FT Raman microscopy of untreated natural plant fibres

SPECTROCHIMICA ACTA

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PART A

SpectrochimicaActa Part A 53 (1997) 2383-2392

FT Raman microscopy of untreated natural plant fibres H.G.M. Edwards a,*, D.W. Farwell a, D. Webster b a Chemistry ’ School

and Chemical of Art,

Design

Technology, and Textiles,

University of Bradford, Molecular Spectroscopy W. Yorkshire BD7 lDP, UK Bradford and Ilkley Community College, Bradford

Group, BD7

Bradford, IAY,

UK

Received4 March 1997; accepted15 April 1997

Abstract The application of FT-Raman microscopy to the non-destructive analysis of natural plant fibres is demonstrated with samples of flax, jute, ramie, cotton, kapok, sisal and coconut fibre. Vibrational assignments are proposed and characteristic features of each material are presented. Samples were not pre-treated chemically before analysis and were used directly from their respective storage collection; the adaptation of the Raman microscopic technique to the identification of specimens of natural fibres in archaeological burial sites is explored for its forensic potential. 0 1997 Elsevier Science R.V. Keywords:

FT-Raman; Microscopy; Fibres; Archaeology

1. Introduction

Natural fibres may be broadly classified into two types, carbohydrate (for example, the cellulose-based cotton and flax) and proteinaceous (for example, the keratotic animal furs generically termed wool and the insect-based secretions termed silk). Plant fibres are of interest archaeologically because they probably represent the earliest textile materials [l]. In Europe, flax (Linium usitatissimum) was the first identified cultivated source of textile fibres, including linens found at Nahal Hemar (7 K years BC) and Fayum (5 K years BC) [2,3]. Until their replacement by cotton in the 18th Century, linen textiles were the predominant fabrics. * Corresponding author. Tel.: + 1274 383 787; fax: + 1274 385 350; e-mail: [email protected]

Vegetable fibres are either long and multiplecelled or short and single-celled; in addition, the long fibres are categorized as hard or soft according to the degree of stiffness and fibre fineness [4]. A very important group of soft fibres, generically known as bast, include flax, hemp, jute and ramie and are obtained from the leaves of tropical plants, or in the special case of coir, from the coarse bristles enveloping the fruit of the coconut palm. A third category of soft fibres is exemplified by the seed-hair and seed-pod fibres, cotton and kapok, respectively. Although natural vegetable fibre characteristics can vary somewhat with the environmental conditions under which the plants grow, their chemical

constitution

consists of mainly cellulose (Fig. 1)

(including hemicelluloses), moisture, lignins and pectins, with residual ash and other organic mate-

1386-1425/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIIS1386-1425(97)00178-9

Fig. 1. Molecular structure of cellulose, based on cl-D-glucose monomer units with b-1,4-glycosidic

rial. A list of the seven plant fibres studied in the present work is given in Table 1, from which it is seen that the content of cellulose ranges from 64-91% and those of water O-12% and the lignins/pectins, O-25%. It is realistic, therefore, to expect that the vibrational Raman spectra of these representative samples of hard and soft plant fibres will exhibit some differences which might be used in their characterisation. Archaeological fibres are usually studied by light and scanning electron microscopy, with identification and assessment of degradation being largely based on physical morphology [5]. There have been relatively few detailed studies made using vibrational spectroscopy; this is ascribed to the presence of significant amounts of free water and hydroxyl groups in the woven fibres and broad absorption bands in the infrared. Despite this, there is an extensive literature which describes the application in general of infrared spectroscopy to the characterization of linen fabrics, in particular, and in some cases applied to archaeological linen samples such as the Shroud of Turin. The spectral quality, particularly of the latter samples, is generally rather poor [6-91, and very few attempts have been made at even a partial vibrational assignment. This may be ascribed to several factors, which include the presence of quite significant amounts of free water and -OH groups in the cellulose component of the linens and restrictions on sample pretreatment and preparation for infrared studies, which limits the facility of presentation of material to the spectrometer. Of even more significance, however, is the fragility and deep brown coloration of some archaeological fabric samples attributed to the cellulose ageing process [lo]. Hence, in several cases, there have been reports of the chemical

links.

