid methyl ester groups in pectins (7, 8). Lignin phenolic groups are weak acids, and aliphatic hydroxyl groups still weaker, and they are not dissociated at common papermaking conditions. Sulphonic acid groups are found only in sulphite pulps, such as CTMP. Wood contains small amounts of proteins which have also positively charged groups. However, their amount is negligible relative to the anionic uronic acid groups. Chemical pulps contain lower amounts of acid groups than mechanical pulps because a large part of the uronic polysaccharides are dissolved and removcd, or degraded in chemical pulping and bleaching. However, chemi-mechanical pulps, such as CTMP, contain many charged groups (9). The total number of acid groups can be determined by acid-base or polyelectrolyte titrations (10). However, such titrations do not provide distinct information about the origin and chemical character of the charged groups, although groups with different acid strengths can be distinguished. Total uronic acid content is traditionally determined by treatment with strong acids and determination of released carbon dioxide (11). Mild cleavage followed by chromatographic analysis is a means to determine the identity of the uronic acid units in wood and pulp samples. Enzymatic hydrolysis combined to e.g. HPLC analysis has been succesfully used for analysis of uronic acids in haft pulps (12). Enzymes are, however, not able to completely degrade hemicelluloses and pectins in wood and mechanical pulp samples (13). Acid hydrolysis is effective, but uronic acid units are degraded to a large extent. Acid methanolysis provides an essentially better protection of the uronic acid units. Acid methanolysis followed by gas chromatographic analysis of the formed methyl glycosides was found to give good yields of hemicellulose sugars, including uronic acids, for both wood and mechanical pulp samples (14). In this study we have applied acid methanolysis and gas chromatography to analyse the hemicelluloses and pectins in Nordic wood and mechanical pulp samples, with the special objective to determine the charged uronic acid groups. The formation of new charged groups in alkaline treatment and peroxide bleaching has also been examined.
WOOD AND PULP SAMPLES Mature, healthy spruce (Picea abies), pine (Pinus silvestris), birch (Betula pubescens) and aspen (Populus tremula) trees growing in the Turku region were felled, discs were cut from the trees and were on the same day placed in a freezer where they were stored at -24°C until analysis. Knot-free parts of the discs, without any reaction wood, were taken for analysis. Small splinters were cut out, fReze-dricd and ground to wood meal in a Cyclo-Tec mill producing particles smaller than about 30 mesh (0.5 mm). Thermomechanical pulp (TMP) was taken after the second refiner in a Finnish paper mill using spruce as wood raw material. The pulp was stored in a freezer until analysis.
Charged groups in wood and mcchanical pulps
111
The TMP was freeze-dried and extracted during 24 h in Soxhlct apparatus with acetone-water (9:1 by vol.). The pulp was furthermore washed extensively with water at 60°Cto obtain clean TMP fibres. Care was taken to avoid loss of fines during washing. The extracted and washed TMP was treated at 2% consistcncy at a constant pH of 11.0 and 60°C for various times. Treatment were made also with addition of 3% hydrogen peroxide. After the treatments, pH was adjusted to 5.5 by addition of sulphur dioxide water. The pulps were then dewatered and washed with distilled water on a Buchner funnel. The drained water was sampled for analysis. Treatment with alkali, and peroxide bleaching was also made at 10% consistency and 60°C with a starting pH of 12. After 60 minutes reaction time, the suspensions were neutralised to pH 5.5 with sulphur dioxide water and the fibres were thoroughly washcd. Fractionation was made of a TMP watcr suspcnsion as follows. TMP, that had been extracted with hexane but not washed, was diluted to 0.5% consistency with distilled water and the suspension was agitated at 60°C with a mixer blade at 150 min-'. The conductivity was adjusted to ca. 1 mS/cm with 1 M NaCI. After stirring for 3 h, the suspension was disintegrated with a household mixer for 2 minutes. The consistency was again adjusted to 0.5%. The fibres were separated from the rest of the suspension by filtration in a Dynamic Drainage Jar.@DJ) equipped with a 100 mesh (0.15 mm) wire. 1000 mL of the suspension was added to the DDJ and the stirring speed was adjusted to 900 mid'. After 10 s the bottom valve was opcncd and about 300 mL of the filtrate was collected. The DDJ was washed and the filtration was repeated. A part of the suspension, now consisting of large fines, small fines, colloidal substances and dissolved substances, was stored in a cold room until the next day. The large fines were removed by filtration in the DDJ, now equipped with a 400 mesh wire (0.045 mm). About 750-1000 mL was filtered and 300-500 mL was collccted. The DDJ was washed and the filtration repeated, if necessary. The small fines wcre separated from the dissolved and colloidal substances by centrifugation at 500 g for 30 minutes. The supernatant was pipetted of. The colloidal substances were removed from the dissolved substances by filtration with a 0.1 pm filter. In an other fractionation procedure, pressurised groundwood (PGW) was fractionated using a Bauer-McNett classifier (SCAN M6-69).The PGW was produced in a Finnish mill from spruce wood and was sampled dircctly after grinding to CSF 45. The fractionation was performed using screens of 30,50, 100 and 200 mesh (0.54.0.29, 0.15 and 0.074 mm, respectively). Thc fines fraction was recovered from the fraction passing the 200 mesh scrcen by filtering on a Buchner funnel with a 400 mesh wire. The filtrate was recycled to recover all fibre material, including the colloidal fines.
ACID GROUP DETERMINATION AND ANALYSIS Hemicellulose and pectin sugar units, including the uronic acid units, wcre determined by acid methanolysis and gas chromatography (GC)(14). The charge of TMP fibres was determined with polyelectrolyte titration. 0.5 g frccze-dried TMP was ripened ovcmight in 29.5 g of distilled water and further suspended with a magnetic stirrcr for 3 hours. 20 g of 0.005 M 1,5-dimethyl-1,5diazaundecamethylene polymethobromide (polybrene) solution was added to the suspcnsion and stirring was continucd for 2 hours. The suspension was centrifuged and an aliquot of the supernatant was titratcd with a MUtek particle charge detector 03 using potassium polyvinyl sulphate (KPVS) as anionic polymer. TMP water samples,
112 Pulp production and processing containing dissolved and colloidal substances, were mixed with polybrene directly in the measuring cell and were then titrated with KPVS.
ACID GROUPS IN WOOD SAMPLES Analysis of spruce sapwood by methanolysis and GC gave the sugar composition shown in Fig. 1. The predominant hemicellulose type in spruce is galactoglucomannan, here seen as large amounts of mannose, glucose and galactose. However, a minor part of the galactose is present in arabinogalactan, and part of the glucose obtained in thc analysis is probably dcrived from starch. Some of the glucosc may also originate from cellulose, although the cellulose is quite stable against acid methanolysis at the conditions uscd, due to inaccessibility and hydrolytic stability of the cellulose glucosidic bonds. Another major hemicellulose in softwoods is 4-O-methyl-glucuronoarabinoxylan, here seen as xylose, arabinose and methylglucuronic acid in the ratio 4.3: 1:0.9. A minor part of the arabinose and galactose is probably present in form of arabinogalactan. Small amounts of an arabinogalactan that also contains glucuronic acid units can be extracted from spruce wood by cold water (15).
Sugar units, mg/g wood 120 100
80
60 40
20 0 Ara
XYl
Gal
Glc
Man
Rha
GlcA
Me GlcA
GalA
Fig. 1. Hemicelluloses and pcctin sugar units in spruce sapwood.
GalA -
-
[4 -0M e G Ic A2
I I
0-Rha 0
- Xyl - Xyl - X y l -
I I A ra Gal I I I Pig. 2. Uronic acid units in softwood hemicelluloses and pectin. I A ra
Charged groups in wood and mechanical pulps
113
Table 2. Amounts of uronic acids in wood samples, in mg/g of oven-dried wood.
Wood Spruce
GlcA
Sapwood (' Heartwood Sapwood Heartwood
MeGlcA
2-3 8-12 2-3 9-13 Pine 1 9 2 10 2 17 Birch 2 15 Aspen (1 range for analysis of three wood samples
GaL4 13-17 15-18 18 16 21 22
Total uronic acids 26-30 28-32 28 28 40 39
The most abundant uronic acid in spruce sapwood, as well as in the other wood samplcs, was galacturonic acid (Table 2). Pectin is made up mainly of galacturonic acid units, but contains also some rhamnose units (Fig. 2). Most of the galacturonic acids are probably methyl esterified in native wood (16), although the degree of methylation is not exactly known. This means that the 4-0-methyl glucuronic (MeGlcA) acid remJns the predominant free acid unit in these woods. Thc glucuronic (GlcA) acid, that is a unit of arabinogalactan,constitutes only a minor part of the uronic acids. Spruce sapwood and heartwood gave a very similar composition of hemicelluloses and pectins, including the amounts of uronic acid units (Table 2). Pine sapwood and heartwood contained about the same amounts of uronic acids as spruce. The hardwoods, birch and aspen, differed considerably from the softwoods, in containing more xylan, and consequently more methyl-glucuronic acid. The pectin contents, seen as galacturonic acid units, were also highcr.
ACID GROUPS IN MECHANICAL PULPS Mechanical pulps are in the Nordic countries produced mainly from Norway spruce. Thermomechanical pulping (TMP) is today the predominant technique. Mechanical pulps are increasingly bleached by alkaline peroxide. Thcre are no extensive chemical reactions in mechanical pulping processes. However, 3-5% of the wood matcrial is dissolved or dispersed into process waters during pulping (7). Acetylatcd galactoglucomannanis the predominant group among the dissolved substances. Acidic hemicelluloses and pectin are, however, dissolved only to a small extent (7). This was observed also now in the fractionation and analysis of a TMP suspension by DDJ filtration, centrifugation and microfiltration (Table 3). The dissolved fraction contained very little glucuronic and methylglucuronic acids. Howevcr, the colloidal fraction containing the so-called microfines, contained remarkably high amounts of both pectin (Rha and GalA) and acidic arabinogalactan (Ara, Gal and GalA). However, this fraction comprised only 0.4% of the total fibre material. These analyses verified that the acidic groups in mechanical pulps are practically the same as in the wood raw material, with the methylglucuronic acid units in arabinoxylan as predominant acid group.
114 Pulp production and processing Table 3. Hemicellulose and pectin sugar units in different fibre fractions of a spruce TMP suspension. Fractionation by DDJ filtration, centrifugation and microfiltration. Data given in mg/g of each fraction. Fibre fraction (mesh)
Ara
Xyl
Gal
Glc
Man
Rha
GlcA
10
54
22
44
108
1.6
1.6
10
10
261
19
69
41
39
86
5
3
13
35
310
21
60
42
34
76
5
1.9
8.5
40
288
52
29
171
104
95
14
36
2.4
33
536
11
4.8
49
109
272'
1
1.7
1.7
16
466
12 58 28 (1 Weight % of total pulp
48
115
2.3
2.0
10
15
290
>loo
81% (' 100-400 7% ~400 8% Coll. 0.4% Diss. 3% Whole pulp
Me GalA GlcA
Tot.
The fines fractions (100-400,and c400 mesh) contained much more arabinose, galactose and galacturonic acid than the coarse fibre fraction (>lo0mesh). The content of glucuronic acid was higher in the larger fines fraction, but not in the smaller fines fraction. The larger fines fraction contained more xylan (Xyl and MeGlcA) than the coarse fibre fraction. Analysis of fibre fractions of PGW separated by Bauer-McNett fractionation showed the same trends in composition (Table 4). Table 4. Hemicellullose and pectin sugar units in different fibre fractions of spruce PGW separated by Bauer-McNett fractionation. Data given in mg/g of each fraction. Fibre fraction (mesh) >30 25% ( I 30-50
Ara
Xyl
Gal
Glc
Man
Rha
GlcA
Me Glc A
GalA
Tot.
10
52
15
43
120
1.2
0.1
5.1
8
255
12
55
23
44
113
2.7
0.6
6.7
9.1
266
11
54
24
44
108
2.2
0.5
5.9
10
259
12
57
31
45
107
2.6
0.5
7.1
11.9
273
18
66
40
47
96
4.1
0.7
7.6
24
303
13 56 25 (1 Weight % of total pulp
45
110
2.2
0.6
6.1
15
274
13% 50-100 13% 100-200 8% c200 Whole pulp
115
Chargcd groups in wood and mechanical pulps
The fines fraction (-
- 20
I-
v,
5 0 400 450 500 5 5 0 600 650
Z w t-
z - 10
0
1 t
l
350
I
I
! I
I
400
t
I
I
I
I
450
I
I
I
I
I
500
I
I
I 1 I
550
I
I
I
I
I
600
I
I
I1
650
W A V E L E N G T H (nm) Fig. 2. Emission spectra of GW treated at 105°C for various times: curve 1,0 h; curve 2, 4 h; curve 3,21 h; curve 4,46 h; curve 5,96 h. Inset: differcnce between the spcctra of GW pulp treated for 96 h and untreated GW pulp.
200 Structure and properties of fibres 50 40
> 30 Iv)
=
W
20
I-
z -
10 0 I
l
l
3 50
1
I
400
I
I
I
I
I
I
I
I
l
l
1
I
l
l
450 500 550 WAVELENGTH ( n m )
1
I
600
I
I
I
650
Fig. 3. Emission spcctra of TMP treated at 105°C for various times: curve 1,0 h; curve 2 , 4 h; curve 3,21 h; curve 4,46 h; curve 5,96 h. Inset: difference between the spectra of TMP treated for 96 h and untreated TMP pulp.
