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Distribution of thermophilic and thermotolerant fungi in a spruce-pine chip pile and their effects on… Ofosu-Asiedu, Albert 1970

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THE DISTRIBUTION OF THERMOPHILIC AND THERMOTOLERANT FUNGI IN A SPRUCE-PINE CHIP PILE AND THEIR EFFECTS ON SOME CONIFEROUS WOODS by ALBERT OFOSU-ASIEDU B . S c , Hebrew U n i v e r s i t y , 1961 M . S c , Hebrew U n i v e r s i t y , 1963 A.I.W. Sc. U.K. 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS  FOR THE  DOCTOR OF PHILOSOPHY DEGREE i n the Department o f Forestry  We a c c e p t t h i s t h e s i s as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1970  ii  ABSTRACT  A study  i n t o the d i s t r i b u t i o n o f t h e r m o p h i l i c and t h e r m o t o l e r a n t  f u n g i i n a s p r u c e - p i n e wood c h i p p i l e F i v e treatments  c o n s i s t i n g of p i n e , s p r u c e , i n c o r p o r a t i o n of wood  i n t o spruce, s t e r i l i z e d a Ptychogaster different  spruce and s t e r i l i z e d  sp. were examined.  moisture  spruce i n o c u l a t e d w i t h  Samples of wood c h i p s b u r i e d a t s i x  From 100 randomly s e l e c t e d c h i p s from each sample  the f u n g i were i s o l a t e d on 2% malt, 45°C.  fines  l o c a t i o n s i n the c h i p p i l e were examined a f t e r 3, 6 and 12  months s t o r a g e p e r i o d s .  and  i n P r i n c e George was c a r r i e d o u t .  Data on temperature  0.5% m a l i c a c i d and 2% agar a t 25°  d u r i n g s t o r a g e and a c i d i t y o f wood c h i p s ,  c o n t e n t and weight l o s s a t t h e time of sampling  f o r the s i x p o s i t i o n s  i n the wood c h i p  were r e c o r d e d  pile.  T h e r m o p h i l i c f u n g i c o l o n i z e d the i n n e r r e g i o n s w h i l e f u n g i i n h a b i t e d the outer r e g i o n s of the wood c h i p p i l e .  thermotolerant Among the t h e r -  mophilic fungi, l i s t e d  a c c o r d i n g t o f r e q u e n c y o f i s o l a t i o n were  Byssochlamys e m e r s o n i i  Stolk-Apinis, Allescheria t e r r e s t r i s  Sporotrichum  thermophile  Humicola l a n u g i n o s a  A p i n i s , Thermoascus a u r a n t i a c u s  ( G r i f f o n a n d Maublanc) Bunce.  f u n g i were A s p e r g i l l u s fumigatus. (Gilman and Abbot) Comb. Nov.  Apinis, Miehe and  The most common t h e r m o t o l e r a n t  F r e s e n i u s and Chrysosporium  pruinosum  iii  F u n g a l d i s t r i b u t i o n was g e n e r a l l y r e l a t e d t o p o s i t i o n i n the wood c h i p p i l e .  Of the a s s o c i a t e d  factors  temperature  (17°-45°C)  was most s t r o n g l y r e l a t e d to f u n g a l d i s t r i b u t i o n , whereas a c i d i t y of wood c h i p s and m o i s t u r e c o n t e n t d i d not v a r y Incubation all  of wood samples on c u l t u r e s demonstrated t h e a b i l i t y o f  t h e common t h e r m o p h i l i c  l o s s of lodge p o l e  g r e a t l y between p o s i t i o n s .  and t h e r m o t o l e r a n t  f u n g i t o cause weight  p i n e P i n u s c o n t o r t a Dougl. v a r l a t i f o l i a ,  p i n e P i n u s ponderosa Laws, and spruce P i c e a g l a n c a samples.  ponderosa  (Moench) Voss sap wood  These weight l o s s e s v a r i e d from 0.65% t o 25% a f t e r s i x weeks  incubation.  Temperature, medium and type of wood a f f e c t e d the a b i l i t y of  the f u n g i to cause weight l o s s .  No s y n e r g i s t i c or a n t a g o n i s t i c e f f e c t s '  e x i s t e d between t h e t h e r m o p h i l i c  fungi.  Chemical a n a l y s i s o f degraded wood i n d i c a t e d t h a t the t h e r m o p h i l i c fungi u t i l i z e d  the a r a b i n o s e f r a c t i o n of the h e m i c e l l u l o s e p r e f e r e n t i a l l y .  iv  In p r e s e n t i n g ments f o r an that  the  I further  the  of B r i t i s h  agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of  representatives.  be  g r a n t e d by  I t i s understood  f i n a n c i a l gain  s h a l l not  be  August,  1970  the Head of my  that  the  require-  Columbia, I agree and  study.  this thesis  for  Department o r by  his  c o p y i n g or p u b l i c a t i o n of  a l l o w e d w i t h o u t my  Department o f F o r e s t r y The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, B.C., Canada  Date:  University  f u l f i l m e n t of  s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  s c h o l a r l y purposes may  for  this thesis in p a r t i a l  advanced degree at  Library  -  written  this  permission  thesis  V  TABLE OF CONTENTS  Page  ABSTRACT  i i  TABLE OF CONTENTS  . v  LIST OF TABLES  ix  LIST OF FIGURES  xi  ACKNOWLEDGEMENTS I II  INTRODUCTION  x i i  .  1  FIELD STUDY OF FUNGAL DISTRIBUTION  6  A  INTRODUCTION  6  B  LITERATURE REVIEW  7  1.  C  Advantages and d i s a d v a n t a g e s o f o u t s i d e wood chip storage  7  2.  Types o f wood c h i p p i l e s  9  3.  E n v i r o n m e n t a l c o n d i t i o n s i n wood c h i p p i l e s  13  4.  Fungi i s o l a t e d  18  5.  Damage i n wood c h i p p i l e s  from wood c h i p p i l e s  21  MATERIALS AND METHODS  24  1.  The e x p e r i m e n t a l wood c h i p p i l e  24  2.  Sampling  27  3.  Treatments  4.  Preparation  5.  Measurement o f e n v i r o n m e n t a l c o n d i t i o n s  positions  27 o f wood c h i p samples  29 30  -  vi  Page  6.  7. D  a.  Temperature i n the wood c h i p p i l e  30  b.  M o i s t u r e c o n t e n t of wood c h i p s  32  c.  A c i d i t y o f wood c h i p s  32  I s o l a t i o n of f u n g i  32  a.  Sampling p r o c e d u r e  32  b.  I s o l a t i o n and g r o u p i n g o f f u n g i  33  D e t e r m i n a t i o n of wood c h i p weight l o s s  RESULTS  36  1.  L i s t of f u n g i i s o l a t e d  36  2.  D i s t r i b u t i o n o f common t h e r m o p h i l i c and thermotolerant  3.  E  35  fungi  36  Environmental f a c t o r s  38  a.  Temperature i n the wood c h i p p i l e  38  b.  M o i s t u r e c o n t e n t o f wood c h i p s  43  c.  A c i d i t y o f wood c h i p s  45  4.  Weight l o s s o f wood c h i p s  46  5.  Evaluation  47  6.  Relationship  o f treatments between the v a r i a b l e s measured  51  DISCUSSION  53  1.  F u n g i i n the wood c h i p p i l e  53  2.  Temperature i n the wood c h i p p i l e  56  3.  A c i d i t y o f wood c h i p s  58  4.  M o i s t u r e i n the wood c h i p p i l e  58  5.  Damage  59  -vii-  Page  III  LABORATORY  INVESTIGATION OF WOOD DEGRADATION  CAUSED  BY THERMOPHILIC AND THERMOTOLERANT FUNGI  62  A  INTRODUCTION  62  B  LITERATURE REVIEW  63  C  GENERAL METHODS  66  D  DEVELOPMENT  E  OF A METHOD FOR THE STUDY OF THE  ABILITY OF THE FUNGI TO CAUSE WEIGHT LOSSES  69  1.  E f f e c t o f media  69  2.  E f f e c t o f wood sample s i z e  75  3.  E f f e c t o f methods o f i n o c u l a t i o n  78  4.  E f f e c t o f d u r a t i o n of i n c u b a t i o n  80  5.  E f f e c t o f temperature  82  6.  Summary  85  WEIGHT LOSSES CAUSED BY THE THERMOPHILIC AND THERMOTOLERANT FUNGI  85  1.  E v a l u a t i o n o f the common i s o l a t e s  85  2.  S u s c e p t i b i l i t y o f lodgepole  p i n e and w h i t e  spruce wood t o a t t a c k by s e v e r a l f u n g a l i s o l a t e s 87 3. F IV  E f f e c t o f mixed i s o l a t e s on weight l o s s e s  DISCUSSION  90 92  CHEMICAL ANALYSIS OF DEGRADED WOOD  100  A  INTRODUCTION  100  B  LITERATURE REVIEW  100  C  MATERIALS AND METHODS  101  D  RESULTS  103  -  viii-  Page  E V  DISCUSSION  104  GENERAL DISCUSSION AND CONCLUSION  105  VI  REFERENCES  111  VII  APPENDIX 1  116  VIII  APPENDIX 2  117  -  ix  -  LIST OF TABLES  Page T a b l e 1.  F i n a l moisture  T a b l e 2.  F i n a l a c i d i t y o f wood c h i p s  45  T a b l e 3.  F i n a l weight l o s s o f samples i n p e r c e n t  46  T a b l e 4.  Mean f u n g a l count f o r d i f f e r e n t s t o r i n g c h i p s f o r t h r e e months  T a b l e 5.  T a b l e 6.  T a b l e 7.  T a b l e 8.  T a b l e 9.  c o n t e n t o f wood c h i p s  44  treatments  after 48  Average number o f f u n g i i s o l a t e d p e r p o s i t i o n t h r e e months s t o r a g e  after 49  F u n g a l counts a t d i f f e r e n t s t o r a g e times a t the different positions  50  F u n g a l counts a t d i f f e r e n t s t o r a g e times w i t h d i f f e r e n t treatments. These a r e averages f o r six positions.  50  Summary o f r e g r e s s i o n a n a l y s i s o f t o t a l weight l o s s on t o t a l f u n g a l count P e r c e n t a g e weight l o s s e s caused sapwood on d i f f e r e n t media  temperature  and 51  to ponderosa p i n e 70  T a b l e 10.  M o i s t u r e c o n t e n t o f samples on each medium  71  T a b l e 11.  P e r c e n t weight l o s s e s o b t a i n e d f o r A. t e r r e s t r i s and T_. a u r a n t i a c u s growing on medium p r e p a r e d from c e l l u l o s e , MgSO4-7H.20 and K2HPO4 w i t h the absence o f y e a s t o r NH4NO3 o r KH2PO4  75  P e r c e n t weight l o s s and a b s o l u t e weight l o s s o f wood caused by t h e r m o p h i l i c f u n g i growing on d i f f e r e n t s i z e s o f ponderosa p i n e sapwood d u r i n g s i x weeks incubation  77  Average p e r c e n t a g e weight l o s s o f wood samples caused by t h e r m o p h i l i c f u n g i u s i n g d i f f e r e n t methods o f p r o v i d i n g inoculum s o u r c e  19  T a b l e 12.  T a b l e 13.  T a b l e 14.  P e r c e n t weight l o s s o f ponderosa p i n e caused by some t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i a t 45°C i n c u b a t i o n 86  Percentage weight l o s s of l o d g e p o l e p i n e and spruce sapwood caused by some t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i a t 45°C a f t e r 6 weeks i n c u b a t i o n Percentage weight l o s s f o r s t a i n e d and u n s t a i n e d s p r u c e i n o c u l a t e d w i t h some t h e r m o p h i l i c f u n g i E f f e c t of i n t e r a c t i o n between A. t e r r e s t r i s , B. e m e r s o n i i and t h e r m o p h i l e on weight l o s s e s of ponderosa p i n e sapwood i n c u b a t e d a t 45°C and 50°C C o n c e n t r a t i o n of v a r i o u s c h e m i c a l components of ponderosa p i n e sapwood a f t e r d e g r a d a t i o n by some thermo p h i l i c fungi. Percentages a r e based on degraded wood  xi  -  LIST OF FIGURES  Page  F i g u r e 1.  E a s t and West view of the c h i p p i l e location^of  c h i p samples  showing  1 to 6.  26  F i g u r e 2.  South view s e c t i o n of the c h i p p i l e  26  F i g u r e 3.  The f o u r s e c t i o n s of the c h i p p i l e which were broken down a f t e r 3, 6, 12 and 24 months  28  F i g u r e 4.  R e t r i e v a l of the c h i p samples  28  F i g u r e 5.  The c h i p p i l e  34  F i g u r e 6.  Byssochlamys e m e r s o n i i growing out of c h i p s on malt and 0.5% m a l i c a c i d agar F u n g a l count  F i g u r e 7. F i g u r e 8.  F i g u r e 9.  34 37  D a i l y temperature c u r v e s f o r the s i x p o s i t i o n s i n the p i l e d u r i n g the f i r s t t h r i t y - f o u r days of s t o r a g e of c h i p s Temperature  39  c u r v e s f o r the s i x p o s i t i o n s i n the  c h i p p i l e d u r i n g 80 weeks of s t o r a g e  of c h i p s  F i g u r e 10.  Average monthly ambient  F i g u r e 11.  The r e l a t i o n s h i p between t o t a l f u n g a l count and m o i s t u r e c o n t e n t of c h i p s  F i g u r e 12.  The r e l a t i o n s h i p between t o t a l f u n g a l count and temperature  F i g u r e 13.  2%  41  temprature i n P r i n c e George  The r e l a t i o n s h i p between t o t a l weight l o s s  42  52 total 52  f u n g a l count and wood 52  F i g u r e 14.  A l l e s c h e r i a t e r r e s t r i s and Thermoascus a u r a n t i a c u s growing on Abrams c e l l u l o s e medium and ponderosa p i n e . T o p , A. t e r r e s t r i s ; bottom, _T. a u r a n t i a c u s 58  F i g u r e 15.  Growth o f A l l e s c h e r i a t e r r e s t r i s and Thermoascus a u r a n t i a c u s on y e a s t - c e l l u l o s e medium and ponderosa pine. Top, A. t e r r e s t r i s ; bottom, T_. a u r a n t i a c u s  73  F i g u r e 16.  Changes i n weight l o s s e s w i t h d u r a t i o n of i n c u b a t i o n  81  F i g u r e 17.  Changes i n weight l o s s e s w i t h changes  84  i n temperature  xii  ACKNOWLEDGEMENTS  The p r o j e c t was  i n t r o d u c e d to me  by Dr. R. W.  Manager, F o r e s t P r o d u c t s L a b o r a t o r y , Vancouver, the study was  B.C.  Kennedy, Programme F i n a n c i a l help f o r  p r o v i d e d by the Canada Department of F i s h e r i e s and  The management of the L a b o r a t o r y has been generous tance d u r i n g the e x e c u t i o n of the p r o j e c t .  For t h i s , my  Forestry.  in i t s assissincere  thanks  go t o the management of the L a b o r a t o r y , e s p e c i a l l y to Dr. Kennedy. author e x p r e s s e s h i s a p p r e c i a t i o n to Dr. R.  S. Smith, Research  Scientist  i n Wood P a t h o l o g y a t F o r e s t P r o d u c t s L a b o r a t o r y , f o r h i s f r i e n d l y and  encouragement throughout  and  t o Dr. J . V. H a t t o n , Research  The  guidance  the study and p r e p a r a t i o n of the m a n u s c r i p t , Scientist  i n Pulping Processes f o r h i s  h e l p i n p r o v i d i n g him w i t h some v a l u a b l e d a t a . T e c h n i c a l h e l p when needed was Mrs.  Christine  Sharman and Mrs.  k i n d l y p r o v i d e d by Mr.  C. B. Johansen.  Eric  I express my  Johnson,  thanks  to  them. My  a p p r e c i a t i o n goes t o Dr. A p i n i s and Dr. C a r m i c h a e l f o r t h e i r  assistance i n identifying  some of the c u l t u r e s .  I thank a l s o Dr. B a r t  van der Kamp, A s s i s t a n t P r o f e s s o r of F o r e s t P a t h o l o g y and my  major a d v i s o r  i n the F a c u l t y of F o r e s t r y f o r h i s k i n d guidance and worthwhile during  the e x e c u t i o n of t h i s work and  suggestions  i n the p r e p a r a t i o n of the m a n u s c r i p t .  xiii  The author e x p r e s s e s h i s a p p r e c i a t i o n t o a l l h i s s t u d e n t who were a s o u r c e of encouragement when t h i n g s became d i f f i c u l t to  h i s wife f o r her patience.  friends and  finally  I  INTRODUCTION  The  s t o r a g e of wood i n the form of wood c h i p p i l e s  p r a c t i c e w i d e l y adopted  by p u l p m i l l s  around the w o r l d .  The method  has many advantages over roundwood s t o r a g e such as reduced c o s t , reduced  s t o r a g e a r e a , the a b i l i t y  to any p r o p o r t i o n e a s i l y , pulp economically.  and  is a  handling  to mix v a r i o u s types of c h i p s  the a b i l i t y  to use s a w m i l l r e s i d u e s f o r  Storage of wood i n c h i p p i l e s a l s o has  certain  dis-  advantages such as the h i g h temperatures  commonly generated  i n the  p i l e w i t h the a t t e n d a n t r i s k of f i r e and  the d e g r a d a t i o n of the wood  chips.  I t has been e s t i m a t e d , f o r i n s t a n c e , t h a t i n the i n t e r i o r of  British  Columbia, annual  by 1987  due  l o s s e s of $5,000,000 to $30,000,000 w i l l  to these f a c t o r s i f c o r r e c t i v e a c t i o n i s not  taken  occur  (Hatton,  Smith and Rogers, 1968). In the i n t e r i o r c o n t o r t a Dougl. spruce r e f e r s  the main s p e c i e s used  v a r l a t i f o l i a ) and  to t r e e s b e l o n g i n g  spruce.  was  s p e c i e s as i t i s found  For the purpose of t h i s  around P r i n c e George B.C.  No  attempt  forms.  The wood i s e i t h e r c h i p p e d a t the p u l p m i l l from as  thesis  the v a r i o u s h y b r i d s formed  made to d i s t i n g u i s h between the v a r i o u s  or e l s e i t a r r i v e s a t the m i l l  (Pinus  to the complex c o n s i s t i n g of P i c e a g l a u c a  (Moench) Voss, P i c e a e n g e l m a n i i P a r r y and between these two  are l o d g e p o l e p i n e  c h i p s produced  f r e s h l y cut logs  from s a w m i l l r e s i d u e .  In  -  contrast mills  Columbia and  of wood, r e p o r t l i t t l e  be  1954).  degradation  to the new  The restricted  i n i t i a t e d by  straw, manure, peat  secondly,  not exceed heat  b e i n g s t o r e d and  the c o a s t  and  depends on  i t occurs  degradation  g e n e r a l l y i n p i l e d o r g a n i c matter  g r a i n (Miche,  1907;  H u l p o i , 1939).  l a r g e enough so t h a t heat  two  i n the  i s not  Isachenko In g e n e r a l  t h e r e must be an adequate s u p p l y  these  cannot  Hence a l a r g e s c a l e  f a c t o r s p l u s the n a t u r e  the e f f e c t s of v a r i o u s e n v i r o n m e n t a l  and two of water  d i s s i p a t i o n does  g e n e r a t i o n u n t i l a h i g h temperature i s reached.  temperature reached  of heat  Firstly,  the p i l e must be  on  storage  Laboratory.  g e n e r a t i o n and  Waksman, Cordon and  c o n d i t i o n s must be met.  species  the s t o r a g e of wood i n c h i p p i l e s  the F o r e s t P r o d u c t s  phenomenon of heat  Mai'chevskaya, 1936;  gained  s i t u a t i o n i n the i n t e r i o r .  