pretreatment of old linens prior to their infrared spectroscopic study, including washing in hot water [lo] and heating with hydrogen peroxide [l 11. A recent study [12] has used infrared spectroscopy to assess the mineralisation of fibres under simulated archaeological conditions. Conventional Raman spectroscopy with visible excitation is prone to fluorescence and gives rise to poor quality vibrational spectra; the presence of fluorescent materials in archaeological samples leached out from grave soil through the ages is a major problem for Raman spectroscopy, but previous work in our laboratories on human skeletal remains [13] from the Romano-British period (ca. 400 AD), dinosaur teeth [14] and the stratum corneum and dermis [15- 171 of the ‘Ice-man’, a 5200-year-old corpse found in a glacial valley in &zal Austria, in 1991 demonstrated the advantage of the near infrared excitation source in FT-Raman spectroscopy. Of particular spectroscopic application in the area of archaeology and museum artefacts, a recent FT-Raman study of ancient linens dating from 600 AD and 1900 BC has been carried out nondestructively using Raman microscopy [18] and the identification of water-soluble, low molecular weight brown coloured extracts from degraded linen has been made [19]. Recently, the characterisation of synthetic polyester fibres from sample spot sizes of 1 nm has been reported [20]. In this work we report the first detailed Raman spectroscopic study of natural plant fibres, representing the hard and soft categories. All the frbres selected are of importance in the study of ancient cultures and it is believed that this work will provide a database for the non-destructive exami-

H.G.M.

Table 1 Composition”

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‘385

of natural plant fibres studied in the present work

--

Fibre Bast-type (soft) Jute Flax

Botanical source

Cellulose (%)

Moisture (x)

Ligning and pectin (X)

Corchorus capsularis Linium usitatissimum

10

25

Boekmeriff

9 0

11

Ramie

65 16 9i

Leaf-type (hard) Sisal

Aguoe

sisalana

II

6

15

Palm-type (hard) Coir

Cocos

nucifera

64

12

22

Seed-pod type (soft) Cotton Kapok

Gossypium hirsutum Ceiba pentandra

90 90

8 5

0

nitlea

_-

1

1

* Typical % compositions are given for average samples; residual material composed of ash and other organic material.

of archaeological fibre and textile material, so providing a chemical spectroscopic adjunct to optical examination.

nation

2. Experimental 2.1. Samples

Samples of the following natural plant fibres were obtained from archival collections: 2.1.1. Ramie

This is a bast fibre produced from the tall, stingless nettle, Boehmeria nivea. It is greenish yellow in colour and its main source of supply is China but the plant is native to other warm, temperate climates such as the Philippines. Degummed ramie is almost pure cellulose. It is superior to other bast fibres in strength and it may be spun, knitted and woven. 2.1.2. Jute

This is a bast fibre produced from the stems of two species of herbaceous annual plants of the genus Corchorus, viz. C. capsularis and C. olitorius. The main source of supply is Pakistan and India. Its colour can range from cream to reddishbrown and darkens with age. Jute has a higher proportion of lignin than any other soft fibre and

also has the lowest cellulose content; this results in a decreased strength and durability, but despite this its diversity of use results in jute being the most important natural plant fibre next to cotton, being used for twine, sacking, rugs and burlap. 2.1.3. Flax

This fibre is an important bast fibre from the dicotyledonous Linium usititissimum plant native to the Middle East. Its colour is pale cream to brown. It has been used for centuries in the manufacture of fine linens and more lately, in admixture with jute, to give hard-wearing fabrics. Two other soft fibres, although not classified as bast, are better described as seed-pod type: 2.1.4. Cotton

This fibre is soft and is studied here as the natural boll, ex-Boone Hall plantation, South Carolina, USA. Cotton is unique among the natural plant fibres in being almost pure cellulose, which can amount to 96% of the dry weight of the fibres. The high-quality of the cotton fabrics produced in the last 200 years displaced linen as the premier high-volume fabric. 2.1 S. Kapok

Kapok is the seed-pod fibre of Ceiba pentandra which is native to the Far East, South America and West Africa. The fibre is of a white or cream colour with a silky lustre.

