Recent Progress on the Structure and Morphology ... - ACS Publications

9 Recent Progress on the Structure and Morphology of Cellulose

Downloaded by PENNSYLVANIA STATE UNIV on June 8, 2012 | http://pubs.acs.org Publication Date: June 1, 1969 | doi: 10.1021/ba-1969-0095.ch009

BENGT

RÅNBY

1

The Royal Institute of Technology, Stockholm, Sweden

Recent progress on the structure and morphology of cellulose is described—e.g.,

the cellulose chain

conformation

with its intrachain hydrogen bonds, the fibrillar morphology of native cellulose containing very thin elementary microfibrils (30-40 A . wide), the acid hydrolysis of cellulose fibers to microcrystalline

cellulose giving rodlike particles

(mi-

celles) of the same width as the fibrils, and the new but controversial

helical model

Penetration measurements

of the cellulose of native

microfibrils.

cellulose fiber walls

using aqueous polymer solutions have given average pore dimensions of 5 A . (cotton fibers) to 10 A . (delignified wood fibers).

Accessibility

measurements

using deuterium

tritium oxide exchange have shown that native probably

is deposited in crystalline form.

and

cellulose

Holocellulose

fibers (delignified wood) are shown to be less accessible than purified wood pulp fibers, and tentative interpretations are given.

T^he ^

chemical structure of cellulose chains was established b y Haworth

and Hibbert more than 40 years ago. Native cellulose occurs i n solid

state and i n partly crystalline form. A schematic model for the native cellulose lattice was worked out about 1930 b y Meyer, Mark, and M i s c h . The basic morphology of native cellulose could not be resolved, however, before

electron microscopy w i t h high resolution was developed and

applied. D u r i n g the last two decades, it has been amply shown and is now generally accepted that native cellulose basically is composed of microfibrils of a width 100 A . or less as was reviewed i n 1956 (12). Visiting Professor of Polymer Chemistry, North Carolina State University at Raleigh, North Carolina and Camille Dreyfus Laboratory, Research Triangle Institute, Durham, North Carolina. 1

139 In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.


140

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A N D T H E I R

A P P L I C A T I O N S

N o interwoven fringe micelles, no membranes, and no networks of cellulose chains seem to occur except when formed from microfibrils, laid down as units i n the morphological structure. Thicker fibrils, lamellae of largely parallel microfibrils, and layers and walls of crossing fibrillar units a l l contain microfibrils of a certain rather well defined thickness and indefinite length. This is not attempted to be a comprehensive review of recent progress on the structure and morphology of native cellulose and related substances. It is limited to aspects supposed to have more direct bearing on the enzymatic degradation. The discussions of the various implications are of necessity brief. Conformation of Cellulose Chains Infrared absorption measurements have shown that the chains i n solid cellulose are to a large extent tied together with hydrogen bonds between hydroxyl groups and oxygen atoms i n the structure (10). I n dry native cellulose, there are no measurable amounts of free hydroxyl groups—i.e., O H groups not engaged i n hydrogen bonds. Cellulose chains i n solution are not considered here, as they represent a state not found i n nature. The cellulose chains i n native crystalline cellulose seem to contain intrachain hydrogen bonds (Figure 1) between O H i n one glucose unit and 0 , i n the following unit as suggested by Hermans i n the 1940's and later supported b y infrared measurements of Liang and Marchessault (7). F o r mercerized cellulose similar intrachain hydrogen bonds are proposed. It is suggested but not proved that these bonds also occur i n non-crystalline celluloses. 3

5

Figure I .

Cellulose molecule with intrachain hydrogen bonds between OH and 0 3

5

Part of an infrared absorption spectrum from a membrane of bacterial cellulose is shown in Figure 2 (16). The absorption bands refer to the stretching frequencies of the hydrogen-bonded O H groups. Free O H groups would give an absorption band at 3640 cm." . A l l O H groups are 1

In Cellulases and Their Applications; Hajny, G., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1969.

9.

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141

Structure and Morphology

apparently hydrogen-bonded i n different but rather specific ways. T w o of the absorption bands are assigned to interchain Η-bonds i n different lattice planes (101" and 101, respectively). The most intense band is assigned to the intrachain Η-bond OH3-O5, i n the 002-planes as previ ously discussed. The infrared band at 3245 cm." with the largest shift from 3640 cm." (free O H groups)—i.e., due the strongest H-bonds—is not yet assigned to a specific hydrogen bond. W h e n native cellulose is wetted with water, an accessible surface layer of the microfibrils is assumed to react. The cellulose lattice, how ever, is not penetrated or otherwise affected by the water i n any way measurable with x-ray diffraction. 1


1

TAPPI

Figure 2.