to p i l e d wood c h i p s , but  such as hay,  c o a s t a l pulp  of wood c h i p s even a f t e r a year of  I t f o l l o w s t h a t the e x p e r i e n c e  applied directly  i n t e r i o r was  i n the i n t e r i o r ,  the P a c i f i c North West, u s i n g d i f f e r e n t  study of the problems a s s o c i a t e d w i t h  and  -  to the l o s s e s s u s t a i n e d by pulp m i l l s  in British  (Wright,  2  The maximum of the m a t e r i a l  c o n d i t i o n s on  the mechanisms  production. V a r i o u s mechanisms f o r the p r o d u c t i o n of heat have been p r o p o s e d .  These are r e s p i r a t i o n of the p l a n t m a t e r i a l , r e s p i r a t i o n of organisms, that u t i l i z e  the s t o r e d m a t e r i a l as a s o u r c e  o r g a n i c substances chip p i l e s  i n the s t o r e d m a t e r i a l s .  i s not a s u b j e c t of t h i s  thesis.  of f o o d , and The  a u t o - o x i d a t i o n of  o r i g i n of heat  production i n  -  3  -  I s o l a t i o n s from such n a t u r a l l y heated p i l e s o f o r g a n i c have y i e l d e d b a c t e r i a , f u n g i and a c t i n o m y c e t e s , a l l w i t h ability  to be m e t a b o l i c a l l y a c t i v e a t h i g h  of organisms have been i s o l a t e d  the p e c u l i a r  temperatures.  from wood c h i p p i l e s .  matter  A l l these  classes  General experience  w i t h wood d e g r a d a t i o n has shown, however, t h a t on wood f u n g i a r e much more important  than e i t h e r b a c t e r i a o r a c t i n o m y c e t e s :  Since,  i t was  impossible  to sample a l l t h r e e groups a d e q u a t e l y i n the e x p e r i m e n t a l c h i p p i l e in  the p r e s e n t  limit  used  study because of l a c k of time and space, i t was d e c i d e d  to  sampling to f u n g i . Most  fungi i n contrast  to b a c t e r i a a r e unable to grow a t •  temperatures above 35°C, a l t h o u g h spores and v a r i o u s  other  r e s t i n g stages  of these f u n g i such as s c l e r o t i a may o c c a s i o n a l l y s u r v i c e exposure to higher  temperatures.  A relatively  temperatures up to 60°C. thermophilic  These have been d i v i d e d i n t o two groups  and t h e r m o t o l e r a n t  For  thesis, thermophilic  T h e r m o t o l e r a n t f u n g i a r e those  which grow a t both 25°C and 45°C on the above medium. to grow a t 25°C b u t n o t a t 45°C a r e c a l l e d m e s o p h i l i c In l a r g e commercial wood c h i p p i l e s , i n t e r i o r region generally  from the o u t e r thermophilic with  exceeds 45°C.  l a y e r s o f such a c h i p p i l e ,  and t h e r m o t o l e r a n t  thermophilic  f u n g i a r e d e f i n e d as  to grow on a 2% malt e x t r a c t , 2% agar, and 0.5% m a l i c  a c i d medium a t 45°C b u t not a t 25°C.  the  called  fungi.  the purpose o f t h i s  those f u n g i a b l e  s m a l l number o f f u n g i can grow a t  fungi.  and t h e r m o t o l e r a n t  fungi  Fungi which a r e a b l e fungi.  the temperature a t t a i n e d i n  I t appears t h e r e f o r e fungal a c t i v i t y  that  apart  i s r e s t r i c t e d to  F o r t h i s r e a s o n t h i s study d e a l s  f u n g i and does not concern i t s e l f  only  with  - l i -  the  a c t i v i t y of m e s o p h i l i c This  f u n g i i n the o u t e r ,  cooler regions  o f the p i l e .  t h e s i s forms a p a r t o f the l a r g e r i n v e s t i g a t i o n mentioned  above and d e a l s w i t h the r o l e of those f u n g i which i n h a b i t the h o t t e r p a r t s o f a wood c h i p p i l e . inhabiting  The c e n t r a l h y p o t h e s i s i s t h a t the f u n g i  the h o t t e r p a r t s o f the c h i p p i l e a r e d i r e c t l y  the l o s s o f wood s u b s t a n c e and t h a t production. Firstly,  t h i s l o s s leads  responsible f o r  to a d e c r e a s e i n pulp  To s u p p o r t t h i s h y p o t h e s i s the f o l l o w i n g w i l l be n e c e s s a r y .  i t must be v e r i f i e d  that fungi are present  i n the h o t t e r  parts  of a c h i p p i l e and t h a t weight l o s s e s a r e i n c u r r e d i n these p o s i t i o n s . S e c o n d l y , i t must be demonstrated t h a t the v a r i a t i o n i n weight l o s s e s between v a r i o u s of f u n g i . due  observed  p o s i t i o n s i n the p i l e can be a t t r i b u t e d to the d i s t r i b u t i o n  T h i r d l y * / i t must be shown t h a t e n v i r o n m e n t a l c o n d i t i o n s  to such f a c t o r s as temperature, m o i s t u r e and a c i d i t y ,  i n the p i l e  i f they can be  c o r r e l a t e d w i t h weight l o s s e s , cannot by themselves r e s u l t i n the observed weight l o s s e s , and t h a t such c o r r e l a t i o n s can be e x p l a i n e d  by the e f f e c t  of these e n v i r o n m e n t a l f a c t o r s on the p r e s e n c e of f u n g i and t h e i r to degrade wood c h i p s . isolated chips be  Fourthly,  i t must be e s t a b l i s h e d  from the c h i p p i l e a r e c a p a b l e o f c a u s i n g  under c a r e f u l l y c o n t r o l l e d  ability  t h a t the f u n g i  weight l o s s e s o f wood  environmental c o n d i t i o n s .  L a s t l y , i t must  shown t h a t a t l e a s t p a r t o f the weight l o s s o f wood i n c u r r e d d u r i n g  storage  i s a t t r i b u t a b l e to the l o s s of components o f the wood which a r e u t i l i z e d as  pulp.  -  The  5 -  t h e s i s i s d i v i d e d i n t o three p a r t s .  The f i r s t  part  d e a l s w i t h the study of f u n g i , e n v i r o n m e n t a l c o n d i t i o n s , and weight i n an e x p e r i m e n t a l c h i p p i l e , of the a b i l i t y last  the second  of commonly i s o l a t e d  losses  i s concerned w i t h the d e m o n s t r a t i o n  f u n g i to cause weight  l o s s e s and  p a r t d e a l s w i t h the c h e m i c a l a n a l y s i s of degraded wood.  the  -  II  A  6 -  FIELD STUDY OF FUNGAL DISTRIBUTION  INTRODUCTION  T h i s s e c t i o n o f the t h e s i s i s concerned w i t h the d e m o n s t r a t i o n that thermophilic hot  regions  regions, fungi.  and t h e r m o t o l e r a n t  of a chip p i l e ,  fungi are present  and a c t i v e i n the  t h a t weight l o s s e s o f wood o c c u r i n these  and t h a t such weight l o s s e s a r e r e l a t e d t o the d i s t r i b u t i o n o f Furthermore, i t d e a l s w i t h the r e l a t i o n s h i p s between  d i s t r i b u t i o n , weight l o s s , and v a r i o u s  fungal  environmental f a c t o r s .  Lastly,  an attempt i s made to show how f u n g a l d i s t r i b u t i o n , weight l o s s e s and environmental conditions The by  develop i n time.  experimental chip p i l e u t i l i z e d  the F o r e s t P r o d u c t s L a b o r a t o r y .  purpose o f t h i s study.  For instance  to be l a r g e enough to a l l o w chips.  f o r t h i s study was d e s i g n e d  I t was n o t planned s o l e l y f o r the samples i n s e r t e d i n t o the p i l e had  experimental pulping  T h i s made the a c c u r a t e  determination  s t u d i e s o f the s t o r e d  o f weight l o s s e s  rather  difficult. The 3,  p i l e consisted of four s e c t i o n s ,  6, 12 and 24 months.  three p e r i o d s  only.  T h i s study c o n t a i n s  to be broken down a f t e r  the r e s u l t s o f the f i r s t  I t was assumed t h a t s i m i l a r c o n d i t i o n s  s i m i l a r p o s i t i o n throughout the whole c h i p  pile.  prevailed at  -  7  -  S e v e r a l t r e a t m e n t s were i n c o r p o r a t e d The  first  two  c o n s i s t e d of bags f i l l e d  chips i n order  i n t o the c h i p  w i t h pure spruce  A third  L a s t l y two  i n c o r p o r a t i o n of a " c o r e " bag  c o n t a i n i n g spruce  a s p e c i e s of P h y t o g a s t e r ,  pine  various  treatment c o n s i s t e d of the i n c o r p o r a t i o n of  " f i n e s " i n t o the sample bag.  treatments c o n s i s t e d of  a thermotolerant  basidiomycete  i n e a s t e r n Canada i n t o the sample bag,  of s t e r i l e  spruce  chips i n a core  the  chips inoculated  chip p i l e s  and  with  isolated  from  a control consisting  bag.  LITERATURE REVIEW  1.  Advantages and  d i s a d v a n t a g e s of o u t s i d e wood c h i p  Compared to roundwood s t o r a g e , s t o r a g e a r e many (Anon, 1961; these It  pure  to determine the e f f e c t of these s p e c i e s on the  factors studied.  B.  and  pile.  the advantages of o u t s i d e  Holekamp, 1962;  Westaway, 1968).  advantages i s the lower c o s t i n h a n d l i n g  i s estimated  t h a t a medium s i z e d m i l l can  u s i n g o u t s i d e wood c h i p s t o r a g e O p e r a t i o n a l advantages as f o l l o w s :  storage  and  storage  save up  C l a r k , 1963)  of  of the m a t e r i a l .  to $50,000 a n n u a l l y  i n s t e a d of roundwood s t o r a g e  (Holekamp, 1962;  One  chip  ( C l a r k , 1963).  have been r e p o r t e d  -  8  -  1.  Reduced f i b e r  2.  Decreased h a n d l i n g  3.  Large manpower s a v i n g .  4.  Twenty to t w e n t y - f i v e p e r c e n t wood y a r d  loss. cost.  increase i n production  from  to wood room.  5.  Reduced maintenance c o s t s .  6.  E l i m i n a t i o n of p r o d u c t i o n l o s s because of wood room-wood y a r d breakdowns.  7.  Increased  bark y i e l d  8.  Improved u n i f o r m i t y i n the d i g e s t e r f u r n i s h .  9.  Greater  s t o r a g e per  and  reduced  fuel costs.  acre.  10.  Less  housekeeping.  11.  Smooth wood y a r d and wood room o p e r a t i o n .  12.  B e t t e r and more a c c u r a t e s p e c i e s  T h i s method, however, has ( A l l e n , 1968;  Hatton,  Smith and  handling.  i t s disadvantages  which a r e as  follows  Rogers, 1968):  1.  R i s k of f i r e r e s u l t i n g  2.  Losses  from the tendency of p i l e s  i n pulp y i e l d due  to heat  to d e t e r i o r a t i o n of c h i p s  up.  during  storage. 3.  Reduction groundwood  4.  Losses  5.  Increased  i n s t r e n g t h p r o p e r t i e s of the c h e m i c a l  and  refiner-  pulps.  i n refiner-groundwood pulp requirements  f o r cooking  brightness. and b l e a c h i n g  chemicals.  6.  Corrosion the low  7.  9  -  problems w i t h c h i p r e c l a i m i n g  pH of s e v e r e l y d e t e r i o r a t e d  Production  of o f f - g r a d e  c h i p s due  m i l l s are using  these d i s a d v a n t a g e s , the  or c o n t e m p l a t i n g u s i n g  advantages outweigh the  2.  the  fact  such as f l y ash and  sand.  t h a t a l a r g e number of the  disadvantages.  Types of wood c h i p  same i n s i z e ,  chips.  t h i s method might i n d i c a t e t h a t  piles  There are as many p i l e forms as are  to  to c o n t a m i n a t i o n of  c h i p p i l e s by a i r borne p a r t i c l e s , Despite  systems due  there a r e m i l l s .  l o c a t i o n or volume.  However, two  No  two  piles  main shapes  are  it in  common use,  the c o n i c a l p i l e s  (Bjorkman and  Haeger, 1963)  and  the  it  rectangular  piles  There i s a l s o no (Hajny, 1966).  (Bjorkman and standard  Recently,  s e c t i o n of the r i n g  design  f o r a system of o u t s i d e  p i l e removed so t h a t the end  transported  ship  instances  round wood i s t r a n s p o r t e d  ( S h i e l d s and  (Bergman and  U n l i g i l , 1968).  two  to the m i l l s by  N i l s s o n , 1966;  storage  form w i t h a surfaces  been advocated  (Croon, 1966).  trucks, t r a i l e r s , r a i l ,  to the m i l l to be  (Blackerby,  chip  resulting  Shields,  There i s l i t t l e  f o r the base of the p i l e  U n l i g i l , 1968).  in a ring  of the p i l e has  barge and  to use  S h i e l d s and  c o n s t r u c t i o n of p i l e s  c o n s t i t u t e the b e g i n n i n g and Chips are  Haeger, 1963;  1967). chipped  agreement as  1958;  In some there  to what  Holekamp,  material  1959;  ti  Annergren, D i l l n e r ,  Haglund and  b u i l t d i r e c t l y on the Under these c o n d i t i o n s  Jagerud, 1965).  ground, and  packed c l a y and  The  e a r l i e s t p i l e s were  sand were used as  2 to 3 f e e t of c h i p s have been l e f t  as  the  base.  the base of  -  a new  pile.  10  -  In some cases hogged f u e l or bark have been used  i n place  of t h i s l a y e r of c h i p s . A hard  s u r f a c e d base such as b l a c k top or c o n c r e t e i s g e n e r a l l y  c o n s i d e r e d to be the most s a t i s f a c t o r y , a l t h o u g h e x p e r i m e n t a l p i l e was  the most e x p e n s i v e .  c o n s t r u c t e d on a p o l y e t h y l e n e s h e e t i n g  An  (Butcher  and Howard, 1968). Chips a r e blown on t o the a r e a by pneumatic c h i p blowers  and  are then spread out by c r a w l e r type t r a c t o r s which compact them a t  the  same time.  Not  a l l the p i l e s a r e m e c h a n i c a l l y compacted and  the c h i p s a r e a l l o w e d  t o compact  The volume and important  experimental p i l e s  two  the e f f i c i e n c y of the p r o c e s s e s which  E x p e r i m e n t a l p i l e s have g e n e r a l l y been s m a l l and  (Butcher and Howard, 1968)  cases  themselves.  the f i n a l h e i g h t of the c h i p p i l e a r e  f a c t o r s determining  p l a c e i n the p i l e .  i n such  varying  from  9 feet  i n Sweden were u s u a l l y l a r g e and  to 145  tall  feet.  (Annergren,  take low The Dillen  it  and Vardheim, 1964;  Annergren, D i l l n e r , Haglund and  volumes of p i l e s  i n Canada have averaged  cords  1968;  (Robinson,  variability  of the r e s u l t s from  The major s p e c i e s used  1965).  The  from 21,000 c o r d s t o a huge 100,000  S h i e l d s and U n l i g i l ,  c h a r a c t e r i s t i c s of the p i l e s must account  Jagerud,  1968).  These v a r y i n g p h y s i c a l  i n p a r t f o r the tremendous  s t u d i e s on c h i p p i l e s . i n c h i p p i l e s on the west c o a s t of  America a r e D o u g l a s - f i r [Pseudotsuga  m e n z i e s i i (Mirb.) F r a n c o ] ,  hemlock [Tsuga h e t e r o p h y l l a (Ref.) Sarg] white  North  western  f i r (Abies c o n c o l o r Gord.  and  G l e n d ) , western  alder  r e d cedar  11  -  (Thuja p l i c a t a Don), p i n e s  (Alnus r u b r a Bong.) and cottonwood  (Populus  (Pinus s p . ) , r e d  t r i c h o c a r p a T o r r . and  Gray) (Wright, 1954; B l a c k e r b y , 1958; H e n s e l , 1958).  In the s o u t h e r n  U.S.A. many p i l e s a r e b u i l t of p i n e s p e c i e s . In spruce  e a s t e r n Canada, balsam f i r (Abies balsamea  (Picea sp.), pine  (L.) M i l l ) ,  (Pinus sp.) and e a s t e r n hemlock (Tsuga  canadensis  (L.) C a r r ) have been s t o r e d as c h i p s ( B l a c k e r b y , 1958; S h i e l d s and Unligil,  1968).  with b i r c h  Pulp m i l l s  i n S c a n d i n a v i a n c o u n t r i e s have  ( B e t u l a sp.) s p r u c e and p i n e s p e c i e s (Annergren,  Vardheim, 1964; Annergren, D i l l n e r , Haglund and Jagerud, and Haeger, 1963; Bergman and N i l s s o n , 1966). have s t u d i e d the b e h a v i o u r  Butcher  experimented D i l l e n and  1965; Bjorkman  and Howard  o f P i n u s r a d i a t a D. Don. i n s m a l l p i l e s  w i n t e r and summer s t o r a g e i n New C o n f l i c t i n g accounts  (1968) during  Zealand.  o f the b e h a v i o u r  o f wood i n o u t s i d e s t o r a g e  a r e common, p o s s i b l y because of the v a r i a t i o n i n r e s i s t a n c e t o d e t e r i o r a t i o n of  the wood of d i f f e r e n t  tree species.  P i l e s o f D o u g l a s - f i r on the west  c o a s t o f North America do n o t seem to s u f f e r even a f t e r  three years of  o u t s i d e s t o r a g e w h i l e a l d e r i s b a d l y d e t e r i o r a t e d a f t e r f o u r months o f storage.  A mixture  o f a l d e r and D o u g l a s - f i r c o u l d s t a n d l o n g e r o u t s i d e  s t o r a g e than a l d e r a l o n e The  (Wright,  1954).  l e n g t h o f time t h a t c h i p s can be kept  s p e c i e s , c l i m a t e and p u l p i n g p r o c e s s  (Hajny,  i n storage v a r i e s  1966; S h i e l d s , 1967).  In the  P a c i f i c Northwest c h i p s have been s t o r e d f o r 2 to 3 y e a r s o r more w i t h little  e v i d e n c e of d e t e r i o r a t i o n  ( B l a c k e r b y , 1958; Burke, 1962).  with  -  It can be  i s concluded  stored f o r lengthy  12  -  that although  many of the west c o a s t  p e r i o d s without  d e t e r i o r a t i o n , such a c o n d i t i o n  might not p r e v a i l w i t h o t h e r  commercial s p e c i e s and  s p e c i e s s e p a r a t e l y might be  necessary.  In the s o u t h e r n  United  States.  i n the s t o r a g e Holekamp  that t e s t i n g  S t a t e s c h i p s of southern  much more r a p i d l y than i n the P a c i f i c Northwest. reflected  Lindgren  U.S.A. and  pines d e t e r i o r a t e  time recommended by workers i n the s o u t h e r n  (1959) recommends summer s t o r a g e  showed t h a t o u t s i d e summer s t o r a g e In Nova S c o t i a , Robinson  United  of t h r e e months, w h i l s t time.  Rothrock, Smith  (1961) s t o r e d s l a s h p i n e c h i p s f o r 5 months i n the  within this l i m i t .  