ANTIFUNGAL EFFICACY OF ENVIRONMENTALLY FRIENDLY WOOD PRESERVATIVESFORMULATED WITH ENZYMATIC-HYDROLYZED OKARA, COPPER, OR BORON SALTS

HO-YONG KIM,y HAN-SEOB JEONG,y BYEONG-CHEOL MIN,y SYE HEE AHN,z SEI CHANG OH,zYOUNG-HO YOON,§ IN-GYU CHOI,yk and IN YANG*k

ySeoul National University, Seoul, South Korea

zDaegu University, Gyeongsan, South Korea

§KCI, Seosan, South Korea

kResearch Institute for Agriculture and Life Sciences, Seoul National University, Seoul, South Korea

(Submitted 19 October 2010; Returned for Revision 17 December 2010; Accepted 28 January 2011)

Abstract—Okara, an organic waste product obtained from soy milk production, was used with copper chloride or sodium borate toformulate new wood preservatives as a substitute for expensive wood preservatives, such as copper-azole–based preservatives andammoniacal copper quaternary. Before formulating the preservatives, okara was hydrolyzed by enzymes (cellulase, pectinase, andprotease) to augment penetration and fix the biocide salts of the preservatives into wood blocks. The preservatives were injected intowood blocks by vacuum pressure to measure the treatability of the preservatives. The treated wood blocks were placed in hot water for3 d to measure leachability. The treatability and leachability of the preservatives were affected by the type and loading amount ofenzymes and the addition of sodium borate into okara-based wood preservative formulations. The treatability and leachability of thepreservatives formulated with copper chloride and okara hydrolysates were 63.38 and 3.15%, and those of the preservatives with copperchloride, okara hydrolysates, and sodium borate were 61.47 and 3.32%, respectively. Despite the hot water leaching, wood blocks treatedwith preservatives formulated with 2% cellulase, pectinase, and protease hydrolyzed okara, CuCl2, and sodium borate showed only1.98% average weight loss against Fomitopsis palustris over 12 weeks. Microscopic observation revealed how okara-basedpreservatives work in wood blocks. Okara has potential as a raw material for cost-effective and environmentally friendly woodpreservatives. Environ. Toxicol. Chem. 2011;30:1297–1305. # 2011 SETAC

Keywords—Okara Wood preservatives Leachability Decay resistance Scanning electron microscope

INTRODUCTION

Copper-based wood preservatives have been used for morethan 200 years. Among them, chromate copper arsenate (CCA)has been used extensively for wood preservation for more than60 years. However, the market has changed drastically in thepast decade because of environmental concerns regarding leach-able components of CCA, such as Cr and arsenate [1]. Chromatecopper arsenate was completely banned in the European Unionand limited to nonresidential uses in the United States [2]. As aresult, CCA alternatives such as ammoniacal Cu quaternary(ACQ), Cu bis-(N-cyclohexyldiazeniumdioxy) (Cu xyligen),amine-Cu formulations [3–5], and Cu-azole–based preservatives(CuAz) [6] have been developed and used in several countries.However, CCA alternatives have been more expensive thanCCA and are characterized by increased leaching of Cu fromwood products [7]. Leached Cu or other components may beharmful to human health and the environment ([8,9]; http://www.ccaresearch.org/Pre-Conference/pdf/Weis.pdf).

Thus, current research on efficient, environmentallyfriendly, and cost-effective wood preservatives have led tothe use of renewable resources for the fixation of variousantifungal salts. For example, lignin [10], tannin [11,12], animalblood [13], and albumin and milk casein [14] have been used toenhance the fixation of antifungal salts in wood structures. Soyprotein also has been used to effectively fix Cu or B salts in anew preservative system that showed good efficacy against

brown-rot fungi [15]. However, such renewable resources aredifficult to implement as fixatives in newly developed preser-vative systems in Korea because of their high cost and rarity.

Okara, a byproduct of soy milk production, was chosen as aninexpensive and readily available fixative for this study. During2007, approximately 700,000 tons of okara were generated fromthe production of 470,000 tons of soy milk in South Korea [16].Okara waste is typically dumped or incinerated but can beobtained at no cost save that associated with delivery. Okarawas used as a fixative in our preservative systems because of itschemical composition. Dry okara contains approximately 20%proteins, and the components can be used to chelate salts toform water-insoluble complexes if properly modified [17].