The OH stretching frequency bands in the infrared absorp tion spectra of bacterial cellulose (16)

Dimensions and Structure of Native Cellulose Microfibrils In electron micrographs, the native cellulose microfibrils are usually seen as bundles of lamellae containing an indefinite number of fibrillar units. A schematic representation of the cross section of a small lamellae of microfibrils is shown in Figure 3 (14). T w o structural features should be pointed out. The cellulose lattice extends through the whole cross section of the microfibrils but the surface layers are supposed to be disordered to some extent because they represent a discontinuity. The microfibrils show a preferred orientation with their lattice planes 101 well aligned parallel with the flat surface of the lamellae—i.e., the tan-


142

C E L L U L A S E S

A N DT H E I R



gential plane of the cell wall or the membrane where they occur. It seems therefore likely that the cellulose microfibrils have a flat cross section. This orientation effect was first found b y x-ray diffraction and later confirmed by infrared absorption measurements. Owing to the prevailing orientation of the lattice planes and the parallel alignment of the microfibrils, it is likely that the cellulose lattice from one microfibril frequently extends into and through adjacent microfibrils as indicated i n Figure 3. But no bundles of microfibrils of preferred size—e.g., 250-300 A . as previously reported ( 5 ) , seem to occur i n native cellulose. Ι00Α.-

i TUT- ' 4+^» Λ . L . . . . . . . . . . . .

101

\{f'{'{t-jfZ£

L

"

4

002 Das Papier

Figure 3.

Schematic cross section of a lamellae of cellulose microfibriU from the secondary wall of a plant cell ( 14)

Along the microfibrils, there are areas of sufficient disorder to allow disintegration by hydrolysis into rodlike particles (micelles) with aque ous, non-swelling strong acid. After washing out the acid with distilled water, aqueous colloidal solutions of the cellulose micelles can be pre pared ( I I ) . The disordered areas of the microfibrils may be native or formed b y mechanical forces, giving deformation beyond the limit of elastic recovery of the microfibrils. The length of the resulting particles after acid hydrolysis (micelles or microcrystals) varies with the pretreatment of the native cellulose and corresponds to the leveling-off degree of polymerization ( L O - D P ) of the hydrocellulose ( 2 ) . Repeated cycles of drying and swelling the native cellulose with water or dilute aqueous caustic soda tend to decrease the length of the particles obtained in subsequent hydrolysis. A t the same time, these treatments are found to increase the crystallinity and decrease the chemical accessibility of the cellulose material (13). The preparation of microcrystalline cellulose by acid hydrolysis of native and regenerated fibers has been studied extensively ( 3 ) and devel oped into a commercial process by Battista et al. ( 4 ) . The resulting products are used as aqueous gels with high water-bonding capacity, inert food and drug additives, viscosity regulators, and stabilizers i n colloidal



9.

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143

solutions. Macrocrystalline celluloses are also used as substrates for enzymatic degradation by fungi and microorganisms. In recent years a new model for the molecular morphology of native microfibrils has been developed by Manley (9). H e has found that the microfibrils have an average width of about 35 A . (or rather 30-40 A . ) and not 60-100 A . as reported 15-20 years ago. The resolving power of the electron microscope has increased i n recent years, and it seems likely that the dimensions reported now do represent some finite morphological unit (Figure 4). Particles observed along the microfibrils on the electron micrographs are taken as evidence for a detailed periodic structure of the microfibrils. This is ambiguous because the specimens were stained —e.g., with uranyl acetate. Both the supporting membrane and the cellulose specimen show grain of the same order of magnitude. Manley has worked out a detailed helical model of folded cellulose chains for the structure of cellulose microfibrils (Figure 5). The helix is assumed to be built as a spiral ribbon of folded chains. The helix could be unwound to a flat ribbon by mechanical disruption. A serious objection to this model is that it does not give a preferred orientation of the (101) lattice planes parallel with the fiber wall, as previously reported from x-ray diffraction and infrared absorption work.

Figure 4.