each  This condition i s  Somsen (1962) g i v e s 16 weeks as the maximum s t o r a g e and  species  southern  of p i n e wood i s f e a s i b l e (1963) recommends two  months  storage. D e t e r i o r a t i o n of c h i p s was of a spruce  and  found to be  balsam f i r c h i p p i l e at a p p r o x i m a t e l y  i n g t h a t complete u t i l i z a t i o n of a p i l e at l e a s t  severe  i n order  to m i n i m i z e r e d u c t i o n s  the pulp a d v e r s e l y  ( S h i e l d s and  Unligil,  should  i n the lower  n i n e months,  occur b e f o r e  e i g h t months  Storage of softwood  f o r 15 or more months i n v o l v e s r i s k s of d e t e r i o r a t i o n to spruce chips  (Bjorkman and  Haeger, 1963)  p i l e s be mixed c o m p l e t e l y  every  and  Forssblad  ( S a u c i e r and M i l l e r ,  d e t e r i o r a t i o n of p i n e c h i p s s t o r e d i n the w i n t e r l e s s than t h a t of summer s t o r e d c h i p s . Sweden (Bergman and  N i l s s o n , 1966)  and  (1965) suggests t h a t  few months to m i n i m i z e t h i s  Experiments i n G e o r g i a  indicat-  i n chip q u a l i t y that could 1968).  1961)  third  affect chips pine chip  risk. i n d i c a t e that  i s one-third  to  one-half  Many of the p i n e c h i p s s t o r e d i n  from the end  of October to the f o l l o w i n g  -  May  13  -  were found to be f r o z e n d u r i n g most of t h i s p e r i o d at the bottom  of  the p i l e .  is  found t h a t s p e c i f i c g r a v i t y  in  samples  3.  Where average s t o r a g e temperature i s c l o s e to f r e e z i n g , i t  affects  l o s s e s a r e v e r y low i n comparison t o l o s s e s  s t o r e d i n the warmer a r e a s of the p i l e .  E n v i r o n m e n t a l c o n d i t i o n s i n wood c h i p  A spontaneous result  level  piles  r i s e i n temperature i n c h i p p i l e s o c c u r s as a  o f p r o c e s s e s which go on i n the p i l e .  T h i s heat  generation  water vapour movement w i t h i n the p i l e , causes changes  e x t r a c t i v e s , and c r e a t e s c o n d i t i o n s f o r a d i f f e r e n t  i n wood  m i c r o f l o r a not n o r m a l l y  a s s o c i a t e d w i t h wood d e t e r i o r a t i o n . S e v e r a l s m a l l e x p e r i m e n t a l c h i p p i l e s have been b u i l t i n the southern United  S t a t e s from s o u t h e r n p i n e c h i p s  L i n d g r e n , 1961;  S a u c i e r and M i l l e r ,  1961;  (Rothrock, Smith and  Somsen, 1962; D a v i s , 1963).  The s l o p i n g s i d e s of these p i l e s were not compacted  compared to the main  body of the p i l e s , however, temperatures i n t h e s e p i l e s showed a degree of u n i f o r m i t y . initial  r i s e of temperature o c c u r r e d .  temperatures. 54°  In the i n t e r i o r compacted  and 63°C.  W i t h i n the f i r s t  p o r t i o n s of the p i l e s , a  No mention was  made of the ambient  two weeks the temperature r o s e to between  D u r i n g the next two to f o u r weeks the temperature dropped  s h a r p l y to about 49°C f o l l o w e d by a g r a d u a l d e c l i n e to about 38°C about f i v e months s t o r a g e . tures rose r a p i d l y at f i r s t , to ambient  rapid  temperature.  In the uncompacted  after  p o r t i o n s of the p i l e s  but o n l y to 38-49°C,  and soon t h e r e a f t e r  temperafell  -  In the  and Haeger  (1963) b u i l t  a softwood p i l e i n  shape of a cone w i t h a base diameter o f 15 meters and a h e i g h t o f 11  meters.  I n the deep i n t e r i o r of the p i l e  ambient The  Sweden, Bjorkman  14 -  the temperature remained  f o r 15 months, the maximum d i f f e r e n c e b e i n g 20.6°C  i n January.  temperature near the p e r i p h e r y f o l l o w e d the a i r temperature. In  two s t u d i e s on l a r g e p i l e s of 7400-8800 c u b i c meters,  Annergren, D i l l e n and Vardheim  (1964) and Annergren, D i l l n e r ,  Haglund  and Jagerud (1965) made o b s e r v a t i o n s on temperature changes. p i l e s were of s p r u c e and one o f b i r c h . to  above  Temperatures  Three o f the  i n the s p r u c e p i l e s r o s e  about 55°C and remained a t t h i s l e v e l f o r 3 to 4 months f o l l o w e d by a  decline.  I n the b i r c h p i l e  the temperature r o s e to 65°C i n the f i r s t  two weeks and remained a t t h i s l e v e l f o r 4 1/2 months a t which p i l e was d i s m a n t l e d .  Annergren, D i l l e n and Vardheim  time the  (1964) and Annergren,  D i l l n e r , Haglund and Jagerud (1965) a l s o c o n f i r m e d the f i n d i n g  that  temperatures near the s i d e s o f the p i l e a r e lower than those near the c e n t r e of  the p i l e .  Ljungqvist  (1965) made an e x t e n s i v e study o f temperature  t i o n i n s e v e r a l commercial p i l e s i n Sweden. spruce  (exact s p e c i e s n o t i d e n t i f i e d  P i l e s o f b i r c h , p i n e and  i n paper) b u i l t  maximum temperature of 69°C, 63°C and 58°C  varia-  i n summer reached  respectively.  A l t h o u g h temperatures i n the i n t e r i o r o f e a s t e r n Canadian c h i p piles  do r i s e v e r y s h a r p l y a f t e r  the f i r s t weeks o f c o n s t r u c t i o n , w i t h  l o n g e r s t o r a g e the temperatures drop to between 30°C and 43°C and U n l i g i l ,  1968).  The drop sometimes o c c u r s v e r y a b r u p t l y  (Shields (Rothrock,  -  Smith and Lindgren,1961;  15  -  S a u c i e r and M i l l e r ,  1961)  but t h i s d e c l i n e has  a l s o been shown to o c c u r more g r a d u a l l y i n the l a r g e r , more compacted i n Sweden (Annergren, D i l l e n and Vardheim,  1964).  T h i s d i f f e r e n c e i n the i n t e r n a l temperatures has been to the l a r g e r volume of c h i p s i n the Swedish  internal  i s believed  The  degree  to be one o f the f a c t o r s r e s p o n s i b l e f o r the  temperature f l u c t u a t i o n s  (Annergren, D i l l e n and Vardheim,  G r e a t e r compaction r e s u l t s i n h i g h e r average  S t u d i e s i n New  attributed  p i l e s which s e r v e s to  i n s u l a t e the i n s i d e of the p i l e from e n v i r o n m e n t a l changes. of compaction  piles  1964) .  temperatures.  Zealand on v e r y s m a l l p i l e s c l e a r l y  illustrate  e f f e c t of volume of c h i p s and compaction on the temperature devleopment the p i l e .  B u t c h e r and Howard  the in  (1968) have shown t h a t t h e r e a r e d i f f e r e n c e s  i n the temperature of w i n t e r p i l e s and summer p i l e s .  The  temperatures  r e c o r d e d i n a w i n t e r p i l e 9 f e e t on a 30 by 50 f o o t base, w i t h s e l f - c o m p a c t e d c h i p s , were v e r y e r r a t i c over the f i r s t  seven weeks o f s t o r a g e .  o c c u r r e d , f o l l o w e d by s e v e r a l major f l u c t u a t i o n s p i l e , which was were compacted by ambient  Reheating  i n temperature.  10 f e e t h i g h on a 70 by 35 f o o t base and i n which  The summer the c h i p s  by c r a w l e r t r a c t o r , had temperatures which were not  conditions.  The r i s e  in internal  temperature was  around  influenced 37°.C to  38 °C f o r most o f the n i n e months of s t o r a g e . I t has been suggested t h a t the r i s e i n temperature which o c c u r s i n the o u t s i d e s t o r e d c h i p p i l e s  i s i n p a r t due to the c o n d e n s a t i o n o f water  vapour on the c h i p s , the p r e s e n c e of f i n e s ,  the p r e s e n c e o f metals and  the  -  m i c r o b i a l a c t i v i t y w i t h i n the p i l e 1968).  No  experiments  16  -  ( S h i e l d s , 1967;  A l l e n , 1968;  have been done on any of these f a c t o r s .  Chalk, In l a r g e  c h i p p i l e s a t r a n s f e r of warm moist a i r from the lower r e g i o n s o f the p i l e to the upper  cooler chips i s believed  to o c c u r .  This  transfer  c r e a t e s a chimney e f f e c t whereby the e v a p o r a t i o n of m o i s t u r e from  the  lower c h i p s r e s u l t s i n c o o l e r temperatures  condensa-  t i o n of water vapour  i n these r e g i o n s w h i l e  on the c h i p s i n the uppor p o r t i o n of the p i l e s  an i n c r e a s e i n temperature  (Rothrock, Smith and L i n d g r e n , 1961;  D i l l e n and Vardheim, 1964;  L j u n g q v i s t , 1965).  Moisture d i s t r i b u t i o n i n chip p i l e s  causes  Annergren,  i s i r r e g u l a r and a tremedous  v a r i a t i o n i n the m o i s t u r e of the wood o c c u r s d u r i n g the s t o r a g e p e r i o d . Bjorkman and Haeger (1963) i n d i c a t e d  t h a t the r e l a t i v e h u m i d i t y measured  i n a p i l e s t o r e d f o r 15 months averaged  98 to  100%.  I t i s g e n e r a l l y a c c e p t e d t h a t the i n t e r i o r of a p i l e i s l e s s m o i s t than the o u t e r r e g i o n s owing to the h i g h temperatures a s s o c i a t e d w i t h the c o r e of the p i l e Annergren,  of the c h i p s to some e x t e n t .  are  (Rothrock, Smith and L i n d g r e n ,  D i l l e n and Vardheim, 1964; F u n g a l a c t i v i t y may  that  Bergman and N i l s s o n ,  1961;  1966).  l e a d to an i n c r e a s e i n the m o i s t u r e c o n t e n t I n c r e a s e d f u n g a l metabolism  p r o d u c t i o n of carbon d i o x i d e and water  r e s u l t s i n the  ( L i n d g r e n and E s l y n , 1961)  as  end  p r o d u c t s of d e g r a d a t i o n of c a r b o h y d r a t e s i n wood. P r e c i p i t a t i o n i s «an important s o u r c e of m o i s t u r e which drastically in  affect  the r a i n f a l l  the b e h a v i o u r of the p i l e .  (Butcher and Howard, 1968).  Small p i l e s  can  r e a c t to changes  The amount of m o i s t u r e i n s m a l l  -  piles  17  -  i n the west c o a s t of the U.S. was not found to be d i r e c t l y r e l a t e d  to the p r e c i p i t a t i o n because the m o i s t u r e content high during storage The  (Wright,  i n i t i a l moisture content  pile  the m o i s t u r e c o n t e n t  (Wright,  uniformly  1954).  the range o f 45 to 50 p e r c e n t , periods  remained  of stored chips i s g e n e r a l l y w i t h i n  green weight b a s i s .  With l o n g e r  becomes more o r l e s s u n i f o r m  1954; S a u c i e r and M i l l e r ,  1961).  storage  throughout the  A d d i t i o n a l moisture i n  the s u r f a c e of the p i l e i s c o n t r i b u t e d by r a i n f a l l o r snow (Holekamp, 1958;  Rothrock, Smith and L i n d g r e n ,  1961; Zak and K r a u t h a u f ,  1964).  High temperatures and m o i s t u r e a r e f a c t o r s which c o n t r i b u t e to brown c h e m i c a l 1964)  s t a i n i n g and a r e r e p o r t e d  (Annergren, D i l l e n and Vardheim,  to r e s u l t i n the d e a c e t y l a t i o n o f h e m i c e l l u l o s e s The  pH o f wood c h i p s g r a d u a l l y d e c r e a s e s d u r i n g s t o r a g e .  a c i d odour i s a v e r y  common phenomenom.  T h i s product  from the d e a c e t y l a t i o n of the h e m i c e l l u l o s e s has  Acetic  i s found to r e s u l t  i n the wood.  Shields  (1970)  shown t h a t the pH o f the wood dropped from 5.92 i n the f r e s h c h i p s to  2.87 i n c h i p s s t o r e d f o r 259 days. of  i n the wood.  microorganisms  appearing  suddenly a t pH 3.50 w h i l e  T h i s pH a f f e c t e d the growth and type  i n the p i l e and the b a c t e r i a l  the f r e q u e n c y  isolations  dropped  o f f u n g i i s o l a t i o n s was h i g h e s t a t  t h i s pH. Annergren, D i l l e n and Vardheim c o l o u r a t i o n of chips  (1964) a t t r i b u t e the brown d i s -  to the pH and the h i g h  that a c e t i c acid i t s e l f  i s not d i r e c t l y  temperatures.  I t i s believed  r e s p o n s i b l e f o r the brown d i s -  -  c o l o u r a t i o n but that other involved  4.  18  -  c h e m i c a l o r enzymatic r e a c t i o n s a r e p r o b a b l y  ( S h i e l d s , 1970).  F u n g i i s o l a t e d from wood c h i p  piles  A wide v a r i e t y of f u n g i , b e l o n g i n g isolated  from c h i p s .  s t a i n the wood.  to a l l c l a s s e s , has been  Some o f these organisms cause wood decay w h i l e  others  A l a r g e group o f organisms has been i s o l a t e d whose  b e h a v i o u r on wood i s as y e t unknown.  The number o f d i f f e r e n t f u n g i i s  g e n e r a l l y much l a r g e r i n c h i p s t o r a g e  than i n round wood s t o r a g e  unit  ( N i l s s o n , 1965) and p r o b a b l y i n f l u e n c e s  ration.  Because o f the h i g h  The Imperfecti  thermophilic  c l a s s and i n c l u d e s  (Lindgren  to s u r v i v e a t h i g h  fungi.  Trichoderma sp., Paecilomyces sp., Graphium  sp., G l i o c l a d i u m  sp., P e n i c i l l i u m s p . , and A s p e r g i l l u s  and E s l y n , 1961; Bjorkman and Haeger, 1963; N i l s s o n , 1965;  Bergman and N i l s s o n , 1966; S h i e l d s and U n l i g i l ,  1968; S h i e l d s , 1970).  of the genera Chrysosporium found i n c h i p s d u r i n g destructive fungi. reported  storage  to cause 33 p e r c e n t  weight l o s s i n l a b o r a t o r y  Another t h e r m o t o l e r a n t  Members  a r e among the most  Chrysosporium l i g n o r u m , a t h e r m o t o l e r a n t  a f t e r f o u r months i n c u b a t i o n 1966).  temperatures.  l a r g e s t group of f u n g i i s o l a t e d b e l o n g s to the F u n g i  sp.,Phialophora sp.  the r a t e and type o f d e t e r i o -  temperatures developed i n a c h i p p i l e a unique  group o f f u n g i i s found which i s a b l e These f u n g i a r e c a l l e d  p e r wood  fungus, was  t e s t s on p i n e sapwood  a t 25°C ( N i l s s o n , 1965; Bergman and N i l s s o n , Fungus Imperfectus commonly i s o l a t e d i s  -  A s p e r g i l l u s fumigatus.  19  -  U n l i k e Chrysosporium  lignorum i t i s not h i g h l y  destructive. The second  commonest c l a s s of f u n g i found  i n chip p i l e s  i s the  Ascomycetes, which i n c l u d e s C e r a t o c y s t i s s p . c a u s i n g s t a i n i n g o f wood, Chaetomium •sp,. A l l e s c h e r i a sp,, Thermoascus sp. and Byssochlamys 1965;  Bergman and N i l s s o n ,  sp, ( N i l s s o n ,  1966).  B a s i d i o m y c e t e s a r e n o t v e r y common i n o u t s i d e c h i p s t o r a g e except i n s m a l l p i l e s where the ambient c o n d i t i o n s a f f e c t of the p i l e ,  the b e h a v i o u r  and i n p i l e s s t o r e d over l o n g p e r i o d s ( N i l s s o n , 1965;  Bergman and N i l s s o n , 1966).  From two s p r u c e p i l e s  r e s p e c t i v e l y , no B a s i d i o m y c e t e s were i s o l a t e d .  s t o r e d f o r 6 and 7 months  From the p i l e s s t o r e d  a l o n g p e r i o d , 13 months, a number o f B a s i d i o m y c e t e s were i s o l a t e d . of these were Fomes annosus and O d o n t i a b i c o l o r  ( N i l s s o n , 1965).  over Two  When  the B a s i d i o m y c e t e s were p r e s e n t t h e i r f r e q u e n c y o f i s o l a t i o n was v e r y low. A heat t o l e r a n t B a s i d i o m y c e t e , P t y c h o g a s t e r s p e c i e s has been i s o l a t e d from e a s t e r n Canadian I t caused at  chip p i l e s  c o n s i d e r a b l y more decay  commonly  ( S h i e l d s and U n l i g i l ,  1968).  of p i n e sapwood when i n c u b a t e d a t 37°C than  27°C. The m a j o r i t y o f the wood-decaying B a s i d i o m y c e t e s  piles  caused w h i t e r o t i n l a b o r a t o r y t e s t s  from wood  ( N i l s s o n , 1965).  Peniophora  g i g a n t e a and P o l y p o r u s s p e c i e s were the most commonly i d e n t i f i e d in  t h i s group  rot.  chip  organisms  a l t h o u g h o t h e r s p e c i e s such as P t y c h o g a s t e r do cause  white  -  Several  20  -  of the B a s i d i o m y c e t e s have caused wood l o s s e s between  20-40% over 3 months (Nilsson-, 1965j Bergman and dangerous i n c h i p s t o r a g e temperatures.  are  N i l s s o n , 1966).  those r o t f u n g i which can  Among these are P o l y p o r u s s p e c i e s  and  Especially  t o l e r a t e high  an unknown B a s i d i o -  mycete both i s o l a t e d i n Sweden ( N i l s s o n , 1965). Few coniferous  piles  Bergman and y e t unknown. and  Phycomycetes have been i s o l a t e d and i s Mucor  (Bjorkman and  N i l s s o n , 1966). Rhizopus has  The  Haeger, 1963;  damage caused by  are F u n g i I m p e r f e c t i .  ( N i l s s o n , 1965).  The  thermophilic  the Phycomycetes i s as (Butcher  Humicola l a n u g i n o s a  Among the  Thermoascus a u r a n t i a c u s  thermophilic  Miehe.  come from softwood c h i p p i l e s S h i e l d s and  Unligil,  Little  f u n g i are Ascomycetes but  Fungi Imperfecti  class includes:  ( G r i f f o n and  Sporotrichum  Maublanc) Bunce  Chaetomium t h e r m o p h i l e La  Most of  the i s o l a t e s of  ( N i l s s o n , 1965;  Bergman and  Touche  these f u n g i have  Nilsson,  1966;  1968).  i s known about the n a t u r e of the  thermophilic  a t t a c k on wood.  I t has  terrestris  r o t of hardwoods  (Bergman and  case i n softwoods.  a  Ascomycetes are A l l e s c h e r i a  t e r r e s t r i s A p i n i s , Byssochlamys e m e r s o n i i ,  the  1965;  Howard, 1968).  t h e r m o p h i l e A p i n i s and  and  Nilsson,  been i s o l a t e d from a P i n u s r a d i a t a p i l e  A l a r g e number of the few  the commonest from  The  been demonstrated t h a t A. N i l s s o n 1967).  n a t u r e of a t t a c k  f u n c t i o n of t h e r m o p h i l i c  fungi i n chip  caused a s o f t  However, t h i s may  i s very piles.  