DRY OVEN
R-. 457 nm V"
TMP
75 70
65 60
55 50 45 40
35 30 0
20
40
60
80
HEATING TIME, min
Fig. 4. Kinetics of the yellowing of TMP at various temperatures (105", 150°C, 165"C, 180°C. 200°C and 230°C): brightness (R,at 457 nm) versus heating timc.
Heat-induced changes in fibre surfaces 20 1 AR 10
-
8 --
6 --
4
_-
2 --
-2 -
250
,
300
350
400
450 500 550 Wavelength, nm
600
650
700
750
Fig. 5 . Kinctics of the yellowing of TMP at 180OC: UV-VIS difference reflectance spectra (unheatcd minus heated) after diffcrent times (up to one hour).
HEATING TEMPERATURE, "C
Fig. 6. Stability of GW and TMP during one hour of heat treatment at different temperatures (ambient to 240°C).
202 Structure and properties of fibres The corresponding UV-VIS difference reflectance spectra of TMP heated at 180°C are shown in Fig. 5. The yellowing maximum is locatcd at 433 nm. The spectra at the beginning of the heat treatment differ considerably from those obtained after prolonged thermal treatment. In the initial stages, there is a significant decreasc in the chromophore content in the UV region at 370-380 nm, as observed previously [3,4]. During heating new chromophores are formed in the long wavelength region and the initial decrease in the chromophore content in the UV region gradually disappears. The brightness stability of GW and TMP is greatly reduced at high temperatures (>130"C). The relative stabilities of the two pulps and their sensitivity to heat can be assessed from Fig. 6. TMP is slightly more stable than GW in accordance with the results of the fluorescence study. At temperatures higher than 130°C. a considerable acceleration is obscrved in the rates of the different colour-forming reactions in GW and TMP.
CONCLUSIONS In an evaluation of the heat stability of mechanical pulps, as for pulps in general, the following practical concepts may be useful. The temperature range is dividcd into three differcnt thermal regions, all of which cause yellowing. The behaviour of the chromophores differs from one region to another, resulting in different types of yellowing with different kinetics. These thermal regions can be roughly defined as follows: The first rcgion comprises moist heat at ambient to modcrate temperaturcs (60"C) for a long time is explained by the formation of nonequilibrium and defective structures in the binder polymeric matrix as a result of both significant increasing of speed of the structure forming processes and partial dissociation of the interpolymer H-bonds between binder's components.
INTRODUCTION The growing interest in technical lignins, by-products of pulp mills, as a raw polymeric matcrial is conditioned by their availability in large scale and their biodegradability. Undoubtedly, this interest is one of the main reasons for considerable scientific progress being made in the development of new ligninbased polymeric products and resins with the purpose of their successful practical application. At present, lignin-based graft co-polymers [1,2], plastics and elastomer blends [3-61, interpolymer complexes [7,8] and the other lignincontaining polymer products [9-121 are known. The lignin-based products can perform various functions such as an adhesive promoters and regulators of the interfacial tension in polymer systems, fillers and reinforcing agents for engineering plastics etc. The application of lignins for partial substitution of synthetic thermosetting resins (UF, PF) in the manufacture of building composite materials such as
298 Wood, fibre and cellulosic materials fiberboard, particleboard, plywood is well known [13-15]. However, the application of lignins in resins is limited due to a significant worsening of the properties of composite materials and the necessity of additional power consumption for their manufacture at a high lignin content. It has been found 1161 that the coupling of lignosulfonate (LS) with a synthetic water-soluble polymer of linear structure with a high molecular mass in concentrated aqueous solutions results in the formation of a modified LS (MLS) which, from the chemical point of view, represents an interpolymer complex. The LS-based interpolymer complex is able to substitute 45% and more of the UF-oligomers in a composite material composition. Advantages of the manufacture of the composite materials obtained with the novel UF-based binder in comparison with the known technology based on traditional UF-resin application are the following: using of the significantly lower temperatures for pressing (20°C - 30"C), using of a lower pressure (0.1 MPa 0.2 MPa), no necessity in any curing agent. If a papermaking waste containing short cellulosic fibers in the form of granules is used as a filler, the composite materials obtained are characterizcd by a density of 450-500 kg/m3 and a heat conductivity coefficient of 0.080-0.11 W/Km [17,18]. In the present work, the results of the study of the effect of fine dispersed filler properties as well as the drying conditions of the composite materials on the UF-MLS binder properties are discussed.
MATERIALS & METHODS Urea-formaldehyde oligomers were used at pH 7.5 in aqueous solution containing 65.0% of dry matter and up to 0.3% of free formaldehyde. An ammonium lignosulfonate was applied for a substitution of a part of the UFoligomers. This LS is characterized by the following: 50.0% of dry matter, 4.8% of ash, pH 4.4, 5.0% of technical sugar. For the purpose of an LS modification, a synthetic water-soluble polymer with an average molecular mass 2 .lo5 was used. The polymer to the LS mass did not exceed 0.1. The modification was carried out by mixing concentrated water solutions of the LS and the polymer for 30 min at a room temperature. The UF-MLS binder was obtained by blending the oligomers with the modified lignosulfonatc in a mass ratio of the UF-oligomers to MLS of 0.67. A papermaking waste containing more than 50 mass % of short cellulose fibers with the minor addition of sawdust was granulated to aggregates with a size of 5-20 mm and applied as a coarse filler. Fractionated milled sand, coal ash, cement dust and wood flour with particles size of less than 1 mm were used as fine dispersed filler. In the composite material UF-MLS binder and filler were blended in a laboratory mixer. The content of the binder in the composite material depended on the kind of filler used. The specimens with a width of 35 mm, length of 155 mm and height of 35 mm were made by pressing of raw blends at 20°C and a pressure of 0.110.12 MPa for 30 min. The drying process of the specimens obtained was carried out at constant temperature. The specimens made were tested in terms of specific gravity, cohesion strength, bending strength both in air-dried state (R,) and wet state (R,) after soaking for 24 h in cold water as well as for water resistance. The water resistance of the composite materials was characterized by
Composite materials from pulp and papcrmaking wastes 299 the y value reprcscnted as a ratio of R, to R,. The strength values reported are averages from three specimens.
RESULTS & DISCUSSION The physico-mechanical properties of a composite material as well as its durability depend significantly on the properties of the binder. It is possible to improve the properties of UF-based binder by creating optimal conditions for its structure formation. One of the promising methods for development of such conditions may be the application of a fine disperscd filler in a binder composition. On the other hand, it is known that the presence of the filler can promote the reduction of the formaldehyde emission and decrease the shrinkage of the composite material obtained. Fig.1 shows the dependence of cohesion strength of an UF-MLS binder containing different fine dispersed fillers. Irrespective of their nature (organic or inorganic), the inclusions of fillers imply an increase in binder cohesion strength. According to the maximum values of cohesion strength, milled sand is the best reinforcing agent. It doubles the cohesion strength of the UF-MLS binder in comparison with a bindcr containing no filler. Cement dust, in fact, is the worst reinforcing agent. Evidently, in this case the lowest values of the cohesion strength of the filled binder are caused by the dust alkali value @H>7) that significantly inhibits the structure forming processes in the UF-MLS binder. Wood flour and coal ash as reinforcing agents occupy an intermediate position between sand and cement dust and are capable of imparting the same value of maximum cohesion strength to the binder. It is shown (see Fig.1) that the maximum value of cohesion strength is reached by the addition of wood flour to the UF-MLS binder in amounts 4, 5 and 6 times lower than in the case of cement dust, coal ash and milled sand, respectively. It is obvious that a high specific surface of wood flour as well as a considerable number of reactive hydroxyl and carbonyl groups on its particle surface causes the sufficient intensification of structure formation process in the binder. The surface functional groups are capable of forming a multitude of hydrogen links with binder macromolecules at the interface, as shown in Fig. 2. As a result of such an interaction, the cohesion strength of the binder is remarkably increased. However, after reaching the crucial concentration of wood tlour in the binder (which is up to 12 mass %), it falls noticeably owing to the thinning of the bindcr interlayer between the filler particles. It is supposed that the increase of speed of the structure forming processes in the UF-MLS binder can be promoted also by the high hygroscopicity of wood flour filler. As shown in Fig.1, for the unreactive fillers such as milled sand and coal ash, the values of the crucial concentration is much more and vary from 60 to 70 mass %. Table 1 shows the water resistance of the composite materials obtained under different drying conditions. The coarse filler, ganulated cellulose waste, was used for making the composite material. The results reported show a significant effect of thc temperature and duration of drying on the adhesive ability of the UF-MLS binder to the fillcr. According to the data, an incrcasc in thc drying time at 20°C causes an increase in the y value which, after 6 h, makes up 50% of the maximum water resistance attained in 24 h. Specimens dried at 60°C
300 Wood, fibre and ccllulosic materials
6
+wood 2,4
-
flour
cement dust +coal
ash
+sand
u
l,o
1
4.0
18
1
1
1
35
1
l
1
l
69
48
Filler content, mass YO
Figure 1.
Dependence of cohesion strength of the binder on a filler content.
0 0 D
’Y
F
L 0
U R
................
OH
w
1
o
........ ................ .............
\
OH
Figure 2.
.............
The scheme of adhesive “bridges” at the interface boundary
P 0
L Y M E
R M A T
R I
X
Composite materials from pulp and papermaking wastes 301 reach Y,,,~ even after 3 h of drying. At the. same time, an increase in the drying time above 3 h at this temperature results in thc deterioration of the water resistance of the specimens. Table 1. Effect of drying conditions on water resistance (y) of composite materials
Water resistance, y = RJR,
l h
3 h
6 h
24 h
-
0.40
0.77
0.15
0.79
0.65
0.57
0.70
0.39
0.12
20°C
60°C
105%
However, the most considerable deterioration of mechanical strength and the water stability is observed for Composite materials dried at 105°C for over 1 h, indicating a worsening of the adhesive properties of the UF-MLS binder. Obviously, an elevated temperature, in combination with prolonged drying leads to speedier dewatering of the composite material resulting in a significant intensification of structure forming processes in the binder polymer matrix. As a rule, the formation of non- equilibrium polymeric structures and the inhibition of the relaxation processes in binder polymer matrix accompany one anothcr. The formation of defective structures at a high temperature (>60°C) in a combination with prolonged drying is also promoted by partial dissociation of the interpolymer H-bonds between UF oligomers and MLS formed the binder complex macromolecules. It has been found that the effect of high temperature and prolongcd drying upon the properties of composite materials with the fine dispersed organic fillers such as wood flour, sawdust, bark and hydrolyzed lignin (characterized by a high hygroscopicity) is even more dramatic than in the case of the coarse organic filler. Hence, the formation of non-equilibrium and defective structures in the W-MLS binder polymer matrix is one of the main reasons of the unsatisfactory physicomechanical properties of the cornpositc materials obtained at elevated temperatures and prolongcd drying.
302 Wood, fibre and cellulosic materials CONCLUSION Thus, to develop the optimum conditions for structure formation of the UF-MLS binder polymer matrix in order to obtain the composite materials with needed physico-mecanical properties, the nature of a filler, its dispersity, surface as well as the water removal rate and duration of dewatering during the drying process should taken into account the composite material is being manufactured.
ACKNOWLEDGEMENTS The authors acknowlcdgc financial support from the Latvian Council of Science (the grants for the rescarch projects).
REFERENCES 1. J. J. Meister, ‘Review of the synthesis, characterization and testing of graft copolymers of lignin’, In: Polymer scicnce and technology: renewable-resource matebals, C.E. Carraher & L.H. Sperling (eds.), Plenum Press, NewYork, 1986, pp. 305-322. 2. W. de Olivera, W. G. Glasser, ‘Starlike macromers from lignin’, In: Lignin: Prope~iesand Materials, ASC Symp. No. 397, W.G. Glasser & S. Sarkanen (eds.), Am.Chem.Soc., Washington, D.C., 1989, pp. 414-435. 3. D. Feldman, M. Lacasse & L. M. Beznaczuk, Lignin-polymer systems and some applications, Prog. Polym. Sci., 1986, 12, 271-299. 4. D. Feldman, D. Banu, C. Luchian & J. Wang, Epoxy-lignin polyblends: correlation between polymer interaction and curing temperature, .l Appl. Polym. Sci., 1991, 42(5), 1307-1318. 5. J. J. Lindberg, T. A. Kuusela & K. Levon, ‘Specialty polymers from lignin’, In: Lignin Properties and Materials, ASC Symp. No. 397, W.G. Glasser & S. Sarkanen (eds.), Am.Chem.Soc., Washington, D.C., 1989, pp. 190-204. 6. Sh. Hirose, H. Hatakeyma, J. Izuta, T. Yoshida, T. Hatakeyma, ‘Synthesis and thermal properties of epoxy resins derived from lignin and lignin-related phenols’, In: Advances in Lignocellulosics Chemistry for Ecologkally Friendly Pulping and Bleaching TechnoIogies, Sa Eur. Workshop, University of Aveiro, Aveiro, Portugal, 1998, pp. 295-297. 7. G. Strom, P. Barla, P. Stenius, The formation of lignin sulphonate polyethyleneimine complex and its influence on pulp drainage, Svensk pappentidn, 1979, 82 (14), 408-414. 8. G. Shulga. Lignin-based interpolymer complexes: obtaining, reactions, properties and prospects for application, Dr.habil.chem.thesis, Latvian State Institute of Wood Chemistry, Riga, Latvia, 1998. 9. B. KoSikova, A. Revajova, V. Demianova, The effect of adding lignin on modification of surface properties of polypropylene, Em-. Polym. 1,1995, 3 1(10), 995-999. 10. B. KoSikova, V. Demianova, ‘Physico-chemical properties of lignin modified polyolefins’, In: Markets for Sulfur-Free Lignin, 3rd Int. Forum, Working Documents, Fribourg, Switzerland, 1996.