Cellulose microfibrils in a fragment of a ramie fiber wall (Manley)

The circular shape of the cross section of the fibril according to Manley, and the cylindrical hole in the middle of the fibril are difficult to reconcile with the measured density of the fiber wall, known to be as high as about 1.5. For pure crystalline cellulose a density of 1.59 is calcu-


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A N D T H E I R



lated from the lattice parameters. T o meet these objections, Manley has indicated that the cylindrical fibrils may be deformed (flattened) to an oval or elliptical cross section i n the fiber walls. Such a deformation seems unlikely, as it would require strong forces and also mean a very extensive deformation of the cellulose lattice. Further evidence is required for the construction of an acceptable model for the native cellulose microfibrils.

b Nature

Figure 5. Structural model of a cellulose microfibril containing folded cellulose chains (9) Porosity of the Cell Walls of the Native Cellulose Fibers Measurements of the pore size distribution of cellulose fibers have been made using a penetration method related to the principle of gel permeation chromatography ( I ) . A cellulose fiber is known to imbibe a considerable amount of water—e.g., for purified native cotton fibers about 0.4 grams of water per gram of dry cellulose at the fiber saturation point. The excess of water is removed b y centrifugation i n a field of about 100 X gravity. If the dry fibers are immersed in aqueous solutions of a polymer with a certain molecular weight, the polymer molecules can penetrate only a fraction of the volume for water penetration. This can be derived from chemical analysis of the polymer concentration i n the solution before and after immersion of the dry fibers i n the solution. The excluded volume for the polymer molecules increases with increasing molecular weight. If a series of polymers with increasing molecular weights are used, the pore size distribution can then be derived from the calculated molecular dimensions of the polymer molecules i n solution (the radius of gyration of the polymer molecules). The measurements of Aggebrandt and Samuelson ( I ) , using polyethyleneglycol as polymer, have shown that the most frequent pore diameter i n cotton fibers is 5 A . Seventy-five percent of the total pore volume ( corresponds to 0.3 m l . per gram of dry fiber) was found i n pores of less than 20 A . diameter. D e lignified wood fibers show two to four times larger pore diameters (15).


9.

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145


These measurements give an indication of the possibilities for enzymes to penetrate a fiber wall as w i l l be described i n later papers during this symposium. Accessibility of Native Cellulose T w o powerful methods have recently been applied to measure the accessibility of native cellulose and wood specimens.


OH

TAPPI

Figure 6. Infrared spectra of deuterated bacterial cellulose (solid line) and rehydrogenated bacterial cellulose (dashed line) (16) Deuterium Oxide. Exchange with deuterium oxide (heavy water) and subsequent infrared analysis can be applied to transparent samples (6, 10, 16). T h i n membranes of bacterial cellulose are ideal as model samples. W h e n the dry membranes are exposed to heavy water as vapor or liquid, the accessible O H groups are exchanged to O D groups. Infrared spectra of a D 0-exchanged and rehydrogenated membrane are given i n Figure 6. Deuterium exchange is recorded as a shift from O H stretching frequencies i n the band at about 3300 cm." to the O D stretching frequency band at about 2500 cm." . The non-accessible O H groups are retained i n the 3300 cm." band. U p o n rehydrogenation, some O D groups are retained (not exchanged to O H groups), which indicates recrystallization and trapping i n the wetting and drying processes. In one series of experiments, the virgin bacterial cellulose gel containing about 0.5% cellulose was purified completely i n heavy water by treatments with dilute N a O D solutions i n D 0 (16). The subsequent infrared measurements showed that about 5 5 % of the O H groups i n the gel cellulose 2

1

1

1

2


146

C E L L U L A S E S

A N D T H E I R


membranes were inaccessible to deuterium exchange. This is interpreted to mean that the bacterial cellulose microfibrils most probably are de posited directly i n crystalline form. The data are collected i n Table I, and they show further that wet (never-dried) membranes are accessible to 3 3 % and dried membranes to 27%. Prolonged treatment with D 0 gives only a minor increase in accessibility. These results for bacterial cellulose are taken to be applicable also to cotton and ramie cellulose fibers which have very closely the same degree of crystalline order as the bacterial cellulose. The microfibrils of cotton cellulose are oriented as spirals around the fiber axis while those of ramie are closely parallel with the fiber axis.


2

Table I.

The Total Accessibility of Bacterial Cellulose Membranes in Heavy Water by Infrared Analysis A Β Total Total accessi amount of bility, resistant % OD,% A-B

m

Membranes purified in heavy water Never-dried membranes Dry membranes Dry membranes (deuteration for 15 hr.)