Among salts that inhibit fungal growth, B salts have recentlyreceived considerable attention because of their low humantoxicity and excellent antifungal efficacy. However, the use of Bsalts has been limited because of their high water solubility. Saltfixation in wood structures may be augmented with the additionof sufficient Cu to B salt preservative formulations [18], as wellas through the application of wax [19] or pyrolytic resins [20] toB-containing wood preservatives. Building on these studies, theobjectives of this study were to formulate wood preservativescontaining okara, Cu, or B; measure the treatability and leach-ability of the preservatives; and determine the efficacy of thepreservatives against fungal decay.

MATERIALS AND METHODS

Preservative formulations

In our preservative formulations, copper chloride (CuCl2 �2H2O, CC) or sodium borate (Na2B4O7 � 10H2O, B) were used

Environmental Toxicology and Chemistry, Vol. 30, No. 6, pp. 1297–1305, 2011# 2011 SETAC

Printed in the USADOI: 10.1002/etc.515

* To whom correspondence may be addressed(dahadad2000@yahoo.com).

Published online 4 March 2011 in Wiley Online Library(wileyonlinelibrary.com).

1297

as antifungal salts. Okara was obtained from CJ Food and usedas a fixative for Cu and B salts. Okara was hydrolyzed byCelluclast 1.5 L FG (cellulase produced from Trichodermareesei, CEL), Pextinex 5 XL (pectinase and arabanase producedfrom Aspergillus niger, PEC), and Alcalase 1.5 L (proteaseproduced from Bacillus microorganism, ALC) to increase thechelating reaction of okara with antifungal salts. The enzymeswere purchased from Enzyme-Tech.

For hydrolysis, okara was dried in a 658C oven for 24 h andthen passed through a grinder. Okara (6 g) was dispersed indeionized water (300ml), and then the pH/temperature of thedispersion was adjusted to 5.5/508C for CEL, 4.5/508C for PEC,and 8.3/508C for ALC. Each enzyme was added into thedispersion, and the mixtures were incubated in a shaker(Lab-Line-Environ-Shaker; Lab-Line Instrument) at 508C for6 h. To examine the effects of the enzyme on the treating andleaching properties and decay resistance of okara-based woodpreservatives (OK-WPs), the three enzymes were used eitherseparately or together. The amount of each enzyme wasadjusted to 0, 2, and 4% (v/w) based on the dry weight ofokara. The CC or B was added to the okara hydrolysates (EOK).In the suspension, the weight ratio of CC to EOKwas 1 to 1, andthat of CC to B was 1 to 0.5. Target retentions of CC, EOK, andB into wood blocks were 16, 16, and 8 kg/m3, respectively.Before the wood blocks were treated, 20ml ammonium hydrox-ide was added to the suspension to improve penetration of thepreservatives by swelling the cell walls of the wood blocks.

Treating wood blocks

Scots pine (Pinus sylvestris) sapwood blocks (2.54� 2.54�2.54 cm) were oven dried at 1058C and weighed. Twelve woodblocks were placed in a plastic bottle with OK-WP formulationsunder vacuum (500mmHg) for 20min and subsequently treatedby 12 kgf/cm2 pressure for 20min in a pressure cylinder.To obtain the dry weights of the wood blocks after treatment,the treated wood blocks were weighed after being air andoven dried at 1058C for 24 h. Treatability is defined as thepercent retention of the preservatives, and the treatability of theOK-WPs was calculated by the difference between the final dryweights before and after treatment.

Leaching procedure

To determine the stability of each OK-WP against leaching,the treated wood blocks were subjected to hot water leaching.Before leaching, the treated wood blocks were placed on a 3-Lextractor and then evacuated with an aspirator in a desiccatorfor 30min, whereby the wood blocks were partially saturatedwith cold water. The partially saturated wood blocks were thenleached in hot water (70� 28C) for 72 h and refreshed bycondensate at a rate of approximately 350ml/h. The hot waterleaching procedure was deemed more effective than cold waterleaching, because it reduced the leaching period and representeda naturally diverse climate because of the repeated refreshingof the hot water. The leached wood blocks were weighedafter being air and oven dried at 1058C for 24 h. Leachabilityrefers to the percentage of preservative leached from treatedspecimens. The leachability of OK-WPs was measured bypercentage weight loss of the treated wood blocks during hotwater leaching.

