Journal of Analytical and Applied Pyrolysis, 21 ( 1993) 1~ 14 Elsevier Science Publishers B.V., Amsterdam

Analysis of leaf and fine root litter from a subtropical montane rain forest by pyrolysis-gas chromatography/mass spectrometry ’

Guido C. Galletti a-*, James B. Reeves III b, Janine Bloomfield ‘, Kristiina A. Vogt ’ and Daniel J. Vogt ’

” Istituto di Microbiologia e Tecnologia Agraria e Forestale, University of Reggio Calabria, 89061 Gallina, Reggio Calabria (Italy) h Ruminant Nutrition Laboratory, United States Department of Agriculture, Beltsville, MD 20705 (USA) ’ School of Forestry and Environmental Studies, Yale University, 370 Prospect St., New Haven, CT 0651 I (USA)

(Received October 10, 1992; accepted in final form February 15, 1993)

ABSTRACT

As a preliminary part of a larger study on litter decomposition in tropical forests, pyrolysis-gas chromatography/mass spectrometry, Fourier transform-infrared spectroscopy (FT-IR) and conventional wet chemistry methods were used to determine the composition of tropical leaf and fine root tissue, and the quality of the tissue acting as a substrate for decay. Wet chemistry fiber analysis of Sierra Palm (Prestoea montana) and Tabonuco (Dacryodes exceka) samples suggested high total lignin contents which were not supported by the other techniques. Results showed a high amount of phenol being produced during the pyrolysis of Sierra Palm, and lower total lignin content than that measured by wet chemistry. Typical FT-IR spectra and pyrograms with peak identification and quantitative data are reported.

Fine root tissue; leaf tissue; lignocellulose; litter decomposition; pyrolysis; pyrolysis-gas chromatography/mass spectrometry; tropical rain forest.

INTRODUCTION

The decomposition of plant litter, i.e. leaves, fine roots and wood, is an essential process in the cycling of carbon and nutrients in ecosystems. The long term fertility of a site is, in part, determined by the quantity and quality of its soil organic matter pool and the way in which nutrients are held or released by humifying materials.

* Corresponding author. ’ Presented at: Pyrolysis ‘92 - Proceedings of the 10th International Conference on Funda- mental Aspects, Processes and Applications of Pyrolysis, Hamburg, Germany, September 28-Qctober 2, 1992.

0165-2370/93/$06.00 0 1993 ~ Elsevier Science Publishers B.V. All rights reserved

G.C. Galletti et al. / J. Anal. Appl. Pyrolysis 27 (1993) l-14

In addition to abiotic climatic factors such as temperature and moisture, substrate quality is considered a key predictive factor in modeling and estimating decay rate [ 11. Total nitrogen, total carbon and lignin content along with abiotic factors have been shown to be strongly correlated to decay rate in many temperate and tropical ecosystems worldwide [2]. In these studies, tissues generally decayed the fastest under abiotic conditions of high temperature and high moisture, and with substrate quality repre- sented by low carbon, low lignin, and high nitrogen concentrations.

Structural polymers (mainly cellulose and lignin), primarily located in the plant cell wall, are routinely measured by crude wet chemistry techniques developed by agricultural scientists interested in the quality of feeds to be digested by ruminants [3-51. Traditionally, cell wall polymers, espe- cially lignin, have eluded accurate and fast chemical characterization owing to their intrinsic association with a tight matrix, which has made it extremely difficult to isolate each component without altering its chemical composition [6]. The techniques, designed for grasses, have been applied by ecologists to fast growing tissues (leaves and roots) of a wide variety of trees and plants in both temperate and tropical ecosystems. Tropical tissues in particular contain large amounts of secondary compounds in- cluding flavonoids, polyphenols and tannins, which in most cases have not been accurately identified and quantified and that could interfere with the measurements.

