514 Volume 57, Number 5, 2003 APPLIED SPECTROSCOPY0003-7028 / 03 / 5705-0514$2.00 / 0q 2003 Society for Applied Spectroscopy

Monitoring Decay of Black Gum Wood (Nyssa sylvatica )During Growth of the Shiitake Mushroom(Lentinula edodes ) Using Diffuse Re� ectanceInfrared Spectroscopy

CHRISTOPHER H. VANEBritish Geological Survey, Kingsley Dunham Centre, Keyworth, Nottingham, NG12 5GG, United Kingdom

Abstract diffuse re� ectance infrared spectroscopy (DRIFT) and el-emental analysis were employed to monitor biodegradation of blackgum wood (Nyssa sylvatica ) during growth of the shiitake mushroom(Lentinula edodes ). Black gum was decayed for up to 4.3 years byL. edodes, during which time it was sampled at 19, 31, and 52months. Biodegraded woods displayed increased % O (w/w) anddecreased % C (w/w) relative to the undecayed control. The DRIFTspectra of decayed black gum showed a decrease in relative inten-sity of absorption bands at 1735 cm21 assigned to carboxyl func-tional groups from xylans and an increase in the absorption bandat 1640 cm21 assigned to conjugated carbonyl groups originatingfrom lignin. Xylan decay was rapid initially but slowed after 19months; however, oxidative decay of the lignin side chains occurredthroughout the 52-month decay period. Overall elemental andDRIFT data show that both polysaccharides and lignin were de-cayed during cultivation of the edible white-rot fungus.

Index Headings: Diffuse re� ectance infrared spectroscopy; DRIFT;Lentinula edodes ; Black gum; Lignin; Polysaccharide; Shiitake.

INTRODUCTION

The shiitake mushroom (Lentinula edodes) accountsfor about 25% of world mushroom production and is cul-tivated on either arti� cial substrate or hardwood logs.1

Subsequent use of spent logs as horticultural compost andorganic fertilizer is partly dependent on the release ofelemental nutrients to plants such as N, which is limitedin residues with high lignin content due to shielding ef-fects.2 Therefore, monitoring changes in chemistry of thedifferent cell wall components during growth can poten-tially provide a useful indictor of the residual quality.Comprehensive wet chemical and analytical methods areavailable for the analysis of polysaccharides and aromaticcomponents of wood, but together they are time consum-ing.3–8 A more attractive strategy for the rapid chemicalanalysis of fresh and decayed wood is to use diffuse re-� ectance infrared spectroscopy (DRIFT).9–31

The effect of the white-rot fungus L. edodes on beechwood under laboratory conditions has been previouslymeasured using Fourier transform infrared spectroscopy.This showed that the fungus preferentially decayed ligninas compared to polysaccharides and suggested that thexylans, as estimated by changes in the absorption bandat 1506 cm21 relative to 1740 cm21, had remained unal-tered.18 However, the methodologies employed in labo-ratory studies are optimal for wood decay and cannot bedirectly extrapolated to commercial log-grown systems,which are subject to different inoculation procedures and

Received 10 May 2002; accepted 6 December 2002.

variable temperature and humidity.1,18 The aim of this re-search was to investigate whether there is any preferentialdecay of wood cell wall components during growth of L.edodes under a commercial regimen.

EXPERIMENTAL

Cultivation of L. edodes . Fifty black gum trees (Nyssasylvatica) were harvested and each tree was cut into ap-proximately 5 logs with a length of 1 m and a width of10 cm; the moisture content of each log was maintainedat approximately 35% (w/w) by the addition of water.The trees were harvested 5 miles from the inoculationsite. Inoculation was achieved by drilling 30 holes perlog at a depth of 2.5 cm in a diamond pattern and packingwith fresh spawn of L. edodes.

After completion of the spawn run (;6 months) thecolonized logs were placed outside where (natural fruit-ing 2–3 times per year) the temperature range was 7.2 to21.1 8C. Samples of black gum sapwood (;5 g dryweight) were removed at 0, 19, 31, and 52 months. Eachsample was washed with sterile distilled water to removesurface mycelium and freeze-dried to remove water.