Chemical analysis

After the stability of OK-WP against hot water leaching wasdetermined, the actual retention of Cu, B, and protein wasmeasured in randomly chosen leached wood blocks using an

inductively coupled plasma (ICP) emission spectrometer(ICPS-1000IV) and Kjeldahl protein/nitrogen analyzer (KjeltecAuto 1035/1038 System) housed at the National Instrumenta-tion Center for Environmental Management (Seoul, SouthKorea). To determine copper and B retention, the leached woodblocks were passed through a grinder, and then the wood sample(0.5 g) was placed in a 100-ml flask. The sample was pretreatedwith 10ml nitric acid and 5ml perchloric acid on a hot plate, at150 to 2008C. Distilled water (50ml) was added to the flask towash the sample, and the sample was filtered by Whatman filterpaper no. 42. The filtrate was measured for Cu and B contents,using the ICP emission spectrometer.

To determine protein content, total N and nonprotein nitro-gen were determined using the Kjeldahl protein/nitrogen ana-lyzer (Kjeltec Auto 1035/1038 System) according to a modifiedKjeldahl method [21]. Protein N was calculated indirectly bysubtracting nonprotein N from total N. The results of Cu, B, andN retentions are the averages of three measurements.

Decay resistance

Decay resistance of the leached wood blocks was evaluatedin accordance with American Society for Testing and Materialsstandard D 1413-05b [22] and Korean Industrial Standard KS F2213 ([23]; http://www.kats.go.kr). The test procedure for thedecay trial followed the American Society for Testing andMaterials standard but used the Cu-tolerant brown-rot fungiFomitopsis palustris (FFPRI 0507) and white-rot fungi Tra-metes versicolor (FFPRI 1030), designated as the decay testfungi in the KS Standard. Fungus samples were provided by theJapanese Wood Research Institute (Tokyo, Japan).

Before the decay trial, the soil culture bottles were insertedinto a decay chamber and sterilized at 1218C for 30min. Fungicultured in glucose 2.5%, malt extract 1%, peptone 0.5%,monopotassium phosphate 0.3%, and magnesium sulfate0.2% were inoculated on the soil surface in the culture bottles.After fungal mycelia covered the surface, wood blocks, steri-lized by ethylene-oxide for 6 h, were placed on the surface, andthe bottles were introduced into the decay chamber. After thesoil block culture was incubated at 26� 18C and 75� 2%relative humidity for 12 weeks, the wood blocks were removedfrom the soil culture bottles. Mycelia on the surface of eachblock were completely removed by brushing. The cleansedwood blocks were air and oven dried at 1058C for 24 h. Eachwood block was weighed to determine the percentage weightloss attributable to exposure to the decay-inducing fungus.

Microscopic observation

After the decay test of the leached wood blocks was com-pleted, the wood blocks were sliced into thin sections by a razorblade. Samples were subjected to X-ray microanalysis bymethods reported previously [24,25]. They were mounted onmetal stubs, using two-sided adhesive carbon tape, and sputtercoated with a thin layer (�20 nm thick) of platinum. Thespecimens were examined with a field emission-scanning elec-tron microscope (Supra 55VP; Carl Zeiss) at an acceleratingvoltage of 3 kV. Random observations were made of differentstructures to identify the existence of complexes such as EOK-CC, EOK-B, EOK-CC-B, or CC-B in the anatomical structureof the specimens. Element composition was determined byusing an energy dispersive X-ray analyzer (EDS, XFlash4000; Bruker AXS Microanalysis) combined with the fieldemission-scanning electron microscope. X-rays were collectedwith a detector fixed at a takeoff angle of 35 degrees, and theirintensities were recorded as counts per second.

1298 Environ. Toxicol. Chem. 30, 2011 H.-Y. Kim et al.

Data analysis

For the decay test, half of the wood blocks were randomlyselected and exposed to F. palustris and the other half toT. versicolor. The effects of each fungus on the treatability,leachability, decay resistance, and Cu contents of wood blockstreated with the OK-WP formulations were examined by thegeneral linear model procedure with the Statistical AnalysisSystem programming package. A 95% confidence level wasused in all statistical tests. If a significant effect was found in avariable at p< 0.05, the data of two samples for the variablewere compared based on Student’s t test at a¼ 0.05.

