Author(s) TAKAHASHI, Munezoh; NISHIMOTO, Koic.

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(1973), 54: 1-8 the Woo Isse Date 1973-3-15 URL http://hdl.handle.

1 Title Utilization of Carbohydrates by Sof globosm KUNZ Athor(s) TAKAHASHI, Mnezoh; NISHIMOTO, Koic Citation Wood research : blletin of University (1973), 54: 1-8 the Woo Isse Date URL Right Type Departmental Blletin Paper Textversion pblisher Kyoto University

2 Utilization of Carbohydrates by Soft Rot Fngs, Chaetomim globosm KUNZ* Mnezoh T AKAHASHI** and Koichi NISHIMOTO** Abstrract--Utilizing ability of the soft rot fngs, Chaetomim globosm KUNZ, to varios mono- and polysaccharides, most of them normally occrring in hardwoods as hemicelllose components, was investigated on fngal growth and consmption rate by shake cltre. Xylose, mannose, glcose, galactose, maltose and crde xylan were available to the fngs, whereas arabinose, glcronic acid and galactronic acid were not tilized at all. Rapid growth and consmption were observed in xylose and xylan media, probably indicating the possible reference to the greater ssceptibility of hardwoods to soft rot. qality of mannose to xylose as carbon sorce, however, casted the propblem on the relation of mannan tilization to the greater resistance of softwoods to soft rot. Introdction It is well known that softwoods are not readily attacked by soft rot fngi as are hardwoods. This is sally attribted to the higher lignin content in softwoods. However, for the completely satisfactory explanation of this phenomenon, different natres of the lignin and hemicellloses in softwoods and hardwoods cold be of importance'!) Frthermore, difference in ability of soft rot fngi to tilize the hemicellloses in softwoods and hardwoods may also be a significant factor for the greater resistance of softwoods or the greater ssceptibility of hardwoods. Wood-deteriorating microorganisms, inclding soft rot fngi, depend pon varios carbohydrates in wood cell walls for accomplishing their metabolic activities. Hemicelllose is one of the principal cell wall constitents and the important carbon sorce as celllose for the wood-decaying fngi. Hardwoods hemicellloses are distinctive for high content of xylans, and softwoods hemicellloses are characterized by a high percentage of mannans. Conseqently, tilization of these polysaccharides and their degradation prodcts by soft rot fngi In the present investigation, the tilizing ability to varios mono- and polysaccharides, many of these normally occrring in hardwoods, of typical soft rot fngs, Chaetomim globosm, has been stdied. xperirnental Carbon sorce and medim The following carbohydrates were added as the sole carbon medim; sorce to the basal ntrient * Presented at the 18th and 19th Meetings of Japan Wood Research Society, Kyoto, April 1968 and Sapporo, Jly ** Division of Wood Biology. ssceptibility of softwoods and hard is one of the major interest in stdying the different woods. -1-

3 WOOD RSARCH No. 54 (1973) D-arabinose, D-xylose, D-glcose, D-mannose, D-galactose, D-glcronic acid, D-galactronic acid, D-maltose, CMC (carboxymethyl celllose sodim salt), and crde xylan extracted from beech wood (Fags crenata BLUM). Abram's soltion was sed as the basal ntrient medim. The composition was as follows; NH 4 NO s 3. g, KHzP g, KzHP 4 2. g, MgS 4 7HzO 2. g, and distilled water 1 ml. Amonts of carbon sorce were 1 g per litre for xylan and 2 g for other carbohydrates. The carbon sorces were sterilized in vapor of Abram's soltion. propylene oxide and added to the atoclaved The experiments were mainly carried ot in 5 ml shaking flasks containing 5 ml of ntrient soltion. Usally three to five flasks per one kind of carbon sorce were sed. Prior to experiments, the mycelia of the test fngs were transferred to Petri dishes containing malt agar sbstrate. Then sterilized cellophane disks of 8 mm diameter were laid on the agar plate. After covering the agar plate and cellophane disks by mycelial development, the disks with mycelia were removed and rinsed in sterilized distilled water. The flasks were inoclated with these mycelial disks and incbated at 28±2 C in a shaker for 21 days. Growth rate and tilizing ability of carbon sorces Growth rate was determined every three days as mycelial dry weight per flask by drying for 2 hors at 9 C after filtering off the ntrient medim. In some experiments with solid agar media containing eqal amont of xylose or glcronic acid as in the ntrient soltion, growth was estimated as the radial growth of the mycelim in millimeters per day. In addition, the ph of each cltre filtrates was measred every three days. The cltre filtrates were analysed for amont of remnant carbohydrates by methods of BRTLANDzl and TOLLNS and KROBR.Zl Samples taken from the xylan-containing soltions were hydrolysed with IN slfric acid for 6 hors at 1 C, followed by netralization with barim hydroxide and passage throgh Amberlite IR-12 and IR-4B. The efflents and washings containing netral sgars were concentrated and analysed for pentose content by method of TOLLNS and KROBR.Zl Usally, Chaetomim globosm KUNZ (Strain No. 859) was sed as test fngs. For the prpose of comparison, Coriols versicolor QUL. (No. 13) and Tyromyces palstris MURR. (No. 57) were occasionally sed as a white rot and a brown rot fngs, respectively. Preparation of crde xylan from beech wood Beech wood sawdst, 4 to 6 mesh, was extracted with ethanol-benzene (1/1). The extractive-free wood (35 g) was sspended in water (3.5 I) and treated with glacial acetic acid (2 ml) and sodim chlorite (8 g) by occasionally stirring for 1 hor at 7-8 C. Then withot cooling, same amonts of additional reagents were added. The heating was contined at the same temperatre range for an additional hor. This treatment was again repeated in the same condition. The chlorite holocelllose was extracted with 5 % sodim hydroxide in nitrogen gas for one over night at room temperatre. The mixtre was filtered with sction into a flask containing an excess of glacial acetic acid. 95 % thanol was added to the filtrate and washings. The reslting precipitate was collected by centrifging, followed by washing repeatedly with 5 % ethanol, ethanol and ether and drying on potassim hydroxide in vaco. A crde xylan was obtained as light grayish powder

