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Bioresource Technology 99 (2008) 2736–2741

Evaluation of waste mushroom logs as a potential biomass resourcefor the production of bioethanol

Jae-Won Lee a, Bon-Wook Koo a, Joon-Weon Choi b, Don-Ha Choi b, In-Gyu Choi a,c,*

a Department of Forest Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Koreab Department of Wood Chemistry and Microbiology, Korea Forest Research Institute, Seoul 130-712, South Korea

c Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, South Korea

Received 6 March 2007; received in revised form 3 July 2007; accepted 3 July 2007Available online 14 August 2007

Abstract

In order to investigate the possibility of using waste mushroom logs as a biomass resource for alternative energy production, thechemical and physical characteristics of normal wood and waste mushroom logs were examined. Size reduction of normal wood(145 kW h/tone) required significantly higher energy consumption than waste mushroom logs (70 kW h/tone). The crystallinity valueof waste mushroom logs was dramatically lower (33%) than normal wood (49%) after cultivation by Lentinus edodes as spawn. Lignin,an enzymatic hydrolysis inhibitor in sugar production, decreased from 21.07% to 18.78% after inoculation of L. edodes. Total sugaryields obtained by enzyme and acid hydrolysis were higher in waste mushroom logs than in normal wood. After 24 h fermentation,12 g/L ethanol was produced on waste mushroom logs, while normal wood produced 8 g/L ethanol. These results indicate that wastemushroom logs are economically suitable lignocellulosic material for the production of fermentable sugars related to bioethanolproduction.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Biomass; Waste mushroom logs; Lentinus edodes; Hydrolysis; Bioethanol

1. Introduction

As energy consumption rises along with global popula-tion, alternatives to fossil resources must be developed.To solve these problems, many researchers have tried toconvert environmentally-friendly biomass into fuel ethanolfor an alternative to fossil fuels (Cheung and Anderson,1997; Lee et al., 2001a,b). In several technologicallyadvanced countries such as the US and Japan, researchand propagation policies support the promotion of alterna-tive future energy sources.

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.07.003

* Corresponding author. Address: Department of Forest Sciences,College of Agriculture and Life Sciences, Seoul National University,Seoul 151-921, South Korea. Tel.: +82 2 880 4785; fax: +82 2 873 2318.

E-mail address: cingyu@snu.ac.kr (I.-G. Choi).

Biomass has received special attention as a new materialfor alternative energy. In particular, lignocellulosic materialsuch as woody biomass is regarded as a promising energysource because it is renewable and consists of abundant car-bohydrates (Sun and Cheng, 2002; Soderstrom et al., 2003).In addition, agronomic residues such as corn stover, sugar-cane waste, wheat or rice straw and forestry residues arealso on the rise as potential materials that can be convertedinto fuel ethanol (Badal et al., 2005; Kitchaiya et al., 2003).

The production of waste mushroom logs, which arewoody residues in shiitake mushroom production, isincreasing annually in Korea (Statistical Yearbook of For-estry, 2005). Some of these logs are consumed in traditionaluses such as industrial fuel for boilers. However, mostwaste mushroom logs which are more than three years pastinoculation are discarded and disposed off by burning inopen fields (Kim and Lee, 1994). It is therefore worthwhileto investigate potential uses for such logs.

J.-W. Lee et al. / Bioresource Technology 99 (2008) 2736–2741 2737

Lentinus edodes, commercially one of the most impor-tant edible basidiomycetes in Korea, is used as spawn forshitake mushroom production, traditionally on freshlycut logs of Quercus mongolica. The fungus produces hydro-lytic and oxidative enzymes responsible for the degradationof wood depending on substrate composition and environ-mental conditions (Hong et al., 1986; Lee et al., 2005; Mataand Savoie, 1998; Sakamoto et al., 2005). Many researchershave reported that the lignin degradation enzymes manga-nese peroxidase and laccase are secreted from L. edodes,while lignin peroxidase apparently does not play a signifi-cant role in its ligninolytic enzymatic system (Buswellet al., 1995; Makkar et al., 2001). White-rot fungi, includ-ing L. edodes, degrade lignin, the mainly ether-linked phe-nylpropanoid biopolymer found in wood cells, eitherselectively or in parallel with polysaccharides (Kirk andFarrell, 1987).

Waste mushroom logs have advantages for conversion tofermentable sugar for bioethanol production because oftheir carbohydrate content and the degradation of lignin(Christopher et al., 2003). Removal of the lignin, whichinhibits enzymatic hydrolysis, and some part of hemicellu-lose from the lignocellulosic biomass, provides an accessiblesurface area for enzyme or chemical reactions for hydrolysis.