RESULTS AND DISCUSSION

Treatability of OK-WP

Table 1 shows the types of each OK-WP formulation andtheir treatability, which represents actual percent retention ofOK-WP in wood blocks. Measured treatabilities of wood blockstreated with CC/EOK and CC/B/EOK were 63.38% and61.47%, respectively. The actual retentions of CC/EOK andCC/B/EOK were much lower than the targeted retentions.These results are probably attributable to the molecular sizesof complexes formed by okara, Cu, or B salts, and an insuffi-cient amount of ammonium hydroxide used as a dissociatingagent in our preservative formulations, which may have madethe complexes too large to penetrate the wood blocks. Theaddition of sodium borate to the CC/EOK suspension alsosignificantly decreased the treatability of the preservatives(p¼ 0.01). This result might be caused by the large molecularsize of the complexes, which formed protein in the okara, Cu,and B salts [14], resulting in CC/B/EOK having a treatabilitylower than that of CC/EOK.

The effect of enzymes used to hydrolyze okara on treat-ability is presented in Figure 1. In the comparison of treatabilitydepending on the use of enzymes, the treatabilities of CC/EOKand CC/B/EOK formulated with either 2% cellulase-EOK(CEL-2) or 2% pectinase-EOK (PEC-2) were significantlyhigher than that of okara hydrolyzed in aqueous solution(CEL-0 or PEC-0, p¼ 0.01). These results could be attributedto the smaller molecular size of okara hydrolyzed by CEL orPEC and suggest that complexes formed by EOK, CC, or Bmight be easily penetrated and fixed into wood blocks. How-ever, the treatability of CC/EOK formulated with 2% protease-EOK (ALC-2) did not differ from that of ALC-0 (p¼ 0.36), andtreatability significantly decreased when the ALC loadingamount was increased from 2 to 4% (p¼ 0.01). Similar resultswere found in CC/B/EOK formulations (Fig. 1). The negativeeffect of ALC on the treatabilities of CC/EOK and CC/B/EOKformulations is probably attributable to ALC EOK, which hasmany functional groups. Hydrolysates react with CC or B andmight subsequently form huge complexes. The complexes mayhave difficulty penetrating wood, resulting in lowered treat-ability.

The treatabilities of OK-WPs formulated with CEL-2, PEC-2, and ALC-2 were compared to examine the effect of enzymetype on the treatability of OK-WP. The treatabilities of OK-WPcontaining CEL-2, PEC-2, and ALC-2 were 66.79, 64.79, and62.82%, respectively. No significant differences in treatabilitieswere found between CEL-2 and PEC-2 (p¼ 0.07) and PEC-2and ALC-2 (p¼ 0.10); however, the treatability of OK-WPformulated with CEL-2 was significantly higher than that ofALC-2 (p¼ 0.04). The difference might be attributable to thehigher digestibility of CEL compared with that of ALC. The

holocellulose content of okara has been reported to be muchhigher than the protein content [17]. Therefore, CEL decreasesthe molecular size of okara more efficiently than ALC, andconsequently, it might contribute to increased treatability.

Leachability of OK-WP

The leachability of each OK-WP formulation is presentedin Table 1. Okara-based wood preservatives were stable againsthot water leaching compared with ACQ or CuAz. The leach-abilities of CC/EOK and CC/B/EOK were 3.15 and 3.32%,respectively, whereas wood blocks treated with ACQ and CuAzreleased 10% of leachates although the leaching test had beenconducted at room temperature [26]. Although the results wereobtained from different wood species, these values suggestthat enzymatic-hydrolyzed okara fixes salts more efficientlyin wood blocks than ACQ and CuAz.Moreover, formulations ofOK-WPs are environmentally preferable wood preservatives,because they present little chance of human exposure to thetoxic chemicals associated with ACQ or CuAz. Adding B to theCC/EOK suspensions negatively affected their leachabilities(p¼ 0.02), probably because of the high water solubility of B.