44.7 39.9 31.4 32.2

12.2 6.9 4.4

—

32.5 33.0 27.0

—

* ( A — B) represents amount (% ) of reexchangeable OD groups. Various attempts to interpret the accessibility data as related to sur face area of microfibrils of measured dimensions have been made. If 10% of the microfibrils are accessible because they contain a disordered cellulose lattice, indicated by rapid hydrolysis i n acid, the remaining 20-25% of the accessible O H groups should be located on microfibril surfaces. Tritiated Water. Exchange with tritiated water and subsequent scintillation analysis of the tritium content is a very useful method for accessibility measurements when the samples are not transparent to IR and visible light. T h i n shives of sapwood from black spruce (a common softwood), and white birch (a common hardwood) where studied before and after delignification by treatment with peracetic acid (17). Also wood cambium from the same softwood was studied. The cambium is the recently formed fiber layer, located close to the bark and not yet lignified. Spruce and birch wood contain about the same amounts of cellulose, 42 and 44%, respectively. Spruce wood has more lignin (28 vs. 18% ), while birch has more hemicellulose (35 vs. 28% ), i n particular more pentosans than spruce (24 vs. 14% ).


Library American Chemical Society 9.

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147

Structure and Morphology Table II.

Percentage Accessibility Accessibility % 9

Sample Sapwood Holocellulose (yield, 83% ) Holocellulose (yield, 77% )


Cambium

Spruce

Birch

60.6 60.5 67.5 64.6

53.4 52.3 54.7 53.8 55.7 55.5

Never-dried Dried Never-dried Dried Never-dried Dried Never-dried

— — 63.5

—

The accessibility of the O H groups i n spruce wood is about 6 0 % while the corresponding value for birch wood is only 5 3 % (Table I I ) . Removal of lignin results in an increase i n the number of accessible O H groups. The O H groups i n lignin are rather few and they are accessible to at least 40% but probably not 100%. Calculations of the carbohydrate accessibility is dependent on the estimates of lignin accessibility (Table III). Pure wood cellulose (the hemicellulose removed) has an accessibility of 58 to 60%. It can be calculated from the data that hemicellulose i n wood, although x-ray amorphous, is only partially accessible to tritium exchange. This is an indication of molecular order in the cellulosehemicellulose arrangement i n the woods—e.g., by ordered hydrogenbonding or some other form of ordered association which makes O H groups inaccessible. This result is well i n line with the finding from infrared measurements on fibers that the hemicellulose chains i n the wood fibers are arranged largely parallel with the cellulose microfibrils ( 8 ) . Table III. Accessibility of Carbohydrates Carbohydrate accessibility, % Lignin accessibility, 40% Sample Sapwood Holocellulose (yield 83%) Holocellulose (yield 77%) Cambium

Never-dried Dried Never-dried Dried Never-dried Dried Never-dried

Lignin accessibility, 100%

Spruce

Birch

Spruce

Birch

63.8 63.7 69.0 66.0 — — 63.5

54.7 52.4 54.7 53.8 55.7 55.5 —

54.7 54.6 65.5 62.5

49.3 48.0 54.3 53.3 55.7 55.5

63.5

Birch wood and birch holocellulose have particularly low accessibility (Table II and III) compared to purified cellulose from birch with an accessibility close to 60%. This is at least to some extent related to