We questioned the reliability of conventional wet chemistry techniques for two reasons: (i) studies on the accuracy and selectivity of the techniques for determining lignin suggested serious methodological problems may exist [7]; (ii) lignin contents reported for fine roots are higher than would have been predicted on the basis of their anatomy and physiology. Commonly reported amounts of lignin in a range of wood types are 25-35% [8], while the greatest amount of lignin ever measured in secondary xylem compres- sion wood, a highly lignified tissue, was 46% [9]. Young, fast growing meristematic tissue such as fine roots would be expected to have little or no lignin [lo], yet values of 20-60% lignin in tree fine roots have been reported in a number of separate studies [ 111.

Accurate estimation of plant tissue chemical composition is extremely important when comparing tissue from different genera and ecosystems, especially since different amounts of interfering substances (e.g. phenols and cell wall bound proteins) may modify the results obtained with each technique. Techniques are now being proposed that use wet chemistry results as calibrations for near infrared (NIR) studies involving the remote sensing of canopies [ 121. If the original results overestimate lignin contents of tissues, use of the NIR approach could cause serious errors.

In the last decade, a number of physical methods have enabled a faster and more accurate elucidation of the molecular composition of cell wall biopolymers. These methods include nuclear magnetic resonance (NMR)

G.C. Galletti et al. / J. Anal. Appl. Pyrolysis 27 (1993) l-14 3

either in solution or in the solid state 113,141, Fourier transform-infrared (FT-IR) spectroscopy [ 153, pyrolysis-mass spectrometry (Py-MS) [ 16- 181 and pyrolysis- gas chromatography~mass spectrometry (Py-GC~MS) [6,19,20-221. These micro-methods of analysis have shown great potential in ecological work because only a small amount of sample is needed for a wide variety of chemical component determinations. In decay studies, in particular, there is often only an extremely small quantity of plant material remaining after a long decomposition period.

Py-CC/MS is based upon a quantitatively reproducible thermal degrada- tion of macromolecules into smaller neutral molecules, usually within a molecular weight range measurable by gas chromatography, and their simultaneous separation and identification by a GC/MS instrument to which the pyrolzyer unit is connected. By this technique, the organic matter composition of forest litter and spruce needles from northern Europe has been exhaustively investigated [23,24].

In the present study, Sierra Palm (Prestoeu montana) and Tabonuco (Dacryodes excetsa) leaves and fine roots, used as the initial tissues to be placed in decomposition litterbags in a subtropical wet forest in Puerto Rico, were analyzed by conventional wet chemistry assays for the cell wall fibrous constituents, and by Py-GC/MS and FT-IR. The objective was to determine the chemical composition of the samples and to evaluate the usefulness and reIiability of different analytical techniques.

MATERIAL AND METHODS

Site description

Samples were collected from the Tabonuco forest zone within the Luquillo Experimental Forest located in the Luquillo Mountains in north- eastern Puerto Rico (65”45’W, 18’17’N) as part of a larger study measuring the decay rates of leaves and fine roots over the forest landscape. The two species used in this study, Tabonuco (Dacryodes excelsa) and Sierra Palm (Prestoea montana), dominate the Tabonuco forest zone, which is classified as a subtropical montane wet forest climate [25] and is typified by a consistently high mean monthly air temperature (21-24”Q high relative humidity (96- 100%) and high rainfall (approx. 4000 mm annually)

FN.

Sample collection and storage

Large amounts of leaves and fine roots of Palm and Tabonuco were collected for a study of decomposition in June, 1989. Tabonuco foliage was collected as senesced leaf litter from the forest floor and air-dried. Because Palm foliage begins to decay while still attached to the tree, Palm leaflets

4 G.C. Galletti et al. / J. Anal. Appl. Pyrolysis 27 (1993) l-14

were collected from the most mature green fronds of individual trees. Fine roots ( <2 mm in diameter) of Tabonuco were collected from above-ground mats while Sierra Palm roots were collected from root systems growing into and in intermittent stream beds. All roots were carefully washed to remove adhering soil and organic matter debris. From these homogenized pools of tissue, subsamples for initial substrate quality analysis were taken. These subsamples were oven dried at 70°C and ground using a Tecator cyclotec mill.