The type and loading amount of the enzymes used to hydro-lyze okara also influenced the leachability of OK-WPs. In theCC/EOK formulations, no significant differences were foundbetween the leachabilities of PEC-0 and PEC-2 (p¼ 0.20) orbetween ALC-0 and ALC-2 (p¼ 0.14). However, as shown inFigure 2, the retention of CC/EOK formulated with CEL-2 wassignificantly higher than that of CC/EOK formulated with CEL-0 (p¼ 0.01). In addition, the leachability of CC/EOK decreasedwhen the loading amount of ALC increased from 2 to 4%(p¼ 0.01). These results indicate that okara hydrolyzed by 2%CEL and 4% ALC might have more functional groups that arechelated with Cu salt than CEL-0 and ALC-0/ALC-2. Hence,the molecular size of the complexes formed in CC/CEL-2 orCC/ALC-4 could properly penetrate into and be retained by thewood blocks and exhibit stability against hot water leaching.

In the CC/B/EOK formulations, the leachabilities of CC/B/CEL-2 and CC/B/PEC-2 were significantly higher than those ofCC/B/CEL-0 (p¼ 0.01) and CC/B/PEC-0 (p¼ 0.01), respec-tively (Fig. 2). The higher leachabilities of CC/B/CEL-2 andCC/B/PEC-2 indicate that EOK unchelated with CC or B mighthave higher leachability.

The leachabilities of CC/B/ALC were not influenced by theincreased loading amount of ALC (Fig. 2). To reveal the effectof enzyme types on the leachability of OK-WP formulations,the leachabilities of OK-WPs formulated with CEL-2, PEC-2,and ALC-2 were compared. The leachabilities of CC/B/CEL-2,CC/B/PEC-2, and CC/B/ALC-2 were 3.21, 3.34, and 3.26%,respectively. No significant differences between the leachabil-ities of CC/B/CEL-2 and CC/B/PEC-2 (p¼ 0.12), CC/B/PEC-2and CC/B/ALC-2 (p¼ 0.18), or CC/B/CEL-2 and CC/B/ALC-2(p¼ 0.36) were found. The effects of enzyme type and loadingamount on the leachability of OK-WP were similar to thosefrom our previous results [25]. However, the leachability valuesof the OK-WPs used in our prior study were much lower thanthose of the OK-WPs formulated with severely hydrolyzedokara, such as 10% or 20% CEL, PEC, and ALC. Thus, theloading amount of enzymes used to hydrolyze okara might besubject to being restricted to less than 4% based on the dryweight of okara.

Decay resistance of leached wood blocks

Decay resistances of leached wood blocks treated with OK-WPs against F. palustris and T. versicolor are shown in Table 1.

New environmentally friendly wood preservatives Environ. Toxicol. Chem. 30, 2011 1299

Table

1.Typeofokara-based

preservatives

form

ulatedin

thisstudyanditstreatability,leachability,effectivenessagainstbrown-rotfungusFomitopsispalustrisandwhite-rotfungusTrametes

versicolor,andtheactual

retentionoftreatedchem

icalsa

Enzymeloadingb

(mlenzyme/gokara,

%)

CEL0

CEL2

PEC

0PEC2

PEC0

PEC2

Control

ALC

0ALC

2ALC

4ALC

0ALC2

ALC4

ALC0

ALC

2ALC

4ALC

0ALC

2ALC4

CC/EOKc

Treatabilityd(%

)60.59

61.09

54.94

64.98

61.34

63.84

66.36

68.64

58.07

66.58

67.95

66.19

-Leachabilitye(%

)3.23

3.61

3.00

3.40

3.27

3.41

3.09

2.89

2.41

2.90

3.28

2.99

-Weightloss

(FPAf ,%)

6.57(2.95)

9.38(2.30)

12.07(4.95)

6.40(2.06)

8.42(4.32)

6.56(2.28)

5.81(0.92)

9.78(2.56)

14.86(5.83)

10.40(3.61)

7.38(2.01)

5.50(1.23)

26.19(0.25)

Weightloss

(TRAg,%)

1.05(0.23)

1.02(0.11)

1.48(0.06)

1.21(0.19)

1.29(0.25)

1.16(0.36)

1.56(0.46)

2.13(0.15)

2.01(0.45)

2.01(0.07)

1.80(0.07)

1.99(0.23)

13.11(2.64)

Copper

contents

(kg/m

3)