fungi's  not  be  the  important i n e l u c i d a t i n g  -  21  -  S p o r o t r i c h u m t h e r m o p h i l e , A_. t e r r e s t r i s utilize  c a l l u l o s e as a s o l e s o u r c e of carbon w h i l s t T_. a u r a n t i a c u s cannot  (Fergus, 1969). sp.  and Humicola s p . can  isolated  No such study has been done f o r v a r i e t i e s o f Byssochlamys  from c h i p  piles.  It i s believed  t h a t these t h e r m o p h i l i c f u n g i a r e p a r t l y  r e s p o n s i b l e f o r the h i g h temperatures u s u a l l y r e c o r d e d i n c h i p p i l e s . r e s u l t s of heat p r o d u c t i o n by f u n g i on wood a r e a v a i l a b l e .  Results  No  from  heat g e n e r a t i o n experiments i n o t h e r o r g a n i c s u b s t r a t e s l i k e hay, straw, compost and g r a i n i n d i c a t e d for  that thermophilic f u n g i are mainly r e s p o n s i b l e  the r i s e i n temperature.  C a r l y l e and Norman (1941) i n f e c t e d  sterilized  straw w i t h A s p e r g i l l u s f u m i g a t u s , a v e r y common fungus i n c h i p p i l e s , and demonstrated hours.  t h a t the temperature c o u l d r i s e from 25°C  to 55°C d u r i n g 38  F e n s t e n s t e i n , Lacey, S k i n n e r , J e n k i n s and Pepys  (1965) have shown  t h a t a l a r g e number of t h e r m o p h i l i c f u n g i can r a i s e the temperature of straw to about 60°C i n a v e r y s h o r t  5.  Damage i n wood c h i p  time.  piles  Chips i n s t o r a g e a r e damaged by the a c t i v i t y such as f u n g i , which  e i t h e r s t a i n o r degrade  the wood and a l s o by c o n d i t i o n s  which develop i n the p i l e such as h i g h temperatures. organisms  of micro-organisms,  Damage by m i c r o -  l e a d s to a r e d u c t i o n o f q u a l i t y and/or q u a n t i t y o f the wood.  -  22  -  A common damage to wood c h i p s i s s t a i n i n g . t i o n of t h i s (Blackerby,  type o c c u r s 1958;  Hensel,  d i s c o l o u r a t i o n and at m i l l s  i n B.C.  i n c h i p s s t o r e d on 1958;  and  of the p i l e o c c u r r e d  1963).  Oregon.  S t a i n i n g occurred  Studies i n Georgia  to a depth of about one  years  confined (Holekamp,  f o o t from the  D e t e r i o r a t i o n due  surface to  (Blackerby,  to two  (Rothrock,  f e e t of uncompacted s i d e s of one  Smith and  Lindgren,  1961;  pile  S a u c i e r and  A softwood p i l e became dark i n c o l o u r a f t e r 9 to 12 months  S h i e l d s and  Unligil  1963)  and  (1968) found  balsam f i r c h i p s s t o r e d i n New A non-fungal  insignificant  w i t h i n one month of s t o r a g e . around r e s i n ducts  The  U.S.A. ( L i n d g r e n  d i s c o l o u r a t i o n was  and  of p i n e c h i p s .  s e v e r a l softwood p i l e s and have been a t t r i b u t e d 1961;  S h i e l d s and  1961)  located primarily  Brownish d i s c o l o u r a t i o n s have been n o t i c e d i n the i n t e r i o r  ( S a u c i e r and M i l l e r  and  found  Lindgren,1961; S a u c i e r and M i l l e r ,  i n the heartwood a r e a s  drying.  storage.  y e l l o w i s h brown or orange d i s c o l o u r a t i o n was  Rothrock, Smith and  Miller,  d i s c o l o u r a t i o n i n spruce  Brunswick a f t e r 4 to 8 months  after  storage  d i d not r e g a i n i t s c o l o u r a f t e r  i n a p i n e c h i p p i l e i n the s o u t h e a s t e r n  E s l y n * 1961^  pile  to  Most of the b l u e ' g r a y f u n g a l d i s c o l o u r a t i o n s i n the p i n e c h i p s were  i n Nova S c o t i a (Robinson,  to occur  to t h r e e  be very minor i n p i n e c h i p s a f t e r 4 months s t o r a g e  month's s t o r a g e  1961).  (1958) r e p o r t e d  e a r l y and was  a f t e r f o u r months s t o r a g e .  s i t u a t e d i n the o u t e r one one  Blackerby  r o t i n softwood c h i p s s t o r e d f o r one  i n d i c a t e d that s t a i n i n g  s t a i n i n g may  the west c o a s t of North America  Burke, 1962).  the o u t e r uncompacted p a r t s of the p i l e . 1958)  L i t t l e deteriora-  to c h e m i c a l  U n l i g i l 1968).  of  changes i n the The  development  -  23  of the brown s t a i n i s p r o g r e s s i v e w i t h  -  i n c r e a s e d s t o r a g e and appears to be  more widespread near the bottom than near the top o f one p i l e  (Young, 1961).  A f t e r 4 months of summer s t o r a g e and 5 months of w i n t e r h e a v i l y s t a i n e d p i n e c h i p s were found and  to be s o f t and brashy  storage,  (Lindgren  E s l y n , 1961; Rothrock, Smith and L i n d g r e n , 1961; S a u c i e r and M i l l e r ,  1961).  T h i s c o n d i t i o n has been a t t r i b u t e d  In one study  i t was found  to the a c t i v i t y of s o f t r o t f u n g i .  t h a t the number o f decay f u n g i i s o l a t e d  from  softwood c h i p s i n the bottom h a l f o f a l a r g e p i l e was g r e a t e r than the number i s o l a t e d from o t h e r p a r t s o f the p i l e Losses  i n wood substance  ( S h i e l d s and U n l i g i l ,  v a r y from one p i l e  i n Sweden and s o u t h e r n U.S.A. i n d i c a t e s .  to another  r e d u c t i o n i n the s p e c i f i c g r a v i t y corresponded  temperature,  to i n c r e a s e i n m o i s t u r e  as work  Minor r e d u c t i o n s i n s p e c i f i c  have been n o t i c e d i n p i n e c h i p s s t o r e d f o r up to two months The  1968).  content  gravity  (Anon, 1961).  to the l o w e r i n g o f  and perhaps to d e p l e t i o n o f  oxygen. A f t e r f i v e month's s t o r a g e S a u c i e r and M i l l e r , wood.  Losses  (Rothrock,  1961) t h e r e was an approximate average l o s s o f 7% o f  i n the compacted c e n t r e s o f the p i l e s were low (about 4 % ) .  Even a f t e r 12 months s t o r a g e , g r e a t e r r e d u c t i o n s outer  Smith and L i n d g r e n , 1961;  (about  9%) o c c u r r e d i n the  t h r e e f e e t o f the s l o p i n g s i d e s of the p i l e where t h e r e was  compaction and where v i s i b l e c h i p d e t e r i o r a t i o n r e s u l t i n g s t a i n and decay f u n g i was e v i d e n t Smith and L i n d g r e n ,  1961).  the l e a s t  from growth o f  ( L i n d g r e n and E s l y n , 1961; Rothrock,  -  Losses  24  -  i n Swedish spruce c h i p p i l e s S e l l e b y , 1965)  (Annergren,  Dillen  Vardheim, 1964;  N o r d i n and  to f i v e p e r c e n t  f o r s t o r a g e p e r i o d s of f o u r to f i v e months.  and  were r e p o r t e d to v a r y from At  two  temperatures  of 20°C to 30°C, l o s s e s of 5 to 10 p e r c e n t o c c u r r e d i n p i n e c h i p s  (Bergman  and N i l s s o n , 1966)  or  no  11 p e r c e n t i n the s p e c i f i c g r a v i t y  of  s t o r e d f o r seven months i n Sweden w h i l e l i t t l e  loss occurred i n frozen chips during this A l o s s of a p p r o x i m a t e l y brown and  d e t e r i o r a t e d balsam f i r and  time.  s p r u c e c h i p s was  determined  samples which had been s o t r e d f o r j u s t over n i n e months near p i l e i n New  Brunswick ( S h i e l d s and U n l i g i l ,  s p e c i f i c g r a v i t y of p i n e (Annergren, 1965)  (Burks, 1962;  D i l l o n and Vardheim, 1964;  1968).  from  the bottom of a  Further decreases  Somsen, 1962; S e l l e b y , 1965)  S e l l e b y , 1965) and b i r c h  spruce  (Selleby,  c h i p s were s l i g h t beyond f i v e months.  C  MATERIALS AND  1.  The  The  METHODS  e x p e r i m e n t a l wood c h i p p i l e  experimental  of t h e r m o p h i l i c and  c h i p p i l e , used  i n the study of the  t h e r m o t o l e r a n t f u n g i , was  distribution  l o c a t e d on the grounds of  I n t e r c o n t i n e n t a l P u l p Company L t d . , P r i n c e George, B r i t i s h Columbia. was  i n the  c o n s t r u c t e d on a l e v e l s i t e and  oriented longitudinally  east-west.  It  the  -  The  chip p i l e matrix  25  -  c o n s i s t e d of w h i t e spruce  i n t o which the c h i p samples were i n s e r t e d .  The  r e s i d u e s s u p p l i e d by  contained  each u n i t b e i n g two  to one.  s t a r t e d on  15  1,080  The  sawmills.  kg.  The  of wood, c h i p s .  b u i l d i n g of the p i l e  the second week of June, The  south  throughout these  pile  The  and  c h i p s were from  ratio  of spruce  took t h r e e weeks and  f a c e of the p i l e was  uniformly  s t u d i e s so t h a t the e f f e c t s of the p r e v a i l i n g c l i m a t o l o g i c a l  used to l e v e l  the p i l e .  Figures  the south  p i l e was 25ft  75ft  (23.2m.) wide a t bottom 2 8 f t .  (7.7m.) h i g h .  t r a c t o r was  The  l e n g t h of the p i l e was  400ft  (124.m). and  views. bags were i n c o r p o r a t e d  For each treatment, sample bags were  i n s i x d i f f e r e n t p o s i t i o n s w i t h i n the p i l e as shown i n the  view of F i g u r e  (2.4m.).  the  (8.6m.) wide  2 g i v e the dimensions of the p i l e i n the e a s t and west  the p i l e d u r i n g c o n s t r u c t i o n .  placed  c h i p s were  pile.  Samples of c h i p s i n "flexmesh" p l a s t i c into  The  T h i s l e v e l l i n g made i t easy f o r the i n s e r t i o n of  the compaction of the  1 and  collection  exposed to the weather  d e l i v e r e d to the p i l e s i t e , then a c a t e r p i l l a r  the top and  was  1968.  pneumatically  at  sawmill  to p i n e  data  the e n t i r e f r o n t f a c e would be c o n s t a n t .  The  pine  2,900 u n i t s of c h i p s ,  c o n d i t i o n s on  samples and  lodgepole  east-west  1.  The  s p a c i n g between the c e n t r e s of any  The  p i l e was  constructed  two  sample bags was  8ft.  to comprise f o u r i n d i v i d u a l s e c t i o n s , each  s e c t i o n c o n t a i n i n g a l l the treatments r e q u i r e d f o r a study  of one  time p e r i o d .  -  F i g . 1.  26  -  East and West view of the chip p i l e showing l o c a t i o n of chip samples 1 to 6.  F i g . 2.  South view section of the chip p i l e .  -  The  f o u r time p e r i o d s  the f i r s t  27  -  6,  12 and  t h r e e were i n v e s t i g a t e d i n t h i s  study.  The  ( F i g . 3) were 3,  sample bags were r e t r i e v e d by  c a r e f u l l y breaking  s e c t i o n of the p i l e a t the r e q u i r e d time i n t e r v a l possibility  ( F i g . 4).  of e n v i r o n m e n t a l changes to the remaining  i n each s e c t i o n were s e p a r a t e d When a s e c t i o n was new  24 months but  f a c e was  by  up  each  To reduce  s e c t i o n s , the  to conform to those  the p o s i t i o n and  s l o p e of  the  i n p l a s t i c bags  and  t r a n s p o r t e d under r e f r i g e r a t i o n to the l a b o r a t o r y i n Vancouver where  2.  chips.  they  used.  Sampling p o s i t i o n s  Each of the f o u r s e c t i o n s of the wood c h i p p i l e the treatments p l a c e d  ( F i g . 3) had a l l  i n s i x d i f f e r e n t p o s i t i o n s which were i n n e r top,  m i d d l e , i n n e r bottom, o u t e r  top,  o u t e r p o s i t i o n s were on the south  3.  samples  of the o l d .  Samples removed from the p i l e were p l a c e d  were s t o r e d a t 1°±0.1°C u n t i l  the  2 0 f t . (6.2m.) l o n g s e c t i o n s of m a t r i x  removed from the p i l e ,  adjusted  only  o u t e r m i d d l e and f a c e of the  o u t e r bottom  inner  ( F i g . 1).  pile.  Treatments  The  treatments were as f o l l o w s :  1.  Sample bags of p i n e  2.  Sample bags of spruce  chips  3.  Sample bags of spruce  c h i p s c o n t a i n i n g a core bag  chips  of f i n e s  The  Fig. 4. Retrieval of chip samples.  -  4.  -  Sample bags of spruce of s t e r i l i z e d  5.  spruce  Each treatment  was  i  c h i p s c o n t a i n i n g a core  chips containing a core  chips inoculated with Ptychogaster  replicated  bag  chips  Sample bags of spruce of spruce  4.  29  bag sp.  twice.  P r e p a r a t i o n of wood c h i p samples  White spruce  and  lodgepole  p i n e were used i n p r e p a r i n g  S u f f i c i e n t sound wood f o r the e n t i r e experiment was l o g s i n the P r i n c e George a r e a .  The wood was  obtained  then chipped  the  samples.  from s e v e r a l  i n the  mill.  I n d i v i d u a l s p e c i e s were w e l l mixed to g i v e homogeneous samples u s i n g the m i x i n g  facilities  These samples were then truck. (26.1 put into  The kg.)  of Rayonier  t r a n s p o r t e d back to P r i n c e George i n a r e f r i g e r a t e d  i n d i v i d u a l samples were?prepared on of c h i p s i n t o each flexmesh bag.  i n a screw-capped b o t t l e , and weighed.  to p r e p a r e  technique  the m a j o r i t y of the samples.  a s m a l l e r "flexmesh"  p l a s t i c bag  chips.  from each  bag,  These s m a l l samples were brought content.  proposed by J.V.Hatton (1970) was The  technique  c o n s i s t s of p l a c i n g  c o n t a i n i n g a t e s t sample i n t o a b i g g e r  c o n t a i n i n g c h i p s so t h a t the s m a l l bag i n f l u e n c e due  the s i t e by w e i g h i n g 58 l b .  Samples were taken  the l a b o r a t o r y to determine the m o i s t u r e A " c o r e ' sample bag"  Any  I n c . i n S h e l t o n , Washington.  e s s e n t i a l l y forms the c e n t r a l  to the core must then pass outward and  affect  the  bag  core. sample  used  -  30  -  The main sample hags weighed between 58 l b . (26.1kg) and 60 l b . (27.kg.) green weight of c h i p s w h i l e the c o r e samples  weighed between 8 l b .  (3.6kg.) arid 10 l b . (4.5kg.). The c o r e bags f o r treatments 4 and 5 were p r e p a r e d by 10 l b . (4.5 kg.) minutes.  s p r u c e c h i p s i n a wooden c o n t a i n e r a t 15 p s i f o r 90  These were put a s e p t i c a l l y i n t o  had been s t e r i l i z e d by d i p p l i n g by a r i n s e i n s t e r i l e water.  " f l e x m e s h " p l a s t i c bags  it  into  which  them i n 5% p h e n o l f o r a number of h o u r s , f o l l o w e d  The c o r e bags i n treatment f i v e were i n o c u l a t e d  w i t h c u l t u r e s of a P t y c h o g a s t e r sp., growing removing  autoclaving  on 2% malt agar, by  carefully  the c o l o n y from the p e t r i d i s h w i t h a s t e r i l e s p a t u l a and the p l a s t i c bag c o n t a i n i n g  the s t e r i l e c h i p s .  putting  The flexmesh  plastic  bags were p l a c e d i n s t e r i l e p o l y e t h y l e n e bags and i n c u b a t e d f o r two and a h a l f months a t room temperature. bag a f t e r  They were then put i n another t h i c k  the i n c u b a t i o n p e r i o d and  transported i n a refrigerated  plastic  truck  to the s i t e of the p i l e .  5.  Measurement of e n v i r o n m e n t a l c o n d i t i o n s  The  e n v i r o n m e n t a l f a c t o r s measured were temperature  i n the wood  c h i p p i l e , m o i s t u r e c o n t e n t of wood c h i p s and a c i d i t y of wood c h i p s .  a.  Temperature  Thermocouples  i n the wood c h i p  were i n s e r t e d  to measure the temperatures g r a d i e n t a c r o s s the p i l e  into  pile  the 2 y e a r s e c t i o n of the p i l e  i n every sample bag  (60 b a g s ) .  The  f a c e to the depth o f 96 i n . (2.4m) was  thermal  1  a l s o measured  -  31  -  on the s o u t h and n o r t h f a c e s by p l a c i n g  thermocouples a t the s u r f a c e  and a t depths of 3, 6, 12, 24, 48 and 96 i n (0.08, 0.15, 0.31, 0.61, and 2.43m). In a l l ,  74 thermocouples were used.  Temperature months a f t e r which  1.22  r e a d i n g s were taken a t d a i l y i n t e r v a l s f o r t h r e e  time t w i c e weekly r e c o r d i n g s were made.  A l l 74  thermocouples were connected to a master c o n t r o l p a n e l s i t u a t e d w i t h i n an i n s u l a t e d hut near the 2 y e a r s e c t i o n o f the p i l e .  High temperatures a r e more f a v o u r a b l e f o r the growth and r e p r o d u c t i o n of t h e r m o p h i l i c f u n g i  (Cooney and Emerson, 1964).  Therefore  the t o t a l number o f f u n g i i s o l a t e d  from a sampling bag a f t e r a g i v e n sampling  p e r i o d c o u l d , i n f a c t , be a r e f l e c t i o n of the temperature regime the bag experienced during  storage.  In d e t e r m i n i n g a measure o f the temperature regime c o r r e s p o n d i n g to each bag, temperature v a l u e s measured a t a l l the bag p o s i t i o n s i n the 24 month s e c t i o n o f the c h i p p i l e were c o n s i d e r e d a p p l i c a b l e to the c o r r e s p o n d i n g positions  i n the 3, 6 and 12 month s e c t i o n s o f the c h i p p i l e ;  average temperature v a l u e s f.or each bag p o s i t i o n were summated p e r i o d d u r i n g which called  the " t o t a l  also  weekly  f o r the  the r e s p e c t i v e bags were i n the p i l e and these summations  temperatures".  These t o t a l  w i t h the t o t a l counts o f f u n g i f o r each bag.  temperatures were then c o r r e l a t e d  -  b.  32  -  M o i s t u r e c o n t e n t of c h i p s  The change i n m o i s t u r e c o n t e n t was  determined by comparing  the  m o i s t u r e c o n t e n t d e t e r m i n a t i o n a t the time t = 0 w i t h those determined a f t e r 3, 6 and 12 months s t o r a g e .  To determine  sample c h i p s were f i r s t weighed and The d i f f e r e n c e i n weight U s i n g the f o r m u l a M.C. Weight o f water  i n chips  i  n  n  ^_  .  