Effect of carbohydrate and nitrogen on hydrogen peroxide formation by wood decay fungi in solid medium

Transcription

1 FEMS Microiology Letters 48 (1987) Pulished y Elsevier FEM Effect of crohydrte nd nitrogen on hydrogen peroxide formtion y wood decy fungi in solid medium Terry L. Highley Forest Products Lortory, Forest Service, US Deprtment ofagriculture, One Gifford Pinchot Drive, Mdison, WI, U.S.A. Received 3 August 1987 Accepted 30 August 1987 Key words: Wood decy; Fungi; Hydrogen peroxide 1. SUMMARY Hydrogen peroxide (H 2 O 2 ) hs een implicted in degrdtion of wood y oth rown-rot nd white-rot fungi. This study found tht low concentrtions of nitrogen nd crohydrtes (celloiose, glucose, xylose nd mnnose) in n gr medium hd little effect on H 2 O 2 production y white-rot fungi. However, low concentrtions of nitrogen nd crohydrtes stimulted H 2 O 2 production y rown-rot fungi. Use of the chromogen 2,2'-zino-di(3-ethyl enzthizoline-6-sulphonic cid) (ABTS) with horserdish peroxidse to detect H 2 O 2 y the fungi ws slightly etter thn detection y the chromogen o-dinisidine with horserdish peroxidse. An uxiliry test to check the role of H 2 O 2 in wood decy found tht hydrogen peroxide-negtive isoltes of the white-rot fungi Phnerochete chrysosporium nd Gnoderm pplntum were unle to decy sweetgum nd southern pine. This rticle ws written nd prepred y U.S. Government employees on officil time, nd it is therefore in the pulic domin nd not suject to copyright. Correspondence to: Richrd Lindeorg, Hed, Pulictions Section, Forest Products Lortory, One Gifford Pinchot Drive, Mdison, WI , U.S.A., Tel. (608) INTRODUCTION The hydroxyl rdicl (OH ) derived from hydrogen peroxide (H 2 O 2 ) hs een proposed to e cellulose-degrding gent in rown-rot decy of wood [1,2]. However, its involvement in cellulose decomposition hs een questioned, primrily ecuse of inility to detect its production y severl rown-rot fungi [3,4]. Hydrogen peroxide is found in most white-rot fungi where it evidently plys n importnt role in lignin degrdtion [5,6]. Glucose oxidse hs een shown to e the predominnt source of H 2 O 2 in lignin-degrding cultures of the white-rotter Phnerochete chrysosporium [7]. Glucose oxidse ctivity in this fungus ppers to e secondry metolic event nd is triggered in response to nitrogen or crohydrte strvtion [8]. Filure to detect H 2 O 2 in certin rown-rot fungi, therefore, could e due to excess nitrogen or crohydrte in the growth medium if glucose oxidse is the source of H 2 O 2. Another reson for inility to detect H 2 O 2 in some rown-rot fungi my e tht the mounts of H 2 O 2 produced re so low tht the chromogens re not sensitive enough to detect it. Muller [9] found tht 2,2 -zino-di-(3-ethylenzthizole-6 sulphonic cid) (ABTS) used with horserdish peroxidse ws 10 times more sensitive thn en /87/$ Federtion of Europen Microiologicl Societies