1.50

4.21

2.10

3.22

3.20

2.88

3.35

3.44

2.97

3.23

4.16

3.90

-Nitrogen

contents

(kg/m

3)

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

-CC/B/EOKh

Treatability(%

)50.15

50.44

51.17

60.63

57.63

59.90

68.98

66.07

64.18

71.92

69.40

67.15

-Leachability(%

)3.09

2.98

2.93

3.30

3.16

3.42

3.21

3.32

3.52

3.59

3.53

3.80

-Weightloss

(FPA,%)

10.57(3.53)

11.34(2.77)

10.11(1.29)

11.66(5.57)

6.86(3.57)

7.57(1.71)

7.03(2.15)

13.11(3.02)

11.92(1.60)

3.49(0.73)

1.98(0.95)

8.34(2.65)

26.19(0.25)

Weightloss

(TRA,%)

1.40(0.15)

1.46(0.25)

1.41(0.27)

1.43(0.16)

1.31(0.40)

1.15(0.39)

1.05(0.10)

1.38(0.14)

1.00(0.18)

1.12(0.12)

1.28(0.18)

1.40(0.10)

13.11(2.64)

Copper

contents

(kg/m

3)

4.25

2.20

2.76

2.34

2.70

2.32

2.30

3.10

3.37

3.60

3.21

3.78

–Boroncontents

(kg/m

3)

0.02

0.01

0.01

0.01

0.00

0.02

0.02

0.06

0.01

0.16

0.10

0.01

–Nitrogen

contents

(kg/m

3)

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

–

aHyphensrepresentnotmeasuredfortreatabilityandleachabilityandnotdetectedforcoppercontents,nitrogen

contentsandboroncontents.Numberin

parentheses

intherowsofweightloss,meansastandarddeviationof

averageofeach

okara-based

woodpreservativeform

ulation.

bEnzymeloadingmeansthat

thepercentageloadingam

ountofeach

enzymeusedin

hydrolysisbased

onthedry

weightofokara.

CEL:Celluclast1

1.5LFG;PEC:Pextinex

15XL;ALC

:Alcalase1

1.5L.

cCopper

chloride/okarahydrolysates.

dTreatabilityrefers

tothepercentageofactual

retentionto

thetarget

retention.

eLeachabilityrefers

tothepercentageofpreservativeleached

from

treatedspecim

ens.

fFomitopsispalustris(FFPRI0507).

gTrametes

versicolor(FFPRI1030).

hCopper

chloride/sodium

borate/okarahydrolyzates.

1300 Environ. Toxicol. Chem. 30, 2011 H.-Y. Kim et al.

The average weight loss of control wood blocks againstF. palustris was 26.19%. A previous study reported that south-ern pine wood blocks treated with CC showed approximately40% weight loss against F. palustris [27]. However, the weightloss of leached wood blocks treated with OK-WP formulationsagainst F. palustris ranged from 1.98 to 14.86% and was muchlower than that of the control wood blocks. Leached woodblocks treated with CC/B/EOK (CEL-2/PEC-2/ALC-2) showedthe best decay resistance against F. palustris, which is a Cu-tolerant fungus. Those treated with CC/EOK (CEL-2/PEC-0/ALC-4) showed the greatest weight loss of all OK-WP for-mulations. These results indicate that the decay resistance ofOK-WP is affected by the addition of B in CC/EOK suspensionsand the hydrolysis conditions of okara. For example, the weightloss of leached wood blocks treated with OK-WP containingCEL-2 was significantly lower than that containing CEL-0(p¼ 0.01). With the exception of CEL, PEC and ALC didnot influence the decay resistance (PEC-0/PEC-2: p¼ 0.26;ALC-0/ALC-2: p¼ 0.21; ALC-0/ALC-4: p¼ 0.09; ALC-2/ALC-4: p¼ 0.12). No significant difference between the decayresistances of CC/EOK and CC/B/EOK (p¼ 0.48) was found.However, the type and loading amount of enzyme and theaddition of B had mutual effects on the decay resistance of OK-

WP (p¼ 0.02). For instance, as shown in Figure 3, when eitherCEL-2 or ALC-2 was used in the formulation of OK-WP insteadof CEL-0 or ALC-0/ALC-4, the decay resistance of leachedwood blocks treated with CC/B/EOK was better than for thosetreated with CC/EOK. Furthermore, the decay resistance of CC/EOK formulated with PEC-0 was better than that formulatedwith PEC-2, but the opposite result was obtained in the CC/B/EOK formulations. These results indicate that the chelatingreaction of B with CC or EOK might vary with the loadingamount of CEL, PEC, and ALC. Thus, the decay resistances ofeach OK-WP were different.