Wet chemistry analysis

Neutral detergent fiber (NDF), acid detergent fiber (ADF), 72% sulfuric acid lignin (ADL) and permanganate lignin were determined according to the methods of Goering and Van Soest [5]. Basically, NDF and ADF are the residues of hot neutral and hot acid detergent extractions, respectively. Permanganate ligin is the substance removed from ADF by treatment with permanganate leaving cellulose as the residue. ADL is the substance remaining after treatment of ADF with concentrated sulfuric acid at room temperature, which should selectively dissolve cellulose and leave lignin as the residue. Dry matter was determined as 105°C oven dried material. Total nitrogen was determined by combustion using an LECO C/H/N elemental analyser.

FT-IR spectroscopy

The infrared spectra of tissues were taken using a Bio-Rad FTS-65 Fourier transform spectrometer from 400 to 4000 cm-’ (4 cm-’ resolution) using diffuse reflectance with samples diluted to 5% with KBr.

Py - GC/MS

An aliquot (l-2 mg) of the sample was pyrolyzed in a quartz tube at 600°C using a CDS Pyroprobe 100 heated filament pyrolyzer coupled to a Varian 3400 capillary gas chromatograph, which was in turn interfaced to a Finnigan MAT ion trap detector (ITD) model 800 mass spectrometer. The pyrolyzate was swept directly (split l/120) into the gas chromatographic column (Supelco SPB-5 (SE54 type), 30 m x 0.32 mm id., 0.25 pm film thickness) operated from 50 to 250°C at S”C/min holding the initial temper- ature for 10 min. The injector was set at 250°C and the GC/ITD transfer line at 220°C. ITD was operated with software release 4.0 and mass spectra were recorded from 40 to 350 m/z under electron impact at 70 eV. Pyrolysis fragments were identified by mass spectra interpretation, and by comparison with available data [21,27,28].

G.C. Galletti et al. /J. Anal. Appl. Pyrolysis 27 (1993) l-14 5

RESULTS AND DISCUSSION

Wet chemical results

The wet chemical results are presented in Table 1. Several points of interest stand out. First, the lignin values are high for both roots and leaves, especially with the 72% sulfuric acid method. High lignin values have been reported in the literature for fine roots using the same or similar techniques [8]. However, fast growing meristematic tissues do not contain a large amount of secondary xylem which is where the greatest amount of lignin is found in the plant cell wall [ 111. Microscopic examination of the fine roots using phloroglucinol, a stain specific for lignin, showed some lignified cell wall but an extremely narrow band of secondary xylem compared with that found in wood [ 291. Lignin values for feeds (which the extraction techniques are designed to measure) are much lower: wheat straw lignin values are 15.2 and 7.6% as determined by permanganate and sulfuric acid methods, respectively [21]. Lignin values for peanut hull, a very hard material compared with the roots examined in the present study, are about 20% by the permanganate method and about 35% by the sulfuric acid method, the latter including some cutin [21].

Because of the apparent overestimation of lignin values, it was decided to investigate the composition of the material in question by other non-chem- ical based methodologies, namely FT-IR spectroscopy and, subsequently, Py-GC/MS.

TABLE 1

Initial wet chemistry data for the tissues used in the decomposition study (mean values f one standard deviation, n = 3). Mean values with the same letter are not significantly different by rows using Tukey’s Studentized range test. (NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, 72% sulfuric acid lignin; N, total nitrogen)

NDF( %)

ADF( %)

Permanganate lignin( %)

ADL( %)

N(mg/g)

Leaves

P. montana

69.1 a

(0.8)

48.5 a

(4.6)

16.1 a

(2.9)

15.9 a

(0.5)

12.0 a

( < 1)

D. excelsa

45.4 b

(0.2)

36.2 b

(1.8)

11.8 a

(1.2)

13.4 b

(0.5)

5.7 b

(<I)

Roots

P. montana

83.0 c

(0.5)

68.0 c

(0.8)

26.8 b

(2.5)

33.7 c

(0.5)

9.0 c

(cl)

D. excelsa

71.5 d

(0.2)

55.3 a

( 1 .O)

28.7 bc

(1.7)

45.1 d

(0.7)

16.0 d

( < 1)

6 G.C. Galletti et al. 1 J. Anal. Appi. Pyrolysis 27 (1993) I- 14

FT-IR results

Figure 1 shows the typical FT-IR spectra obtained for Sierra Palm leaves: (a) control (no treatment), (b) ADF, (c) permanganate lignin, and (d) ADL. Similar spectra were recorded for the other tissues but are not presented here since the results were comparable with those for the Palm leaves.