2 374 zidine in the detection of H 2 O 2 in cteri. The purpose of this study is to evlute the influence of nitrogen nd crohydrte source nd concentrtion on H 2 O 2 production y rown-rot nd white-rot fungi using the highly sensitive chromogen ABTS. 3. MATERIALS AND METHODS 3.1. Fungl strins nd culturl conditions The fungl strins used re listed in Tle 1. Two strins of P. chrysosporium (ME-446 nd ME-461) nd Gnoderm pplntum (MAD-708 nd 7823-S) were used ecuse the originls strins (ME-446 nd MAD-708) lost their ility to produce H 2 O 2 during the course of the study. Fungl cultures were mintined on 2% (w/v) mlt-gr slnts t 4 C until used. Tues contining medi for detection of H 2 O 2 production were inoculted with plug from mlt-gr tues nd incuted t 27 C nd 70% humidity Comprison of chromogens The sl slts medium contined: NH 4 NO 3, 2 g; KH 2 PO 4, 2 g; MgSO 4 7 H 2 O, 0.5 g; CCl 2, 0.1 g, thimine, 1 mg; nd minor elements [10]; 15 g of gr were dded to 900 ml of the sl slts medium contining 10 g of celloiose. After utoclving t 121 C for 15 min, the medium ws cooled to 50 C. Horserdish peroxidse 20 mg (Sigm, Type II) nd 300 mg of ABTS or o-dinisidine were dissolved in 100 ml of distilled H 2 O, filter-sterilized nd dded septiclly to the gr medium; 3-ml portions were poured into pre-sterilized test tues. To determine the effect of wood cell wll polymers on H 2 O 2 formtion, xyln (Sigm), cellulose (Hercules; cotton type A-600), Tle 1 Comprison of chromogens in detection of H 2O 2 y white-rot nd rown-rot fungi grown on medium of high crohydrte nd nitrogen Leucogyrophn rizonic Ginns, RLG-9902-Sp. L. olivscens (Berk. et Curt) Ginns et Weres., FP Sp. Coniophor puten (Schum.: Fr.) Krst., MAD-515 Pori plcent (Fr) Cke., MAD-698 Gloeophyllum treum (Pers.: Fr.) Murr., MAD-617 Lentinus lepideus Fr., MAD-534 P. cronic Overh., FP Sp. A. lpin (Litsch.) Giln.: Ryv., FP Sp. Antrodi serilis Fr., FP R Firopori villntii (DC: Fr.) Prm Serpul incrsst (Berk. et Curt) Donk, MAD-563 Letiporus sulphureus (Bull.: Fr.) Bond. et Sing., Sh-27-R Fomitopsis melie (Underw.) Giln., FP Sp. Phellinus contiguus (Pers.: Fr.), HHB-7051-Sp. Phnerochete chrysosporium Burds., ME-446 Gnoderm pplntum (Pers.: S. F. Gry) Pt. (MAD-708) Xyloolus frustultus (Pers.: Fr.) Boid., FP S Coriolus versicolor (L: Fr.) Quél., MAD-697 Ph. ferrugineofuscus (Krst.) Bourd, L Sp. Spongipellis unicolor (Schw.) Murr., RGL-7638-Sp. Colortion of medium using chromogen ABTS o-dinisidine 0 = no colortion; 1 = very slight colortion; 2 = slight colortion; 3 = moderte colortion; 4 = hevy colortion.

3 375 or hemlock swdust were lyered onto the gr surfce Effect of nitrogen nd crohydrte on H 2 O 2 production The rown-rot nd white-rot were tested for H 2 O 2 production on the ove medi with 1% (w/v) celloiose nd ABTS with vrious nitrogen sources: 0.2 nd 0.02% NH 4 NO 3, 0.02 nd 0.2% csein mino cids (Difco), 0.02% yest nitrogen se (Difco) nd 0.2% mlt extrct. Hydrogen peroxide ws lso determined using the ove sl slts medi with 0.02% NH 4 NO 3 nd the following crohydrte sources: None, 0.1% celloiose, 0.1 nd 1.0% glucose, 0.1% xylose nd 0.1% mnnose Decy tests The stndrd ASTM D 2017 soil-lock method [11] ws used to compre the ility of H 2O 2 negtive nd H 2O 2-positive isoltes of P. chrysosporium nd G. pplntum to decy sweetgum nd southern pine locks. Following incution, test smples were removed from ottles, conditioned t 27 C nd 70% humidity, nd weighed. Weight loss ws used s n estimte of decy susceptiility. 4. RESULTS AND DISCUSSION With high crohydrte nd nitrogen in the medium, ABTS-peroxidse ws only slightly etter in detection of H 2 O 2 production y the fungi thn ws o-dinisidine-peroxidse (Tle1). How- Tle 2 Effect of nitrogen source nd concentrtion on H2O2 L. rizonic L.olivscens C. puten P. plcent G. treum L. lepideus P. cronic P. xnth A. serilis F. villntii S. incrsst L.surfureus P. melie Ph. contiguus P.chrysosporium G. pplntum X. frustultus Ph.ferrugineofuscus S. unicolor C.versicolor Colortion ofmedium Nitrogen source formtion y white-rot nd rown-rot fungi H2O2 detected with ABTS. 0 = no colortion; 1 = Very slight discolortion; 2 = slight colortion; 3 = moderte colortion; 4 = hevy colortion. c % NH4NO3; % NH4NO3; % csein mino cids; % csein mino cids; % yest nitrogen se; % mlt extrct.