In the case of wood blocks exposed to T. versicolor, theweight loss of the control wood blocks was 13.11%. Leachedwood blocks treated with OK-WP formulations showed excel-lent decay resistance regardless of the addition of B in CC/EOKsuspensions and the hydrolysis conditions of okara. For exam-ple, most leached wood blocks treated with OK-WP formula-tions showed less than approximately 2% weight loss and werenot covered by mycelia after being exposed to T. versicolor for12 weeks. These results indicate that, as formerly suggested[19], the hyphal growth of T. versicolor might be inhibited byCu or B salts retained in the leached wood blocks treated withOK-WP formulations. Thus, OK-WP showed excellent decay

Fig. 1. Effects of the typeand loadingamountof enzymesused for thehydrolysis ofokaraon the treatabilities ofCC/EOK(top) andCC/B/EOK(bottom).Differentcapital letters over columns indicate significant difference at p¼ 0.05 (least significance difference test). CC/EOK, copper chloride/okara hydrolyzates; CC/B/EOK, copper chloride/sodium borate/okara hydrolysates; CEL, Celluclast, 1.5 L FG; PEC, Pextinex 5 XL; ALC, Alcalase 1.5 L.

New environmentally friendly wood preservatives Environ. Toxicol. Chem. 30, 2011 1301

resistance against T. versicolor. Based on these results, OK-WPformulations can be prepared with CEL-2/PEC-2/ALC-2,CC, and B to protect wood blocks effectively against bothF. palustris and T. versicolor.

Chemical analysis

The actual retentions of Cu, B, and nitrogen in leached woodblocks treated with OK-WP are shown in Table 1. The Cucontents ranged from 1.50 to 4.25 kg/m3, which was sufficient toprotect wood blocks against T. versicolor. The Cu content was

not affected by the addition of B in CC/EOK, use of PEC,increase of ALC loading amount, or type of enzymes. However,a significant difference was seen between the Cu contents ofCEL-0 and CEL-2 (p¼ 0.04). Specifically, the Cu content ofCEL-0 (2.81/kg �m3) was lower than that of CEL-2 (3.48 kg/m3). This indicates that wood blocks treated with OK-WPcontaining CEL-2 retain more Cu through hot water leachingthan those treated with CEL-0. Therefore, OK-WP formulatedwith CEL-2 could be used to protect wood blocks from thegrowth of decay fungi.

Fig. 2. Effects of the type and loading amount of enzymes used for the hydrolysis of okara on the leachabilities of CC/EOK (top) and CC/B/EOK (bottom).Different capital letters over columns indicate significant difference at p¼ 0.05 (least significance difference test). CC/EOK, copper chloride/okara hydrolyzates;CC/B/EOK, copper chloride/sodium borate/okara hydrolyzates; CEL, Celluclast 1.5 L FG; PEC, Pextinex 5 XL; ALC, Alcalase 1.5 L.

Fig. 3. Interaction effects of the addition of sodium borate in CC/EOK suspension and the loading amount of cellulase (left), pectinase (middle), and alcalase(right) on the decay resistance of OK-WP formulations against Fomitopsis palustris. CC/EOK, copper chloride/okara hydrolysates; OK-WP, okara-based woodpreservatives; CEL, Celluclast, 1.5 L FG; PEC, Pextinex 5 XL; ALC, Alcalase 1.5 L; B, sodium borate.

1302 Environ. Toxicol. Chem. 30, 2011 H.-Y. Kim et al.

Most leached wood blocks treated with CC/B/EOK con-tained 0.01 to 0.02 kg/m3 B (Table 1). However, the B contentsof leached wood blocks treated with CC/B/EOK, particularlyCEL-2/PEC-2/ALC-0 and CEL-2/PEC-2/ALC-2, were over0.1 kg/m3, and OK-WP, as shown in Table 1, had much betterdecay resistance against F. palustris than any of the other CC/B/EOK formulations. These results indicate that B in the leachedwood blocks treated with OK-WPs might be the most importantfactor protecting wood blocks against F. palustris.