The degradation of mushroom log components bysecreted enzymes from L. edodes provides advantageousconditions for the physical, chemical and biological conver-sion of biomass. Biomass with these conditions maydemand mild pretreatment or no pretreatment for hydroly-sis. Consequently, waste mushroom logs are thought to bea promising source for producing ethanol by fermentationbecause they are cheaper, more easily hydrolysable.

In this study, we evaluated the potential of waste mush-room logs as a biomass resource for the production of bio-ethanol based on the physical and chemical characterizationof waste mushroom logs.

2. Methods

2.1. Biomass material

Q. mongolica, as both normal wood and waste mush-room logs three years after shiitake mushroom cultivation,was obtained from a mushroom farm located in Hwasung-

si, Gyeonggi-do in South Korea. The normal wood wasreduced to 40 mesh wood powder using a milling machine.Waste mushroom logs were washed with sterile distilledwater to remove surface mycelium and ground to a powderof 40 mesh. The powders were dried and the moisture con-tent reduced to less than 10%. The wood powders wereused for physical and chemical analysis as well as acidand enzymatic saccharification.

2.2. X-ray diffraction analysis

The measurement of crystallinity in the normal woodand waste mushroom logs was carried out by powder High

Resolution X-ray Diffractometry (HR-XRD, Bruker D8DISCOVER, Germany) as described by Segal (Segalet al., 1959). Crystallinity (%) was defined as [(I002�Iam)/I002] · 100, where I002 is the crystalline peak of the maxi-mum intensity at 2h between 22� and 23� for cellulose I,and Iam is the minimum intensity at 2h between 18� and19� for cellulose I. The measurement conditions were40 Kv, 40 mA and a scanning speed of 0.75/min.

2.3. Analysis of biomass components

The chemical analysis of biomass was performed usingTAPPI test methods. Insoluble and soluble lignins (Klasonlignin) were determined by acid treatment. Acid soluble lig-nin was determined by UV-spectrometry at 205 nm for sol-uble fraction after insoluble lignin extraction using H2SO4.Moisture content (T207 om-88), cellulose (TAPPI UsefulMethod 249, Wise method), ash (T211 om-93), alkaliextractives (T212 om-93) and Klason lignin (T222 om-88)of waste mushroom logs were compared to comparablevalues for normal wood.

2.4. Energy consumption for size reduction of biomass

A chipper was used for size reduction of normal woodand waste mushroom logs. Dimension lumber was pre-pared in pieces of 8 cm · 8 cm · 100 cm. The samples weredried prior to milling to a water content in the range of 4–7%. Specific energy requirements were calculated by timeconsumption in relation to amount of biomass. This energycorresponds only to that required for size reduction, notfor movement of the mill alone.

2.5. Analysis of nitrobenzene oxidation products of biomass

The change in lignin structure was analyzed by nitro-benzene oxidation of biomass. Three mg of normal woodand waste mushroom logs, respectively, and 4 ml of 2 MNaOH were mixed with 250 ll nitrobenzene, and the mix-ture was reacted at 170 �C for 2 h (Iiyama and Lam,1990). 3,5-Dimethoxy phenol was used as an internal stan-dard. After addition of the internal standard, the mixturewas extracted with 20 mL of dichloromethane, and theaqueous fraction adjusted to pH 1–2 with 4 M HCl. Theaqueous fraction was extracted again with dichlorometh-ane and this extraction step was repeated three times.The phenolic components obtained from lignin in the sol-vent fraction were trimethylsilysilated with 50 ll of pyri-dine and N,O bis(trimethylsilyl) trifluoro acetamide(BSTFA) of 50 ll at 60 �C for 2 h. The derivatives wereapplied to a HP 6890 Series GC System equipped with aDB-5 capillary column (60 cm · 0.25 mm, 0.25 lM film,J&W Scientific). GC conditions were as follows: injectortemperature 250 �C, detector temperature 280 �C and tem-perature program 160–250 �C with a heating rate of 2 �Cper min.

Fig. 1. Change in the crystallinity value of normal wood (dotted line) andwaste mushroom log (solid line) by HR-XRD.