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Because of its buoyancy and moisture resistance, it has diverse uses as a material for insulation and life-jackets. Examples of hard fibres studied here were: 2.1.6. Sisal

A leaf fibre obtained from Agave sisalana and agave variants from America, Africa and Asia. Of a white or cream colour, the fibre is strong and finds use in cordage. 2.1.7. Coir

A hard fibre enveloping the fruit of the coconut palm, Cocos nucifera, Coir is brown in colour and because of its strength, flexibility and resistance to salt water, it is used for marine applications and heavy domestic work. 2.2. FT-Raman

spectroscopy

FT-Raman spectra were excited using a Bruker IFS66 optical bench with FRA106 Raman module attachment and Nd3+/YAG laser excitation at 1064 nm with a liquid-nitrogen cooled gerrnanium detector. In the macroscopic mode, sample areas of 100 urn diameter were examined using low laser powers of 50 mW or less to minimise sample degradation; for this application, bundles of fibres were used. In the microscopic mode, a Raman microscope attachment with objective lenses of 40 x or 100 x magnification was used to examine single fibres; for this application, a minimum sample illumination area of about 8 urn using the 100 x objective was available.

3. Results and discussion The Raman spectra of the seven examples of hard and soft fibres studied in the present work have been arranged as follows for convenience of presentation: the soft bast fibres, jute, rarnie and flax are stack-plotted in Fig. 2; the hard fibres, coir and sisal are shown in Fig. 3 and the seedpod derived soft fibres, kapok and cotton are shown in Fig. 4. Cotton is composed predominantly of cellulose of high molecular weight, typically 6 x lo5 and

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larger, consisting of long chains of I>,-glucose units joined by p-1,bglycosidic links (Fig. 1). The rigidity of the cellulose structure in cotton conferred by the anhydroglucopyranose units with their fi-1,4-glycosidic (COC) links and intramolecular hydrogen-bonding is believed to be responsible for the resistance of these fabrics to environmental extremes and archaeological burial conditions which would result in the destruction of other natural biopolymers. From a vibrational spectroscopic standpoint, however, the molecular rigidity imposes several constraints which give a rather different picture from typical model compounds. namely the ‘monomer unit’, D-glucose, and related disaccharides such as cellobiose. sucrose and maltose; hence, there is little virtue in attempting to transfer vibrational spectroscopic information from a-D-glucose to assist in the assignment of cellulose. The vibrational assignments in Table 2 follow from those of our previous work on cotton [21]; other assignments from the literature [22,23] are based on an extension of these. Some of the most characteristic, strongest features in the Raman spectra of cotton are the vibrations of the p-1,4glycosidic ring linkages between the cx-D-glucose units in cellulose. Generally, the major features of the natural fibres are very similar in the three sets of spectra (Figs. 2-4), but on closer inspection there are several minor differences between, in particular, the hard, soft bast and seed-pod (cotton, kapok) fibres. For example, the r(CH) stretching regions of the six specimen materials studied here are very similar to each other; this is not an unexpected result and a method will be described later which utilises this similarity as a basis for the comparison of the relative intensities of vibrational bands for different fibres in the skeletal region. However, jute is the only natural fibre of those studied found to have a mediumweak intensity feature at 1736 cm - ‘, characteristic of an ester v(C=O) vibration. Previously, it has been noted [ 181 that adulteration of modern linens with jute to provide stiffness and strength can be identified in the Raman spectrum using this feature, which is absent from flax from which true linens are made.

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I

I

I

I

I

3200

3150

3100

3050

3000

1

I

I

I

I

1700

1600

1500

I

I

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I

I

1

1

I

I

I

I

2800

2750

2700

2650

2600

I

2950 2900 2850 Uavenumber cm+

1

I

1900 1300 1200 1100 1000 900 800 Uavenumber

2387

I

I

I

700 600 500

I

I

I

400 300

cm-'

Fig. 2. FT-Raman spectra of soft bast fibres, (i) jute; (ii) ramie; and (iii) flax: (a) 2600-3200 cm- ’ region; (b) 200-1700 cm-’ region: 1064 nm excitation, spectral resolution 4 cm- I, 2000 scans accumulated, laser power 40 mW.