initially was  screen.  One  i n a Wiley M i l l  i  i  J  calculated.  (Model ED-5)  gram of wood meal (oven-dry b a s i s ) was  f o r 15 seconds w i t h 25-ml d i s t i l l e d water  (of pH 6.0  to  stirred - 6.5)  and  (Model 7 ) .  I s o l a t i o n of f u n g i  a.  Sampling  procedure  A random sample of 100 bag.  ^  A c i d i t y o f wood c h i p s  determined w i t h a C o r n i n g pH meter  6.  . ^  the p e r c e n t a g e m o i s t u r e c o n t e n t was  F i v e to lOg of c h i p s were ground pass a 1-mm  i n the c h i p s .  % =  r  c.  then o v e n - d r i e d to c o n s t a n t w e i g h t .  r e p r e s e n t s the a c t u a l weight of water  — — — r ~ -7—, — — r ~ 7 ^ — 100 Oven dry weight o f c h i p s s  the m o i s t u r e c o n t e n t the  c h i p s was  These were s u r f a c e s t e r i l i z e d  through a f l a m e .  A p i e c e was  s t e r i l e bone f o r c e p s . sterilized  taken from each 60 l b . sample  i n d i v i d u a l l y by q u i c k l y p a s s i n g them  removed from the c e n t r e of the c h i p s u s i n g  T h i s p i e c e was  s p l i t into  two h a l v e s , and  then s u r f a c e  a g a i n and p l a t e d on a c i d malt agar composed of 2% m a l t , 2% agar  -  and  0.5% m a l i c a c i d .  33 -  One p i e c e on one p l a t e was i n c u b a t e d  the o t h e r p i e c e on another p l a t e a t 25°C. p i e c e s of c h i p s The  were s u b - c u l t u r e d  and c o l o n i e s growing on them  onto 2% malt 2%agar s l a n t s .  remained was removed and d i s c a r d e d . of the agar p r e v e n t e d  At  Each p e t r i d i s h c o n t a i n e d t e n  (Fig. 6).  p l a t e s were examined d a i l y  b.  a t 45°C and  The p a r t o f t h e colony  This process  was r e p e a t e d  until  that drying  further isolation.  I s o l a t i o n and grouping  of f u n g i  the end o f the sampling, a l l the s l a n t c u l t u r e s were examined  under t h e b i n o c u l a r m i c r o s c o p e and were grouped i n t o broad c a t e g o r i e s , mainly g e n e r i c .  The s l a n t s were then counted and r e c o r d e d .  A f t e r a thorough e x a m i n a t i o n o f a s e l e c t e d number o f c u l t u r e s from each group the remainder of the tubes were d i s c a r d e d .  These r e p r e s e n t a t i v e  c u l t u r e s were then examined u s i n g a phase c o n t r a s t microscope and i d e n t i f i e d wherever  possible.  Group 1.  T h i s group was comprised of a s p e r g i l l u s type  no p e r f e c t s t a g e . the i s o l a t e s Group I I .  The f o l l o w i n g c a t e g o r i e s were used.  The c u l t u r e was  light  f u n g i which had  green to g r e e n .  The m a j o r i t y o f  i n t h i s group were A s p e r g i l l u s fumigatus .  T h i s group was made up o f a l l types  conidiophores.  Some produced ascocarps  fungus was Byssochlamys  emersonii.  while  of f u n g i which had p e n i c i l l a t e others  d i d not.  The most common  -  34  -  F i g u r e 6. Byssochlamys e m e r s o n i i growing out of c h i p s on 2% malt and 0.5% m a l i c a c i d agar.  -  Group I I I . mycelium.  35  -  T h i s group c o n s i s t e d o f a s p e c i e s w i t h white  thread-like  I t had no a s e x u a l s t a g e , grew s l o w l y i n c u l t u r e and had l i g h t  brown a s c o c a r p s .  The fungus i s s t i l l  u n i d e n t i f i e d b u t resembles  Byssochlamys. Group IV.  T h i s group was made up o f a s i n g l e f a s t growing s p e c i e s  was i d e n t i f i e d as Chrysosporium pruinosum.  which  The . c u l t u r e was cream i n  c o l o u r and powdery on t o p . Group V.  The hyphae o f the f u n g i i n t h i s group were h y a l i n e and had no  conidiopho res.  The c l e i s t o t h e c i a which were produced i n l a r g e numbers were  r e d d i s h brown and somewhat a n g u l a r .  T h i s fungus grew v e r y f a s t  i n culture.  Group V I .  T h i s group was made up o f f u n g i which had w h i t e t o cream  mycelium.  They produced h y a l i n e c o n i d i a which were p y r i f o r m to o v a l .  Some produced b l a c k c l e i s t o t h e c i a .  I t c o n t a i n e d b o t h A l l e s c h e r i a and  Sporotrichum. Group V I I .  T h i s group c o n t a i n e d  t h e y e a s t l i k e organisms.  i n f r e q u e n t l y i s o l a t e d and were p r e s e n t m a i n l y i n t h e samples  They were stored f o r three  months.  7.  D e t e r m i n a t i o n o f wood c h i p weight  loss  The weight l o s s e s were determined by comparing oven-dry weight o f c h i p samples 3, 6 and 12 months s t o r a g e .  a t s t o r a g e time t = 0 w i t h those measured a f t e r  The weight l o s s e s were e x p r e s s e d as p e r c e n t o f  o r i g i n a l oven-dry weight of wood u s i n g ' i-Qo  0 r x  igi  n a x  the c a l c u l a t e d  the f o r m u l a p e r c e n t weight l o s s =  ovendry weight of wood - ovendry weight of wood a f t e r O r i g i n a l ovendry weight o f wood.  test  -  D  36 -  RESULTS  1.  L i s t of fungi i s o l a t e d  The  f o l l o w i n g most commonly o c c u r r i n g  A s p e r g i l l u s fumigatus Allescheria  f u n g i were  identified:  Fresenius  terrestris  Apinis  Cephaslosporium t e r r e s t r e  A s e x u a l s t a t e o f A. t e r r e s t r i s . Byssochlamys e m e r s o n i i  Stolk-Apinis.  Chrysosporium pruinosum Humicola l a n u g i n o s a  (Gilman and Abbot) Comb. Nov.  ( G r i f f o n and Maublanc) Bunce.  Sporotrichum thermophile Thermoascus a u r a n t i a c u s  Apinis. Miehe.  In a d d i t i o n to the f u n g i l i s t e d  above, 21 d i s t i n g u i s h a b l e  f u n g i i s o l a t e d from the p i l e were n o t i d e n t i f i e d .  2.  D i s t r i b u t i o n o f common t h e r m o p h i l i c  The B_. e m e r s o n i i  most common t h e r m o p h i l i c ( F i g . 7), which o c c u r r e d  p i l e where temperatures were h i g h . increased  w i t h time o f s t o r a g e .  and t h e r m o t o l e r a n t  fungus o c c u r r i n g  fungi  i n the p i l e was  mainly i n the i n t e r i o r r e g i o n  of the  The numbers of i s o l a t e s o f t h i s fungus  CO  CO  -n —i.  to  TJ  0  (J) to o o c  H  •^4  0 z  rs  rt  m  O  c+  TO  o X* TO  — N J C O K O l O N I O O O  ooo  oooooog  F U N G A L / l O O  —  C O U N T CHIPS  ~ F U N G A L / l O O  ooc  C O U N T CHIPS  000000ooog^o F U N G A L / l O O  C O U N T CHIPS  -  A. t e r r e s t r i s  38  -  and S_. thermophile  ( F i g . 7) were commonly  isolated  from the i n n e r r e g i o n of the p i l e and were most common i n the samples stored  f o r t h r e e months.  T h e r e a f t e r , t h e i r numbers decreased  with  time of  storage. _T. a u r a n t i a c u s  (Appendix 1) d i d not occur as f r e q u e n t l y as the  o t h e r f u n g i but would grow f a s t out of the wood i f i t were p r e s e n t . erratically  isolated  from b o t h  A. fumigatus pile  i n n e r and o u t e r r e g i o n s of the p i l e .  ( F i g . 7) was c o n f i n e d to the o u t e r r e g i o n of the  and appeared i n l a r g e numbers.  position.  I t was  I t f r e q u e n t l y o c c u r r e d i n the o u t e r top  I t s numbers remained s t a b l e throughout  the p e r i o d o f s t o r a g e of  the c h i p s . (Z. pruinosum than from p i n e c h i p s . top and o u t e r m i d d l e  (Appendix 1) was more commonly  p o s i t i o n s of the p i l e .  remained s t a b l e throughout  Environmental  a.  from  The h i g h e s t number o f i s o l a t i o n s came from  from the o u t e r bottom p o s i t i o n .  3.  isolated  spruce  outer  I t was o c c a s i o n a l l y i s o l a t e d  The numbers of i s o l a t e s of C^. pruinosum  the d u r a t i o n of s t o r a g e of the c h i p s .  factors  Temperature i n the wood c h i p  pile  Changes i n temperature i n the s i x p o s i t i o n s i n the p i l e days and 80 weeks a r e shown i n F i g u r e s 8 and 9 r e s p e c t i v e l y .  f o r 34  °F  140.0-  130.0120.0-  110.0-  100.0-  90.0UJ DC 80.0D h < CE 70.0UJ Q. UJ 60.0h 50.0-  1  INNER BOTTOM POSITION OUTER BOTTOM POSITION INNER MIDDLE POSITION OUTER MIDDLE POSITION INNER TOP POSITION OUTER TOP POSITION  40.0- 4.4  30.0- 1.1  20.0- -6.7  F i g . 8.  D a i l y temperature curves f o r the s i x p o s i t i o n s i n the p i l e during the f i r s t t h i r t y - f o u r days of storage of c h i p s .  10.0- •12.2  j  i  ~T~  6  I  10  14  18  D A Y S  —J— 22  26  I 30  I 34  -  40  -  The i n n e r r e g i o n o f the p i l e had h i g h e r the o u t e r r e g i o n . the  temperatures  In the i n n e r r e g i o n , the h i g h e s t  top f o l l o w e d by the middle and the bottom.  than  temperatures were a t  The r i s e i n temperature  at  the top was v e r y r a p i d and a t t a i n e d the maximum temperature o f 60.6°C  in  19 days.  Two weeks l a t e r  The  the temperature had dropped  to 49.4°C.  temperature t r e n d was the same f o r the i n n e r m i d d l e p o s i t i o n  as the i n n e r top p o s i t i o n but temperatures were never as h i g h .  The maximum  temperature was 54.4°C and was a t t a i n e d t h i r t e e n days a f t e r the b u r i a l o f the bags. in  The drop i n temperature was g r a d u a l ,  going  to 46.1°C  two weeks. Low temperatures were r e c o r d e d  f o r the i n n e r bottom p o s i t i o n of  the p i l e compared to the o t h e r i n t e r i o r r e g i o n s . to  from 54.4°C  a t t a i n a maximum temperature o f 42.5°C.  A longer  Temperatures  time was  required  were s t a b l e i n  t h i s p a r t o f the p i l e . In the o u t e r  regions  d e a l , and except f o r the o u t e r the i n i t i a l  o f the p i l e  top p o s i t i o n s , temperatures never r o s e  temperatures o f the p i l e .  The h i g h e s t  the o u t e r r e g i o n was 29.4°C a t the o u t e r Temperatures which  ambient  top p o s i t i o n .  Temperatures  t h r e e months  were low d u r i n g  and e a r l y s p r i n g but s t a r t e d to r i s e a g a i n  though not a t t a i n i n g the v a l u e s  above  temperature r e c o r d e d i n  were r e l a t i v e l y h i g h f o r the f i r s t  t h e r e was a r a p i d drop.  winter  temperatures f l u c t u a t e d a g r e a t  o f the p r e v i o u s  the l a t e  after  fall,  i n the summer months,  summer.  In the w i n t e r  temperature a f f e c t e d the i n n e r top p o s i t i o n more than the o t h e r  months  op  o  C  140.0130.0INNER BOTTOM POSITION OUTER BOTTOM POSITION INNER MIDDLE POSITION OUTER MIDDLE POSITION INNER TOP POSITION OUTER TOP POSITION  120.0-  110.0-  100.090.0-  LU CC D 80.0h < DC 70.0LU CL  ^> 60.0-  LU h  50.0-  40.030.020.0Temperature curves f o r the s i x p o s i t i o n s the chip p i l e during 80 weeks of storage chips.  10.0-  o.o  5.0  10.0  15.0  20.0  25.0  30.0  \  35.0  40.0  45.0  WEEK  T 50.0  T"  55.0  60.0  65.0  70.0  75.0  80.0  in of  F i g . 10.  Average monthly ambient temperature in Prince George.  -  p o s i t i o n and the i n n e r middle  43  -  temperatures  were sometimes h i g h e r  than  those o f the i n n e r top p o s i t i o n . Temperatures i n the o u t e r r e g i o f o l l o w e d the ambient f o r most of the time  ( F i g . 9, 1 0 ) . F r e e z i n g temperatures  were  temperature common.  Temperature r i s e was n o t i c e d i n the summer b u t was o n l y marked i n the o u t e r top p o s i t i o n o f the p i l e  b.  (Fig.  9).  M o i s t u r e c o n t e n t of wood c h i p s  No d e f i n i t e p a t t e r n o f m o i s t u r e treatments  o r f o r the p i l e as a whole.  was h i g h l y v a r i a b l e  content e x i s t e d  The m o i s t u r e c o n t e n t of the samples  (Table 1 ) .  Spruce always had a h i g h e r m o i s t u r e i n the o u t e r p o s i t i o n s had h i g h e r m o i s t u r e inner p o s i t i o n u n t i l position,  f o r a l l the  content  content  the m o i s t u r e  i n n e r p o s i t i o n s i n the l a t e r  The c h i p s  than the c h i p s i n the  the "wet l e n s " e f f e c t developed  thus b i a s i n g  than p i n e .  a t the i n n e r top  c o n t e n t v a l u e s of the c h i p s i n the -  time p e r i o d s .  been adequate f o r f u n g a l development.  M o i s t u r e , g e n e r a l l y , must have  -  T a b l e 1.  44  -  F i n a l m o i s t u r e c o n t e n t of wood  3 months % M. C.  Treatment  chips.  6 months % M. C.  12 months % M. C.  P i n e (1) 42.6  39.2  46.7  33.6  38.1  39.7  Outer samples 1, 3, 5  64.7  66.2  65.6  Inner samples 2, 4, 6  58.6  56.4  72.0  Outer samples 1, 3, 5  70.8  63.2  63.9  Inner samples 2, 4, 6  65.6  67.5  72.9  Outer samples 1, 3, 5  64.9  61.6  66.4  Inner samples 2, 4, 6  61.3  76.7  74.4  Outer samples,  1, 3, 5  Inner samples 2, 4, 6 Spruce  (2)  Sterilized  s p r u c e (4)  I n o c u l a t e d s p r u c e (5)  -  c.  45  -  A c i d i t y o f wood c h i p s  Measurements o f a c i d i t y o f wood c h i p s were taken o n l y f o r the spruce and p i n e treatments first  sampling  f o r samples i n the o u t e r p s o t i o n s o f the  p e r i o d (Table 2 ) .  T a b l e 2.  Storage  except  F i n a l a c i d i t y o f wood c h i p s  Outer 1, 3, 5 Position  Months  Spruce  Pine  periods  0  Inner 2, 4, 6 Position  4.80  Outer 1, 3, 5 Position  Inner 2, 4, 6 Position  5.40  4.80  5.40 5.00  4.60  3 6  4.96  4.75  5.37  4.93  12  5.01  4.81  5.57  5.30  Mean  4.92  4.74  5.45  5.16  Changes i n pH were not v e r y marked b u t g e n e r a l l y f o l l o w e d the i n i t i a l pH o f s p r u c e and p i n e which i n d i c a t e d h i g h e r than p i n e . outer  t h a t the pH o f spruce was  The pH o f the i n n e r samples was lower  than t h a t o f the  samples. The  change i n pH w i t h d u r a t i o n of s t o r a g e was e r r a t i c .  went down a f t e r s t o r i n g months and c o n t i n u e d  pH g e n e r a l l y  the wood f o r t h r e e months but r o s e a g a i n a f t e r s i x  to i n c r e a s e .  The b e h a v i o u r  o f the i n n e r samples o f  -  s p r u c e d i d not  follow  months' s t o r a g e and  4.  the  general  -  pattern.  s t a r t e d to i n c r e a s e  Weight l o s s e s of wood  The  46  pH  only  region.  significant three  six  chips  weight l o s s e s of wood c h i p s  This  to the  at twelve months.  are  Weight l o s s e s were lower i n the o u t e r r e g i o n inner  d e c r e a s e d up  d i f f e r e n c e was  summarized i n T a b l e of  significant  d i f f e r e n c e between t r e a t m e n t s .  the p i l e than i n a t 5%.  There was  Weight l o s s e s  to s i x months, the d i f f e r e n c e b e i n g s i g n i f i c a n t  3. the no  increased  a t 5%.  from  Although  weight l o s s e s a t twelve months were lower than weight l o s s e s a t s i x months, the d i f f e r e n c e was  not  T a b l e 3.  statistically  significant.  F i n a l weight l o s s of samples i n p e r c e n t  Position  Percent 3 months  weight loss 6 months 12 months  Outer: 1 , 3 , 5  1.1  2.0  1.9  Inner: 2 , 4 , 5  2.4  4.0  3.2  -  5.  The  -  E v a l u a t i o n o f treatments  The philic  47  treatments were a n a l y z e d  and t h e r m o t o l e r a n t  on the t o t a l count o f thermo-  f u n g i i s o l a t e d a t 45°C f o l l o w i n g , i n c u b a t i o n .  t o t a l count of f u n g i i s the number o f f u n g i i s o l a t e d  from one hundred  chips. The  treatments  (Tables 4, 7) g e n e r a l l y had no e f f e c t on the  d i s t r i b u t i o n o f f u n g i i n the p i l e . period  I t was o n l y a t the s i x months sampling  t h a t d i f f e r e n c e s e x i s t e d between t r e a t m e n t s .  spruce w i t h P t y c h o g a s t e r  sp had no e f f e c t on the d i s t r i b u t i o n o f the f u n g i .  There were a l s o no d i f f e r e n c e s between p i n e and The  Pre-inoculating  l e n g t h of storage  of chips  e f f e c t on the d i s t r i b u t i o n o f the f u n g i .  spruce.  (Tables 6,7) had a s i g n i f i c a n t Fungal p o p u l a t i o n  increased  the t h r e e months p e r i o d to the s i x months p e r i o d and remained  from  constant  between the s i x months and the twelve months p e r i o d s . The  p o s i t i o n o f the samples i n the p i l e  (Table 5) p l a y e d  dominant r o l e i n the changes i n numbers of the f u n g a l p o p u l a t i o n . of the f u n g i were i s o l a t e d of the p i l e i n the f i r s t months s t o r a g e  from the i n n e r bottom and o u t e r  t h r e e months.  positions.  months  storage.  Most  top p o s i t i o n s  E x a m i n a t i o n o f the c h i p s a f t e r s i x  showed a l a r g e i n c r e a s e i n the t o t a l f u n g a l p o p u l a t i o n i n  the i n n e r upper and the i n n e r m i d d l e p o s i t i o n s a l t h o u g h all  a  increases occurred at  Changes i n t o t a l f u n g a l p o p u l a t i o n d i d n o t o c c u r  after  twelve  -  The using  48  -  e v a l u a t i o n o f the treatments was c a r r i e d out i n two s t e p s  the a n a l y s i s of v a r i a n c e .  The a n a l y s i s of the t h r e e months samples  was done s e p a r a t e l y because a treatment which was n o t examined i n the subsequent time p e r i o d s was i n c l u d e d i n t h i s a n a l y s i s . was f o r the f o u r treatments a t the t h r e e  Table  4.  time  periods.  