The region 3000-3500 cm-’ is dominated by a broad absorption (O-H stretching) of little diagnostic significance. The carbonyl absorption bands at 1724 and 1659 cm-’ are typical of esters and proteins, respectively, while the aromatic skeletal vibration at 1603 cm-’ is assigned to lignin. The absorption bands in the 1050 cm-’ region (C-O stretching), typical of alcohols or phenols, and in the 600-500 cm-’ region (C-H out-of-plane deformation), typical or aromatics, are also significant.

Some spectral variations are evident when examining the different cell wall fractions. The changes in the 1724 (esters), 1659 (proteins) and 1603 cm-i (lignin) bands were as expected from studies performed on other lignocellulosic materials [21], i.e. ADF was greatly reduced in protein and esterified materials and enriched in lignin, while permanganate residue lost the lignin and residual protein, and ADL retained both lignin and protein. The changes in the 1050 and 600-500 cm-’ bands were not as clear and no further conclusions can be drawn.

The question, then, is what these spectra can tell us about the composi- tion of these samples and, specifically, whether the lignin values found using chemical procedures are reasonable considering the FT-IR spectra. Absorp- tion bands assignable to carbohydrates, proteins and lignin, i.e. the main constituents of plant cell wall, could be found in the FT-IR spectra of Sierra

Fig. 1. FT-IR spectra from 4000 to 400 cm-’ at 4 cm-’ resolution for Sierra Palm leaf (a) control, (b) acid detergent fiber (ADF), (c) permanganate lignin residue and (d) 72% sulfuric acid lignin (ADL).

G.C. Gulletti et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) I- 14 I

Palm and Tabonuco leaves and roots. On a relative basis, such bands did not appear to differ markedly from those found in li~ocellulosic by-prod- ucts such as hay, hulls and straw [21], whose lignin values as measured by wet chemistry techniques were much lower than those of the tissues in this study. Since protein was extracted with lignin in both the permanganate and the acid detergent methods, the high amount of nitrogen in the fine roots could add to the overestimation of lignin (Table 1).

These observations seem to corroborate the earlier suggestion that lignin values obtained using conventional chemical analysis are too high. How- ever, a larger set of samples and appropriate chemometric techniques would possibly be needed for FT-IR alone to prove that lignin values were overestimated by conventional wet chemistry methods or to determine the exact factors contributing to the overestimations. To further identify these factors, all tissues were analysed using Py-GC/MS.

Py - GC/MS results

Figure 2 shows the pyrograms of (a) Sierra Palm leaves and (b) Ta- bonuco leaves. The root samples gave rise to similar patterns, the only differences being the relative abundances of the peaks and not their chemi- cal nature. Only those peaks that produced an interpretable mass spectrum above the background noise were considered. The identifications proposed

(4

RIC

(b) ’ ’ 12’00 . ’ ’ 20bo ’ ‘SCAN’

Fig. 2. Pyrograms of (a) Sierra Palm leaf and (b) Tabonuco leaf.

8

TABLE 2

G.C. Galletti et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) l-14

Proposed identification of the pyrogram peaks

# Scan mw Formula Name

1 137 2 14.5 3 154 4 170 5 182 6 212 7 301 8 326

9 347

10 361 11 380 12 467

13 760

14 768

15 789 16 917

17 967 18 992

19 1021

20 1105 21 1140

22 1152

23 1243

24 1254

25 1264

26 1273 27 1321

28 1343 29 1406 30 1443

31 1477

32 1495

33 1529 34 1554

35 1562

36 1580 37 1609 38 1638 39 1702 40 1730 41 1795 42 1807 43 1855

44 1863 45 1885

44 _

co* _ _ _ 102 W-&O, 60 C&,0, 74 WW, 82 G&O

_ - _ - 102 C&O, 84 GH,O, 96 W-LO, 84 GH,O, 98 GH,ON,(?) 98 GH,O, 86? CJ%OA‘3

110 C&O, 110 G H,O% 94 C&O

114 GH,O, _ _ 128? GH,,O,(?) 114 CsH@, 108 GHsO _ _ 124 GH,O, _ _ 126 C,H,oO, 114 G&O, 100 CJ,O, _ _ _ _ 138 GH,oO, 144 GJW, 120 GH,O _ _