2738 J.-W. Lee et al. / Bioresource Technology 99 (2008) 2736–2741

2.6. Determination of released sugars by hydrolysis

In acid hydrolysis, a biomass of 0.5 g was hydrolyzedwith 5 ml of 72% sulfuric acid at room temperature for1 h. The acid was diluted to a final concentration of 3%and heated at 121 �C for 1 h. The residual material wascooled and filtered through a porous glass filter. The quan-tification of glucose, mannose, galactose, xylose and arab-inose as monosaccharides in the soluble fraction wasdetermined by high-performance anionic exchange chro-matography (HPAEC) (Dionex Bio-LC50 system;Sunnyvalle, CA, USA). Carbo Pac PA10 Columns(4 · 250 mm) and an ED50 pulsed amperometic detector(PAD) were used. Analysis was performed with 3 mMNaOH as the mobile phase at 0.8 mL/min for 45 min.

The enzymes used for enzymatic hydrolysis were Cellu-clast 1.5 L (Novo Co., Denmark), cellulase prepared by aculture solution of Trichoderma reesei, and Novozym 188(Novo Co., Denmark) as b-glucosidase. A biomass of 1 gwas transferred to a 250 ml Erlenmeyer flask, and 100 mlof 50 mM sodium acetate buffer (pH 5.0) was added. Next,appropriate amounts of cellulase (350 EGU/g) and b-glu-cosidase (376 IU/g) were added. The flask was put in ashaking incubator at 50 �C and 150 rpm and incubatedfor 48 h and 72 h (Soderstrom et al., 2005). After hydroly-sis, incubation supernatants of 2 ml were centrifuged andfiltered through a 0.45 lM filter, and the solution was ana-lyzed for monosaccharides as previously described in acidhydrolysis. Hydrolysis experiments were performed intriplicate.

2.7. Simultaneous saccharification and fermentation (SSF)

To compare ethanol yield, normal wood and wastemushroom logs were pretreated with 1% H2SO4 at 120 �Cfor 1 h, respectively. Biomass collected after pretreatmentwas separated by filtration into a solid residue and liquid.The solid residue was thoroughly washed with water andthen dried. For fermentation, Saccharomyces cerevisiae(ATCC 24858) was cultured on YPD medium (yeastextract 1%, peptone 2%, dextrose 2%) at 30 �C, 180 rpmfor 24 h. After pretreatment, in the SSF process, 15 g ofhydrolyzate and 50 mL of nutrient medium were mixedand then 50 mM sodium citrate buffer (pH 5.5) was added.Nutrient medium components were (NH4)2HPO4 (1.0 g/L),MgSO4 Æ 7H2O (0.05 g/L) and yeast extract (2 g/L).

The mixture of hydrolyzate and nutrient medium wasautoclaved at 120 �C for 15 min and, then commercial cel-lulases, Celluclast 1.5 L (350 EGU/g) and Novozyme 188(376 IU/g) were added to the reaction mixture. Then, S.

cerevisiae was inoculated at a cell concentration ofOD600 = 2. SSF was started by adding enzymes and S.

cerevisiae, and then incubated at 37 �C in a shaker at180 rpm. Samples were withdrawn after 6 h, 12 h, 24 h,36 h and 48 h, and analyzed for ethanol yield.

Ethanol concentration was determined using am HPLC(Hewlett Packard, HP 1100, USA) equipped with an Amin-

ex HPX-87P column (Bio-rad) and refractive index detec-tor (RID). The mobile phase was 0.01 N H2SO4, the flowrate of the mobile phase was 0.5 mL/min, and the analysiswas done under isocratic mode.

3. Results and discussion

3.1. Physical characteristics

3.1.1. X-ray diffraction analysis of biomass

The crystallinity value of normal wood was estimated at49%, while that of waste mushroom logs was only 33%(Fig. 1). The decrease in crystallinity value suggests thatcrystalline cellulose has been disrupted by cellulasessecreted by L. edodes. These findings agree with the obser-vation that cellulases from L. edodes are activated in thepresence of crystalline cellulose (Lee et al., 2001a,b; Honget al., 1986). The fungal treatment may make the b-gluco-sidic bonds of cellulose more easily accessible to cellulase inenzymatic hydrolysis. Therefore, waste mushroom logsdecrystallized by secreted cellulases from L. edodes maybe suitable biomass on application of acid or enzymatichydrolysis for the production of sugars.

3.1.2. Energy consumption

In order to produce ethanol from lignocellulosic bio-mass, size reduction of the biomass is required. Energyconsumption needed for size reduction was estimated bychipper (Laura and Gerardo, 1989). Energy consumptionof normal wood was compared with that of waste mush-room logs. Waste mushroom logs (70 kW h/ton) requiredsignificantly less energy than normal wood (145 kW h/ton) for size reduction, resulting in an energy savings of51.72%. Waste mushroom logs required less energy dueto the decrease of crystallinity and lignin content bysecreted enzymes from L. edodes. It has been suggested thatfungal treatment by L. edodes disrupts the wood structureby partially breaking down the lignin and carbohydratecomplex (Messner and Srebotnik, 1994). Based on energyconsumption, waste mushroom logs are an economicallyfeasible source for bioethanol production.