Mean f u n g a l count f o r d i f f e r e n t treatments a f t e r s t o r i n g c h i p s f o r t h r e e months.  Treatments  F u n g a l count  Pine  44  Spruce  55  Fines  The second a n a l y s i s  incorporated  i n t o spruce  65  Sterilized  spruce  53  Inoculated  spruce  43  In the t h r e e months s t o r a g e  (Table 4) d i f f e r e n c e s between  treatments were not s i g n i f i c a n t b u t d i f f e r e n c e s between p o s i t i o n s were s i g n i f i c a n t a t -5%.  No s i g n i f i c a n t  (Table 5)  i n t e r a c t i o n e x i s t e d between t r e a t -  ments and p o s i t i o n s .  A Duncan's m u l t i p l e range t e s t  (Table 5) c a r r i e d out on the mean  f u n g a l counts f o r p o s i t i o n s showed s i g n i f i c a n t d i f f e r e n c e s a t 5% between f u n g a l counts from i n n e r bottom, o u t e r  top p o s i t i o n s and a l l the o t h e r  -  positions. by  49  -  The h i g h e s t count of f u n g i was  from i n n e r  followed  the o u t e r top p o s i t i o n .  T a b l e 5.  Average number of f u n g i i s o l a t e d per p o s i t i o n t h r e e months s t o r a g e . *  Position  Inner top  24  Outer bottom  28  Inner M i d d l e  39  Outer  middle  45  Outer  top  79  two p o s i t i o n s  d i f f e r e n t a t 5%  and  interactions treatments  99  means s i d e s c o r e d  by  the same l i n e a r e not s i g n i f i c a n t l y  level.  In the second positions  after  Mean F u n g a l Count  Inner bottom  * Any  bottom p o s i t i o n  analysis  significant difference  time but not between t r e a t m e n t s . between a l l combinations  (Table 6).  a t 5% e x i s t e d  There were s i g n i f i c a n t  of these f a c t o r s  e x c e p t time  and  between  -  T a b l e 6.  50 -  Fungal counts a t d i f f e r e n t different positions.  Positions  s t o r a g e times a t the  Storage p e r i o d i n months  3  6  Outer bottom  32  67  Inner bottom  99  119  108  Outer middle  48  68  73  Inner m i d d l e  41  112  100  Outer  top  79  113  105  Inner top  25  118  100  12 •• ' 73  The p o p u l a t i o n o f f u n g i changed w i t h time o f s t o r a g e b u t n o t w i t h the t r e a t m e n t s .  The s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the  treatments s t o r e d f o r 6 months but n o t f o r 3 o r 12 months  T a b l e 7.  (Table 7).  F u n g a l counts a t d i f f e r e n t s t o r a g e times w i t h d i f f e r e n t treatments. These a r e average f o r 6 positions.  Treatments  Storage p e r i o d i n months  3  6  12  Pine  44  82  92  Spruce  55  107  93  spruce  65  116  96  Inoculated spruce  53  94  92  Sterilized  -  6.  Relationships  51  -  between v a r i a b l e s  measured  F i g u r e 11 shows the r e l a t i o n s h i p between t o t a l f u n g a l and m o i s t u r e c o n t e n t . was no s i g n i f i c a n t  A simple r e g r e s s i o n  analysis  indicated  c o r r e l a t i o n between these v a r i a b l e s  (r  that there  = 0.014).  F i g u r e 12 shows the r e l a t i o n s h i p between t o t a l f u n g a l and  total  fungal  temperature.  correlation  temperature and weight l o s s  T a b l e 8.  A multiple (R  2  regression  analysis  = 0.34) between f u n g a l  total  on the d a t a  count,  total  (Table 8 ) .  Summary o f r e g r e s s i o n a n a l y s i s o f t o t a l temperature and weight l o s s on t o t a l f u n g a l count  variable  R-'  TQ  temperature and weight l o s s  0.34  17.8  *  temperature  0.29  28.9  *  The f i n a l pH o f the wood c h i p s any o f the o t h e r f a c t o r s measured. I t i s concluded that are  count  F i g u r e 13 shows the r e l a t i o n s h i p between  count and weight l o s s .  showed s i g n i f i c a n t  count  related.  variable.  ^  ( T a b l e 2) was n o t r e l a t e d t o  pH v a r i e d  from 4.74 to 5.57.  temperature, weight l o s s and f u n g a l  I t i s n o t c l e a r which o f these f a c t o r s  count  i s the independent  -  52 -  162.9 •  U J O ^ 130.3 -  DC 3 Z H ill h  97.7 65.1 •  o-z 32.5 -  * u  18  33 48 63 78 93  T O T A L  11.  108 123 138 153 168 183  F U N G A L  C O U N T  The relationship between total fungal count and moisture content of chips.  4533 -  lil  3782 •  cc  <D h 0 < h cc  LU Q.  3031 2280 1530  h  17  31 44 58 71  T O T A L  12.  86 99  113 127 141 155 168  F U N G A L  C O U N T  The relatinship between total fungal count and total temperature.  7.7 6.2 -  4.7 3.2 1.8 •  17  31 44  T O T A L  13.  58 72  86 99 113 127 141 155 168  F U N G A L  C O U N T  The relationship between total fungal count and wood weight loss.  -  E  53  -  DISCUSSION  1.  F u n g i i n the c h i p wood p i l e  T h i s p a r t o f the study i n d i c a t e s t h a t t h e r m o p h i l i c thermotolerant The  f u n g i occupy d i f f e r e n t  thermophilic  thermotolerant  o f the c h i p p i l e  f u n g i m a i n l y i n h a b i t the i n n e r r e g i o n s  f u n g i a r e found i n the o u t e r  d i s t r i b u t i o n a l and s u c c e s s i o n a l tenperature  regions  "preferences"  Bergman and N i l s s o n  regions  (0.29) i s shown between f u n g a l  group.  from  correlation  T h i s was n o t a s p e c i f i c  In the p r e s e n t  study a c o r r e l a t i o n  i s o l a t i o n s a t 45°C and temperatures i n the  (Table 8 ) . The  t o t a l number of f u n g i does n o t d e c r e a s e w i t h l e n g t h o f  o f c h i p s even a f t e r s t o r a g e  o f the c h i p s  f o r twelve months.  u n f a v o u r a b l e temperatures from the s i x months' s t o r a g e months p e r i o d  d i d not a f f e c t  the p o p u l a t i o n  period  of the f u n g i .  the numbers of f u n g i from the s i x months' sampling p e r i o d months sampling p e r i o d was not s i g n i f i c a n t . of S h i e l d s  Any  result  (1967, 1968) showed a s i g n i f i c a n t  c o r r e l a t i o n based on any f u n g a l  storage  of the p i l e .  fungi.  between f u n g i and the temperatures i n the p i l e .  chip p i l e  (Fig. 7).  w h i l s t the  changes of f u n g i i n the p i l e  o f the d i f f e r e n t  and  (1970), who  The low  to the twelve  The r e d u c t i o n i n to the twelve  This f i n d i n g i s contrary  showed t h a t the numbers of f u n g i d e c r e a s e d  to t h a t  sharply  -  with  stonage time and  one-half  it  -  to n e g l i g i b l e v a l u e s  months' s t o r a g e of the c h i p s .  sharp d e c l i n e , but being  even f e l l  54  a t the h i g h  could s i g n i f y  i t might imply  No  a f t e r seven  explanation  i s offered for this  t h a t the c h i p s were s t e r i l i z e d  temperatures f o r t h a t l e n g t h of time. t h a t the low  and  after  Alternatively,  i s o l a t i o n temperatures which S h i e l d s used  were r e s p o n s i b l e f o r h i s f a i l u r e to i s o l a t e the  t h e r m o p h i l i c f u n g i which  would have been p r e s e n t  t h a t p e r i o d of  initially  i n the c h i p s even a f t e r  The  p o p u l a t i o n of f u n g i i n the h o t t e r r e g i o n of the p i l e i s  low  and  comprised of A.  terrestris,  S_. t h e r m o p h i l e  and  emersonii.  With s t o r a g e beyond t h r e e months, the p o p u l a t i o n of  increases.  The  emersonii  e n t i r e i n c r e a s e i s accounted f o r by  population.  decreases,  and  probably  The  p o p u l a t i o n of A.  f o l l o w s A.  frequency  of the  of o c c u r r e n c e  p i l e and initial the  i t may  S_. In  t h r e e f u n g i i s confirmed  by  succession,  fungi.  No  A l t h o u g h i n comparison to A.  high  and  succession.  In t h i s study  the  of  the  high  throughout  s u c c e s s i o n a l p a t t e r n i s shown f o r <Z. i n the o u t e r  top and m i d d l e p o s i t i o n s .  fumigatus i t s numbers were low,  areas where i t appeared.  not h e a v i l y degraded, and  The  Bergman  p o p u l a t i o n of t h i s fungus remained at the same l e v e l  the d e c a y i n g  thermophile  t e r r e s t r i s and J3. t h e r m o p h i l e .  not be r e p l a c e d by o t h e r  t h r e e sampling p e r i o d s .  those  fungi  fumigatus i s an e a r l y c o l o n i z e r of the o u t e r r e g i o n s  pruinosum except t h a t i t o c c u r s  in  t e r r e s t r i s and  (1966) but no mention i s made of t h e i r A.  B.  i n c r e a s e i n _B.  they a r e r e p l a c e d by B^. e m e r s o n i i .  t h e r e f o r e , B_. e m e r s o n i i  Nilsson  storage.  The  c h i p s i n these  i t is stable  a r e a s , however,  u n l i k e the f i n d i n g s of Bergman and  a c t i v i t y of t h i s organism must have been low  Nilsson  i n the  were (1966),  spruce-pine  -  c h i p p i l e a t P r i n c e George.  55 -  The cause of t h i s low d e c a y i n g  c l e a r but may be due to c o m p e t i t i o n The  from f u n g i l i k e A. f u m i g a t u s .  to the a c t i v i t y o f the t h e r m o p h i l i c f u n g i i n the  Indeed a s i g n i f i c a n t c o r r e l a t i o n was found between f u n g a l  and weight l o s s e s o f wood not  i s not  h i g h wood s u b s t a n c e l o s s i n the i n n e r r e g i o n o f the c h i p  pile i s attributed partly pile.  activity  ( F i g . 13 T a b l e  8) a l t h o u g h  counts  t h i s c o r r e l a t i o n was  as s t r o n g as the c o r r e l a t i o n between f u n g a l counts and temperature  ( F i g . 12 T a b l e The  8) i n the p i l e . t h e r m o p h i l i c and t h e r m o t o l e r a n t  f u n g i through t h e i r  metabolic  a c t i v i t y must be p a r t l y r e s p o n s i b l e f o r thermogenesis i n the c h i p  piles.  Thermogenesis through f u n g a l a c t i v i t y has n o t been shown i n wood, b u t thermogenetic c a p a b i l i t i e s have been shown f o r A. fumigatus i n o t h e r materials  ( C a r l y l e and Norman, 1941). The  i n n e r bottom r e g i o n p r o b a b l y  had the h i g h e s t  because the temperature was m i l d and s t a b l e . stability erant but  thought t h a t the f o u n d a t i o n  Ptychogaster the c h i p s .  thermotol-  S h i e l d s and U n l i g i l  (1968)  o f o l d c h i p s c o n t r i b u t e d to the g r e a t e r  region.  s p . does n o t spread  I t was n o t i s o l a t e d  (1968) i s o l a t e d P t y c h o g a s t e r  out o f the inoculum bag i n t o  from the c h i p samples.  S h i e l d s and U n l i g i l  s p . from balsam f i r and spruce  i n the upper and m i d d l e l a y e r s o f the p i l e . from o t h e r p i l e s  counts  Because o f the m i l d n e s s and  A s i m i l a r o b s e r v a t i o n was made by  numbers o f f u n g i i n t h a t  isolated  fungal  t h i s a r e a o f the p i l e can harbour both t h e r m o p h i l i c and  fungi.  they  plant  ( S h i e l d s , 1967).  chip  samples  T h i s fungus has not been I t i s p o s s i b l e that  Ptychogaster  -  sp. i s not a b l e  to compete w i t h  s p e c i e s of f u n g i . Unligil  56  -  the t h e r m o p h i l i c  Its inability  and  thermotolerant  to compete i s i n f e r r e d by S h i e l d s and  (1968) who wrote t h a t the fungus cannot stand  temperatures above  50°C.  2.  Temperature i n the wood c h i p  pile  A sharp r i s e i n temperature o c c u r s of most c h i p p i l e s .  s h o r t l y a f t e r the c o n s t r u c t i o n  T h i s g e n e r a l l y takes two weeks to a t t a i n maximum  temperature, and, depending upon the s p e c i e s o f wood which i s p i l e d , the volume o f the p i l e and the e x t e n t from 37°C to 69°C  1965; S h i e l d s and U n l i g i l ,  A s i m i l a r sharp r i s e o c c u r r e d  i n P r i n c e George.  vary  (Bjorkman and Haeger, 1963; Annergren, D i l l o n and  Vardheim, 1964; L j u n g q v i s t , Howard, 1968).  o f compaction, the temperature w i l l  1968; B u t c h e r and  i n the s p r u c e - p i n e  I n t h i s s t u d y , however, i t was n o t i c e d  chip  pile  t h a t the temperature  r i s e was not the same f o r a l l the p o s i t i o n s i n the i n n e r r e g i o n s  of the  p i l e , nor d i d i t take the same time f o r a l l the p o s i t i o n s to a t t a i n maximum temperatures.  The time taken f o r these p o s i t i o n s to a t t a i n maximum  temperature v a r i e d from two to t h r e e weeks.  The p a t t e r n o f the d e c l i n e i n  temperature i s the same as g i v e n by o t h e r workers  (Bjorkman and Haeger, 1963;  Annergren, D i l l e n and Vardheim, 1964; S h i e l d s and U n l i g i l ,  A l t h o u g h d i f f e r e n c e s i n temperature occur  1968).  between p i l e s  composed  of d i f f e r e n t wood s p e c i e s , no d i f f e r e n c e s were n o t i c e d between samples o f s p r u c e and p i n e .  Ljungqvist  (1965) found t h a t as much as 5°C d i f f e r e n c e can  -  e x i s t between p i n e and as D o u g l a s - f i r T h i s may  spruce  (Hensel,  be due  57  1958)  -  piles.  There a r e , however, s p e c i e s  where no  rise  to h i g h e x t r a c t i v e content  i n temperature o c c u r s of the wood which  such during  storage.  inhibits  the growth of f u n g i . Temperatures i n the o u t e r r e g i o n of the P r i n c e George wood c h i p p i l e were much lower than those Smith and  Lindgren,  1961;  different  locations.  ( F i g . 10)  and  f o r southern  S a u c i e r and M i l l e r ,  the compaction i n a l l these can be accounted f o r by  reported  1961;  pine species  Davies,  cases must have been the same.  1963)  (Rothrock, although  Such a d i f f e r e n c e  the d i f f e r e n c e s i n the ambient temperatures of  Comparison of the ambient c l i m a t i c  the temperature changes  shows t h a t the ambient c o n d i t i o n s had  conditions  ( F i g . 9) i n the wood c h i p p i l e a s t r o n g e f f e c t on  the  studied  the changes i n  temperature. S e a s o n a l changes a f f e c t e d the p i l e i n P r i n c e George more than other studies i n d i c a t e . winter ambient  months, but (Rothrock,  Generally,  the o u t e r r e g i o n s  freeze during  even when the i n n e r temperatures drop they Smith and  Lindgren,  1961;  do not  S a u c i e r and M i l l e r ,  the  the approach  1961;  II  Bjorkman and  Haeger, 1963;  S h i e l d s and  Unligil,  1968).  of P r i n c e George, the i n n e r r e g i o n s of the p i l e s had  In the c o l d weather  temperatures near  freezing. The  second r i s e i n temperature  the maximum i s not  ( F i g . 9) a f t e r  common w i t h most p i l e s .  i n summer months (Butcher  and  Howard, 1968).  the d e c l i n e from  T h i s happens i n s m a l l e r T h i s second r i s e  piles  i n temperature  -  i s probably  due i n i t i a l l y  perhaps w i l l ,  58 -  to i n c r e a s i n g e x t e r n a l temperature.  i n time, b r i n g temperatures to l e v e l s which w i l l  resumption o f t h e r m o p h i l i c f u n g a l a c t i v i t y .  A c i d i t y o f wood c h i p s  The  during storage  warm whether.  ,  r e s u l t s on the pH o f wood c h i p s a r e a t v a r i a n c e  f i n d i n g s of s e v e r a l authors  a l l o w the  T h i s i s shown by F i g u r e 9  where the second r i s e i n temperature began w i t h . t h e  3.  This  with  who r e p o r t e d a r e c t i l i n e a r d e c r e a s e o f pH  (Annergren, D i l l e n and Vardheim, 1964; S h i e l d s ,  No attempt a t c o r r e l a t i n g pH w i t h  1970).  time o f s t o r a g e was p o s s i b l e i n t h i s  study  s i n c e no s e t p a t t e r n e x i s t e d f o r changes i n pH. The  drop i n pH i s a t t r i b u t e d to the p r o d u c t i o n  of a c e t i c a c i d  as a r e s u l t o f d e a c e t y l a t i o n o f the h e m i c e l l u l o s e s i n the wood D i l l o n and Vardheim, 1964; S h i e l d s , 1970). of h e m i c e l l u l o s e s must have o c c u r r e d  (Annergren,  A l t h o u g h some d e a c e t y l a t i o n  i n the p i l e under study,  the low wood  substance l o s s c o u l d n o t have l e d to l a r g e a c e t i c a c i d p r o d u c t i o n . i n pH i n t h i s p i l e w i l l n o t a f f e c t t h e r m o p h i l i c and t h e r m o t o l e r a n t  4.  Moisture  The said  Changes  the a c t i v i t y and s u c c e s s i o n o f the  fungi.  i n the wood c h i p  pile  extreme v a r i a t i o n i n m o i s t u r e of c h i p s d u r i n g s t o r a g e i s  to be r e s p o n s i b l e f o r the d i s t r i b u t i o n o f f u n g i , and the r i s e i n  temperature i n the i n n e r top p o s i t i o n o f the c h i p p i l e  (Rothrock,  Smith  -  and  Lindgren,  1961;  -  S a u c i e r and M i l l e r ,  Annergren, D i l l e n and 1966).  59  Vardheim, 1964;  the d i s t r i b u t i o n of f u n g i .  The samples.  of f u n g a l  Haeger,  Bergman and  Nilsson,  outer  samples, i t had  Moisture  i n the wood must have been  activity.  average m o i s t u r e content  However, m o i s t u r e content  of the c h i p s was  above 100%  to the f i n d i n g s of Bjorkman and The  p i l e i s due rainfall.  h i g h m o i s t u r e content  to the m e l t i n g  was  above 50%  common i n the  quicker  Haeger  (Butcher  and  This i s  (1963).  of the wood i n the i n n e r top of  of the i c e and  snow a f t e r the w i n t e r  Howard, 1968).  