4 TAKAHASHI, NISHIMOTO: Utilization of Carbohydrates by Chaetomim globosm The content of lignin and ronic acid of the sample were determined according to T APPI Standards T 13 m-54 and BROWNING's method,s) as Klason lignin and ronic anhydride, respectively. Reslts and Discssion Growth of Chaetomim globosm on varios carbon consmption rate of some of these sorces are shown in sorces other than crde xylan and Fig. 1 and Fig. 2. Xylose. mannose, -1 _3 - (,/) /::;) - --/ -;'2 c: e ụ... 7 Q) a:: c: >- Q) C. 5 " ::J CIl IOO > c: 4 3 glcose o mannos8 A galactose 6. maltose X xylose Fig. 1. ;C<---O--O--O---D Wiii=====::Q.:-=-=-=_--=9=====-k:=4>--4>-;=;_ \8 21 Incbation Period (day) glcose D. maltose arabinose o mannose X xylose glcronic acid A galactose - em C <> galoctronlc acid Mycelial dry weight of Chaetomim globosm grown in ntrient medi:n with varios carbon sorces. Fig Incbati on Peri od (d a y ) Consmption rate of five carbon sorces by Chaetomim globosm grown in ntrient medim. glcose, galactose and maltose were available to Ch. globosm. Among these sorces, however, the fngal growth on galactose was apparently slower, althogh the final mycelial yield was almost eqivalent to other sorces. COCHRAN4) reviewed the carbon ntrition of fngi and conclded that galactose was sed by most fngi bt was not sally so good sore as glcose. From the data obtained, it was sggested that adaptive mechanism was at work in tilization of galactose by Ch. globosm and this fngs also might not be particlar in its metabolic activity to galactose. There are many reports that glcose is the good carbon sorce for many fngi inclding wood-deteriorating fngi and that xylose and mannose are eqivalent to glcose. 5,6) In the present investigation, however, glcose was somewhat inferior to xylose and mannose in daily growth rate. In the metabolism of glcose by fngi, three different pathways have been established, namely MP (mbden-meyerhof-parnas) pathway, HMP (hexose monophosphate) pathway and D (ntner-dodoroff) pathway. Mannose and galactose are converted into - 3

5 WOOD RSARCH No. 54 (1973) frctose 6-phosphate via phosphorylated derivatives of these sgars and glcose, and incorporated into the MP and HMP pathways,7) whereas xylose is converted into xyllose 5 phosphate via xyllose and connected with HMP pathway to prodce frctose 6-phosphate. 8 ) Rapid growth and rapid consmption in xylose medim seems to reflect active isomerization. phosphorylation and other reactions, leading the major participation of HMP pathway in Ch. globosm. On the other hand, the reason for the speriority of mannose to glcose, thogh the extent was little, is nknown. Or, it may be de to a contamination with metals or growth factors. Xylose and mannose are the main constitents of wood hemicellloses. Percentage of xylose in extractive-free basis is ranging 12 to 26 in hardwoods and 5 to 1 in softwoods, whereas that of mannose is less than 3% in hardwoods and abot 1 % in softwoods. g ) As mannose was the good carbon sorce as xylose for Ch. globosm, tilizing ability of mannan shold be investigated in relation to the higher resistance of softwoods, thogh the lower mannanase activity than the xylanase activity in cltre filtrates from Ch. globosm has been reported by KILICH.l) Change of economic coefficient in five carbon sorces are shown in Fig. 3. conomic coefficient is one of the methods to determine the efficiency of conversion of carbon sorce to mycelim. It is defined by the formla; mycelimdry weight (g) x 1 amont of carbon compond consmed (g) Fig. 3. c Q) 5 ::: 4 Q) o U 3 oc 8 2 w 1 t::,. o /./...--r--q- mannose A. galactose t::,. maltose X xylose ol..-_...l-_-"-_...l.l--_...l-_-i-_--l_ o Incbation Period (day) fficiency of conversion of five carbon sorces to mycelim of Chaetomim globosm. conomic coefficient can be seen by inspection to be maximal when respiratory carbon dioxide and solble metabolic prodcts are minimal in qantity. Very low vales reflect probably the prodction of significant amonts of solble componds in high-carbohydrate media.m As mentioned above, xylose and mannose were reflecting rapid growth on these sorces. tilized more rapidly than other sorces, However, economic coefficient of xylose maintained the moderately same level throghot the incbation period, whereas that of mannose increased rapidly in early period and then decreased gradally. This sggests that in case of mannose transition from accmlation of metabolic prodct to mycelial formation was abrpt. Consmption rate of maltose was slower compared to other sorces, however, economic coefficient of the sorce was generally kept in higher level and mycelial yield was eqivalent