Table 1Determination of lignin, holocellulose, ash and alkali extractives ofnormal wood (cultivation period: 0) and waste mushroom logs (cultivationperiod: 3 year)

Cultivationperiod (year)

Solublelignin (%)

Insolublelignin (%)

Holo-cellulose (%)

Ash(%)

Alkaliextractives(%)

0 0.6 17.9 61.7 0.2 19.73 1.2 15.7 58.7 0.7 24.3

Chemical components are based on oven dry weight of biomass.

Table 3Yields of monosaccharides and total sugar of normal wood and waste

J.-W. Lee et al. / Bioresource Technology 99 (2008) 2736–2741 2739

3.2. Chemical characteristics

The chemical components of normal wood and wastemushroom logs are shown on Table 1. During the cultiva-tion of L. edodes, holocellulose and lignin degraded almostsimultaneously.

L. edodes growth degraded lignin corresponding to aweight loss from 17.9% to 15.7%, due to enzymes relatedto lignin degradation such as manganese peroxidase andlaccase (Crestini et al., 2000). This result corresponds to areport that the lignin network covering the holocelluloseis broken down by fungal treatment (Sawada et al.,1995). However, the content of acid-soluble lignin in wastemushroom logs was higher than that of normal woodbecause lignin was degraded to acid-soluble phenolic com-pounds of low molecular weight by ligninases. The contentof alkali extractives increased 19.7–24.3% after inoculationwith L. edodes, implying that carbohydrate and lignin deg-radation products of low molecular weight were producedby extracellular enzymes of L. edodes during cultivation.

The yield of nitrobenzene oxidation products is pre-sented on Table 2. The sum of the amounts of syringalde-hyde and syringic acid was noticeably lower in the wastelogs. However, the sum of the amounts of vanillin andvanillic acid was only slightly reduced. The decrease in S/G ratio upon fungal degradation can be explained by theenhanced susceptibility of syringyl moieties to degradationbecause they have fewer aryl–aryl bonds and a lower redoxpotential than their guaiacyl counterparts (Tai et al., 1983).

Collectively, these chemical characterizations of wastemushroom logs indicate that such logs have better poten-tial for hydrolysis than normal wood, and may require onlymild or no pretreatment before hydrolysis.

Table 2Yields of nitrobenzene oxidation products from normal wood and wastemushroom logs

Cultivation period(year)

Nitrobenzene oxidation(mg/g)

Total(mg/g)

S/Gratio

Guaiacyltype

Syringyltype

VN VA SD SA

0 27.41 1.35 60.85 3.07 92.68 2.223 22.95 1.05 27.92 3.30 44.62 1.36

Vanillin (VN), vanillic acid (VA), syringaldehyde (SD), syringic acid (SA),S/G ratio ((SD + SA)/(VN + VA)).

At least three replicates were used in all analytical deter-minations, although only the average values are presentedhere. All results were statistically tested by standard devia-tion and variation coefficient, not exceeding 5% of thiscoefficient.

3.3. Sugar yield

Condition of optimal sugar production was also differ-ent because normal wood and waste mushroom logs haddifferent component ratio. Normal wood required highertemperature, pressure and longer reaction time than thoseof waste mushroom log for hydrolysis. In this study, sugaryield was investigated on the same condition for compari-son of normal wood and waste mushroom logs.

The yields of glucose and other monosaccharides basedon dry weight biomass after acid and enzymatic hydrolysisare shown on Table 3. Depending upon the type of sub-strate, significantly different yields were obtained for bothglucose and hemicellulose monosaccharides. Waste mush-room logs released approximately 136 mg/g glucose,61 mg/g xylose, 2.7 mg/g galactose, 1.7 mg/g mannoseand 1.3 mg/g arabinose per biomass dry weight by acidhydrolysis. The highest sugar yield of hydrolysis on thesubstrates was 40.64%, when using acid hydrolysis onwaste mushroom logs. In enzymatic hydrolysis, the sugaryield after 72 h of enzymatic reaction was not very differentfrom the 48 h reaction, and so results from the 48 h reac-tion are shown. The sugar yield of waste mushroom logs(32.02%) was higher than that of normal wood (24.81%).The high yield of xylose obtained from waste mushroomlogs by enzymatic hydrolysis indicates that hemicelluloseswere converted to an easily degradable structure by fungaltreatment with L. edodes. Waste mushroom logs in bothhydrolysis processes had greater production of glucoseand other monosaccharides than did normal wood. Thefaster conversion of waste mushroom logs to fermentablemonosaccharides could be due to reduced crystallinityand lignin content following cultivation of L. edodes (Hsu

mushroom logs by HPAEC after acid and enzymatic hydrolysis usingH2SO4 and commercial enzymes