than the top, c o n s e q u e n t l y  in a l l samples  and  Changes i n r a i n f a l l have a f f e c t e d the m o i s t u r e c o n t e n t  i n small p i l e s  highly  a minimal  from the i n n e r top r e g i o n , when they were s t o r e d f o r 12 months. similar  1963;  of the c h i p s i n t h i s study was  v a r i a b l e , e s p e c i a l l y between i n n e r and  adequate f o r the support  Bjorkman and  N i l s s o n , 1965;  A l t h o u g h the m o i s t u r e content  e f f e c t on  1961;  The  the to  of  chips  edges of the p i l e dry  the i n n e r top i s w e t t e r than the  out  outer  sides.  5.  Damage  No  s e r i o u s damage as diagnosed by v i s u a l i n s p e c t i o n was  the c h i p s d u r i n g George.  the o u t s i d e s t o r a g e  S t a i n i n g of the c h i p s was  f o r a p e r i o d of 12 months a t P r i n c e  minimal and weight l o s s e s were  Moderate s t a i n i n g of the c h i p s o c c u r r e d months.  done to  i n the o u t e r  These c h i p s were mouldy, w i t h v e r y  little  low.  c h i p s s t o r e d f o r twelve  blue s t a i n i n g present.  -  S t a i n i n g of balsam f i r and  s p r u c e has  twelve months' s t o r a g e , w i t h Unligil,  1968).  The  two  60  little  -  been r e p o r t e d  blue  staining occurring  spruce-pine and  and  d i s c o l o u r e d to any  e a s t e r n Canada (Annergren, D i l l e n  extent.  and  found t h a t owing to the low  In t h i s  respect  the  from p i l e s  Vardheim, 1964;  i n Sweden  Shields,  1970)  pH of the c h i p s , they were brown  discoloured. The  not  and  of the p i l e were l i g h t  p i l e a t P r i n c e George behaved d i f f e r e n t l y  where i t was  (Shields  f i n d i n g s are i n agreement.  A l t h o u g h c h i p s from the i n n e r r e g i o n s brown, they were not  a f t e r s i x to  increase  l o s s e s may  weight l o s s e s of p i n e and  s p r u c e samples were low  from the s i x to twelve months' sampling p e r i o d .  not have i n c r e a s e d  weather c o n d i t i o n s which  Bergman and  Nilsson  c h i p s s t o r e d i n the warmer p a r t s of the p i l e l o s t High weight l o s s e s o c c u r pile.  Saucier  and  southern pine p i l e Bergman and  Nilsson  Miller  spruce-pine  and  Nilsson  weight third  could  (1966) showed t h a t  1% per month d u r i n g  i n the i n n e r , h o t t e r r e g i o n s  of  than i n the s i d e s d u r i n g both summer and w i n t e r (1966) found lower wood l o s s e s i n the c e n t r e of i n winter  storage  storage.  of  storage. a  the i n n e r samples l o s t more.  p i l e i n P r i n c e George behaved l i k e the w i n t e r  (1966) s t u d i e d .  pine  the  (1961) found lower wood l o s s e s i n the c e n t r e  summer s t o r e d c h i p p i l e w h i l e The  The  did  from the second sampling p e r i o d to the  sampling p e r i o d because of the i n c l e m e n t have a f f e c t e d f u n g a l a c t i v i t y .  and  p i l e which Bergman  -  The c o n c l u s i o n  61 -  from these f i n d i n g s i s t h a t the weight  are a f f e c t e d by e x t e r n a l c o n d i t i o n s .  This i s p o s s i b l e i n a small  where the i n s i d e i s a f f e c t e d by e x t e r n a l c o n d i t i o n s 1968).  However, i n a b i g p i l e  a f f e c t e d by e x t e r n a l c o n d i t i o n s tested only  losses pile  (Butcher and Howard,  the i n s i d e i s w e l l i n s u l a t e d and i s n o t (Bjorkman and Haeger, 1963).  Of the f a c t o  temperature and d i s t r i b u t i o n of m i c r o organisms a r e r e l a t e d  and t h e r e f o r e  c o u l d be r e s p o n s i b l e  f o r weight l o s s e s .  -  III  62  -  LABORATORY INVESTIGATION OF WOOD DEGRADATION CAUSED BY  A  THERMOPHILIC AND  THERMOTOLERANT FUNGI  INTRODUCTION  Recent accounts from wood c h i p s thermotolerant  during  (Bergman and  storage  N i l s s o n , 1966)  have i n d i c a t e d t h a t the t h e r m o p h i l i c  p a r t of t h i s study i t was  thermotolerant  temperatures e x i s t .  shown t h a t the  thermophilic  f u n g i were common i n the i n n e r r e g i o n of the  a r e l a t i o n s h i p between the wood substance l o s s and  fungi. of the  R e l a t i v e l y few thermophilic  affecting  spruce-pine  f u n g i to degrade wood and  The  Bergman and  these  ability the  These f u n g i caused low weight l o s s e s and  the experiments performed u s i n g  ( N i l s s o n , 1965;  There  the number of  s t u d i e s have been made i n t o the  thermotolerant  t h i s phenomenon.  r e s u l t s of  c o u l d be  and  laboratory  In  and  c h i p p i l e , where the h i g h e s t weight l o s s e s of wood a l s o . o c c u r r e d . was  and  f u n g i might p l a y a r o l e i n wood c h i p d e t e r i o r a t i o n ,  e s p e c i a l l y i n areas of the c h i p p i l e where h i g h the f i r s t  of f u n g i i s o l a t e d  factors the  these f u n g i were a l s o v a r i a b l e  N i l s s o n , 1966;  S h i e l d s and  Unligil,  r e a s o n f o r the v a r i a t i o n i n the r e s u l t s of  t h a t the b a s i c f a c t o r s g o v e r n i n g the a b i l i t y  degrade wood have not been t h o r o u g h l y i n v e s t i g a t e d .  1968).  the p r e v i o u s  of these f u n g i The  studies to  methods t h a t have  been used i n the e a r l i e r work were borrowed from those methods d e s i g n e d f o r the  i n v e s t i g a t i o n of wood decay caused by  caused by Ascomycetes and  Fungi  B a s i d i o m y c e t e s and  Imperfecti.  s o f t r o t of wood  -  63  -  T h i s s e c t i o n i s concerned w i t h the d e m o n s t r a t i o n t h a t common t h e r m o p h i l i c  and  thermotolerant  c h i p p i l e are c a p a b l e of c a u s i n g a c c o m p l i s h t h i s i t was this a b i l i t y . two  parts.  first  first  f u n g i i s o l a t e d from the wood  weight l o s s e s of wood.  thesis i s therefore subdivided  to cause weight The  f a c t o r s which were c o n s i d e r e d  of the common  i n the development of a  of t h e r m o p h i l i c  to cause weight l o s s e s of wood were the  and  thermotolerant  fungi  following:  1.  The  e f f e c t of the c u l t u r e medium  2.  The  e f f e c t of wood sample s i z e  . 3.  The  e f f e c t of method of i n o c u l a t i o n  4.  The  e f f e c t of d u r a t i o n of  5.  The  e f f e c t of  incubation  temperature  LITERATURE REVIEW  The thermophilic Nilsson  into  losses.  method f o r t e s t i n g the a b i l i t y  B  to  d e a l s w i t h the development of a method of t e s t i n g ,  the second d e a l s w i t h the d e m o n s t r a t i o n of the a b i l i t y isolates  In o r d e r  n e c e s s a r y to develop a method to t e s t f o r  T h i s s e c t i o n of the  The  the  e f f e c t of medium on weight l o s s e s of wood caused and  thermotolerant  (1965) and  Bergman and  f u n g i has Nilsson  been l i t t l e s t u d i e d .  by In Sweden,  (1966) used malt s o l u t i o n i n  -  64  -  v e r m i c u l i t e , w h i l e i n Canada, S h i e l d s and block b u r i a l Cooney and  to study the a c t i v i t y of  Emerson (1964) have p o i n t e d  thermophilic four  test  to be  1.  Y e a s t s t a r c h agar  2.  Y e a s t g l u c o s e agar  3.  Oatmeal agar  4.  Czapek agar  been p o i n t e d  yeast  they found  these  g l u c o s e agar were p a r t i c u l a r l y  out  fungi.  t h a t l a r g e numbers of m i c r o f u n g i  from c h i p s because of the c h i p s i z e  critical  in affecting  (1953), i n l a b o r a t o r y  ranged from two Unligil,  Nilsson,  to volume r a t i o and  s t u d i e s of n a t u r a l d u r a b i l i t y  In the p r e v i o u s  S h i e l d s and  (Bergman and  are 1966).  this  the r a t e a t which they are d e t e r i o r a t e d .  the amount of decay i n c r e a s e d  as  of wood, found  f u n g i on wood,  months to t h r e e months (Bergman and  1968).  f i r s t month of i n c u b a t i o n  Bergman and  that  month when the experiment was  to i n c r e a s e  terminated.  incubation  reduced.  incubation  Nilsson,  (1967) i n c u b a t e d  every month t i l l  1966;  C^. l i g n o r u m  showed t h a t the weight l o s s e s s t a r t i n g  continued  s o f t r o t f u n g i the u s u a l  Nilsson  may  Findlay  the volume of the t e s t s p e c i e s was  s t u d i e s of t h e r m o p h i l i c  on p i n e sapwood a t 40°C and  by  the media t e s t e d  s m a l l wood c h i p s have l a r g e s u r f a c e a r e a  periods  f u n g i on wood.  the d i f f i c u l t y of e s t a b l i s h i n g ,  Of  f o r the growth of t h e r m o p h i l i c I t has  be  soil  useful:  favourable  The  (1968) used the  thermophilic  out  f u n g i on l a b o r a t o r y media.  Y e a s t s t a r c h agar and  isolated  Unligil  from  the  fifth  In s t u d i e s of weight l o s s e s  period  i s s i x weeks (Duncan,  the  caused  1953;  -  -  65  Savory, 1954). Bergman and  Nilsson  (1966, 1968)  have t e s t e d some t h e r m o p h i l i c they c o u l d  a t t a c k and  and  and  S h i e l d s and  thermotolerant  Unligil  f u n g i and  have shown t h a t  cause weight l o s s e s i n wood samples i n the  laboratory.  The weight l o s s e s of c o n i f e r o u s  s p e c i e s were lower than the weight  of hardwood s p e c i e s  the f u n g i would not  thermophilic birch  and  most of  f u n g i caused c o n s i d e r a b l y  a t t a c k hardwoods, w h i l s t c a u s i n g globosum caused e x t e n s i v e i n softwoods  nitrogen,  (Savory, 1954).  i n the wood.  E s l y n , 1969).  C_. globosum, and  The  The and  I t was  the e x t e n t  d i s t r i b u t i o n of many f u n g i . factor.  increased  Nilsson  d i d not  Chaetomium  cause a weight l o s s  the a v a i l a b i l i t y of n u t r i e n t s , e s p e c i a l l y  shown t h a t i m p r e g n a t i o n of the  t e s t wood  the weight l o s s e s s u b s t a n t i a l l y (Savory,  A d d i t i o n of n u t r i e n t s a l t s i n c r e a s e d  Temperature i s a v e r y  generally  r o t i n softwoods.  extensively  a b i l i t y of the s o f t r o t f u n g i to cause  of decay appeared to be  amount of n u t r i e n t s a v a i l a b l e to the  the most c r i t i c a l  shown t h a t s o f t r o t f u n g i  damage to hardwoods but  w i t h Abram's s o l u t i o n i n c r e a s e d  and  spruce.  h i g h e r weight l o s s i n aspen  l i t t l e or no  weight l o s s e s i n wood i s a f f e c t e d by  by  attack  losses  samples. Work on s o f t r o t of wood has  1954;  (1968)  the r a t e of decay  proportional  to  the  fungus.  important e c o l o g i c a l f a c t o r a f f e c t i n g the  For a t h e r m o p h i l i c Increasing  the  the r a t e of growth and  fungus, temperature may  temperature up  be  to the optimum  decay of wood by  fungi.  Bergman  (1967) showed t h a t the weight l o s s e s i n b i r c h , aspen, s p r u c e  -  66  -  and p i n e wood caused by C_. l i g n o r u m i n c r e a s e d r a p i d l y w i t h i n c r e a s i n g temperature and were h i g h e s t a t the optimum temperature f o r growth, was  40°C.  t h a t S^  A. t e r r e s t r i s behaved  similarly.  Nilsson  (1965) demonstrated  t h e r m o p h i l e a t i t s optimum temperature of 45°C caused up  weight l o s s  to wood d u r i n g  which  to 7%  two months i n c u b a t i o n .  U n l i k e the t h e r m o p h i l i c f u n g i most B a s i d i o m y c e t e s a r e a c t i v e a t lower temperatures.  Henningsson  of the many i s o l a t e d  from b i r c h and aspen pulpwood had o p t i m a l growth  decay a c t i v i t y  above 30°C.  (1967) found t h a t o n l y t h r e e B a s i d i o m y c e t e s  He a l s o showed t h a t low temperature f u n g i have  a c o m p a r a t i v e l y low decay a c t i v i t y and  t h a t many f u n g i w i t h h i g h e r temperature  optima were more a c t i v e a t low temperatures than the low temperature  C  and  fungi.  GENERAL METHODS  T e s t wood p i e c e s  (1/16 x 3/4  x 1 3/4  i n . ) 0.2  x 1.9  were c u t from ponderosa p i n e sapwood so t h a t the l o n g a x i s was the g r a i n of the wood.  x 4.4  cm.  parallel  The p i e c e s were q u i c k l y impregnated w i t h water  to and  then c o n d i t i o n e d a t a c o n s t a n t temperature of 22.2°C and r e l a t i v e h u m i d i t y of 50 ± 2% f o r seven days.  The i n i t i a l  c o n d i t i o n e d weights then were  measured. The t e s t p i e c e s were e i t h e r s t e r i l i z e d autoclaved.  i n ethylene oxide or  They were then p l a c e d on "S" shaped g l a s s rods on the f u n g a l  c u l t u r e s growing i n p e t r i d i s h e s on a medium. w i t h the medium.  The wood had no d i r e c t  Three p i e c e s were put i n each 9 cm. p e t r i  dish.  contact  -  67 -  The samples were i n c u b a t e d a t 45°C f o r s i x weeks. the c u l t u r e s from d e s s i c a t i n g  the r e l a t i v e h u m i d i t y  To p r e v e n t  i n the i n c u b a t o r was  kept h i g h . A f t e r i n c u b a t i o n the s u r f a c e s o f the wood samples were c l e a n e d o f a l l mycelium and were weighed w h i l e wet. a t the same temperature weights  were  The t r e a t e d wood was a g a i n c o n d i t i o n e d  and r e l a t i v e h u m i d i t y  f o r seven days when f i n a l  taken.  Except f o r one experiment Abrams c e l l u l o s e medium was used t h i r t y minutes a t 121°C.  where d i f f e r e n t media were s t u d i e d ,  i n a l l studies.  Media were a u t o c l a v e d f o r  S i x t y - m i p o r t i o n s were d i s p e n s e d i n t o p e t r i d i s h e s  and a f t e r c o o l i n g were i n o c u l a t e d w i t h d i s k s of i n o c u l a . i n c u b a t e d a t 45°C f o r about d e s i g n was the randomized used  complete  design.  the fungus.  The g e n e r a l  The a n a l y s i s o f v a r i a n c e was  to a n a l y s e the r e s u l t s . The f u n g i used  from  f o u r days to e s t a b l i s h  The p l a t e s were  i n these experiments  the s p r u c e - p i n e c h i p s from  had p r e v i o u s l y been i s o l a t e d  the P r i n c e George e x p e r i m e n t a l p i l e .  Much  a t t e n t i o n was p a i d to the f o l l o w i n g because of t h e i r abundance i n the p i l e : 1.  15. e m e r s o n i i  2.  A.  fumigatus  3.  A.  terrestris  4.  S^. t h e r m o p h i l e  5.  C_. pruinosum  I  i g u r e 14. A l l e s c h e r l a t e r r e s t r l s and Thermoascus a u r a n t i a c u s growing on Abrams c e l l u l o s e medium and ponderosa p i n e . Top, A. t e r r e s t r i s ; bottom, T. a u r a n t i a c u s .  -  D  69  -  DEVELOPMENT OF A METHOD FOR THE STUDY OF THE ABILITY OF THE FUNGI TO CAUSE WEIGHT LOSSES OF WOOD  1.  E f f e c t o f media  S t a n d a r d methods a r e a v a i l a b l e f o r t e s t i n g t h e a b i l i t y o f f u n g i , e s p e c i a l l y t h e B a s i d i o m y c e t e s , t o degrade wood (BS 838 t e s t 1 9 6 1 and ASTM ?  D1413-61, 1961),  These t e s t s have been based on t h e c a r e f u l s t u d y o f  media w h i c h s e r v e as t h e s u b s t r a t e  f o r t h e growth o f t h e f u n g i .  No s t u d y o f t h i s n a t u r e i s a v a i l a b l e f o r t h e t h e r m o p h i l i c  fungi.  T h i s s t u d y i s i n t e n d e d . t o e v a l u a t e t h e e f f e c t o f growth media on t h e a b i l i t y of thermophilic  f u n g i t o cause w e i g h t l o s s e s o f wood.  The f o l l o w i n g  media, t h e c o m p o s i t i o n o f w h i c h appear i n Appendix- 2, were used: 1.  Abrams medium  2.  2% m a l t  3.  Y e a s t - s t a r c h medium YpSS  4.  A b r a m s - c e l l u l o s e medium  5.  2% m a l t - c e l l u l o s e  6.  Yeast-cellulose  agar  medium  medium Y S c n  T a b l e 9.  Medium  P e r c e n t a g e weight l o s s e s caused  Fungus  % Wt. loss  S.D.  % Wt. loss  Control  1.42  0.24  0.93  Thermoascus aurantiacus  3.46  0.40  3.41  Humicola  3.10  0.41  Sporotrichum thermophile  3.52  Allescheria terrestris  3.64  sp.  2% Malt  Abrams cellulose  ,, Abrams  to ponderosa p i n e sapwood on d i f f e r e n t media.  2% M a l t 1% c e l l u l o s e  YpSs  %  YpSc.  Wt.  %  Wt.  % Wt. loss  S.D.  % Wt. loss  S.D.  loss  S.D.  loss  S.D.  1.15  0.15  0.85  0.52  0.50  0.35  0.97  0.52  0.28  1.61  0.16  1.64  0.43  2.63  0.83  4.08  0.33  3.44  0.51  2.82  0.22  3.21  0.57  3.80  0.57  3.72  0.42  0.64  3.82  0.45  1.21  0.92  2.50  0.69  2.69  2.84  3.67  1.01  0.80  4.39  0.46  3.51  1.10  3.31  0.89  3.90  0.88  3.71  0.81  S.D. 0.24  -  Table 10.  A b r  M o i s t u r e c o n t e n t of samples on each medium  Abrams cellulose  m g  /o  /a  2% malt  . 2 % malt 1% c e l l u l o s e  /a  /a  YpSs  YpSc.  /o  /Q  M.C.  S.D.  M.C.  S.D.  M.C.  S.D.  M.C.  S.D.  M.C.  S.D.  M.C.  S.D.  Control  22.1  2.2  62.5  42.9  48.5  23.2  50.3  7.1  42.0  19.0  34.8  14.9  Thermoascus aurantiacus  36.4  5.0  33.5  3.8  65.0  14.2  53.2  13.4  91.3  32.7  37.5  6.0  Humicola  30.3  1.8  33.3  3.2  39.1  6.4  44.0  9.0  44.2  9.8  43.0  6.8  Sporotricum thermophile  35.6  7.2  33.0  7.4  178.1  9.9  187.6  15.7  96.7  34.7  46.7  16.7  Allescherla terrestris  31.5  6.6  41.3  7.4  103.4  34.7  140.5  59.3  55.5  20,1  49.5  13.3  sp.  -  72  -  The f i r s t three media normally are used f o r the study of fungal decay and the other three were made by adding 1% c e l l u l o s e to the f i r s t two media and replacing starch with c e l l u l o s e i n the t h i r d medium. The following fungi were used: A. t e r r e s t r i s , Humicola sp., and T. aurantiacus.  