Composite materials from pulp and papermaking wastes
303
11. N. G. Lewis, T. R. Lantzy, ‘Lignin in Adhesives’, In: Adhesives h m renewable nsouzces, ACS Symp. No. 395, P.W. Hemingway & A.II. Conner (eds.), Am.Chem.Soc., Washington, D.C., 1989, pp. 13-26. 12. J. J. Meister, ‘Chemical modification of lignin’, In: Chernicd modification of lignocellulosic rnaten-ds, D.N.-S.Hon (eds.), Marcel Dekker, New York, 1996 pp. 129-157. 13. K. G. Forss, A. Fuhrmann, Finnish plywood, particleboard and fibreboard made with a lignin-based adhesive, Forest P d . 1,1979, 29 (7), 39-43. 14. H. H. Nimz, G. Hitze, The application of spent liquor as an adhesive for particleboard, Cellulose Chem. Technology, 1980, 14(3), 371-382. 15. A. L. Wotten, T. Sellers, Md. Tahir Paridah, Reaction of formaldehyde with lignin, Forest Prod. 1,1988, 6, 45-46. 16. G. M. Shulga , G. M. Telisheva, A. V. Soms, V. H. Lapsa, T. E. Betkers, A. B. Zezin, V. A. Kabanov, ‘Lignosulfonate-based compositions as chemical admixtures in a manufacture of building materials’, In: Application of chemical admixtures in manufkture of cellulose, pap4 timber and building boards h m wood fibrcs, Int. conf., Federation of Scientific and Technical Cooperation, Bulgaria, Sofia, 1990, pp. 30-31. 17. V. H. Lapsa, T. E. Betkers, Thermo- and sound insulating materials, Papermaking industry (Russian), 1990, 4, 21-22. 18. G. Telysheva, G. Shulga, V. Lapsa, T. Betker, A. Zezin, Modified lignosulfonate-based complex binder for building boards, Cellulose. Paper. C d board (Russian), 1994, 4-5, 23-24.
CELLULOSE COMPOSITE MATERIALS AS SORBENTS SORPTION AND RHEOLOGICAL PROPERTIES
-
Sorin Ciovica', Bruno Liinnberg', and Kurt Liinnqvist' 'Abo Akademi University, Laboratory of Pulping Technology, FI-20500 Turku/Abo, Finland
'Cellomeda Oy, vkistdkatu 6 A, FI-20520 Turku, Finland ABSTRACT
In the last few years sorbent cellulose sponges with a tridimensional porous structure obtained by the viscose process have become more important. Their physical properties depend on the characteristics of the initial dissolving pulp, the viscose procedure and coagulation parameters, as well as on the regeneration conditions. Some propcrties can be significantly changed by reinforcing with cotton or man-made cellulose fibres. The properties of viscose sponges made of low-grade dissolving pulps obtained by the IDE and the soda pulping processes were compared with those of a commercial rayon-grade sulphite dissolving pulp. The influence of the amount and orientation of the cotton reinforcing fibres is also presented. A method for evaluation of the water absorption in the sponges and their retention ability was developed and used to test the sponges. All sponges tested absorbed water and reached the water saturation, i.e. the maximum water absorption (at equilibrium) in about 20 s, and this behaviour seemed to follow the sponge structure. The maximum water absorption of the sponges made of birch soda and IDE pulps was 10 - 15 g water/g dry sponge, as the corresponding sponge density was 0.12 - 0.08 g/cm3. A commercial pulp sponge with a density of 0.05 g/cm3 (reinforced by 20 % cotton fibres) absorbed 25 g water/g dry sponge. The birch IDE and soda pulp sponges had a specific compressibility much lower than the corresponding wheat straw pulp sponges, while reinforcement of the commercial pulp sponge with 20 % cotton fibres resulted in a superior compressibility. Microscopy confirmed a preferential orientation of the sponge cavities perpendicularly to the main sponge surface. INTRODUCTION
The state of engineering of biodegradable materials compatible with living tissues is in remarkable progress. Absorbent materials with a porous structure formed by cavities have been studied on a large scale, especially for medical purposes, e.g. as carriers of drugs, bases for cell growth and as implants for tissue regeneration /1-13/. The materials have also been developed for ion-exchange and mass transfer purposes /3, 14-16/. Absorbent materials are characterized by a tridimensional structure of cavities, which is a prerequisite for liquid absorption in the absorbent /17/. The studies in this area have been focusing on synthetic and natural polymers considered biocompatible and biodegradable, e.g. cellulose and its derivatives, modified proteins, alginates /13, 181, copolymers of polylactic and polyglycolic acids 111, polyvinylpyrrolidone, polyvinylalcohol, polyurethanes efc. The structure of cavities is generally created by the
306 Wood, fibre and cellulosic materials introduction of an inert pressurised gas (usually C02) into the polymer solution or melt 11, 19-201, the gas then expanding or separating. In this study, cellulose was converted to viscose and the cavities were created by solid salt crystals mixed with the viscose /4/. The porosity of the cavity-separating cellulose walls is due to the fibrillar structure of the polymers used. Irregular packing of microfibrils creates micropores with 2-7 nm diameters and mesopores with 50-150 nm diameters, as macrofibrils correspondingly create macropores with 1-10 pn diameters. The macropores can be dimensionally modified and controlled by epichlorohydrin-crosslinkingin the case of cellulose /3/, while the mesopore and micropore domains mainly remain unchanged. Chemical reactions as well as drying and freezing affect the hydrogen bond density and thus introduce structural changes to the pores making their specific propcrties particularly suitable for separation, ion-exchange and protein-chromatography procedures. Cellulose properties in general as well as biocompatibility and biodegradability might further be modified and improved by introduction of suitable substituents to the alcoholic hydroxyl groups. The simultaneous hemostatic and tissue-regencration properties of oxycellulose have been studied in many projects 111, 13, 21-22/. Regenerated cellulose with a homogeneous molecular weight distribution and a relatively low DP is supposed to be more suitable for living organisms from the viewpoints of resorption ability and being inert. The behaviour of absorbents obtained with carboxymethyl or hydroxyethyl cellulose crosslinked with cpichlorohydrin /15/ has been studied for this purpose and for structural and rheological properties /16/. The importance of cellulose viscose sponges obtained by coagulation of the viscose in presence of solid electrolytical salts has been emphasized /3,4, 24/. The low mcchanical strength of these sponges when not reinforced may be a drawback for their utilisation in general /l/. Therefore cellulose sponges still require a lot of optimisation of the pore and cavity size distribution for absorption properties, and further development work for better biodegradability.
EXPERIMENTAL Cellulose properties
The dcgree of substitution and viscose filterability are necessarily not required at highquality levels for the production of regenerated cellulose sponges. The hemicellulose content does not in this case seem to be as important as in fibre spinning, because the sponges can be easily reinforced by external cotton fibres. Subsequently, low-grade dissolving pulps might be introduced for this purpose. A new IDE pulping concept developed for both wood and non-wood raw materials was expected to provide a reactive cellulose, unbleached or partly bleached, because of the smooth delignification. Birch and winter wheat straw pulps wcre produced by the IDE pulping process developed in Finland /25-28/ and bleached by a DED-sequence. The pulps appeared degradable as a result of electron-beam treatment, especially at reduced doses, and showed subsequently a high reactivity in the xanthation /24/. The IDE and soda pulp properties are given in Table 1.
Cellulose composite materials as sorbents 307
15:88, 4, TAPPI T203 om-88 Sponge preparation General
The blcached pulps were converted into viscose solutions and finally transformed into sponges .on a laboratory scale. The viscose composition and the ripening conditions corresponded to those applied in common mcmbrane technology. Viscose coagulation and cellulose regcneration were initiated by intensive mixing of the viscose solution into Glauber salt crystals. When a gel structure appeared, the cellulose-salt conglomerate was immersed into hot water (90-95’C), where the cellulose regeneration was completed and the salt rcmoval finished by repeatcd washing with distilled watcr of the same temperature. Cellulose-salt ratio
The cellulose-salt ratio was varied from 22 up to 44 g cellulosekg Glauber salt crystals for optimisation of the cavity structure of the sponge. Provided that the cellulose can be properly distributed in all experiments, irrespective of the cellulose rate, decreasing cellulose would imply thinncr and evidently more “fibrillated” intercavity walls of the sponge with subsequently quicker absorption of water. In this context the cellulose-salt conglomerate was compressed (I 00-250 bar) to reduce the air introduced during thc mixing procedure, which would imply bctter control of the sponge density. Reinforcement
Cotton fibres were introduced in variable proportions (20-60 YO)into the viscose to form a reinforcing fibre network in the final sponge. The aim was to determine the changes in strength and density of the final sponges. The cotton fibres were oriented by certain treatments of the viscose-fibre-salt conglomerate.
308 Wood, fibre and cellulosic materials Sponge characterization Water absorption Viscose sponges of certain fixed dimensions were immersed repeatedly into distilled water of room temperature for short periods of time with intermediate weighing to determine the water absorption rate. The water absorption rate and the maximum water absorption obtained were then related to the sponge density and other characteristics. The data obtained provided some information on the functional properties of the sponges. Routine measurements of the water absorption were also performed, but then the sponges were kept immersed in water until the absorption achieved the maximum absorption corresponding to the state of equilibrium. The sponges were removed from the bath and initially blotted with filter papers on both sides to remove loose surface water and weighed. The water retained and related to the dry sponge weight was reported as the “initial water retention” (IWR, g watertg dry sponge). The sponges were further pressed for 10 s with a weight corresponding to 150 g/crn’ of sponge volume and the “pressed water retention” (PWR, g watertg dry sponge) was reported. Compressibility The specific compressibility oSp was measurcd on the sponges and reported together with the sponge stiffness. Also the yield point was determined. For clarification of the cavity orientation and distribution further compressibility tests were conducted on differently cut sponges as to represent cross-cut, parallel and length section surfaces relative the main sponge surface; cross-cut and length section surfaces are perpendicular to the main surface.
RESULTS AND DISCUSSION Ccllulosc-salt ratio and compression A number of sponges were prepared from the explored birch and straw IDE and soda pulps bleached by the DED-sequence. The cellulose-salt ratio was kept constant and no pressure was applied as to reduce included air. But sponges were produced from a commercial sulphite dissolving pulp with variable cellulose-salt ratio and by doing the compression. The data are compiled in Table 2.
In general, the birch pulps provided better sponges with lower density and higher initial water retention than the straw pulps and the commercial pulp. Fig. 1 shows that the cellulose-salt ratio to a high extent determined the sponge density, which implies that the cellulose viscose was distributed between the salt crystals as expected. The less cellulose, the lower was the sponge density, and the higher was the water retention, as indicated in Fig. 2. It seems that the sponge density was the most important factor for the water retention ability; the commercial pulp sponges followed the same curve in Fig. 2 as well. Table 2 shows however that increased compression slightly increased the sponge density.
Cellulose composite materials as sorbents 309 Table 2. Sponge prcparation parameters (cellulose-salt ratio and compression) and results on sponge density and watcr absorption and retention on compression. ~~
Sample no.
Cellulose
ell./Glaube pressure, salt mtio, bar
& 1 2 3 4 5 6 7 8 ' 9 10
I'
0.8
Birch IDE Birch Soda Straw IDE strawsoda Borregaard begaard Bomgaard Borregaard Bonregaard
I)orregaard
+Sponge
25 25 25 25 25 22 22 22 40 42
-
100 200 250 200 200
0.085 0.122 0.227 0.297 0.144 0.207 0.214 0.217 0.327 0.357
Nater retention, g waterlg sponge Initial 'reSsed(PWR) at W) 150 an3 sponge 'WR PWIWR% 16.5 8.6 52 11.6 7.5 65 6.1 4.7 78 4.2 3.6 85 12.8 8.0 63 4.0 7.3 55 7.9 4.6 59 8.2 4.8 62 6.8 5.4 80 5.7 5.1 89
-
density
-A-PwR/IwR
0.6
0.4 0.2
20 30 40 g celluloselkg Glauber salt
Figure 1. Effect of cellulosc-salt ratio on sponge density.
I
0,05
0,l
0,16
0.2
0,25
0.3
Sponge density, glcrn'
Figure 2. Effect of sponge density on watcr retention.
Water absorption
Fig. 3 is showing the water absorption rate of the sponges made of the commercial sulphite pulp included in the study. It implies that the water is absorbed quickly in the beginning and achieves the equilibrium phase; the initial absorption rate indicates the absorptivity and the equilibrium level of absorption the ability to retain watcr. Both are
3 10 Wood, fibre and cellulosic matcrials important characteristics for porous materials. In comparison with the initial water retention values (IWR) given in Table 2 (for corresponding but not the same samples) the sponges revealed a water absorption not fully explicable by the parameters accepted, i.e. the cellulose-salt ratio and the compression.
]+Sample
0 0
60
120
180
10 240
I 301
The, I
Figure 3. Water absorption rate and maximum absorption in commercial pulp sponges. Sponge reinforcement
The commercial pulp sponges were reinforced with variable proportions of cotton fibres introduced in the viscose so as to strengthen the final sponge. The results are compiled in Table 3. It is evident that the sponge density was dependent on the proportion of cotton fibres charged from 20 to 60 %. The high charge resulted in about 0.07 gkm3 density, as the low charge gave only 0.05 g/cm3, which also provided the best water retention (IWR) whatsoever, about 25 g water/g dry sponge. - The orientation however did not change the sponge density with the same magnitude as did the cotton fibre charge, but slightly increasing though. Sponge compressibility
The samples 1-4 (Table 2) representing unreinforced sponges were testcd for compressibility and stiffness, see Table 4. There was a significant difference in specific between the sponges made of birch and straw pulps. The birch IDE compressibility (ap) pulp sponge (sample 1) showed a low stiffness, as the birch soda pulp sponge (sample 2) was much stiffer and simultaneously stronger. The corresponding straw pulp sponges (samples 3 and 4) had a higher rigidity and a yield point significantly dependent on the pulping process. The cotton reinforced sponges reported in Table 5 had a much higher spccific compression resistance, and in comparison with the sponges made of regenerated cellulose the elastic domain only was significantly larger.