Elemental Analysis. The organic carbon, hydrogen,and nitrogen content of black gum prior to and after treat-ment with L. edodes was determined in quadruplicate us-ing a Carlo Erba 1106 elemental analyzer. Ash contentwas determined gravimetrically following combustion ofthe dry powdered wood at 650 8C for 18 h. Blanks andsamples were interchanged in order to account for pos-sible instrumental drift. Oxygen content was determinedby difference.

Diffuse Re� ectance Infrared Spectroscopy. Spectrawere obtained using a Bio-Rad FTX3000MX series FTIRspectrometer (Digilab Div., Bio-Rad Laboratories, Cam-bridge, MA) and a diffuse re� ectance attachment (PikeTechnologies Inc. Madison, WI). Woods (10 mg) and 210mg of KBr (FT grade, Aldrich Chemical Co.) were � nelyground for 45 s in a Wig-L-Bug ball mill. The homog-enized mixtures were immediately transferred to thestainless steel sample holder, their surface smoothed witha razor blade, and DRIFT spectra recorded. Diffuse re-� ectance infrared spectrometry was determined from4000 to 500 cm21 with 2 cm21 resolution, and 100 scanswere determined in each acquisition. Sample spectra werereferenced against a powdered KBr matrix at the sameinstrument setting. The baseline was corrected to the re-gions at 800, 2000, and 3800 cm21,16 and peak area in-tegration was obtained with the integration limits going

APPLIED SPECTROSCOPY 515

FIG. 1. DRIFT spectra for (a) undegraded control black gum (Nyssasylvatica), (b) black gum degraded for 19 months by L. edodes, (c)black gum degraded for 31 months by L. edodes, and (d ) black gumdegraded for 52 months by L. edodes.

TABLE I. Wavenumbers and tentative assignments for predominant vibrations in black gum wood.

Wavenum-ber

(cm 21) Tentative assignment Structural polymer

3338 OH stretch of phenolic OH and or aliphatic OH Cellulose, xylans, lignin2930 Asymmetric CH stretch of –CH2– Lignin2840 Symmetric CH stretch of –CH2– Lignin1735 –C5O stretch of H-bonded –COOH (unconjugated) Xylans, cinnamic acids1640 Aromatic C5C stretch and/or asymmetric –COO2 stretch Lignin, cellulose1594 Aromatic C5C Lignin1504 Aromatic C5C Lignin1458 Aliphatic C–H deformation of CH2 and CH3 Lignin1421 C alkyl–O ether stretch of interunit methoxyl and b-O-4, aromatic C5C Lignin1371 C–H deformation symmetric Cellulose1322 C aryl–O ether stretch of intraunit methoxyl and b-O-4 Lignin1238 C–O stretch and OH deformation of COOH and or C aryl–O ether stretch of intraunit

methoxyl and b-O-4Cellulose, xylans, lignin

1151 C–O–C bridge stretching (asymmetric) Cellulose1044 C–O stretch skeletal vibrations Cellulose, Lignin898 Out-of-phase ring stretching (asymmetric) Cellulose, Lignin781 Aromatic CH out-of-plane bending Lignin

to the constructed baseline. The integration areas mea-sured were: 898 cm21 (921–880), 1047 cm21 (1096–994),1235 cm21 (1266–1215), 1327 cm21 (1339–1311), 1371cm21 (1385–1360), 1421 cm21 (1430–1410), 1456 cm21

(1471–1448), 1504 cm21 (1522–1494), 1594 cm21 (1605–1572), 1640 cm21 (1650–1630), and 1735 cm21 (1799–1702).