6 TAKAHASHI, NISHIMOTO: Utilization of Carbohydrates by Chaetomim globosm to other sorces. This indicates the presence of maltase and the effective se of maltose for mycelial formation. However, occrrence of maltose in natre, as a prodct of starch hydrolysis, is very scarce. A glcose B xylose C man nose I6 I6 I I I I V o o day day day galactose maltose F CMC I6 :r:6. :c I 1 I o o day day day G arabinose H glcronic galactronic 7 7 acid 7 acid I6 :r:6 :r: I I I o o day day day J crde xylan 7 V 7 I 6 :r: 6 Fig. 4. K crde xylan V 7 :r:6 L crde xylon \r I 1 I o o o day day day Change in ph vale of ntrient medim with varios carbon sorces dring corse of incbation (A-J; Chaetomim globosm, K; Coriols versicolor, L; Tyromyces palstris)

7 WOOD RSARCH No. 54 (1973) Arabinose occrs as the hemicelllose component, thogh the content is very low. In contrast to xylose, this sgar was not tilized at all by Ch. globosm, probably indicating the lack of metabolic pathway-arabinose-hiblose--hiblose 5-phosphatexyllose5-phosphate.J2) Inability to tilize both isomers of arabinose, especially to D-arabinose, has been freqently reported in many fngi.13) Glcronic acid and galactronic acid, occrring in wood as a fraction of glcronoxylan and galactoglcomannan, were also not tilized at all. However, as ph vales in these ronic acid media were very low throgh the incbation period (Fig. 4), the inability to tilize these sorces seems to be related to impermeability of carbon sorces cased by low ph level. the experiment with solid agar medim, containing glcronic acid as the sole carbon sorce and adjsted to ph 6.5 by sodim hydroxide, poor growth, thogh better than no carbon medim, was observed, bt it was apparently lesser than Coriols versicolor and Tyromyces palstris (Fig. 5). From these reslts, it was sggested that tilizing ability of Ch. globosm to the ronic acid in wood hemicelllose was very low. In 9 8 Chaetomim globosm 7 Coriols versicolor 6 X Tyromyces palstris r'\ t;,..;;o:::::: Cl " '' '''' '- o Incbation Period (day) Fig. 5. Radial growth of three fngal species grown on solid agar medim with glcronic acid as sole carbon sorce (Broken Iine shows the diameter of Petri dish.). Growth on CMC was also very poor bt the ph vale of the medim was not low as those of glcronic acid and galactronic acid (Growth at third day was not measred de to the difficlty of separating the mycelim from the carbon sorce.). As a celllolytic activity of Ch. globosm, in the present investigation, was not estimated enzymatically, the reason for sch poor growth is ncertain. Althogh Ch. globosm is ndobtedly the trly celllolytic fngs and CMC is one of the commonly sed sbstrates to determine the celllolytic activity, carboxymethyl grop or sodim in the sbstrate may be spressive to the growth of the fngs. Degree of sbstittion and degree of polymerization of CMC sed in this experiment were.5 to.7 and 3 to 45, respectively. Growth, consmption rate and economic coefficient of three fngal species in xylancontaining medim are shown in Figs. 6 to 8. The contents of lignin and ronic anhydride of this sorce were 6.25 % and 5.56 %, respectively. As mentioned in xperimental, determination of pentosan was carried ot with the netral fraction from hydrolyzed cltre filtrates. Ths, correction to the frfral yield from ronic acids was not made. Maximm mycelial yields of test fngi were eqivalent as shown in Fig. 6. However, Ch. globosm grew more rapidly and spported the same level of growth in longer time period than Co. versicolor and Ty. palstris. Conseqently, economic coefficient in Ch. globosm - 6 -