_ _

140

150 154 164 _

131 152

Carbon dioxide Unknown Unknown Acetic anhydride Acetic acid Hydroxypropanone 1,4-Pentadien-3-one Unknown Unknown Pyruvic acid methyl ester (3H)-Furan-2-one 7 2-Furaldehyde t

(SH)-Furan-2-one t (lH)-4-Methanolimidazole (?) (3H)-5-Methylfuran-2-one t Tetrahydrofuran-2-one t (?) SMethyl-2-furaldehyde t ( 1 H)-2-Methylpyrimidin-4-one Phenol 5,6-Dihydro-4-hydroxy-(2H)-pyran-2-one t Unknown Tetrahydrodimethylpyranonet (?) n,n-Dihydro-n-hydroxypyranone t

4-Methylphenol * Unknown 2-Methoxyphenol *

Unknown 3-Hydroxy-2-methyl-(4H)-pyran-4-one t n,n-Dihydro-n-hydroxypyranone t (3H)-Dihydro-4-methylfuran-2-one t Unknown Hydrocarbon (?) 4-Methyl-2-methoxyphenol * 1 :4,3:6-Dianhydro-[a-D]-glucopyranose t 4-Vinylphenol * Unknown Unknown Unknown 3-Methoxy-2-hydroxyphenol * Unknown 4-Vinyl-2-methoxyphenol * 2,6-Dimethoxyphenol * 4-( 2-Propenyl)-2-methoxyphenol * Unknown 3-Methylindole 4-Hydroxy-3-methoxybenzaldehyde *

G.C. Galletti et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) l-14

TABLE 2 (continued)

# Scan mw Formula Name

46 1895 47 1903 48 1919 49 1956 50 1966 51 1988 52 1995 53 2019 54 2086 55 2097 56 2107 57 2146 58 2190 59 2203 60 2275 61 2298 62 2319 63 2331 64 2346 65 2374 66 2447 67 2476 68 2522 69 2532 70 2555 71 2579 72 2638 73 2656 74 2682

164

168 164

_

162 182 180

180

194 194 182 _ _

194 _

210

Cd,,% _ _

GH,,O, G,H,A _ _

C,H,oO, C,oH,,O, C,oH,,O, _ C,oH,,O, _ C,, HMO, C,,H,,O, C,H,oO, _ _ C,,H,,O, _

C, I HMO, _

c&4-( 1 -Propenyl)-2-methoxyphenol * Unknown Unknown 4-Methyl-2,6-dimethoxyphenol * tram-4-( 1 -Propenyl)-2-methoxyphenol * Unknown Unknown 1,6-Anhydro-[p-D]-glucopyranose t 4-Ethyl-2,6-dimethoxyphenol * 4-( 2-Propanone)-2-methoxyphenol * Unknown 4-Vinyl-2,6-dimethoxyphenol * Unknown 4-( 2-Propenyl)-2,6-dimethoxyphenol * cis-4-( 1-Propenyl)-2,6-dimethoxyphenol * 4-Hydroxy-3,5_dimethoxybenzaldehyde * Unknown Unknown tram-4-( 1 -Propenyl)-2,6-dimethoxyphenol * Unknown

4-( 2-Propanone)-2,6-dimethoxyphenol * Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown

* Compounds ascribed to lignin. 7 Compounds ascribed to polysaccharides.

in Table 2 are based on published [21,23,24,27] and unpublished [28] studies were relevant mass spectra are also reported. Finally, Table 3 presents the quantitative analysis for each sample expressed as peak percentage relative to the entire pyrogram.