Cellulose composite materials as sorbents 3 11 ose.
The rheological characteristics depended on the sample orientation towards the main surface of the sponge. The strength, stiffness and elastic behaviour of the sponge in the cross-cut direction suggestcd a prevalent orientation of the cavities perpendicularly to the main surface.
Sponge
a,,,
sample
m/m2
Stiffness, m/m3
kNlm2
no.
1 2 3 4
'
Yield Point aEL, oEL,
300 428 1097 1754
7.35 41.5 165 179
300 375 665 887
% asp 100 00 61 51
Post-Yield Point EEL,
aVL
%E,~
kNh2
100 48 23 36
418 89 1 1547
OVB
%asp
98 81 88
%E,~
90 72 85
3 12 Wood, fibre and cellulosic materials
Position
Stiffness,
a,,
GN/m3
in spongc MN/m2
cross-cut parallel-
length-
7.46 2.53 5.21
5.88 3.91 5.16
average , 4.79
,
Yield Point oEL,
GEL,
m/m2 2.97 1.7 1.19
4.61 , 2.10
%or,, 50 44 34 ,
43
,
Post-Yield Point EEL,
OVD
%ESP
mum2
15 31 17
4.44 2.97 3.12
76 76 59
54 77 45
23
3.41
71
62
OVE,
% osp
%ESP
Microscopic structure
Microscopic investigation of birch IDE pulp sponges revealed thin intercavity walls with fibrillar and porous structure which would maintain a good liquid penetration, Fig. 4. The birch soda pulp sponge again had bulkier and thus less fibrillar intercavity walls, Fig. 5 , which also was supported by the highcr specific compressibility and stiffncss. The straw pulp sponges made of IDE pulp, Fig. 6, and soda pulp, Fig. 7, compared in common structure with the respective birch pulps. The microscopic structure of the reinforced sponges made of a commercial softwood sulphite pulp with 20% cotton fibres charged for reinforcement, Figs. 8-10, clearly indicated a homogeneous, fibrillar structure of the intercavity walls, and with most cavities visible in the parallel section (Fig. 9, meaning that they were mainly oriented perpendicularlyto the main sponge surface).
CONCLUSIONS Cellulose sponges used in various medical and clinical products must be pure and strong enough and able to absorb sufficient amounts of water. However, to absorb water and to retain it even under certain pressure, the sponges must have cavities and porosities formed by the salt crystals having the right size distribution and being present in suitable proportions. Accordingly, the intercavity ccllulose walls are formed thin and fibrillar enough to let liquids penetrate sufficiently. All these properties require studies to clarify the sponge forming mechanisms. In this work, it was observed that the sponge density being the most important factor for the absorption properties evidently followed the cellulose-salt ratio, as the applied pressure with the aim to displace the air from the coagulating cellulose-salt conglomerate did not much affect the density. Another very important factor was the cotton fibre charge for reinforcement of the sponge, and it seemed that a charge of 20 % might be optimum. The new IDE pulping concept was tested, and birch and straw were cooked and semibleached (DED) and finally converted to viscose and sponges. The birch IDE pulp sponge had a low density and subsequently a high water absorption ability.
Cellulose composite materials as sorbents 3 13
Fig. 4. Birch IDE pulp sponge.
Fig. 5. Birch soda pulp sponge.
3 14 Wood, fibre and cellulosic materials
.
Fig. 6. Straw IDE pulp sponge.
Fig. 7. Straw soda pulp sponge.
Cellulose composite materials as sorbents 3 15
Fig. 8. Reinforced sponge; cross-cut section.
Fig. 9. Reinforced sponge; parallel scction.
3 16 Wood, fibre and cellulosic materials
Fig. 10. Reinforced sponge; length section.
REFERENCES 1. D.J. Mooney. D.F.Baldwin, N.P.Suh, J.P.Vacanti, R.Langer: “Novel approach to fabricate porous sponges of poly (D,L-lactic-co-glycolicacid) without the use of organic solvents”, Biomaterials, 1996 17 (14) 1417-1422. 2. E.P.Goldberg, Y.Yaacobi, J.W.Burns, M.Staples et al.: “Water soluble polymers for tissue protection during surgery”, Polymer Preprints, 1989 30 ( 2 ) 359. 3. S.Tasker, J.P.S.Badya1: “Influence of Cross-linking upon the macroscopic pore structure of cellulose”, J.Phys.Chem., 1994 98: 7599-7601. 4. O.Pajulo, B.Llinnberg, K.Lonnqvist, J.Viljanto: “Development of a high grade viscose cellulose sponge”, The XYVII Congress of the European Society for Surgical Research (ESSR), Turku-Finland, May 23-26, 1993. Abstract Book, P-156. 5. R.Langer, J.P.Vacanti: “Tissue engineering”, Science, 1993 260 920-926. 6. A.G.Mikos, A.J.Thorsen, L.A.Czerwonka et al.: “Preparation and characterization of poly(L-lactic acid) foams”, Polymer, 1994 35 1068-1077. 7. D.J.Mooney, S.Park, P.M.Kaufmann et al.: “Biodegradable sponges for hepatocyte transplantation”, J BiomedMater.Res., 1995 29 959-966. 8. A.G.Mikos, G.Sakarinos, M.D.Lyman et al.: “Prevascularization of porous biodegradable sponges”, Biotech.Bioeng,, 1993 42 716-723. 9. D.J.Mooney, P.M.Kaufmann, K.Sano et al.: “Transplantation of hepatocytes using porous, biodegradable sponges”, Transplant Proc., 1994 26 3425-3426. 10. L.Freed, G.Vunjak-Novokovic, R.Biron et al.: “Biodegradable polymer scaffolds for tissuc engineering”, Biotechnology , 1994 12 689-693.
Cellulose composite materials as sorbents 3 17 1 1. T.M.Tierney: “Control of bleeding after prostalectomy with special reference to use of oxidized regenerated cellulose”, J. of Urologv, 1964 91 (4) 400-401. 12. R.W.Post1ethwait:“Studies of new absorbable hemostatic material”, Bull Sot.
Internationale de Chirurgie, 1962 (3) 243-250. 13. J.R.Matthew, R.M.Browne, J.W.Frame, B.G.Millar: “Subperiosteal bchaviour of alginate and cellulose wound dressing materials”, Biomaterials, 1995 16 (4) 275-278. 14. F.A.L.Dulien: “Porous media: Fluid Transport and Pore Structure ”,Academic Press, New York, 1979. 15. L.Westman, T.Lindstr6m: “Swelling and mechanical properties of cellulose hydrogels. I. Preparation, characterization, and swelling behavior”, JAppI.PolymSci. 1981 26 (8) 25 19-2532. 16. T.Lindstrom, J.Tulonen, P.Kolseth: “Swelling and mechanical properties of cellulosc hydrogels. Part VI. Dynamic mechanical properties”, Holzforschung 1987 41 (4) 225230. 17. W.0ppemann: “Superabsorbierende Materialen auf Cellulosebasis”, Pupier 1995 49 (12) 765-769. 18. M.Rosdy, L.-C.Clauss: “Cytotoxicity testing of wound dressing using human keratinocytes in culture”, J.Biomed.Mater.Res.1990 24 363-377. 19. C.B.Park, D.F.Baldvin, N.P.Suh: “Ccll nucleation by rapid prcssure drop in continous processing of microccllularplastics”, Polymer Eng.Sci. 1995 35 432-440. 20. D.F.Baldvin, M.Shimbo, N.P.Suh: “The role of gas dissolution and induced crystallization during microcellular polymer processing: a study of poly(ethy1ene terephtalate) and carbon dioxide systems”, J.Eng.Mater.Tech. 1995 117 62. 21. A.Lebendiger, G.F.Gitlitz, E.S.Hunvitt, G.H.Lord, J.Henderson: “Laboratory and clinical evaluation of a new absorbablc hemostatic material prepared from oxidized regenerated cellulose”, Surg. Forum, 1959 10 440-443. 22. E.S.Hurwitt, J.Henderson, G.H.Lord, G.F.Gitlitz, A.Lebendiger: “A new surgical absorbable hemostatic agent”, American J. of Surgery, 1960 100 439-446. 23. A.Holst: “Quellflibige Cellulosederivate, deren Eigenshaften und Anwendungen”, Papier, 1978 32 (10 A) V7-Vl3 24. S.Ciovica, B.Ltinnberg, K.Ltinnqvist: “Dissolving pulp by the IDE concept”, Cellulose Chem. Technol., 1998 32 (3-4) 279-290. 25, K.Ebeling, K.Henricson, T.LaxCn, B.Lonnberg: Method of producing pulp, Patent Application, 1992. 26. M.Backman, B.Lonnberg, K.Ebeling, K.Henricson, T.LaxCn: “Impregnation Depolymerization- Extraction pulping”, Puperi j u Puu, 1994 76 (10) 644-648. 27. L.Robcrtsen, B.Lonnberg, K.Ebeling, K.Henricson, T.LaxCn: “IDE pulping. The impregnation stage”, Paperi j a Puu , 1996 78 (3) 96- 101 . 28. T.E.M.Hultholm, K.B.LBnnberg, K.Nylund, M.Finel1: “The IDE process: a new pulping concept for nonwood annual plants”, in: Proceedings of Pulping Corference, Chicago, Oct. 1-5, 1995, Book 1, TAPPI Press (1995): 85.
NEW CARBOHYDRATE POLYMER DERIVATIVES FROM RENEWABLE BIORESOURCES TARGETED FOR INDUSTRIAL APPLICATION Charles J. Knill, Sabinr F. Rahmrn & John F. Kennedy Birmingham Carbohydrate & Protein Technology Group (BCPl'G), Chernbiotech Laboratories, University ofBirmingham Research Park, Vincent Drive, Birmingham B15 2SQ, UK
ABSTRACT
The results of preliminary investigations into the derivatisation and characterisation of selected renewable carbohydrate polymers, via esterification using cyclic anhydride reagents under basic conditions, are presented. Carbohydrate substrates utilised for such investigations included glucose, maltose, cellobiose, maltodextrin, cotton cellulose and a range of starches of different botanical origin (namely wheat, potato, maize, waxy maize, sago and tapioca). Cyclic anhydride reagents used for esterification purposes included succinic, octenylsuccinic, dodecenylsuccinic, octadecenylsuccinic, maleic, citraconic (methylmaleic), 2,3-dimethylmaleic, and glutaric anhydrides. Triethylamine was utilised as the base and the resultant carbohydrate ester derivatives were isolated as the triethylamine salt of the corresponding carboxylate group liberated via ring-opening of the cyclic anhydride upon ester formation. Successful esterification was confirmed by FT-IR spectroscopic analysis via observation of an ester carbonyl absorbance in the 1750-1720 cm-' region, and carboxylate ion anti-symmetrical and symmetrical stretching signals in the 1610-1550 cm" and 1420-1300 cm-' regions of the spectra, respectively, such peaks being absent in the spectra of the corresponding underivatised substrates. 'H-Nh4R spectroscopic analysis was employed for the determination of average DS via elucidation of the ratio of the integration values for carbohydrate backbone (pyranose ring) protons and ester side chain protons, respectively. Preliminary results demonstrate that this simple room temperature reaction system can be utilised to produce multifunctional ester derivatives of varying DS from a wide variety of carbohydrate substrates. INTRODUCTION
Carbohydrate polymers (polysaccharides) represent a diverse group of renewable bioresources that provide a complex and essentially inexhaustible substrate library for chemical derivatisation. Modification of their physicochemical properties by chemical derivatisation is an important factor in their industrial production and application. Chemical modification of polysaccharides is based upon hydroxyl group chemistry and a modified polysaccharide can be defined as one whose hydroxyl groups have been altered by chemical reaction, e.g. by oxidation, alkylation, esterification, etherification or by cross-linking '. The most important chemical modifications are those of a 'nondegradative' type, where substitution of the free hydroxyl groups takes place. The physicochemical properties of such a modified polysaccharide are largely dependent upon the degree of substitution (DS), which is defined as the average number of substituted hydroxyl groups per anhydroglucoseunit.
'
320 Wood, fibre and cellulosic materials Such chemical modifications affect hydrogen bonding, charge interactions and hydrophobic character thereby altering the nature of the interactions between the polysaccharide chains. For the majority of industrial applications only a low DS value (< 0.2) is required to significantly change the properties of polysaccharides. In the case of amylose and cellulose (linear polysaccharides), there are three hydroxyl groups available for substitution, namely those attached to the C2, C3 and C6 carbon atoms, the C1 and C4 hydroxyl groups being involved in the glycosidic linkages that form the polysaccharide backbone. Thus, the maximum theoretical average DS value, and maximum DS value for a single anhydroglucose monomer unit, is three ’. Obviously, this value is reduced in the case of a branched polysaccharide (e.g. amylopectin), since a branched anhydroglucose unit has a maximum theoretical DS value of two, thus the higher the. degree of branching the lower the maximum theoretical average DS value. Polysaccharide esters are generally prepared in two ways. Aqueous reactions under controlled pH generally produce low DS value esters (< 2), whereas non-aqueous processes can produce higher DS value esters (up to -3). Over the years, considerable attention has focused upon the synthesis of polysaccharide derivatives with particular emphasis given to the investigation of starch and cellulose derivatives. Many diverse starch ester derivatives have found industrial application 3=1. The polysaccharide esters of greatest commercial value are those that provide enhanced hnctional properties compared with the native polysaccharides, such as improved water resistance, stability and improved film forming ability ’. Reactions of cellulose nearly always take place under heterogeneous conditions, i.e. solid cellulose is usually suspended in a liquid reaction medium. The cellulose itself is heterogeneous in nature as different parts of the fibrils have different accessibility to the same reagent. This can often lead to the formation of non-uniform products. There are a number of In this study the lithium solvent systems in which cellulose can be dissolved chloridddimethylacetamide (LiCVDMAC) solvent system was utilised.