The extent of unsaturation in the fibres can also be measured from the v(C=C) stretching band near 1602 cm - ‘; flax and ramie are clearly very different from the other fibres here in that they have no detectable v(C=C) scattering in this region. Flax, with a medium intensity band at 1578 cm-’ characteristic of aromatic ring quadrant

stretching has some aromatic content. Other features, such as those at about 1460 and 1378 cm- * are assignable to b(CHJ modes and these are common to all fibres studied here. One of the most significant differences between the observed FT-Raman spectra of the natural fibres is provided by the intensity ratio, R, of the

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Fig. 3. FT-Raman spectra of hard fibres, (i) coir; and (ii) sisal: (a) 2600-3200 cm- ’ region; (b) 250-1700 cm- ’ region: 1064 nm excitation, spectral resolution 4 cm - ‘, 2000 scans accumulated, laser power 40 mW.

unresolved v(CH) band profile around 2900 cm - I and the strong band at 1096 cm - ’ assigned to the v(COC) glycosidic link stretch, where R = 1'096/ 12900. It should be noted that the true relative intensities of the 1096 and 2900 cm - r features for the samples are not demonstrated in Figs. 2 and 3 and Fig. 4, which are designed to show the quali-

tative differences in the stack-plot spectra. For example, with a normalised band intensity of the 1096 cm - ’ feature for cotton, R = 0.75,whereas the R-values for the samples of the two modern and ancient Egyptian linens are 1.12 and 1.08, respectively; these experimental R values have an error of & 0.03, which means that they are sign&

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Fig. 4. FT-Raman spectra of soft seed-pod fibres, (i) kapok; and (ii) cotton: (a) 2600-3200 cm ’ region; (b) 250- 1700 cm- ’ region: 1064 mn excitation, spectral resolution 4 cm - ‘; 2000 scans accumulated, laser power 40 mW.

cantly different for the linen and cotton samples and are essentially the same for the modern and ancient linen sample. The R-values for the fibres studied in the present work, and R’-values, which represent a symmetric:antisymmetric band intensity for the v(COC) glycosidic links in cellulose, are presented in Table 3.

Relative intensity measurements carried out on the spectra of the natural fibres studied in the present work show that the intensity ratios R' for the 1121 and 1096 cm - ’ doublet are remarkably constant (Table 3) except for the kapok and coir fibres, with an average value of 0.74 + 0.03; this probably reflects the correct assignment of these

H.G.M.

2390 Table 2 Wavenumbers \I cm-’

ramie

3260 w, br

2968 mw 2950 w 2895 m

and vibrational v cm-

’ jute

3222 w, br

Edwards

assignments v cm-’

3240

for natural

flax

v cm

w, br

3070

VW

2968

mw

2970 mw

2935 m 2894 m 2850 mw,

2893 m 2848 mw,

ct al. / Spectrochimica

plant I coir

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fibres v cm -I

sisal

v cm wk

ka-

’ cotton

18cm

3336 w 3292 mw,

3275 w, br

3256 w, br

2383-2392

br

Approximate assignment of vibrational mode II free v(OH). COH drogen-bonded

hy-

3062 mw

sh

2990 2968

sh

2936 m 2896 m 2850 mw,

sh

2936 m 2891 m 2850 mw

2938 m 2900 m 2846 mw,

2720

2729 VW

sh

w mw

2920 mw 2894 s 2867 m, sh

2820 w 2718 1736 1662 1603

1479 1462 1411 1378 1360 1338 1320 1293 1280

m w, sh w m w mw w mw w

2720

VW mw w m 1578 mw, 1518 w 1470 mw

1463 1417 1378 1360 1333

m w m mw m

VW

w

1655 VW 1602 ms

1692 nw

1601 m

1458 m 1412 w 1380 mw

1460 nw 1410 w 1380 m

1463 nwm

1339 m 1310 w

1334 mw

1280 m

1260 w

2739 w 2718 w

v(CW v(CH) v(C=O)

1601 m

v(C=c) v(C=c)

br

1378 m 1339 mw

1380 w 1367 w 1338 m

1478 1470 1408 1379

m mw, w s

1320 mw, 1293 mw 1280 vw

1296 w 1280 w

vr

sh

sh

1150 w

1202 w 1152 m

1120 mw

1119 m

1121 ms

1095 mw

1094 ms

1092 m

1096 s

1040 w

1038 mw 1000 w

1062 1040 992 975 904

1058 w 1036 w 997 mw

1150 mw

1147 w

1122 m

1120 m

1122 m

1121 mw

1096 ms

1096 ms

1096 ms

1065 1040 996 970

1065 1038 999 980

1065 w

w w w w

w mw w w

993 VW 914 mw,

897 mw

896 mw

br

914 w 901 w

w w w VW w

900 mw,

893 w

6(CH,) d(CG,); 6 (CW WW 6 (CH,) 6 KH,) 6(CH,)

scissors 6(COH)

twisting

6 (CH,) WX) twisting 6(COH) out-ofplane 6(COH); G(CCH) v(CC) ring breathing, asymmetric v(COC) glycosidic, symmetric v(COC) glycosidic, asymmetric v(CO), 2” alcohol v(CO), 1” alcohol