RESULTS AND DISCUSSION

Spectroscopic Characterization of BiodegradedBlack Gum Wood. The DRIFT spectra of black gum

control (0 months) and after 19, 31, and 52 months cul-tivation with L. edodes are shown in Figs. 1a–1d, andtentative wavenumber assignments are presented in TableI. After 19 months of treatment with L. edodes, the peakat 1735 cm21 decreased in intensity relative to otherbands (Fig. 1b). A change in intensity of the absorptionband at 1735 cm21 relative to that at 1504 cm21 was mea-sured by the 1735/1504 cm21 ratio.18 The 1735/1504 cm21

ratio of the control was 0.94; this decreased to 0.69 aftercultivation for 19 months and remained unchanged after31 and 52 months (Table II). The fall in 1735/1504 cm21

values in the L. edodes decayed samples can be explainedby a decrease in xylans. These � ndings are contrary tothe constant intensity of the absorbance band at ;1740cm21 assigned to xylans observed throughout 98 days ofgrowth of L. edodes under laboratory conditions.18 How-ever, the xylan degradation observed in this current in-vestigation is consistent with 13C NMR and neutral sugarstudies of naturally biodegraded woods, which demon-strated that xylans were decayed to a greater extent thancellulose or lignin.3 For cultivation times greater than 19months, the apparent degradation of xylans must be in-hibited or their loss must occur at a similar rate to thatof lignin. On the other hand, the observed decrease in1735/1504 cm21 values could also be attributed in part toa loss of cinnamic acids, which contain similar function-ality. Thermochemolysis with tetramethylammonium hy-droxide (TMAH) of wheat straw during commercialgrowth of the Oyster mushroom (Pleurotus ostreatus) for63 days revealed a decrease in cinnamic acid derivatives,namely trans-3-(4-methoxyphenyl) acrylic acid, methylester, and trans-3-(3,4-dimethoxyphenyl) acrylic acid,methyl ester.32 Unfortunately it is not possible fromDRIFT data acquired in this study to determine changesin lignin content in black gum woods treated with L. edo-des due to masking of aromatic skeletal vibrations at1594 cm21 caused by the large increase in the peak at1640 cm21 (Figs. 1a–1d). Although lignin content of ex-tractive free woods can be determined as the residue re-maining after treatment with 72% H2SO 4 (Klason Lig-nin), it has been reported that fungal decay increases lig-nin solubility in acid and can yield low values.33 For these

516 Volume 57, Number 5, 2003

TABLE II. Relative intensity ratios determined from FT-IR ab-sorption bands for fresh and L. edodes-decayed black gum.

Decay time(months)

1735/1504cm21

1504/1640cm21

0193152

0.800.690.690.69

0.940.460.410.27

TABLE III. Time-dependent changes in elemental compositions inblack gum degraded by L. edodes.

Decaytime

(months) % N % C % H % O C : N

0193152

0.1 6 0.040.1 6 0.030.1 6 0.010.3 6 0.01

46.8 6 0.0244.3 6 0.0744.6 6 0.0440.6 6 0.01

6.0 6 0.0025.9 6 0.015.6 6 0.275.4 6 0.12

47.149.749.653.8

468443446135

reasons, both DRIFT spectroscopy and solubility-basedgravimetric determinations of lignin content in fungallydecayed woods can be misleading. However, in this cur-rent study the carbon and hydrogen elemental analysis offresh (C 5 46.8%, H 5 6.0%) and decayed black gum(e.g., 52 months: C 5 40.6%, H 5 5.4%) suggested thatthe L. edodes decayed woods have a relatively higherlignin content than the fresh counterpart because ligninhas a higher C/H ratio than do polysaccharides (TableIII). These changes in elemental composition con� rmedthat polysaccharides were decayed by L. edodes and thissupports the notion that the fall in 1735/1504 cm21 valueswas caused by a decrease in xylan content during fungalgrowth. The increase in intensity of the absorption bandat 1640 cm21 relative to the absorption band at 1504 cm21