’.
MATERIALS & METHODS The methodologies utilised to synthesise the carbohydrate ester derivatives were based upon those described by McCormick and Dawsey ’. Carbohydrates undergo a nucleophilic acyl substitution reaction with cyclic anhydrides in the presence of a base (triethylamine, TEA, in the case of the work presented) to produce ester derivatives that also contain carboxylate hnctionality (figure 1). Carbohydrate substrates
Glucose, maltose and cellobiose were purchased from Sigma-Aldrich Company Ltd., Poole, UK. Maltodextrin (from maize starch, DE 11-14) was supplied by Roquette, Greenwich, UK. Cotton cellulose was supplied by Bundesforschungsanstalt fir Forst und Holzwirtschaft (BFH), Hamburg, Germany. Wheat, potato, maize and waxy maize starches were supplied by Cargill PIC, Tilbury, UK. Sago and tapioca starches were supplied by CRAUN Research Sdn. Bhd. (CRSB), Kuching, Sarawak, Malaysia.
New carbohydrate polymer derivatives
carbohydrate
321
succinic anhydrlde
u+oh&CH2CH3)3
J ()-o/,AX0
carbohvdratesuccinate (trieihyiamine salt)
-0J
LO
Figure 1. Nucleophilic acyl substitution of carbohydrates using cyclic anhydrides. Preparation of solvent exchanged swollen cellulose
Cotton cellulose (20 g) was stirred overnight in deionised water (500 ml). The mixture was suction filtered to remove excess water. The cellulose was added to methanol (500 ml), stirred for 1 hour, then filtered gently under suction. This procedure was repeated four times using methanol (4 x 500 ml). The cellulose was then added to DMAC (500 ml) and stirred for 1 hour. The mixture was filtered gently under suction and the procedure repeated five times with DMAC (5 x 500 ml). The cellulose was purged with nitrogen overnight to produce solvent exchanged cellulose. Preparation of cellulose solution (in LiCVDMAC)
DMAC (1 L) was heated to 80°C using an oil bath. Anhydrous lithium chloride (LiCl, 84 g) was gradually added to the DMAC with continuous gentle stirring until complete dissolution was achieved to afford a 9% wlw solution. The solution was allowed to cool to room temperature prior to use. LiCVDMAC (9% wlw, 1 L) was added to solvent exchanged swollen cellulose with gentle swirling, the mixture purged with nitrogen, and left overnight in a nitrogen atmosphere. DMAC (500 ml) was added to the mixture and it was purged with nitrogen and regularly swirled over several days to produce a highly viscous opaque solution (with a cellulose concentration of- 13.3
a).
Synthesis of carbohydrate ester derivatives
Carbohydrate substrates (5 g) were dissolveddispersed in LiCVDMAC (100 ml) at 80°C (or 200 ml of celluloseLiCV DMAC solution was used). The specific cyclic anhydride (figure 2) was stirred into the mixture until it dissolved (1:l molar equivalent with available hydroxyl groups). Heating was stopped. At 50°C triethylamine (TEA) was gradually added with stirring. Almost immediately the product precipitated out. DMAC was then added to the reaction mixture, which was swirled and left so that the precipitate settled to the bottom of the reaction vessel. The solvent was decanted off and the product washed firther with tetrahydrofiran (THF) until all the solvent soluble impurities were removed by suction filtration. Derivatives wcre then oven dried (6OOC) to remove residual THF.
322 Wood, fibre and cellulosic materials
no o
0
succinic anhydride
a
oBo oEo
o
maleic anhydride
.
methyimaleic anhydride [CitraCOnlC anhydride1
2,3dimethylmaleic anhydride
\
Peten-1-yl succinic anhydride
0 A O J % Pdodecen-I -yl succinic anhydride
glutaric anhydride
(CH315( CH=CH)CHj
.
octadecenyl succinic anhydrlde (mixture of isomers)
Figure 2. Cyclic anhydride reagents utilised for carbohydrate derivatisation (all purchased from Sigma-Aldrich Company Ltd., Poole, UK). Fourier transform infrared (FT-IR) spectroscopic analysis
FT-IR spectroscopic analysis of the synthesised carbohydrate ester derivatives was performed using a Nicolet Avatar 360 FT-IR Spectrometer fitted with a Graseby Specac Golden Gate attenuated total reflectance (ATR) sampling accessory. The Golden Gate sampling accessory is composed of a small diamond embedded in a carborundum plate. A small portion of sample is layered onto the diamond and contact achieved using a sapphire anvil tightened to a predetermined torque value, which depends on the nature of the sample under investigation. All resulting FT-IR spectra were manipulated using Omnic software. Proton nuclear magnetic resonance ('11-NMR)spectroscopic analysis Carbohydrate ester derivativcs (30 mg) were dissolved in deuterated dimethylsulphoxide (d6-DMS0,l ml), and a few drops of trifluroacetic acid (TFA) added to each sample in order to move the signals from any remaining unsubstituted hydroxyl protons upfield to a higher ppm value (i.e. out of the range of interest), so that subsequent integration of the desired signals could be performed using WIN'NMR software to facilitate calculation of average DS values. 'H-NMR spectra of the synthesised carbohydrate ester derivatives were collected using a Brtiker AMX 400 M H Z NMR spectrometer. Samples soluble in d6-DMSO were run using an automated sampling system at room temperature. Other samples were run manually at 80°C in order to ensure complete solubilisation. Some samples were insoluble in available deuterated solvents and could not be analysed All of the chemical shifts are reported in parts per million (ppm) using tetramethylsilane(TMS) as an internal standard..
New carbohydrate polymer derivatives
323
RESULTS
FT-IRspectroscopic analysis Confirmation that esterification had been successfbl was obtained by comparison of the FT-IR spectra of the carbohydrate ester derivatives with those of the unmodified substrates. In all cases, the FT-JR spectra of the synthesised carbohydrate derivatives contained peaks that corresponded to a carboxylate carbonyl C-0 antisymmetrical stretch (- 1610-1550cm-I), a carboxylate carbonyl C-0 symmetrical stretch (- 14201300 cm-I), and an ester carbonyl C-0 stretch (- 1750-1720cm-'). The presence of these three peaks, along with a relative reduction in hydroxyl group peak (- 3500-3200 cm-') and increase in C-H stretching peaks (- 2800-3000 cm-') compared with the unmodified substrate spectra, that confirmed esterification had been successfid. Several bands corresponding to N-H stretches for the TEA salt could also be observed (- 2400-2600 cm-') and the carbohydrate ring ether C-0 stretching (- 11501070 cm-I). An example FT-IR spectrum (for the TEA salt of maltodextrin succinate) is displayed in figure 3. All peaks were identified from absorbence values quoted by Williams and Fleming lo.
---
maltodextrin
;
q
4000
,.,".....,-.
. ..,. ....-, .... .
3 W
succinate
~
, ..,,
3030
.
. ......,...... .. . ,..,..... 2500 MM) wawnunbso(m1)
triethylamine salt
-.--..-
.-..I--
1 W
I..,.. ~
.. *---, -.- ".. I
1WXl
I
Figure 3. FT-IR spectrum of maltodextrin succinate (triethylamine salt).
*
I
500
324 Wood, fibre and cellulosic materials 'H-NMR spectroscopic analysis The resultant 'H-NMR spectra of the carbohydrate ester derivatives were integrated using WINNMR software in order to calculate the average DS values from the ratio of the starch backbone proton signals to the ester substituent protons. The starch backbone protons generally occur as a complex multiplet in the region 3-6 ppm. The signal from any unsubstituted hydroxyl groups has been shifted upfield (> 9 ppm) out of the range of interest by the addition of TFA and is usually observed as a very broad low peak. Two example NMR spectra, for yellow maize starch succinate (TEA salt) and sago starch glutarate (TEA salt) are displayed in figures 4 and 5 , respectively. In the case of the succinate derivative (figure 4) the CH2 groups (a) are equivalent, both being adjacent to a carbonyl group, and hence appear as a single triplet (- 2.4 pprn). In the case of the glutarate derivative, the CH2 groups (b) are equivalent, both being adjacent to a carbonyl group, and hence appear as a single triplet (- 2.2-2.3), however the CH2 group (a) is not adjacent to a carbonyl group and therefore appears as a pentet at 1.7 ppm. Groups next to a carbonyl group are normally located at a higher ppm value because of the electron withdrawing influence of a carbonyl group. Average DS values (calculated from the NMR integration ratios of carbohydrate backbone protons to ester side chain protons) for selected carbohydrate ester derivatives are presented in table 1.
-
0
0
--II
II
().--O-C-CH2-CH2-C-O-
a
starch
a
succinate
+
HN(CH2CH3)3
b c
triethylamine salt
starch backbone protons
Figure 4.
1
H-NMR spectrum of yellow maize starch succinate (triethylamine salt).
New carbohydrate polymer derivatives
starch
glutarate
triethylamine salt
starch anomeric (Cl) protons
Figure 5.
1
Table 1.
Average DS values for selected carbohydrate ester derivatives.
H-NMR spectrum of sago starch glutarate (triethylamine salt).
Carbohydrate ester derivative
Maltose succinate Maltodextrin succinate Yellow maize starch succinate Waxy maize starch succinate Sago starch succinate Wheat starch succinate Tapioca starch succinate Sago starch maleate Yellow maize starch maleate Sago starch glutarate Yellow maize starch glutarate [all TEA salts]
Average DS 1.09 0.51
2.36 0.20 0.18 1.23 0.74 0.91 0.78 3 .OO 2.97
325
326 Wood, fibre and cellulosic materials CONCLUSIONS
Preliminary results demonstrate that this simple organic phase reaction system can be utilised on a homogeneous or heterogeneous basis to synthesise a variety of multifunctional carbohydrate ester derivatives with a wide range of DS values. Many of the carbohydrate esters produced could be analysed using 'H-NMR, however there were a few derivatives that could not be characterised using this technique as they were insoluble in available deuterated solvents. Further investigations are required to carry out NMR analyses using alternative solvents in which the carbohydrate esters are soluble. The starch or cellulose backbone of the derivatives could be partially hydrolysed using DCI (in deuterated DMSO) in order to make the sample more soluble. Ester derivatives prepared using cyclic anhydride derivatives have the added bonus of carboxylate functionality, and therefore their physicochemical properties can be modified by simply changing the counter ion. The derivatives presented in this work have all been isolated as the TEA salt, however, they could easily be converted to the free acid or sodium salt which would have a considerable effect on their solubility profiles. Selected derivatives will be assessed to determine suitability for specific industrial applications, e.g. as surfactants and their suitability as potential surface coating components. A more detailed paper covering other derivatives, oustanding characterisations and assessment of physicochemical properties (e.g. solubility profiles, film formation, etc) will be presentcd at a hture Cellucon conference. REFERENCES 1. G. Fleche, Chemical modification and degradation of starch, In: Starch Conversion
2.
3.
4. 5.
6. 7.
Technology, G. M. A. Van Beynum and J. A. Roels, J. A. (eds.), Marcel Dekker, New York, 1985, pp. 73-99. M. W. Rutenberg & D. Solarek Starch derivatives: production and uses, In: Starch: Chemistry and Technology, 2"dEd, R. L. Whistler, J. N. BeMiller & E. F. Paschal1 (eds.), Academic Press, Orlando, 1984, pp. 3 11-388. J. W. Mullen & E. Pascu, Starch studies: preparation and properties of starch triesters, Ind. Eng. Chem., 1942,2f?, 1209-1207. J. W. Mullen & E. Pascu, Starch studies: possible industrial utilisation of starch esters, Ind. Eng. Chem., 1943,s, 381-384. M. M. Tessler & R. L. Billmers, Preparation of starch esters, J. Environ. Polym. Deg., 1996,4, 85-89. I. A. Wolff, D. W. Olds & G. E. Hilbert, Triesters of corn starch, amylose and amylopectin: properties, Ind. Eng. Chem., 1951,a, 91 1-914. I. A. Wolff, D. W. Olds & G. E. Hilbert, Mixed esters of amylose, Ind. Eng. Chem., 1957,48,1247-1248.
T. P. Nevell & S . H. Zeronian, Cellulose Chemistiy and its Applications, Ellis Horwood, Chichester, 1985. 9. C. L. McCormick & T. R. Dawsey, Preparation of cellulose derivatives via ringopening reactions with cyclic reagents in lithium chloride/ N,N-dimethylacetamide, Macromolecules, 1990,2,3606-3610. 10. D. H. Williams & I. Fleming, SpectroscopicMethods in Organic Chemistry, 5IhEd., McGraw-Hill, London, 1995. 8.
THERMAL AND VISCOELASTIC PROPERTIES OF CELLULOSE- AND LIGNIN-BASED POLYCAPROLACTONES H Hahkeyama', T Yoshida', S Hirose' and T Ihtakeyama' I Fukui
Universiv of Technology,3-6-1 Cakuen, Fukui, Fukui 910-8505,Japan. 'National Insrirure ofMaterials and Chenrical Research, 1-1 IIigashi Tsukuba, Ibaraki 305-8565 Japan.