8 TAKAHASHI, NISHIMOTO: Utilization of Carbohydrates by Chaetomim globosm IOO 12 g 11 "' :7' "a; 3: 8 > >. :: /;\!j o, Q. X-- X 9 In.2- c a- 8 - a: 7 C 6 ::J In c 5 4 Chaetomim globosm o Coriols versicolor 3 X Tyromyces palstris Choetomim globosm o Coriols versicolor X Tyromyces palstris 3 Fig Incbotion Period (day) Mycelial dry weight of three fngal species grown in ntrient medim with crde xylan as sole carbon sorce. Fig Incbation Period (doy) Consmption rate of pentose by three fngal species grown in ntrient medim with crde xylan as sole carbon sorce. 1: 4 :-Q; 3 8 " 2 o c o w lo X Tyromyces polstris '--_...L-_--1"--_...l..._--'-.l.-_...L-_--I... o Incbation Period (day) Fig _ f ----;;/' o Coriols versicolor o Choetomim globosm fficiency of conversion of crde Fig. xylan to mycelim of three fngal species ::J = 6 5 :: ' F 2 c3 1"'""""':;o..= Chaetomim globosm o Coriols versicolor X Tyromyces palstris Incbation Period (day) 9. Radial growth of three fngal species grown on solid agar medim with xylose as sole carbon sorce (Broken line shows the dimeter of Petri dish.). kept higher level throghot the incbation period, althogh the progressive increase of consmption rate showed fairly similar pattern to those of other fngi. Radial growths of these fngi on solid agar medim containing xylose as the sole carbon sorce were almost same to fifth day, bt after that day Ch. globosm was sperior to other fngi (Fig. 9). As shown in Fig. 4, the ph vale of each ntrient medim of which carbon sorce was available to test fngi exhibited a similar pattern of transition, that is to say, a considerably - 7 -

9 WOOD RSARCH No. 54 (1973) rapid fall and recovery of the vale dring corse of incbation. Particlarly in xylose- and xylan-containing media inoclated with Ch. globosm, sch trend was more prominent. pattern of transition in ph vale of ntrient medim has been sally obsedved in many fngi and seems to reflect the accmlation of some acidic intermediate prodcts. In the present experiments, growth factors, e.g. thiamine-hci, biotin, nicotinic acid, etc., were not added at all to the ntrient medim. Sch Then, rapid decline of mycelial yield, observed in the two species of basidiomycetos fngi, may be related to a defficiency of growth factors or to some inadeqacy of medim composition. Ths, metabolic activity of Ch. globosm to the hardwood hemicelllose was not discssed here comparing those. of other wood-decaying fngi. However, considering the reslts with the ability of Ch. globosm to tilize xylose and xylan, this soft rot fngs has possibly an active pathway to metabolize these mono- and polysaccharide, indicating the possible reference to the greater ssceptibility of hardwoods to soft rot fngi. References 1) BAILY, P. J., W. LIS and R. ROSCH, Biodeterioration of Materials, 546, edited by A. H. WALTRS and J. J. LPHlCK, Amsterdam, New York and London, lsevier (1968). 2) T. MIWA and A. YASUMURA, Seibts-gak Zikken-ho Koza (in Japanese), 6F, 13, Nakayama Shoten (1955). 3) B. L. BROWNING, Methods of Wood Chemistry, II, 633, New York, London and Sydney, Interscience (1967). 4) V. W. COCHRAN, Physiology of Fngi, 61, New York, John Wiley and Sons (1958). 5) Ibid., 6. 6) D. PRLMAN, The Fngi, J, 481, edited by G. C. AINSWORTH and A. S. SUSSMAN, New York and London, Academic Press (1965). 7) V. W. COCHRAN, Physiology of Fngi, 62. 8) H. W. DOLL, Bacterial Metabolism, 188, New York and London, Academic Press (1969). 9) T.. TIMML, Wood Science and Technology, 1, 45 (1967). 1) G. KILlCH, P. J. BAILY and W. LIS, Wood Science and Technology, 4, 273 (197). 11) D. PRIMAN, The Fngi, I, ) H. W. DOLL, Bacterial Metabolism, ) B. HNINGSSON, Stdia Forestalia Secica, No. 52 (1967). -8-

Tricarboxylic Acid Metabolism Studies in the Ovary Throughout the Menstrual Cycle. S. J. Behrman, M.D., M.R.C.O.G., and Gregory S. Duboff, M.S., D.Sc.

Tricarboxylic Acid Metabolism Stdies in the Ovary Throghot the Menstral Cycle S. J. Behrman, M.D., M.R.C.O.G., and Gregory S. Dboff, M.S., D.Sc. THE VARIOUS ENDOCRINE CHANGEs dring the hman menstral cycle