1240 VW

1152 mw

ester

P (CH,) P (CH,)

br

v(COC) in plane, symmetric v(COC) in plane, symmetric

826 w 610 w 566 w

602 w

639 w 600 VW

635 w 600 w

609 w 566 w 540 VW

G(CCH)

twisting

v(COC) in-plane

glycosidic,

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Table 2 (continued) v cm i ramie

519 496 458 436 378

mw w m m m

1’ cm - ’ jute

520 496 457 436 379

mw w mw mw m

v cm-i flax

v cm-’

coir

v cm-i

sisal

v cm-’ kapok

521 w

519 w

515 w

521 mw

459 w 436 mw 379 mw

456 w 434 w 377 mw

450 w

456 w 432 w 378 mw, br

374 m

v cm-’ cotton

Approximate assignment of vibrational mode

519 492 459 436 380

r(COC) glycosidic r(COC) glycosidic v(CC0) ring i#(CCO) ring SCCCC) ring, symmetric

mw w m

m ms

360 mw 347 mw 331 mw

329 w

330 w

308 w

Table 3 Relative band intensities for natural fibres: R’ = I’12’/1’096, R = po96,poo

R’ = pnl,po96

Flax Ramie Jute Cotton Kapk Coir Sisal Linen, modern Linen. ancient Egyptian”

6(CCC) ring 6(CCC) ring, twisting

252 VW 171 mw 115 mw

z(COH) r(COH)

303 w

bands to v(COC) glycosidic link stretching modes in the cellulose, which forms the basis of all samples studied here. However, an examination of the intensity ratios of the 1096 and 2900 cm-’ bands, R, reveals a more complicated picture. Although the ancient Egyptian and modern linens have nearly the same R-value, namely 1.10 f 0.02, those of the natural fibres and cotton occupy a wider range of values, namely 0.32-1.63. This is to be expected, since the R-value must reflect

Sample

346 mw 320 w

0.72 0.70 0.77 0.79 0.89 0.96 0.73 0.76 0.75

R = I’096/12900

0.85 1.63 0.92 0.75 0.35 0.32 0.91 1.12 1.08

* Egyptian XIIth Dynasty mummy wrapping from ‘Tomb of the Two Brothers’, ca 2000 y BC, excavated by Flinders Petrie in 1906 at Der Rifeh, Nile Valley [18].

contributions to the spectrum in each sample from pure cellulose (cotton) to cellulosic material with varying content of waxy and lignin material in the natural fibres themselves. It is interesting from Table 3 that the two natural fibres whose R’-values deviate significantly from 0.74 are kapok and coir-it is these fibres which also have anomalously low R-values of 0.35 and 0.32, respectively. It is tempting to suggest that a possible explanation of these results is a significantly different cellulosic structure for the kapok and coir whose fibres contain 90 and 64% cellulose content, respectively (Table 1); a cellulose structure of different rigidity would be expected to affect not only the symmetric:antisymmetric stretching bands of the p-(1,4)-glycosidic linkage, but also perhaps a different scattering factor compared with the other cellulosic fibres studied here e.g. cotton and jute, which resemble closely the cellulose compositions of kapok and coir, yet whose R'- and R-values are 0.79, 0.77 and 0.75, 0.92, respectively. The natural fibres in the present work have been studied without any pre-treatment either of a chemical or mechanical nature; this minimises the introduction of changes in cellulose structure through processing or storage. Degradation of cellulose would be expected to produce a decrease in the 1’o96 component through fission of the