'Otsuma Women's Universiv, 12 Sanbancho, Chiyoda-ku, Tokyo 102- 8357, Japan ABSTRACT Polycaprolactone (PCL) derivatives were newly synthesized from cellulose acetate (CA) and lignins such as alcoholysis lignin (AL) and &aft lignin (KL). Thermal and viscoelastic properties of the obtained polycaprolactoncs (CA-, AL and KLPCL's) were studied. PCL's were synthesized by the polymerization of E-caprolactone (E-CL) which was initiated by each of the OH groups of cellulose and lignin molecules. The amount of E-CL was varied from 1 to 25 mols / each OH group. A marked change in bascline due to glass transition was obscrvcd in each DSC curve. Tg decreases with increasing CUOH ratio in PCL's, since caprolactonc chains act as soft segments in PCL molecular chains. CA-PCL showed a-,fl-and y-dispersions in dynamic mcchanical analysis. Thermal degradation temperatures (Td's) of PCL's increascd with increasing CL / OH ratio. From this rcsult, it is considered that the thermal degradation of CA-, A L and KLPCLYs with increased PCL chain length occurs with more difficulty than in plant cornponcnts such cellulose and lignin.
INTRODUCTION Since natural polymers are biodegradable and can be circulated in the ecological system, various research groups '-I3) have tried extensively to synthesize polymers which can be derived from plant components such as saccharides and lignin. We have synthesized various types of polyurethancs (PU's) which have plant components in the PU molecular chains. We have also found that their mechanical and thermal properties can be controlled by appropriate molecular design. The PU's were biodegraded by microorganisms when they were placed in soil .?'In our rcccnt study, ") polycaprolactone (PCL) derivatives were synthesized from saccharides such as glucose, fructose and sucrose. Polyurethane sheets were also prepared from the above PCL derivativcs by the reaction with diphenylmcthanc diisocyanate (MDI). In the present study, polycaprolactone (PCL) derivatives wcrc synthcsized from cellulose acctate (CA) and lignins such as alcoholysis lignin (AL) and Kraft lignin (KL). Molecular properties of the obtained polycaprolactoncs (PCL's) were studied by differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), thcrrnogravimetry (TG) and TGFourier transform infrared spectromcty (FTIR).
328
Wood, fibre and cellulosic materials
EXPERIMENTAL Materials and sample preparation AL and KL were kindly provided by Repap Co. and Westvaco Co. The provided lignins were used without further purification. Cellulose acetate ( C A acetyl content, 39.87 %; M, = 6.32~10~; M A , ,= 2.27), E-caprolactone (E-CL) and dibutyltin dilaurate (DBTDL) were commercially obtained. PCL's were synthesized by the polymerization of E-CL which was initiated by each of the OH groups of cellulose and lignin molecules. The amount of E-CL was varied from 2 to 20 mol / each OH group in the case of CA-PCL and from 1 to 25 mol / each OH group in the case of AL-PCL and JLPCL. The polymerization was carried out for 12 hr at 150 "C with the presence of a small amount of dibutyltin dilaurate (DBTDL).
Measurements Differential scanning calorimetry (DSC) was performed using a Seiko DSC 220 at a heating rate of 10 "C / min under a nitrogen flow (flow rate = 30 ml/min). Sample mass was ca. 5mg. Aluminum open pans were used. The samples were heated to 120 "C and quenched to -150 "C. DSC heating curves were usually measured at 10 "C/min. However, when the coordination with the heating rate of DMA is needed, DSC heating curves were measured at 2 "C/min. The melting temperature (Tm),melting enthalpy (M,,,), cold crystallization temperature (T=),glass transition temperature (Ta and heat capacity gap (AC,,) were determine by the method reported previously "). Dynamic mechanical properties of PCL's derived from C A (CA-PCL's) were measured using a Seiko dynamic mechanical analyzer DMS 210 equipped with a stretching module. Sample size as follows: width 8 mm, length 20 m m and thickness 0.2 0.7 mm. Temperature was varied from -150 to 100 - 150 "C. The maximum temperature was chosen dcpending on rigidity of each sample. The heating rate was 2 "C/min. Frequency was varied at 0.1, 1.0, 2.0,3.0, and 5.0 Hz, respectively. Dynamic modulus (E'), dynamic loss (E") and tan S were calculated using the equipped software. Activation energy of each dispersion was calculated using the equipped software and/or manual calculation. Thermogravimetry (TG) was carried out in nitrogen flow (flow rate = 200 ml/min) using a Seiko TG 220 at a heating rate of 20 "C / min in the temperature range Gom 20 to 800 "C. Sample mass was ca. 5mg. TG curves and derivatograms were recorded. Mass loss was calculated according to the equation: [(m,/mm)/m20]x 100 (%) where m, is mass at temperature T and mm is mass at 20 "C. Gasses evolved by thermal degradation were analyzed by TG-FTIR using simultaneously a Seiko TG 220 and a JASCO FTnR-420. The heating rate was 20 "C/min in the temperature range from 40 to 800 "C. The flow rate of carricr nitrogen gas was 100 ml/min and the sample weight was 7 to 10 mg. The gas transfer system was maintained at 270 "C. The resolution power of FI'IR was 4 cm-', The number of integration was ten and the data incorporation time was 30 s.
-
Thermal and viscoelastic properties of polycaprolactones 329 RESULTS AND DISCUSSION Figs. 1 shows the phase diagram of CA-PCL's with various CUOH ratios from 2 to 20. As shown in Fig. 1, the glass transition temperature (Tg ) of CA part (Tg1 )in CA-PCL is observed in the initial stage. The Tg of CA observed in this study is 147 "C. It bccomes difficult to detect when CUOH ratio exceeds 15 mol/mol. Tg of PCL part (Tg2) in CA-PCL decreases with increasing CUOH ratio when that in CA-PCL's is below 10, since PCL chains act as soft segments in CAPCL. This softening effect of caprolactone chains was enhanced progressively with increasing chain length of CL chains when CUOH ratio is below 10. However, as also shown in Fig. 1, in the case of CA-PCL's with CWOH ratio over 8, melting of crystalline region is observed. This suggests that CAPCL's with long PCL chains (CUOH ratio is over 8) becomes rigid, since the long PCL chains take regular arrangement, form crystalline structure and restrict the main chain motion. This causes the increase of Tg of CA-PCL(Tg2), when CUOH ratio is over 10. The melting temperature (Tm) of CA-PCL increases with increasing CUOH ration when it is over 8. Fig. 2 shows the phase diagram of A L and KLPCL's with various CUOH ratios from 1 to 25. As shown in Fig. 1, Tg of A L and KLPCL's decreases with increasing CUOH ratio when CUOH ratio is below 10. This softening effect of caprolactone chains was enhanced progressively with increasing chain length of CL chains when CWOH ratio is below 10. However, Tg increases with increasing CUOH ratio when CUOH ratio becomes over 10. Melting of crystalline region of A L and KLPCL's is observed when CWOH ratio is over 2. The above results suggest that the incorporation of PCL chains into cellulose and lignin structures leads to the enhancement of molecular motion of PCL derivatives when the chain length of PCL is below a certain critical value which is specific to cellulose or lignin core structure. However, when the PCL chain becomes over a certain length, a prominent peak of melting of crystals is observed at a specific temperature which is characteristic to each core plant component such cellulose and lignin, respectively in the DSC curve, and the Tm increases with increasing PCL molecular chain. The above facts suggest that the long PCL chain easily takes regular molccular arrangements and form crystalline region. Fig. 3 shows DMA heating curves of CA-PCL with CUOH ratio of 5 measured various frequencies. The dynamic modules E' and tan 6 are clearly observed. From the high to low temperature side, each E' decrease is designated as a-dispcrsion and fl-dispersion, respectivcly. Tan 6 shows a large and broad peak at around -20 "C and a small and broad peak bclow -100 "C. Fig. 4 shows the relationship between frequency and reciprocal temperature corresponding to a-, fl- and y-dispersions. All CA-PCL samples show a-dispersions at almost the same temperature region, but the temperature region of fl-dispersion depends on each sample. The y-dispersion is observed as a small peak at around -120 when CWOH ratio is 5. Fig. 5 shows the relationship between CUOH ratio, thermal degradation tempcrature (Td) and mass residue (WR) of CA-PCL. The Td of CAPCL increased from ca. 350 "C to 390 "C
330 Wood, fibre and cellulosic materials
0
80
-
40
-
0
I-'
0 -
5
0
.15
10
20
25
C V O H Ratio/(mol/mol)
Figure 1. Phase diagram for CA-PCL A; T g 1 , A ; T g 2 , 0 ; Tm
-10
' 0
5
10 '
15
20
25
CL / OH Ratio/(mol/mol)
Figure 2. Phase diagram for A L and KLPCL's 0 ; KLPCL Tgl B; KL-PCL Tm 0 ;ALPCL Tgl 0;AL-PCL T m
30
Thermal and viscoelastic properties of polycaprolactones 33 1 1. ox 10'
0. 40
I . ox 10'
0. 30
. I
,"
'0
1.0XlO'
4
0.20
1.OXlO'
9
0. t o
1. ox 10' -110.0
0. 00
50. 0
-10.0
110.0
Figure 3. E" and tan 6 for CA-PCL (CUOH ratio = 5 moYmol) Numerals in the figure show frequency
1.5
-2
1
0.5
v
'Mz o 0
-0.5
-1 -1.5 2
3
4
5
6.
7
1000/T / K-'
Figure 4. Arrhenius plots for CA-PCL,(CUOH ratio = 5 moYmol)
332 Wood, fibre and cellulosic materials 400
20
380 15
360
;F
10
340 320
0
300
I
I
0
5
I
-
5
I
10 15 20 C Y O H Ratio/ (mol/m ol)
' 2"
0
25
Figure 5. Change of T,and WR with CUOH ratio for CA-PCL ;T d
0;W R
with increasing CUOH ratio. From this result, it is considered that the thermal degradation of CAPCL with increased chain length seems to occur with more difficulty, since the ratio of thermally unstable cellulose structure in CAPCL decreased comparatively with increasing CUOH ratio. The WR at 450 "C decreases with increasing CUOH ratio, suggesting that the CA part in CAPCL constitutes a significant part of the residual products. Fig.6 shows the relationship between CUOH ratio, thermal degradation temperature (Td ) and mass residue (WR) of A L and KLPCL. The Td of CAPCL increased with increasing CUOH ratio. This suggests that the thermal degradation of A L and KL-PCL's seems to occur with more difficulty with increased chain length, since the ratio of thermally unstable lignin structure in lignin PCL derivatives decreased comparatively with increasing CUOH ratio. The WR at 450 "C decreases with increasing CUOH ratio, suggesting that the lignin part constitutes a significant part of the residual products of A L and K L PC L Fig. 7 shows the stacked FTIR spectra of gases evolved at various temperatures during the thermal degradation of AL-PCL (CIJOH ratio = 20). The main peaks observed for the samples are as follows: wavenumber assignment; 1126 cm-' (vC-0-), 1260 cm" (v-C(=o)C), 1517 and 1617 cm" (vC=C), 1718 cm.' (vC=O), 2345 cm-' (vCO,), 2892 cm" (vC-H) and 3700 cm'l (vH,O).
Thermal and viscoelastic properties of polycaprolactones 333 400
1
390
-
380
-
,o \
370
-
360
-
I2
350
-
I 40 40
A
--
p
-
340
30 a? \
20 a 5 10
0 0 0
5
1155 20 CL / OH Ratio/(mol/mol) 10
25
30
Figure 6. Change of Tdand WR with CUOH ratio for AL and KLPCL's A; KLPCL TJT, A; ALPCL TfC, ;KLPCL WW%, 0;ALPCL WR/%
Figure 7. TG-FTIR stacked curves for ALPCL
334 Wood, fibre and cellulosic materials
0.1 u)
m Q
0.05
0 0
20
10
30
CL / OH Ratio/(mol/mol)
Figure 8. Relationship between IR absorption intensity for AL-PCL and CUOH ratio OiC-O-C, O;C=O, O;CO,, A;CH
0.15
0.1 u)
A-
m Q
0.05
-
-+%+
A
v
0
+
A-
30
20 C V O H Ratio/(mol/mol)
0
10
Figure 9. Relationship between IR absorption intensity for KLPCL and CUOH ratio
0 ;C-0-C,
M; C=O,
+; CO,,
A;CH
Thermal and viscoelastic properties of polycaprolactones 335 Figs.8 and 9 show the changes of intensities of characteristic IR absorption peaks of evolved gases from AL- and KL-PCL‘s. The changes of IR absorption intensities for A L and KLPCL’s are almost similar. The evolution of CO, gas is prominent, as shown in Fig. 8. However, the IR absorption intensity of CO, gas from lignin based PCL’s do not show the PCL chain length dependency. This suggests that the cvolution of CO, gas occurs randomly and is not specific to the chemical structure. On the other hand, the IR absorption intensities of C-OX, C=O and CH increase prominenlly with increasing CL/OH ratio. This suggests that gascs having C-0-C, C=O and CH groups are evolved from PCL chains of lignin-based PCL’s. The above facts well accord with the decrease of the mass residue, WR, with increasing PCL chain length.