At a first glance (Table 2), the pyrolysis of both Sierra Palm and Tabonuco tissues appeared to produce the fragments typically obtained from plant cell walls [21,27]. Accordingly, the pyrograms were subdivided into: (a) an initial area with carbon dioxide, acetic anhydride, acetic acid and other low molecular weight fragments, some of which remained uniden- tified, that represent some of the smallest moieties obtainable by pyrolysis and are therefore of little diagnostic interest; (b) a second section with

10 G.C. Galletti et al. 1 J. Anal. Appl. Pyrolysis 27 (1993) l-14

TABLE 3

Quantitative analysis of the pyrogram peaks (relative %). For peak identification, see Table 1

# P. montana

leaf root

D. excelsa

leaf root

1 6.13 3.46 4.68 6.05 2 0.94 1.13 0.62 1 .oo

3 4.81 4.59 3.13 3.81

4+5 16.55 14.15 21.39 17.59 6 2.12 2.25 1.31 1.73

7 tr. tr. tr. tr.

8 2.50 2.23 2.03 2.32

9 0.35 0.42 0.16 0.55

10 1.47 1.49 0.89 1.27

11 0.41 0.39 0.76 0.23

12 0.90 0.84 1.53 0.50

13 0.05 0.08 0.06 0.06

14 0.18 0.20 0.19 0.12

15 1.21 1.19 1.15 1.51

16 0.70 0.82 1.21 0.38

17 0.29 0.26 0.51 0.24

18 6.79 13.05 0.28 0.47

19 2.50 2.84 3.70 1.27

20 1.28 0.63 0.50 0.48

21 0.17 0.17 0.21 0.22

22 0.47 0.61 0.31 0.26

23 0.15 0.42 0.18 0.18

24 0.45 0.40 0.66 0.11

25 0.60 1.25 0.65 2.76

26 0.54 0.48 0.31 0.37

27 0.30 0.30 0.79 0.09

28 0.23 0.21 0.21 0.25

29 0.42 0.48 0.32 0.32

30 1.97 4.39 1.05 1 .oo

31 1.85 2.08 0.99 0.23

32 0.59 1.17 1 .oo 2.83 33 0.56 0.81 0.89 0.27

34 0.41 0.15 0.44 nd.

35 0.42 0.41 0.49 0.60

36 0.24 0.15 0.20 nd.

37 0.73 0.74 2.10 0.10

38 1.86 1.52 1.15 0.64

39 0.73 0.51 2.59 0.36

40 2.22 3.37 2.98 6.03

41 1.38 1.37 0.92 1.29

42 0.16 0.26 0.30 0.78

43 0.47 0.52 0.27 0.14

44 tr. tr. nd. nd.

G.C. Galletti et al. /J. Anal. Appl. Pyrolysis 27 (1993) I --I4

TABLE 3 (continued)

D. excelsa

leaf root leaf root

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

0.17 0.25 0.27 0.57

0.08 0.17 0.15 0.48 0.17 0.18 0.11 0.04

0.42 0.24 0.78 0.51 0.21 0.21 0.37 nd. 0.45 1.29 1.05 6.64 0.32 0.27 nd. nd. 0.27 0.46 0.19 nd.

13.85 9.93 19.51 12.15 0.16 0.09 0.22 0.17

0.15 0.54 0.31 0.65 0.78 0.59 1.25 0.27 2.67 2.02 2.27 1.92 0.31 0.31 0.32 1.10 0.57 0.40 0.34 0.39 0.41 0.27 0.58 0.74 0.51 0.44 0.35 0.56 0.42 0.51 0.41 0.92 0.09 0.07 0.13 nd. 2.03 2.16 2.12 4.24 0.46 nd. 0.27 nd. 0.44 0.25 0.49 0.82 0.14 1.87 nd. nd. 2.77 0.88 1.35 0.17 0.30 nd. 0.21 nd. 0.30 0.42 0.18 0.21 1.26 0.13 0.68 nd. 0.29 nd. nd. nd. 0.25 0.23 0.13 0.49 1.11 1.38 1.27 2.52

compounds of furanic and pyranic nature, such as furaldehydes, furanones and pyranones, which can be ascribed to the structural polysa~ha~des of the cell wall; (c) a third segment containing phenolics attributable to lignin. Some protein markers are also evident in the pyrograms, such as 4- methylphenol (peak 23) which is known to be produced by pyrolysis of both lignin and tyrosine, and a trace of 3-methylindole (peak 44), a product of the pyrolysis of tryptophan. A number of the identified compounds (Nos. 11, 12, 15, 16, 18, 19, 23, 25, 34, 44-46, 50 and 55, Table 2) have been detected by Hempfling and Schulten [23] in forest humus.