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v(COC) glycosidic linkages and an increase in the 12goo feature due to new v(CH) modes arising from the decomposition products; this could be a useful parameter for the study of ancient fibres. The similarity of the R' and R' values for 4000year-old and modern linens here indicates that the ancient fibres are in extremely good condition chemically (Table 3). The FT-Raman spectroscopic technique seems to be suitable for the non-invasive examination of archaeological fabrics and ancient fibres and may provide a method for the distinction between a number of highly morphologically damaged materials. From these preliminary studies of the v(CH) stretching region and v(COC) and 6(CH,) regions of the Raman spectra, the ageing of ancient cellulose-based materials could be investigated at the molecular level. It is well known that under acidic [24] conditions, hydrolysis and oxidation of the /3-glycosidic link between anomeric C, and 0 occurs, resulting in a shortening of the cellulose chains and in the formation of oxycelluloses. Also, cellulose is readily attacked by the cellulolytic enzymes of micro-organisms [25]; the enzymatic cleavage of cellulose is catalysed by cellulase. Firstly, en&-p - 1,4 glucanases break the p-1,4 bonds, then exo-p-1,4 glucanases remove cellobiose units from the chain ends (cellobiose consists of two glucose units linked by a glycosidic link), finally P-glucosidases break the glycosidic link in cellobiose to form flucose [24]. In conclusion, the results of our study of natural, untreated plant fibres using FT-Raman spectroscopy are seen to provide a method for the non-destructive characterisation of these materials and indicates future possibilities for the application of this technique to archaeological fabrics.

References [l] E.J.W. Barber, Prehistoric Textiles, Princeton University Press, Princeton, NJ, USA, 1991, pp. 12.

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PI D. Zohary, M. Hopf, Domestication of Plants in the Old World, ch. 4, Oil and Fibre Plants. Clarendon Press. Oxford. 1988. [31 E.J.W. Barber. Neolithic textiles in europe and the Near East, Archeomaterials 4 (1990) 63. [41 A. Standen (Ed.). Kirk-othmer’s Encyclopedia of Chenucal Technology, 2nd ed.. vol. 9. Wiley, New York. 1965, p. 171. PI Identification of Textile Materials, 6th ed.. The Textile Institute. Manchester, 1970, p. 16. I61 E.J. Jumper, R.W. Mottern, Appl. Optics 19 (1980) 1909. [71 SF. Pellicori. Appl. Optics 19 (1980) 1913. PI J.S. Accetta, J.S. Baumgart, Appl. Optics 19 (1980) 1921. [91 R. Gilbert. M.M. Gilbert. Appl. Optics 19 (1980) 1930. UOl Th.N. Kleinert, Holzforschung 26 (1972) 46. [ill D. Nikolova, I. Svetkova, S. Pishmanova. Izv. Khim. 24 (1991) 382. 1121R.D. Gillard, S.M. Hardman, R.G. Thomas, DE. Watkinson, Stud. Conserv. 39 (1994) 132. Fat-well, C.A. Roberts, A.C. P31 H.G.M. Edwards, D.W. Williams, in: J. Bertie, H. Wieser (Eds.), Proceedings of the 9th International Conference on Fourier-Transform Spectroscopy, Calgary, Canada, SPIE. Washington, USA. 2089, 1993. pp. 256. u41 H.G.M. Edwards, A.C. Williams, D.W. Farwell, Biospectroscopy 1 (1995) 29. s51 A.C. Williams, H.G.M. Edwards, B.W. Barry, Biochim. Biophys. Acta 1246 (1995) 98. 1161B.W. Barry, A.C. Williams, H.G.M. Edwards, Pharm. J. 254 (6828) (1995) 217. [I71 H.G.M. Edwards, A.c. Williams, D.W. Farwell, B.W. Barry, F. Rull Perez, J. Chem. Sot. Faraday Trans. 91 (1995) 3883. WI H.G.M. Edwards, E. Ellis, D.W. Farwell, R. Jannaway. J. Raman Spectrosc. 27 (1996) 663. 1191 H.G.M. Edwards, M.J. Falk, Appl. Spectrosc. (in press), PO1I.P. Keen, G. White, P.M. Fredericks, Proceedings of the 2nd Australian Conference on Vibrational Spectroscopy, Brisbane, October, 1996, p, 87. 1211H.G.M. Edwards. D.W. Farwell, A.C. Williams, Spectrochim. Acta Part A 50 (1994) 807. [22] J.G. Grasselli. B.J. Bulkin, Analytical Raman Spectroscopy, Wiley Interscience, New York, 1991. [23] P.R. Carey, Biochemical Applications of Raman and Resonance Raman Spectroscopies, Academic Press, New York, 1992. [24] J.M. Cardamone, KM Deister, A.H. Osareh, Degradation and Conservation of Celluloses and esters, in: N.S. Allen, M. Edge, C.V. Horie (Eds.), Polymers in Conservation, Royal Society of Chemistry, London, 1991, p. 111. [2S] H.G. Schlegel, General Microbiology, Cambridge University Press, Cambridge, 1986, p. 407.