The protein contents of the leached wood blocks treated withOK-WPs were 0.01 kg/m3 regardless of the factors examined inthis study (Table 1). This means protein could be used to formcomplexes with Cu or B salts by chelating reactions in woodblocks. The complexes might play roles in retaining the salts inwood blocks through hot water leaching and augment theantifungal property of leached wood blocks treated with OK-WP. From the results of the chemical analysis, OK-WPs for-mulated with CEL-2, PEC-2, and ALC-0/ALC-2 might beeffective in retaining Cu or B salts in wood blocks againstleaching.

Microscopic observation and analysis

Scanning electron microscope observation and energy-dis-persive X-ray spectroscopy (EDS) analysis were used to iden-tify the antifungal complexes in the OK-WPs treated andleached wood blocks. Complexes were not detected in control

wood blocks by scanning electron microscopy observation(Fig. 4, top left). However, when the leached wood blockstreated with CC/EOK formulations were observed, variousspherical agglomerates were found in the cell lumen (Fig. 4,top right). The spot analysis using scanning electron micro-scopy–EDS proved that the agglomerates contained Cu and Cloriginating from CC (Fig. 4, bottom). In leached wood blockstreated with CC/B/EOK, several areas in which pits in the celllumen were clogged by some materials were easily detected(Fig. 5, left). The spot analysis confirmed the presence of Cu inareas of leached wood blocks (Fig. 5, right), but the existence ofB in the same areas could not be identified. This might beattributable to the high water solubility of B and the fact that itwas leached from the wood blocks treated with CC/B/EOK,perhaps resulting in concentrations of B that were too low to bedetected by scanning electron microscopy–EDS. The micro-scopic observation and analysis of wood blocks treated withOK-WPs confirmed that complexes formed by protein-Cu, andprobably protein-B, existed in the leached wood blocks treatedwith OK-WP formulations and contributed to their good decayresistance against both F. palustris and T. versicolor.

CONCLUSIONS

Although the actual retention of OK-WPs was lower than thetarget retention, the treatability of OK-WP might be improvedby properly using enzymes in hydrolyzing okara and increasing

Fig. 4. Scanning electronmicroscopic image andcorresponding spectrumof controlwoodblock (top left) and the leachedwoodblocks treatedwithCC/EOK(top rightand bottom). CC/EOK¼ copper chloride/okara hydrolyzates. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com]

New environmentally friendly wood preservatives Environ. Toxicol. Chem. 30, 2011 1303

the amount of ammonium hydroxide used as a dissociationagent.When the leachability of OK-WPwas compared with thatof ACQ and CuAz, which were extensively used as woodpreservatives, it was comparably stable against hot waterleaching. In the results of the decay resistance test, the weightloss of leached wood blocks treated with OK-WP formulationsagainst F. palustris, a copper-tolerant brown-rot fungus, wasmuch lower than that of the control wood blocks. The CC/B/EOK (CEL-2/PEC-2/ALC-2) showed excellent decay resist-ance against F. palustris compared with other OK-WP formu-lations. Most leached wood blocks treated with the OK-WPformulations showed less than approximately 2% weight lossagainst T. versicolor. From the chemical analysis of the leachedwood blocks treated with OK-WP formulations, complexescontaining Cu, B, and protein were identified in the woodblocks. The SEM observation and EDS analysis of the leachedwood blocks treated with OK-WP formulations visually con-firmed that the complexes formed by protein–copper, andprobably protein–B, exist in the cell lumens and pits of leachedand decayed wood blocks. The complexes might have con-tributed to the wood blocks treated with OK-WP formulationshaving good decay resistance against F. palustris andT. versicolor. Based on these results, OK-WPs can effectivelyprotect wood against decaying fungi and could be used as anenvironmentally preferable and cost-effective wood preserva-tives. However, further research on OK-WP, such as a long-term field test comparing decay resistance with that derived byACQ and CuAz, as well as tests against termite resistance andmetal corrosion, are required to determine its applicability.

Acknowledgement—This study was supported by the Technology Develop-ment Program for Agriculture and Forestry, Ministry for Agriculture,Forestry and Fisheries, Republic of Korea.

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