More remarkable features appear when examining the quantitative data summarized in Table 3. For example, phenol (peak 18) ranged from 6.79 to

12 G.C. Galletti et al. / J. Anal. Appl. Pyrolysis 27 (1993) l-14

13.05% respectively in Sierra Palm leaves and roots. These percentages are abnormally high when compared with both the Tabonuco samples and other lignin-containing materials [ 6,18,2 1,301 including plant litter in forest humus [23] where phenol has either been only a minor constituent or not been detected at all. Further preliminary Py-GC/MS experiments aimed at investigating the origin of this compound showed that (i) it was still present in acid detergent fiber residues, and (ii) it was removed by hot alkali extraction. These observations ruled out that phenol was present in the samples as a free molecule and suggested that it may be included in an alkali-hydrolyzable structure, or it may be the pyrolysis product of a flavonoid. For instance, 1,2_dihydroxybenzene (catechol) is the main pyrol- ysis product of condensed tannins and catechin [ 311, and compounds of similar structure having a phenolic instead of a catechol moiety yield phenol under pyrolysis [32]. It is therefore legitimate to suspect that in the present samples phenol was derived not from lignin but from other phenolic substances [ 331.

When quantities of fragments were summed, the percentages of phenolic fragments were 22.01 and 30.65 for Sierra Palm leaves and roots respec- tively, and 16.42 and 32.16 for Tabonuco leaves and roots respectively. Such figures, although they include phenol which might not be derived from lignin, fall well within the range of values reported elsewhere [21] for other lignocellulosic materials, including soybean hull (the corresponding phenolic percentage of the total pyrogram being 9.2%), wheat straw (20.4%) peanut hull (35.8%) and rice hull (37.1%). However, the permanganate lignin determination in these samples led to results ranging from 5.7% for the soybean hull to 13.3% for wheat straw, 14.7% for rice hull, and 21.8% for peanut hull, which are on average lower than the results reported in Table 1 for Sierra Palm and Tabonuco roots, i.e. 26.8 and 28.7%, respectively. This is surprising, considering that the tissues in the above feeds are more lignified than the fine roots and leaves in question.

CONCLUSION

Py-GC/MS and FT-IR corroborated the earlier comments about the seemingly unreasonably high lignin values obtained using conventional wet digestion techniques for both Sierra Palm and Tabonuco fine root tissues. The differences in lignin content between roots and leaves (about twice as high in roots as in leaves) obtained by permanganate oxidation were similar to the differences in percentage of phenolic fragments measured by Py-GC/MS. However, a large portion of the phenolic material from Sierra Palm samples was accounted for by phenol which is difficult to ascribe to lignin. Therefore some of the “lignin” measured by the wet chemistry techniques, especially in the Palm tissue, is probably of some other chemical composition.

G.C. Gall& et al. 1 J. Anal. A&. Pyrolysis 27 (1993) L-14 13

With respect to pure phenolic percentages, those obtained by Py-CC/ MS for the Palm and Tabonuco leaves, and fine roots are near the upper limit of those found for lignocellulosic materials such as wheat straw and peanut hulls, which could expain the high lignin values reported by wet chemistry techniques. However, wheat straw and peanut hulls were both lower in permanganate lignin and much more “woody” in nature than the leaves and fine roots used in this study. The phenolic substances in the Palm and Tabonuco tissues could result in higher wet chemistry lignin values both by interfering with the extraction of cellulose, protein and/or lignin during the permanganate oxidation or sulfuric acid digestion, and by co-extracting with lignin and thereby being measured as lignin. This could explain some of the discrepancies and shows the need for more accurate lignin measurements.

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