CONCLUSIONS The decrease of the Tg of cellulose- and lignin-based PCL’s with increasing CWOH ratio is caused by the fact that the PCL chains act as soft segments. When thc PCL chain becomes over a certain length, PCL chains take regular arrangements and crystallization occurs. Melting of crystalline structure occurs at a specific temperature which is characteristic to each core plant component such as cellulose and lignin. Since thermal degradation temperatures (Td’s) of PCL derivatives increased with increasing C L / OH ratio, it i s considered that the thermal dcgradation of CA-, AL- and KLPCL’s with increased chain length seems to occur with more difficulty than in plant components such as cellulose and lignin. The results obtained by TG-FTIR analysis of CA-PCL showed that gases with OH, CH, G O ,C-0-C groups were mainly evolved by thermal degradation.
REFERENCES
1. V.P. Saraf and W.G. Classer, J. Appl. Polym. Sci., 1984,29,1831. 2. V.P. Saraf and W.G. Glasscr, J. Appl. Polym. Sci., 1984,30,2207. 3. K. Nakamura, R. Morck, A. Reimann, K.P. Kringstad and H. Hatakeyama, Polym. Adv. Technol., 1991,2,41. 4. K. Nakamura, T. Hatakeyama and H. Hatakeyama, Polym. Adv. Technol., 1992,3,151. 5. H.Yoshida, R. Morck, K.P. Kringstad and H. Hatakeyama, J. Appl. Polym. Sci., 1990, 40, 1819. 6. K. Nakamura, Y. Nishimura, T. Hatakeyama and H. Hatakeyama., Preparation of Biodegradable Polyurethanes Derived from Coffee Grounds, in Proceedings for International Workshop on Environmentally Compatible Matcrials and Recycling Technology in Tsukuba, Japan, Novembcr 15-16,1993, pp. 239 7. H. Hatakeyama, S. Hirose, K. Nakamura and T. Hatakeyama, New types of Polyurethanes Derived from Lignocellulose and Saccharides, in Cellulosics: Chemical, Biochemical and Material Aspects, J . F. Kennedy, G. 0. Phillips and P. A. Williams, Eds., Ellis Horwood, 1993, pp. 381. 8. H. Yoshida, K. Kobashigawa, S. Hirose and H. Hatakeyama, Molecular Motion of Biodegradable Polyurethanes Derived from Molasses, in Proceedings for International Workshop on Environmentally Compatible Materials and Recycling Tcchnology in Tsukuba, Japan, Novembcr, 1993, pp. 15.
336 Wood, fibre and cellulosic materials 9. N. Morohoshi, S . Hirose, H. Hatakeyama, T. Tokashiki and K. Teruya, Sen-i Gakkaishi, 1995,51,143. 10. T. Nakamura, Y. Nishimura, P. Zctterlund, T. Hatakeyama and H. Hatakeyama, Thermochimica A m , 1996, 2821283,433. 11. P. Zetterlund, S. Hirose, T. Hatakeyama, H. Hatakeyama and A-C. Albertsson, Polymer International, 1997,42, 1. 12. M. J. Donnely, Polymer Internalional, 1992,37,297. 13. H. Hatakeyama, K. Kobashigawa, S . Hirose and T. Hatakeyama, Macromol. Symp., 1998,130,127. 14.T. Hatakeyama and F. X. Quinn, Thermal Analysis, John Wiley & Sons, Chichester 1994, pp.65.
INDEX absorption, of water 305-17 Acetobacter xylinurn 3-12,23, 122 acid group determination 111-19 acid-base characteristics, of fibres 188 adhesive bridges to wood 291-303 aerobic incubator 4-10 aging, of fibre surfaces 197-203 alditol acetates 227 algal cellulose 34-8 alkaline pulping 91-4 anion exchange 26 anthraquinone 91-102 application, of polymer derivatives 31926 bacterial cellulose 3-16 bacterial endoglucanases 81-86 beating, of fibres 249-60 beating, pulp fibre 159-65 biobleaching 56 bioresources 319-26 black liquor 149-57 blackening, of fibres 235-45 bleaching 149-57 bleaching, enzymic 55-60 bleaching, oxygen 95-102 bleaching, ozone 137-47 caprolactones 327-36 carbohydrate, oil palm trunk 227-34 carbohydrates, wood 91-102, 126 carboxyl groups 129-35 catalysis, of delignification 103-7 cellobiose 93 cellulase activity 71 cellulases, in pulp processing 69-80 cellulose acetate 327-36 cellulose binding domains 72 cellulose II, supramolecular structure 33-8 cellulose polymorphs 121-7 cellulose, algal 34-8 cellulose, bacterial 3-12, 36, 37
cellulose, composite material 305-17 cellulose, crystallinity of 39-44 cellulose, from oil palm waste 13-17 cellulose, sago 19-22 cellulose, specific mass 39-44 cellulose-based polycaprolactones 327-36 cellulosic filament 3-12 carboxymethyl cellulose 81-6 charged groups 109-19 chemical derivatives, of polymers 31926 chemical pulp 75 chemistry, of adhesive bridges 291-303 chemometrics 33-8 chlorine dioxide 149-57 Cladophora 33 composite, sorbants 305-17 13CP-CP/MAS-NMR33-8, 39-44, 121-7 crystallinity, of cellulose 39-44 cultivation, of Acetobacterxylinum 3-12 curl index 141 degree of polymerisation 92 de-inking 76 delignification 91, 103-7 derivatives, of polymers 319-26 dielectric constant, of paper 267-75 distribution coefficients 100 dry fractionation, of fibres 261-66 drying stress, of paper 255 elastic modulus, paper 249-60 elastic properties, of fibre 267-75 electrical resistance 272 endoglucanases 81-6 enzyme assay 62 enzymes, in pulp processing 69-80 enzymic bleaching 55-60 enzymic hydrolysis 27, 30
338
Index
eucalypt fibres 181-96 fibre blackening 235-45 fibre length 142 fibre structure 209-25 fibre surfaces 197-203 fibre, flax structure 169-79 fibre, hardwood 181-96 fibre, in paper 235-45, 249-60 fibre, softwood 209-25 fibres properties 137-47 fibres, beating 159-65 fibres, dry fractionation of 261-6 fibres, from palm trunk 227-34 fibres, viscose-Kraft mixtures 267-75 filament, cellulosic 3-12 flax, fibre structure 169-79 fluorescence, of fibre surfaces 197-203 fractionation, dry 261-6 FTIR, of flax fibre 169-79 fungal endoglucanases 81-6 GC analysis 27 glucomannans, in paper 277-88 grass, fibre 261-6
handsheets, of viscose-Kraft mixtures 267-75 hardwood fibres 181-96 heat, effect on fibres 197-203 HPLC 25 industrial biopolymers 319-26 kink index 141 Kraft fibre, in paper 249-60 Kraft lignin, derivatives, 327-36 Kraft pulp 277-88 Kraft, surface energy 169-79 Kraft, viscose mixture fibres 267-75 light microscopy, of pulp fibres 205-8 lignin, of hardwood 185 lignin-based polycaprolactones327-36 mechanical pulping 73
mechanical pulps 109-19 mercerisation 33 metabolism, in pulping 95-102 methylate 26 microcrystalline cellulose 13-17, 19-22 molasses 23-31 monosaccharide analysis 25 morphology, of fibre surfaces 197-203 morphology, of flax fibre 169-79
NMR 39-44 NMR, of flax fibre 169-79 odour transfer 152 oil palm wastes 13-17 oil palm, trunk fibres 227-34 oligosaccharide analysis 25 optical properties, of fibre surfaces 197-203 organic solvents 91-4 organosolve pulping 91, 103-7 oxygen bleaching 95-102 oxygen-acetone delignification 103-7 ozone 149 ozone bleaching 137-47 palm trunk, fibres 227-34 paper density 267-75 paper industry 61-68 paper properties 144 paper, fibres 249-60 paper, from grass, 261-6 paper, Kraft fibres in 249-60 paper, processing 69-80 paper, structure 235-45 paper, wood resin in 277-88 paper, strength 277-88 papermakingfibres 109-19 papers, supercalendered 235-45 pectic acids, in paper 277-88 peroxyacetic acid 149-57 phenol groups 129-35 polycaprolactones327-36 polymorphism, cellulose 121-7 polysaccharides, in paper 277-88 pores, in flax fibre 169-79
Index 339 porous cellulose composites 305-17 protein assay 62 pulp fibre 159-65 pulp industry 61-68 pulp, cellulases in 69-80 pulp, for reinforcement 209-25 pulp, from grass, 261-66 pulp processing 69-80 pulp, sponges from 305-17 pulp, surface energy 169-79 pulp, wood 55-60 pulping conditions 261-66 pulping, alkali 91-102 pulping, effect on crystallinity 39-44 pulps, Kraft 129-35, 137-47, 150-7, 163-5 pulps, polymorphs in 121-7 pulps, woods and mechanical 109-19 reed canary grass 261-6 reflectance, of fibre surfaces, 197-203 reinforcement pulp 209-25 renewable bioresources 319-26 resin, wood 277-88 rheology of wood 291-303 rheology, of cellulose composites 30517 rupture surfaces, in wood 291-303 sago cellulose 19-22 scanning electron microscopy 7, 9 SEM, of flax fibre 169-79 SEM, of wood rupture 291-303 shrinkage, of paper 255 size-exclusion chromatography 55-60 softwood fibre, strength 209-25 softwood Kraft pulp 249-60 solubility parameter 48 specific mass, of cellulose 45-51 specific viscosity 85, 86 sponges, cellulosic 305-17 strength, of fibres 205-8 structure, in paper 235-45 structure, of cellulose II 33-8 structure, of fibre 209-25 structure, of pulp 305-17
sugar cane molasses 23-31 supercalendered papers 235-45 supramolecular structure 33-38 supramolecular structure, of flax 16979 surface chemistry, of hardwood fibres 181-96 surface energy, of hardwood fibres 169-79 surface extractives, pulp 185 surfaces, in wood 291-303 swelling, of fibres 205-8 swelling , of pulp fibres 205-8 tear index 145 tensile index, of paper 277-88 tensile strength 144 thermal properties 197-203, 327-36 thermomechanical pulp 115,277-88 thermophilic endoglucanases 81-6 thermostable xylanases 61-8 two-phase equilibria 95-102 UV-VIS reflectance, of fibre surfaces 197-203 viscoelectric properties 327-36 viscometric measurement 3, 6 viscose-Kraft fibre, mixtures 267-75 viscosity, specific 85,86 water absorption, of fibre 267-75 WAXS, of flax fibre 169-79 wet pressing of fibres 249-60 wide angle x-ray diffraction 3, 5 wood carbohydrates 91-102 wood pulps 55-60, 109-19 wood resin 279-88 wood, rupture surfaces 291-303
XPS, of hardwood fibres 181-96 xylan 61-8 xylanase 58, 61-8 zero-span index 142, 143 Zoogloea sp. 23-31
CELLULOSIC PULPS, FIBRES AND MATERIALS
CELLULOSIC PULPS, FIBRES AND MATERIALS Editors: JOHN F KENNEDY Director of the Birmingham Carbohydrate and Protein Technology Group, School of Chemistry, The University of Birmingham, and Chembiotech Laboratories, The University of Birmingham Research Park, Birmingham, UK and Professor of Applied Chemistry, The North East Wales Institute of Higher Education, Wrexham, Clwyd, Wales, UK
GLYN 0 PHILLIPS Chairman of Research Transfer Ltd Professorial Fellow and formerly Executive Principal of the North East Wales Institute of Higher Education, Wrexham, Clwyd, Wales, UK Formerly Professor of Chemistry, The University of Salford, UK PETER A WILLIAMS Head of the Multidisciplinary Research and Innovation Centre, and The Centre of Expertise in Water Soluble Polymers, and Professor of Polymer and Colloid Chemistry, The North East Wales Institute of Higher Education, Wrexham, Clwyd, Wales, UK Guest Editor: BRUNO LONNBERG Professor of Pulping Technology, and Head of Laboratory of Pulping Technology, Faculty of Chemical Engineering, Abo Akademi University, AbolTurku, Finland
W O O D H E A D P U B L I S H I N G LIMITED
Published by Woodhead Publishing Ltd Abington Hall, Abington, Cambridge CB 1 6AH, England www.woodhead-publishing.com First published 2000 0 2000, Woodhead Publishing Ltd The authors have asserted their moral rights
Conditions of sale All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. While a great deal of carc has been taken to provide accurate and current information, neither the authors, nor the publisher, nor anyone else associated with this publication shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. British Library Cataloguing in Publication Data A cataloguc record for this book is available from the British Library.