4 376 ever, only 2 out of the 13 rown-rot fungi produced detectle H 2O 2 on the ABTS medium. With the white-rot fungi, H 2O 2 ws detected in 5 out of 7 fungi. Addition of xyln, cellulose, or hemlock swdust to the medium did not chnge the numer of fungi producing detectle H 2O 2 (not shown). Nitrogen concentrtion hs een found to hve profound effect on H 2O 2 production nd ligninolytic ctivity y the white-rot fungus, P. chrysosporium [12]. Cultures of P. chrysosporium grown in low nitrogen medium exhiit high levels of H 2O 2 production, wheres cultures grown under identicl conditions in high nitrogen medium show little H 2O 2 production [12]. In the present study, low nitrogen in the medium contining 1% celloiose did not stimulte H 2 O 2 production y the white-rot fungi (Tle 2). Source of nitrogen lso hd little effect on H 2O 2 production y the white-rot fungi. P. chrysosporium produced slight mounts of H 2O 2 only on the medi contining 0.2% csein mino cids or 0.2% yest extrct. Xyloolus frustultus ws the only white-rot fungus not producing detectle H 2O 2 on ny of the nitrogen sources. Vrying the nitrogen source nd concentrtion in the 1% celloiose medium gve vned response in H 2O 2 production y the rown-rot fungi (Tle 2). Seven out of 13 of the rown-rot fungi still did not produce detectle H 2O 2 using different sources of nitrogen. Tle 3 shows H 2O 2 production y the fungi using different concentrtions nd sources of crohydrte with 0.02% NH 4NO 3 s the nitrogen source. In this study H 2O 2 production y the white-rot fungi ws ffected little y crohydrte Tle 3 Effect of crohydrte source nd concentrtion on H2O2 formtion y white-rot nd rown-rot fungi Colortion of medium Crohydrte source None 0.1% 0.1% 1.0% 0.1% 0.1% 0.2% celloiose glucose glucose mnnose xylose mlt extrct L. rizonic 0 L. olivscens C. puten G. treum 0 L. lepideus P. cronic P. xnth A. serilis F. villntii 1 S. incrsst L. sulfureus F. melie P. plcent P. chrysosporium (ME-446) P. chrysosporium (ME-461) G. pplntum (MAD-708) 0 G. pplntum (7823-S) 2 X. frustultus C. versicolor H2O2 detected with ABTS. 0 = no colortion; 1 = very slight colortion; 2 = slight colortion; 3 = moderte colortion; 4 = hevy colortion.

5 377 Tle 4 Decy of wood y H2O2 positive nd negtive isoltes H2O2 production Weight loss (I) of wood Sweet- gum Pine G. pplntum (MAD-708) - G. pplntum (7823-S) P. chrysosporium (ME-446) - P. chrysosporium (ME-461) , = H2O2 detected; - = H2O2 not detected. Blocks removed t 10 weeks. concentrtions even though Kelly nd Reddy [7] found tht crohydrte strvtion triggered H 2 O 2 production in the white-rot fungus P. chrysosporium (Tle 3). In the present study, y vrying the crohydrte source nd concentrtion, H 2 O 2 ws detected in ll ut 2 of the rownrot fungi, G. treum nd L. rizonic. As whole, H 2 O 2 production y most of the rown-rot fungi ws stimulted y low sugr concentrtion. In some cses, H 2 O 2 ws produced without crohydrte dded to the medium. Hemlock swdust ws dded to the low nitrogen medium contining no other cron source to see if it would stimulte H 2 O 2 formtion in G. treum, L. rizonic, nd X. frustultus. The swdust did not induce production y the ove fungi. Since production of H 2 O 2 hs een linked to ligninolytic ctivity of white-rot fungi, the decy ilities of the H 2 O 2 -negtive nd H 2 O 2 -positive isoltes of P. chrysosporium nd G. pplntum were compred. The H 2 O 2 -negtive isoltes were unle to decy pine nd sweetgum, ut the H 2O 2-positive isoltes redily decyed wood (Tle 4). This oservtion provides circumstntil evidence of H 2O 2 involvement in degrdtion of wood y white-rot fungi. However, dditionl degrdtive studies with H 2O 2-positive nd H 2O 2 negtive isoltes re needed. ACKNOWLEDGEMENT The use of trde, firm, or corportion nmes in this puliction is for the informtion nd convenience of the reder. Such use does not constitute n officil endorsement or pprovl y the U.S. Deprtment of Agriculture of ny product or service to the exclusion of others which my e suitle. REFERENCES