148

RECEIVED

C E L L U L A S E S

A N DT H E I R

APPLICATIONS

the relative inaccessibility of the xylan portion. This could partly be a result of strong internal hydrogen bonding between acetyl groups and neighboring O H groups. It could also be partly attributed to regularly arranged hydrogen bonds between cellulose microfibrils and xylan chains. Taken together, the data for O H group accessibility of wood, holocellulose, and purified wood cellulose fibers indicate that the carbohydrate part of the wood is regularly organized on the molecular level—e.g., b y hydrogen bonding between cellulose and hemicellulose. This molecular order reduces the accessibility of the x-ray amorphous hemicellulose por tion to tritium exchange and would be of importance also for the accessi bility to enzymatic degradation. Native wood is known to be less accessible to enzymatic degradation than purified wood cellulose fibers. Acknowledgment The author is indebted to R. St. J. Manley for mailing a review paper (including F i g . 4) available before publication. Literature Cited (1) Aggebrandt, L . , Samuelson, O., J. Appl. Polymer Sci. 8, 2813 (1964). (2) Battista, Ο. Α., Ind. Eng. Chem. 42, 502 (1950). (3) Battista, Ο. Α., Coppick, S., Howsmon, J. Α., Morehead, F. F., Sisson, W. Α., Ind. Eng. Chem. 48, 333 (1956). (4) Battista, Ο. Α., Smith, P. Α., U. S. Patent 2,978,446 April 4, 1961). (5) Frey-Wyssling, Α., Mühlethaler, Κ., Fortschr. Chem. Org. Naturstoffe 8, 1 (1951). (6) Lang, A. R. G . , Mason, S. G . , Can. J. Chem. 38, 373 (1960). (7) Liang, C . Y., Marchessault, R. H., J. Polymer Sci. 37, 385 (1959). (8) Liang, C. Y., Bassett, Κ. H., McGinnes, Ε. Α., Marchessault, R. Η., TAPPI 43, 1017 (1960). (9) Manley, R. St. J., Nature 204, 1155 (1964). (10) Mann, J., Marrinan, H . J., Trans. Faraday Soc. 52, 492 (1956). (11) Rånby, Β., Acta Chem. Scand. 3, 649 (1949). (12) Rånby, B., Rydholm, S. Α., " 'Polymer Processes,' High Polymers," C. E . Schildknecht, E d . , Vol. 10, Chapt. IX, p. 351, Interscience, Ν. Y., 1956. (13) Rånby, Β., "Fundamentals of Papermaking Fibers," F. Bolam, E d . , p. 55, Cambridge, 1957. (14) Rånby, Β., Das Papier 18, 593 (October 1964). (15) Stone, J. E., Scallan, A. M . , Pulp Paper Mag. Can. 69, 288 (1968). (16) Sumi, Y., Hale, R. D . , Rånby, Β. G . , TAPPI 46, 126 (1963). (17) Sumi, Y., Hale, R. D., Meyer, J. Α., Leopold, B., Rånby, Β. G . , TAPPI 47, 621 (1964). October 28, 1968.


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Discussion K . Selby: " W e are talking now about accessibility, and crystallinity in cotton, so I thought you might like to see some results we got on the course of solubilization of cotton by cellulases. W e were rather surprised to find this follows a first order reaction with respect to substrate. This cotton sample was solubilized by Trichoderma viride cellulase up to about 35% solubilization (Figure A ) . The next one (Figure B ) is a similar curve from the P. funiculosum cellulase that I was talking about this morning; and the final one (Figure C ) contains those two curves and also another obtained with a concentrate of Trichoderma viride cellulase which is producing up to 97% solubilization. N o w the point is that that reaction is either first order all the way, or, i n some cases, it departs slightly from it as solubilization proceeds. W e were a little bit wide-eyed about this; what this is all about, we don't really know. But what is of considerable interest in this context is that we have established very very carefully that this reaction is first order over this critical range up to 3 5 %

Time, hr.

Figure A.

Solubilization of cotton by T. viride cellulose

solubilization. W e have done a lot of determinations here because this is a log plot. What we are saying is that, if there were any departure from uniform accessibility and reactivity as suggested in the old theories involving the idea of amorphous regions, we must surely have a break somewhere in this curve. W e are interested i n the fact that this is a straight line."


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B. Rânby: "I have not seen these data reported before. I n the hydrolysis b y acid we know that it is an initial phase which takes something like 10% away from the cellulose very quickly and then the rate of reaction is much slower. Therefore, the reaction is not first order throughout. Referring to D r . Selby's report apparently the enzyme is 0-24


0-20 Negative log ·ΐ6 (proportion of cellulose remaining) 0-12 0

0-08

OfH

ι

0

20

ι

l

40 60 Time, hr.

ι

I

80

KX)

Figure B. Solubilization of cotton by P. funiculosum cellu lose "197 -95 Solubilization -90 % -80 -70 -50 -20 400 Time, hr Figure C.

Solubilization of cotton by cellulase

attacking the cellulose microfibrils evenly whether they are disordered or not i n some way. The material is appearing as homogeneous to the enzyme. That is my preliminary conclusion."



9.

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151

K . Selby: "The attack of mineral acid on cotton does not always follow the same course—i.e., a rapid solubilization of the first 10%). Jeffries, Roberts, and Robinson ( I ) have recently shown that the size of the rapidly hydrolyzed fraction varies with acid concentration and deduce that "two clearly defined fractions are not present in the structure" but that "reaction is at an accessible surface, the accessibility depending on the concentration of the hydrolyzing acid." The conclusion would therefore seem to be that the structure has very low accessibility to cellulase (as to the weakest mineral acid). The interesting fact still remains, however, that as the degradation proceeds there is no sudden change i n accessibility or reactivity that would suggest the early disappearance of a more accessible fraction." Literature Cited (1) Jeffries, Roberts, and Robinson, Text. Res. J. 38, 234 (1968).


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