Treatment Monomeric sugars (%) Total sugars (%)

Ara Gal Glu Xyl Man

Acid-normal 0.19 0.28 18.20 10.11 0.14 29.82Acid-waste 0.26 0.54 27.20 12.30 0.34 40.64Enzyme-normal 0.44 0.70 20.35 2.97 0.36 24.81Enzyme-waste 0.62 0.79 22.95 7.16 0.50 32.02

Yield of monomeric sugars are based on oven dry weight of biomass.Arabinose (Ara), galactose (Gal), glucose (Glu), xylose (Xyl), mannose(man), acid hydrolysis of normal wood (acid-normal), acid hydrolysis ofwaste mushroom logs (acid-waste), enzymatic hydrolysis of normal(enzyme-normal), enzymatic hydrolysis of waste mushroom log wood(enzyme-waste).

2740 J.-W. Lee et al. / Bioresource Technology 99 (2008) 2736–2741

and Penner, 1991). Additionally, these conditions allowedmore favorable access of enzymes and acids to potentialcleavage sites for hydrolysis (Baker et al., 1997). Manyresearchers reported that sugar yield from biomass wasaffected by hydrolysis factors such as temperature andretention time (Szczodrak, 1987; Sharma et al., 2002;Leathers, 2004). In this study, a higher yield of total sugarscould be obtained by optimizing conditions on enzymatichydrolysis. Acid hydrolysis was not performed at optimalcondition. The highest sugar yield will be obtained whenhydrolysis are performed at optimal condition.

The larger amounts of glucose and other monosaccha-rides that can be obtained from waste mushroom logsmay be advantageous for ethanol production because glu-cose and other monosaccharides can be converted to etha-nol with higher yields (Dien et al., 2003).

As the result of chemical components on Table 1, wastemushroom log generated loss of holocellulose. In acid andenzymatic saccharification, the increase of glucose concen-tration was relatively lesser than that of monosaccharidesinduced from hemicellulose after acid and enzyme hydroly-sis. However, glucose and monosaccharides induced fromhemicellulose are fermentable sugars for ethanol produc-tion. Therefore, the results confirm that waste mushroomlogs are more easily hydrolysable to glucose as well asmonosaccharides induced from hemicellulose.

3.4. Ethanol fermentation from biomass

The concentration of ethanol increased with time,regardless of carbon source, until 24 h of SSF reactiontime. The maximum ethanol concentration was 12 g/Land 8 g/L on waste mushroom logs and normal wood at24 h, respectively (Fig. 2). After 24 h, ethanol concentra-tion was slightly lower on both carbon sources. Wastemushroom logs produced more ethanol than normal wood,implying that waste mushrooms logs could be a good mate-rial for bioethanol production as an alternative energysource.

Fig. 2. Ethanol production by S. cerevisiae from normal wood and wastemushroom log depending on SSF reaction time after pretreatment using1% H2SO4. Square: waste mushroom logs, Triangle: normal wood.

4. Conclusion

This study shows that waste mushroom logs showpotential for development as an economical alternativeenergy source because of easy conversion to fermentablesugars by various hydrolysis methods and low energy con-sumption during size reduction. Additionally, waste mush-room logs were ready for hydrolysis with only mild or nopretreatment because of efficient pretreatment by extracel-lular enzymes of L. edodes during cultivation, leading towith lower lignin content and crystallinity. Finally, ethanolproduction was higher on waste mushroom logs than onnormal wood.

These results suggest that waste mushroom logs are apotential biomass resource for bioethanol production asan alternative energy source.

As further study, heterogeneity of waste mushroom logsis regulated and problems about collection as well as con-veyance of biomass must be resolved for practical applica-tion in a real industrial case.

Acknowledgements

We are grateful for the graduate fellowship provided bythe Ministry of Education through the Brain Korea 21 Pro-ject. This work was also supported by the Korea EnergyManagement Corporation and Korea Forest ResearchInstitute.

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