was measured by the 1504/1640 cm21 ratio (Table II).18

The 1504/1640 cm21 value for fresh black gum was 0.94;then, after 19 months of treatment, this decreased to 0.46.With longer cultivation times (31 and 52 months) the1504/1640 cm21 values decreased further to 0.41 and0.27, respectively. The evident increase in the absorptionat 1640 cm21 probably originates from an enrichment ofconjugated carbonyl groups in biodegraded lignin (TableI). Increases in conjugated carbonyl groups from ligninalso occur in beech wood when decayed by L. edodesunder laboratory conditions. The systematic accumula-tion of oxygen from 47.1% to 53.8% with decay sug-gested that the biodegraded black gum woods had beentransformed in part via an oxidative process (Table III).A similar relative intensity increase of the band at 1322cm21 assigned to COO2 ions was also observed withgrowth of L. edodes (Fig. 1).18 These data support thehypothesis that lignin side-chain oxidation had been me-diated by L. edodes. The formation of side-chain oxida-tion products during degradation of phenolic model com-pounds by manganese peroxidase extracted from L. edo-des has been previously reported.34,35 These studiesshowed that biodegradation proceeded via the formationof benzyl alcohol, aldehyde, and carboxylic acid, in ad-dition to minor demethylation of methoxyl groups at-tached to aromatic rings. A similar increase in lignin unitswith ketone and aldehyde functional groups due to oxi-dation at the Ca atom has been observed with progressivenatural decay of Calluna vulgaris shoots and in soils un-der Scots pine by pyrolysis gas chromatography massspectrometry (Py-GC/MS).36,37 Organic matter treatedwith fungi can contain residual mycelium32 and elevated% N content. Here the absence of absorption bands at1542 cm21 C–N stretching (amide II) suggested that thecontribution of fungal biomass is minor. This is supportedby the moderate % N (wt/wt) content of the decayedwoods as compared to the fresh analogue (Table III).There is a decrease in C:N values with increasing decay

time (Table III). Fresh black gum had a C:N value of468; this decreased to 443 and 446 for 19 and 32 months,respectively, and then after 52 months decay decreasedto yield a C:N value of 135 (Table III). An importantchemical characteristic of mature composts is a low C:Nvalue of ;25; therefore, logs decayed for up to and in-cluding 52 months require further microbial degradationand or mixing with other constituents prior to use as hor-ticultural compost or organic fertilizer. This � nding is im-portant because application of organic matter with highC:N values to soils can cause a reduction in availabilityof N for plants due to activity of soil microbes.2

The spectra of decayed black gum woods show an in-crease in the 779 cm21 band with cultivation time, whichmay be associated with loss of polysaccharides duringmushroom growth, thereby enriching the black gum res-idue in aromatic units. The changes in spectra suggestedthat xylans and cellulose were decayed in preference tolignin; however, even after 52 months of decay some ofthe polysaccharide component remains. These resultscontrast with previous laboratory-based studies, which re-ported the preferential decay of lignin as compared topolysaccharide components during growth of L. edodes.18

The different pattern of decay by L. edodes on black gummay be explained by (1) the long decay period of up toand including 52 months used in this study, (2) differ-ences in wood chemistry between black gum and beechwoods, and (3) the effect of growing shiitake on logsoutdoors as compared to wood blocks in the laboratory.

CONCLUSION

The main chemical transformation during commercialgrowth of L. edodes on logs of black gum was the decayof polysaccharides. Xylan degradation was rapid up toand including 19 months of growth and then slowed. Acomplete loss of polysaccharides was not observed, pos-sibly because of shielding from fungal attack by the re-maining lignin. The lignin was partially degraded by ox-idative cleavage of Ca–Cb bonds, but preferential deg-radation of lignin as compared to polysaccharides suchas that previously reported in laboratory studies of white-rot fungi was not detected.18 It is concluded that L. edodesdegraded black gum via simultaneous degradation of cel-lulose, xylan, and lignin. The chemical composition ofthe residue remaining after 52 months of shiitake growthis not suf� ciently mature for use as horticultural compostor organic fertilizer without further treatment.

ACKNOWLEDGMENTS

The author is indebted to Tom Kimmons and co-workers, Ozark Spe-ciality, Shirley, AR, USA, for collection of fresh and decayed blackgum, and thanks Ian Harrison, British Geological Survey, for helpful

APPLIED SPECTROSCOPY 517

comments regarding the manuscript. This paper is published by per-mission of the Executive Director, British Geological Survey (NERC).

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