ISBN 1 85573 421 4
Printed in Great Britain by Antony Rowe Ltd, Chippcnham, Wiltshire
Contents xi
Preface PART 1: NEW SOURCES, STRUCTUREAND PROPERTIES OF CELLULOSE . 1. Continuous harvest of cellulosic filament during cultivation of Acetobacter Xylinum . S Tokura, H Tamura, M Takai, T Higuchi and H Asano Oil palm (EZueis guineensis) wastes as a potential source of cellulose 2. M A M Noor and H Sarip Isolation and characterisation of sago (Metroxylon Sugu) cellulose . 3. A Mohd Zahid, M D Modh Zulkali and B M N Azemi A highly cellulosic exopolysaccharideproduced from sugarcane 4. molasses by B Zoogloea sp . M Paterson-Beedle. L L Lloyd, J F Kennedy, F A D Melo and V Medeiros The supramolecular structure of cellulose 11. Studies with 5. 13C-CP/MAS-NMRand chemometrics H Lennholm Effects of pulping on crystallinity of cellulose studied by solid state 6. NMR T Liitia, S L Maunu and B Hortling 7. On the specific mass of cellulose and the cellulose-water system . J Chirkova, B Andersons and I Andersone
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PART 2: APPLICATION OF ENZYMES TO PULP, FIBRJ3S AND CELLULOSE . 8. Application of size-exclusion chromatography to enzymatic bleaching of wood pulp . T Eremeeva, M Leite, T Bykova, A Treimanis and U Viesturs Thermostable xylanases and their potential application in paper 9. and pulp industries . M K Bhat, S Kalogiannis, N A Bennctt. P Biely, D E Beevcr and E Owen 10. Cellulases in pulp and paper processing L Viikari, T Oksanen. A Suurnakki, J Buchert and J Pere 11. Mode of action of thermophilic bacterial and fungal endoglucanases on carboxymethyl celluloses . M K Bhat, S Bhat. N J Parry, J F Kennedy, C J Knill, D E Beever and E Owen
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vi
Contents
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PART 3: PULP PRODUCTION AND PROCESSING. 12. The effect of anthraquinone on wood carbohydrates during . alkaline pulping in aqueous organic solvents M F Kiryushina, M I Ermakova, A S Olefirenko, E-M Bennacer, T G Fedulina, A B Nikandrov and M Ya Zarubin 13. Two phase equilibria of metal ions in pulping unit operations: . from impregnation to oxygen bleaching J Karhu, P Snickars, L Harju and A Ivaska 14. Catalysis of oxygen-acetone delignification I Deineko and I Deineko 15. Charged groups in wood and mechanical pulps . B Holmbom, A V Pranovich, A Sundberg and J Buchert 16. The investigation of cellulose polymorphs in different pulps using I3C CPMAS NMR S Maunu, T Liitia, S Kauliorniiki, B Hortling and J Sundquist 17. Characterization of carboxyl and phenol groups in kraft pulps at different temperatures . J Karhu, P Forslund, L Harju and A Ivaska 18. Effect of ozone bleaching on the fibre properties of pine and birch kraft pulp A Seisto, K Poppius-Levlin and A Fuhrmann 19. Studies on the use of black liquor evaporation condensates a t different bleaching stages . K Niemela, R Saunamaki and R Rasimus 20. Evaluation of pulp fibre beating B Lonnberg, T Lundin, K Harju and P Soini
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PART 4: STRUCTURE AND PROPERTIES OF FIBRES . 21. Study of flax fibre structure by WAXS, IR and "C NMR spectroscopy, and SEM N E Kotelnikova, E F Panarin, R Serimaa, T Paakkari, T E Sukhanova and A V Gribanov 22. Evaluating the surface energy of hardwood fibres using the Wilhelmy and inverse gas chromatography methods . W Shen, Y J Sheng and I H Parker 23. Heat-induced changes in fibre surfaces I Forsskihl, T Korhonen and H Tylli 24. Investigation of spruce pulp fibres by swelling experiments and light-microscopy . B Hortling, T Jousimaa and H-K Hyvarinen 25. Role of softwood fibre form and condition on its reinforcement capability . K Ebeling 26. Compositional analysis of oil palm trunk fibres . P F Akmar, M N M Yusoff, J F Kennedy and C J Knill . 27. Fibre blackening in supercalendered papers T Koskinen
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. 205 . 209
. 227 .
235
Contents vii PART 5: PAPER FIBRE PRODUCTION AND PROPERTIES 28. Kraft fibers in paper effect of beating . K Niskanen 29. Effect of dry fractionation on pulping conditions and fibre propertics of reed canary grass M Finell, B Hedman and C-A Nilsson 30. Electrical propertics of viscose-kraft fibre mixtures . S Simula and K Niskanen 31. Effects of retained wood resin and polysaccharidcs on . paper properties A Sundberg. B Holmbom, S Willfor and A Pranovich
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PART 6: WOOD, FIBRE AND CELLULOSIC MATERIALS 32. Appearance of rupture surfaces in wood . K NygHrd, R Gyllenberg, B Lonnberg and G Gros 33. Composite materials from pulp and papermaking wastes V Lapsa, T Betkers and G Shulga 34. Cellulose composite materials as sorbents sorption and . rheological properties S Ciovica, B LZjnnberg and K Lonnqvist 35. New carbohydrate polymer derivatives from renewable bioresources targeted for industrial application . C J h i l l , S F Rahman and J F Kenncdy 36. Thermal and viscoelastic properties of cellulose- and lignin-based polycaprolactones H Hatakeyama, T Yoshida, S Hirose and T Hatakeyama
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Index.
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. 267 . 277 . 289 ,
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MJ
THE CELLUCON TBUST incorporating
CELLUCON CONFERENCES International Educational Scientific Meetings on Cellulose, Cellulosics and Wood
Cellucon Conferences as an organisation was initiated in 1982, and Cellucon '84, which was the original conference, set out to establish the strength of British expertise in the field of cellulose and its derivatives. This laid the foundation for subsequent conferences in Wales (1 986), Japan (1 988), Wales (1 989), Czechoslovakia (1 990)' USA (1991), Wales (1992), Sweden (1993)' Wales (1994), Finland (1998), and Japan (1999). They have had truly international audiences drawn from the major industries involved in the production and use of cellulose pulp and fibre derivatives of cellulose, plus representatives of academic institutions and government research centres. This diverse audience has allowed the cross-fertilisation of many ideas, which has done much to give the field of cellulose in its diverse forms the higher profile that it rightly deserves. Cellucon Conferences are organised by The Cellucon Trust, an official UK charitable trust with world-wide objectives in education in wood and cellulosics. The Cellucon trust is continuing to extend the knowledge of all aspects of ccllulose world-wide. At least one book has been published from each Cellucon Conference as the proceedings thereof. This volume arises from the 1998 conference held in TurkdKbo, Finland and the conferenccs planned to be hcld in Japan, Walcs, etc, will generate further useful books in this area. THE CELLUCON TRUST TRUSTEES AND DIRECTORS
Prof. G.O. Phillips (Chairman) Prof. J.F. Kennedy (Deputy Chairman and Treasurer) Prof. P.A. Williams (Secrctary General)
Research Transfer Ltd, UK The North East Wales Institute, UK, and The University of Birmingham, UK The North East Wales Institute, UK
THE CELLUCON TRUST is a registered charity, UK Registration No: 328582 and a company limited by guarantee, UK Registration No: 2483804 with is registered offices at Chembiotech Laboratories, The University of Birmingham Research Park, Vincent Drive, Birmingham, B15 2SQ, UK.
ACKNOWLEDGEMENTS w i 4 W y - w ’
The 10 Intcrnational Cellucon Conference
CELLUCON ‘98
This book arises from the International conference - CELLUCON ’98 - which was held at the Mauno Koivisto Centre, Biocity, TurkdAbo, Finland. This meeting owed its success to the invaluable work of the Organising Committee and its generous sponsors. SPONSORS OF CELLUCON 98 City of TurkdAbo, Finland DataCity Centcr (since August 1999: T u r k Technology Centre), TurkdAbo, Finland
PULP FOR PAPERMAKING Fibre & Siirface Properties & Other Aspects of Cellulose Technology
Abo Akademi University, TurkdAbo, Finland
MEMBERS OF TIlE ORGANISING COMMITTEE - CELLUCON ’98 Prof. Bruno Lonnberg, Laboratory of Pulping Technology, Abo Akademi University, TwkdAbo, Finland (Chairman)
h4s Outi Rapila, Laboratory of Pulping Technology, Abo Akademi University, TurkdAbo, Finland (Conference Secretary) Ms Agneta Hermansson, Abo Akademi University, TurkdAbo, Finland (Treasurer) Prof. Raimo A h , Laboratory of Applied Chemistry, University of Jyviskyla, Jyviskylii, Finland Prof. John F. Kennedy, Birmingham Carbohydrate and Protein Technology Group, School of Chemistry, The University of Birmingham, Birmingham, UK Dr Charles J. Knill, Birmingham Carbohydrate and Protein Technology Group, School of Chemistry, The University of Birmingham, Birmingham, UK
Prof. Jar1 Rosenhoh, Department of Physical Chemistry, Abo Akademi University, TurkdAbo, Finland Prof. Per Stcnius, Laboratory of Forest Products Chemistry, Helsinki University of Technology, ESPOO,Finland Prof. Jorma Sundqvist, Finnish Pulp and Paper Research Institute (KCL), ESPOO,Finland Prof. Liisa Viikari, VTT Biotechnology, ESPOO,Finland
THE CELLUCON CONFERENCES
1984 Cellucon '84 UK
CELLULOSE AND ITS DERIVATIVES Chemistry, Biochemistry and Applications
1986 Cellucon '86 UK
WOOD AND CELLULOSICS Industrial Technology, Biotechnology, Structure and Properties
1988 Cellucon '88 Japan
CELLULOSICS AND WOOD Fundamentals and Applications
1989 Cellucon '89 UK
CELLULOSE: SOURCES AND EXPLOITATION Industrial Utilisation, Biotechnology and PhysicoChemical Properties
1990 Cellucon '90 Czechoslovakia
CELLULOSE New Trends in the Complex Utilisation of Lignocellulosics (Phytomass)
1991 Cellucon '91 USA
CELLULOSE A Joint Meeting of: ACS Cellulose, Paper and Textile Division, The Cellucon Trust, and 1I* Syracuse Cellulose Conference
1992 Cellucon '93 UK
SELECTIVE PURIFICATION AND SEPERATION PROCESSES
1993 Cellucon '93 Sweden
CELLULOSE AND CELLULOSE DERIVATIVES Physico-Chemical Aspects and Industrial Applications
1994 Cellucon '94 UK
CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS The Chemistry and Processing of Wood and Plant Fibrous Materials
1998 Cellucon '98 Finland
PULP AND PAPER MAKING Fibre and Surface Properties and other Aspects of Cellulose Technology
1999 Cellucon '99 Japan
RECENT ADVANCES IN ENVIRONMENTALLY COMPATABLE POLYERS
2000 Hyaluronan 2000 Wales
ASPECTS OF HYALURONAN
The procecdings of each conference were formerly published by Ellis Honvood, Simon and Schuster International Group, Prcntice Hall, Campus 400, Maylands Avenue, Hemel IIempstead, Herts, HP2 7EZ and from 1993 are published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB 1 6AH.
PREFACE
The 10’ Cellucon Confercnce was held in thc city of TurkuIAbo, Finland, 1417‘ December 1998 at the Mauno Koivisto Centre, Biocity, in the immediate neighbourhood of the universities in Turku. The conference on “Pulp for Papermaking - Fibre and Surface Properties and other aspects of ccllulose tcchnology” was organised by Abo Akademi University, Faculty of Chemical Engineering, Laboratory of Pulping Technoloa. The background to the organisation of the conference is worth noting. Prof. Glyn 0. Phillips, Chairman of The Cellucon Trust, asked me during a previous Cellucon Conference in Bangor, Wales, 1994, whether Finland would be interested in hosting the Conference in 1997. My answer was not definite, and I wanted to explore the possibilities and the interest in my country, although I realised that thc Confercnce would fit very wcll into the Turku environment. AAcr a number of exchanged E-mails bctwecn Prof. John F. Kennedy (Dcputy Chairman and Treasurer of The Cellucon trust) and myself, we finally ended up with a confcrence datc in Dccember 1998, which maybe was risky. However, I considered that time as suitable, because the City of Turku recently had dcclared itsclf the Christmas Town of Finland, and it would perhaps be an experience for many of the delegates to see the winter and the “kamos” (darkness) almost without any daylight.
The first day, 14Ih Dccember, was a true winter day with beautihl snow fall and decorative illumination of the Aura River and the Porthan Square, when delegates invited by the City of Turku went to the reception in the House of Brinkkala, welcomed by Ms Cay Sev6n, head of the networking activities of the City. Rut winter wcather is very changeable, and a tmible rain storm struck the conference delegates when they were rushing to the technical sessions next morning to be in time for thc opening ceremony and the inaugural speech by Rector Gustav Bjorkstrand of Abo Akadcmi University. Twenty-six oral presentations wcre given in five sessions on “Engineering of fibrcs for papcrmaking”, “Fibre surface properties”, “Biochemical processes in pulping”, “Pulping and bleaching” and “Cellulose”, and in addition thcre wcre as many prcsentations in a special “Poster Session”. All this within two days (13’’ and 16Ih Decembcr)! The Banquet Dinner at Turku Castle entertained by Count Per Brahe, Governor General of Finland in the l?‘ century, and the Countess Kristina Katarina Stenbock, was very enjoyable and the atmosphere was most rclaxing. The last day, 17’ December, conference delegates made a tour of the Laboratories of Forest Products Chemistry, Paper Chemistry, Physical Chemistry and Pulping Tcchnology in the Gadolinia Building of Abo Akademi University. This book marks the end of the 10“ Cellucon Conference, but hopefully it will recall pleasant memories fiom Turku and Finland, and also provide scientific benefits and ncw creative idcas for the more than one hundred delegates who attendcd the meeting and othcr readers of thc proceedings. Personally, I hope to makc new friends and renew old acquaintanccs with delegates at futurc Cellucon Confcrences.
xii Preface This book contains an excerpt of the presentations given at the 10"' Cellucon Conference on "Pulp for Papermaking - Fibre and Surface Properties", and the contents of six parts actually represent a wide range of important research areas of cellulose properties (Part l), application of enzymes (Part 2), pulp production and processing (Part 3), fibre properties (Part 4), paper properties (Part 5 ) and cellulosic materials (Part 6). Since the contents of these proceedings cover the most important subprocesses of the pulp and papermaking process, fkom pulping of differcnt raw materials by delignification and mechanical refining to biochemical and mechanical treatments of the pulp fibre materials by enzymes and beating, and to paper and its properties, this volume will evidently provide a base for innovative research, and is certainly worth reading. Prof Bruno Lonnberg Abo Akadcmi University, TurkdAbo, Finland Chairman of the Cellucon '98 Organising Committee