thermophile  Each treatment was r e p l i c a t e d twelve times.  Media had an a f f e c t on the a b i l i t y of the fungi to cause weight losses of ponderosa pine sapwood (Table 9). between media and between fungi.  There were s i g n i f i c a n t differences  No single medium was the best f o r a l l the  fungi and there was s i g n i f i c a n t i n t e r a c t i o n between fungi and media. The  two media r e s u l t i n g i n the greatest wood weight losses were  Abrams-cellulose and yeast-cellulose media.  The addition of c e l l u l o s e  generally improved the effectiveness of the fungi to cause weight losses to ponderosa pine sapwood.  Almost a l l fungi gave low weight losses when  grown on malt agar medium. On some media the moisture content of the samples (Table 10) was high and an average moisture content above 100% was measured i n some samples incubated on malt-cellulose medium.  S.. thermophile caused high  weight losses at higher moisture contents than the other fungi. In the test of the e f f e c t of media on the weight losses caused by thermophilic medium.  fungi, i t was shown that fungi d i d not behave independently of  There were "preferences"  "preference"  for media and the most pronounced was the  of A. t e r r e s t r i s for Abrams-cellulose medium and T. aurantiacus f o r  y e a s t - c e l l u l o s e medium.  -  73  -  F i g u r e 15. Growth of A l l e s c h e r i a t e r r e s t r i s and Thermoascus a u r a n t i a c u s on y e a s t - c e l l u l o s e medium and ponderosa pine. Top, A. t e r r e s t r i s ; bottom, T. a u r a n t i a c u s .  -  The yeast  7ft -  d i f f e r e n c e s between these  from A b r a m s - c e l l u l o s e  two media were the absence o f  and ammonium n i t r a t e and potassium  phosphate from y e a s t - c e l l u l o s e medium.  S i n c e the s e a r c h was f o r a medium  which would be s u i t a b l e f o r a l l the f u n g i , i t was f e l t of these components might g i v e the b e s t medium. to t e s t the b e h a v i o u r  E i g h t media were t e s t e d . magnesium s u l p h a t e , d i p o t a s s i u m The  composition The  that a  combination  An experiment was s e t up  o f the two f u n g i A. t e r r e s t r i s  to the t h r e e c o n s t i t u e n t s and t h e i r  dibasic  and T_. a u r a n t i a c u s  combinations. Each medium had a b a s a l c o m p o s i t i o n o f  monobasic phosphate, c e l l u l o s e and agar.  o f the media appear i n the appendix. 2.  two f u n g i behaved d i f f e r e n t l y  to the d i f f e r e n t media ( T a b l e 1 1 ) .  D i f f e r e n c e s between f u n g i and between media were s i g n i f i c a n t . significant  There was  i n t e r a c t i o n between f u n g i and medium.  E x c l u s i o n of yeast t e r r e s t r i s while decreasing  i n c r e a s e d the weight l o s s e s caused by A. t h a t caused by _T. a u r a n t i a c u s .  When  d i b a s i c phosphate was removed the a c t i v i t y o f A. t e r r e s t r i s was while having  no e f f e c t on the performance o f T_. a u r a n t i a c u s ,  potassium depressed  The r e a c t i o n  of the f u n g i to the e x c l u s i o n o f ammonium n i t r a t e was s i m i l a r to the e x c l u s i o n of y e a s t .  The removal o f a l l t h r e e components from the medium had  a more pronounced e f f e c t on A. t e r r e s t r i s  than on _T. a u r a n t i a c u s .  -  75 -  P e r c e n t weight l o s s e s o b t a i n e d f o r A. t e r r e s t r i s and T_.  T a b l e 11.  a u r a n t i a c u s growing on medium p r e p a r e d 7H 0 and K HPO. w i t h  from c e l l u l o s e , MgSO^  the absence o f y e a s t o r NH NO  A. t e r r e s t r i s % Wt. l o s s  Composition  T.  o r KH PO  aurantiacus % Wt. l o s s  Complete medium  3.89  4.46  Without y e a s t  4.42  3.85  Without KH P0. 2 4  3.25  4.31  Without NH.NO. 4 3  4.32  4.03  Without y e a s t & KH^O^  3.78  3.94  Without y e a s t & NH N0  2.16  3.71  Without KH P0. & NH.N0 2 4 4 3  3.91  4.17  None  1.92  3.60  o  4  o  3  o  jT. a u r a n t i a c u s was g e n e r a l l y n o t as s e n s i t i v e to changes i n t h e n u t r i e n t composition  o f the medium as A. t e r r e s t r i s and b e n e f i t e d most from  the i n c o r p o r a t i o n o f a l l t h r e e components i n t o t h e medium. A f t e r these experiments,  A b r a m s - c e l l u l o s e medium was s e l e c t e d  f o r the r e s t o f the study.  2.  E f f e c t o f wood sample  size  Two sample s i z e s s e l e c t e d f o r t h i s study were s i m i l a r i n s i z e to c h i p s and the t h i r d s i z e was chosen to be t h e same as t h a t i n the ASTM  -  D1413-61 t e s t .  7tf -  The i n f l u e n c e of sample  caused by t h e r m o p h i l i c  f u n g i was  investigated.  The s i z e s of samples were 0.2 1 3/4  i n s . ) , 0.4  (3/4 x 3/4  x 2.5 x 5.1 cm.  x 3/4  s i z e on weight l o s s e s of wood  x 1.9  x 4.4  cm.  (1/16 x 3/4  (1/8 x 1 x 2 i n . ) and 1.9  x 1.9  i n ) , and were c u t so t h a t the long a x i s was  the g r a i n o f the wood. large hereafter.  parallel  placed  on the c u l t u r e .  sapwood.  cm  to  The  The f u n g i used i n t h i s  experiment were A. t e r r e s t r i s , 13. e m e r s o n i i and S_. t h e r m o p h i l e . ment was  x 1.9  The s i z e s w i l l be r e f e r r e d to as s m a l l , medium and  They were a l l c u t from ponderosa p i n e  t a n g e n t i a l s u r f a c e was  x  Each t r e a t -  r e p l i c a t e d s i x times. Sample s i z e (Table 12) i n f l u e n c e d  wood and the p e r c e n t a g e weight l o s s of wood.  the a b s o l u t e There were  weight l o s s of significant  d i f f e r e n c e s between the p e r c e n t a g e weight l o s s i n the d i f f e r e n t s i z e s , The p e r c e n t greater higher  weight l o s s v a l u e s  f o r the s m a l l and medium s i z e d p i e c e s  than those f o r the l a r g e p i e c e s , percent  losses occurred  or l a r g e samples.  were  except f o r S_. t h e r m o p h i l e where  i n the medium s i z e d samples  The r e a c t i o n of the f u n g i was  than i n the s m a l l  independent of the s i z e o f  wood. Sample s i z e (Table i n such a way pieces.  t h a t the s m a l l p i e c e s  l o s t l e s s weight  weight l o s s o f wood  than the medium and  The d i f f e r e n c e i n weight l o s s between d i f f e r e n t s i z e s of b l o c k s  significant.  The r e a c t i o n to the d i f f e r e n t s i z e s of samples was  on the sample, of sample.  used.  since a s i g n i f i c a n t  large was  dependent  i n t e r a c t i o n e x i s t e d between f u n g i and s i z e  The r e s u l t s of t h i s experiment d i d not l e a d to a change i n the  s i z e o f samples still  12) a f f e c t e d the a b s o l u t e  and i n a l l subsequent experiments the s m a l l s i z e p i e c e s were  T a b l e 12.  P e r c e n t weight l o s s and a b s o l u t e w e i g h t l o s s o f wood caused by t h e r m o p h i l i c f u n g i growing on d i f f e r e n t s i z e s o f ponderosa p i n e sapwood samples d u r i n g s i x weeks i n c u b a t i o n .  P e r c e n t weight  (0.2 x 1.9 x 4.4) (0.4 x 2.5 x 5.1) Small  Medium  Absolute weight l o s s  loss  i n grams  (1.9 x 1.9 x 1.9) (0.2 x 1.9 x 4.4) (0.4 x 2.5 x 5.1)(1.9 x 1.9 x 1.9) Large  Small  Medium  Large  Control  0.25  0.51  0.31  0.001  0.009  0.009  Allescheria terrestris  3.61  3.67  3.21  0.019  0.063  0.090  Byssochlamys emersonii  3.58  3.56  2.82  0.019  0.061  0.078  Sporotrichum thermophile  3.08  3.65  2.98  0.017  0.061  0.084  -  3.  7§  -  E f f e c t o f methods o f i n o c u l a t i o n  T h i s study was designed to i n v e s t i g a t e the e f f e c t o f d i f f e r e n t methods o f p r o v i d i n g  the inoculum s o u r c e on the weight l o s s e s o f wood  caused by t h e r m o p h i l i c  fungi.  The u s u a l method o f p r o v i d i n g  inoculum i n  wood decay s t u d i e s c o n s i s t s o f growing the fungus on the b a s a l medium f o r a p e r i o d o f time b e f o r e unsuitable studies  the t e s t b l o c k s  f o r soft r o t studies  the wood, p l a n t e d  a spore suspension.  are-planted.  T h i s method i s  (Duncan, 1953; Savory, 1954).  on Abrams medium, i s d i r e c t l y  In s o f t r o t  inoculated  with  A l t h o u g h these two methods have been e x t e n s i v e l y  i n studies of microfungi  from wood c h i p s , no i n f o r m a t i o n  e f f e c t o f the d i f f e r e n t methods of p r o v i d i n g  used  i s a v a i l a b l e on the  inoculum s o u r c e on t h e weight  losses. The  f o l l o w i n g methods were t e s t e d :  1.  I n o c u l a t i o n o f wood samples.  2.  I n o c u l a t i o n o f medium, w i t h wood samples s u b s e q u e n t l y planted  3.  on the medium on the same day.  I n o c u l a t i o n o f medium, w i t h wood samples s u b s e q u e n t l y planted  on the c u l t u r e a f t e r one week.  Two f u n g i , A. t e r r e s t r i s and jB. e m e r s o n i i were used and the method was the same as o u t l i n e d under C-General methods. was r e p l i c a t e d s i x t i m e s .  Each treatment  The r e s u l t s a r e shown i n T a b l e 13.  -  T a b l e 13.  79 -  Average p e r c e n t a g e weight l o s s o f wood samples caused by thermophilic  fungi using  d i f f e r e n t methods o f p r o v i d i n g the  inoculum s o u r c e .  Methods o f p r o v i d i n g  inoculum  A. t e r r e s t r i s % Wt. l o s s  J3. e m e r s o n i i % Wt_. l o s s  2.87  2.91  D i r e c t on wood Inoculate  medium-wood p l a n t e d  same day  2.65  2.91  Inoculate  medium-wood p l a n t e d  a f t e r one week 3.24  3.03  The  method o f p r o v i d i n g  the inoculum s o u r c e had an e f f e c t on  the weight l o s s e s caused to the wood. were s i g n i f i c a n t .  The e x p r e s s i o n  D i f f e r e n c e s between these methods  of these d i f f e r e n c e s was l i n k e d t o the  fungus used i n the t e s t . Both f u n g i caused h i g h e r weight l o s s e s i f the medium was l a t e d a week b e f o r e  the wood samples were p l a n t e d .  With both f u n g i  was no s i g n i f i c a n t d i f f e r e n c e s i n weight l o s s i f the wood was d i r e c t l y o r i f the medium was i n o c u l a t e d on  i t on the same day.  Inoculating  wood samples was c o n s i d e r e d  inocu-  inoculated  and the wood samples were  the medium a week b e f o r e  there  planted  p l a n t i n g the  the b e s t method f o r i n o c u l a t i n g the wood samples  and was used i n a l l subsequent experiments.  -  4.  8Q  -  E f f e c t of d u r a t i o n of i n c u b a t i o n  L i m i t e d i n f o r m a t i o n i s a v a i l a b l e on the e f f e c t s of d u r a t i o n of i n c u b a t i o n on the weight l o s s e s caused by t h e r m o p h i l i c and fungi.  The i n c u b a t i o n p e r i o d s  f o r previous  thermotolerant  s t u d i e s were a r b i t r a r i l y  chosen.  T h i s i n v e s t i g a t i o n was conducted to determine how d u r a t i o n of i n c u b a t i o n a f f e c t e d weight l o s s e s caused by the t h e r m o p h i l i c and t h e r m o t o l e r a n t The  f u n g i used i n the study were A. t e r r e s t r i s , ]3. e m e r s o n i i ,  (3. pruinosum and £>\ t h e r m o p h i l e . from two to twelve weeks.  Every  S i x i n c u b a t i o n p e r i o d s were used v a r y i n g two weeks, a whole s e t o f treatments was  taken o u t , c o n d i t i o n e d and weighed. The  and  Each treatment was r e p l i c a t e d s i x times.  r e s t of the method has been o u t l i n e d b e f o r e The  under C-General methods.  weight l o s s e s were p l o t t e d a g a i n s t p e r i o d o f i n c u b a t i o n  the r e s u l t s appear i n F i g u r e 16.  c o r r e c t e d d a t a by d e d u c t i n g the t r e a t e d samples. during  fungi.  the f i r s t  A l l the graphs have been drawn from  the weight l o s s e s i n the c o n t r o l samples from  Rapid weight l o s s e s were caused by a l l of the f u n g i  s i x weeks w i t h  low weight l o s s e s i n the f i r s t  the e x c e p t i o n  of _C. pruinosum, which produced  f o u r weeks f o l l o w e d by a r a p i d i n c r e a s e from  the f o u r t h week to the s i x t h week. G e n e r a l l y , no a p p r e c i a b l e i n c r e a s e s i n weight l o s s e s after  the s i x t h week except f o r A. t e r r e s t r i s which a t t a i n e d a s t a b l e  condition after of  occurred  the e i g h t h week.  Weight l o s s e s went up s l i g h t l y  f o r three  the f u n g i i n the t w e l f t h week, but t h i s c o u l d be the r e s u l t of a g e n e r a l  drop i n m o i s t u r e c o n t e n t  of the wood s i n c e the samples s t a r t e d to dry o u t .  Byssochlamys  emersonii DURATION  Chrysosporium OF  INCUBATION  IN  W E E K S  pruinosum  -  82 -  F l u c t u a t i o n s i n weight l o s s e s were common f o r samples i n o c u l a t e d w i t h J3. t h e r m o p h i l e .  These f l u c t u a t i o n s were o p p o s i t e to  changes i n m o i s t u r e c o n t e n t of the samples. S i n c e the a c t i v i t y o f most o f the f u n g i had s t a b i l i z e d s i x weeks i n c u b a t i o n ,  this  after  time was chosen as the i n c u b a t i o n p e r i o d f o r  the r e s t o f t h e e x p e r i m e n t s .  5.  E f f e c t o f temperature.  I t was shown i n the f i r s t affected  p a r t of t h i s study t h a t  temperature  t h e d i s t r i b u t i o n o f t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i i n t h e  chip p i l e .  Wood s u b s t a n c e l o s s was a l s o h i g h e r i n t h e i n n e r r e g i o n o f t h e  p i l e where temperatures were h i g h .  T h i s experiment was conducted to  i n v e s t i g a t e t h e e f f e c t of temperature on t h e weight l o s s e s caused by t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i . Temperatures the f u n g i .  The temperature i n t e r v a l was 5°C.  was n o t used. experiment.  r a n g i n g from 25°C to 60°C were used f o r i n c u b a t i n g I n a few c a s e s 35°C i n c u b a t i o n  A l l c u l t u r e s were i n c u b a t e d a t 45°C b e f o r e the s t a r t o f t h e The i n c u b a t i o n p e r i o d was f o u r weeks and s i x samples were, used  i n each e x a m i n a t i o n .  -  -  83  The f o l l o w i n g f u n g i were t e s t e d . A.  terrestris  A. fumigatus _B. e m e r s o n i i (]. pruinosum S^. t h e r m o p h i l e _T. a u r a n t i a c u s The r e s u l t s appear i n F i g u r e 17.  A l l the graphs have been  drawn from c o r r e c t e d d a t a by d e d u c t i n g the weight l o s s e s i n the c o n t r o l samples  from the t r e a t e d weight For  losses.  a l l the f u n g i , i n c r e a s e s i n temperature l e d to i n c r e a s e s i n  weight l o s s e s caused by them u n t i l the optimum temperature was reached and then weight l o s s e s d e c r e a s e d . the  Except f o r J3. e m e r s o n i i and _T. a u r a n t i a c u s ,  h i g h e s t weight l o s s e s o c c u r r e d a t 40°C.  These two f u n g i caused  their  h i g h e s t weight l o s s a t 50°C. All  the f u n g i were a c t i v e a t 25°C except f o r j3. e m e r s o n i i and  _T. a u r a n t i a c u s , which showed no a c t i v i t y temperature a t which different  organisms.  a t 25°C b u t s t a r t e d a t 30°C.  The  the a c t i v i t y o f the organisms s t o p p e d , v a r i e d f o r C^. pruinosum a c t i v i t y  ended a t the maximum temperature  50°C, w h i l s t  t h a t of JJ. e m e r s o n i i and T_. a u r a n t i a c u s ended a t o r a l i t t l e  above 60°C.  The o t h e r t h r e e f u n g i had the same l i m i t a t i o n  at  maximum  a temperature o f 55°C. The i n c r e a s e i n a c t i v i t y  was  of a c t i v i t y  from the minimum to the optimum  r a p i d but the d e c l i n e i n a c t i v i t y  from optimum to the maximum was g r a d u a l .  -  84  -  543-  /  2-  1 25  30  35 40 45  Thermoascus  50 55 60  0  aurantiacus  6  5  5-  4  4-  3  3-  2  2-  ^  , , , \ ,  , _l  25  I  I  I  30 35 40  Sporotrichum  I  45  I  \  30  35 40  45  Byssochlamys  6r  1  25  V  I  50 55 60  thermophile  \  50 55  60  emersonii  r  1 25  J L 30 35 40  Aspergillus  45  50  55 60  fumigatus  65432-  1 -  /  0 — _L 25  30 A.  35 40  45  terrestris  50 55 60  25  30  35 40 45  Chrysosporium  T E M P E R A T U R E (°C)  F i g . 17.  Changes i n weight losses with changes i n temperature.  50 55  pruinosum  60 '  -  except f o r C^. pruinosum  85 -  and _T. a u r a n t i a c u s .  The p r o d u c t i o n o f a s c o c a r p s was g r e a t l y reduced a t temperatures above 45°C and stopped c o m p l e t e l y a t temperatures beyond  55°C.  Mycelia  p r o d u c t i o n was s p a r s e a f t e r 55°C except f o r J3. e m e r s o n i i and T_. a u r a n t i a c u s . Samples,  g e n e r a l l y , showed a m o i s t u r e d e f i c i t  and c h a r r i n g  after  f o u r weeks i n c u b a t i o n a t 60°C.  6.  Summary  I t appears from these r e s u l t s e v a l u a t i n g the a b i l i t y  t h a t the most s u i t a b l e method f o r  o f t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i to cause  weight l o s s e s o f wood w i l l be by growing  the t e s t fungus on Abrams  c e l l u l o s e medium f o r one week a t temperatures between 40° and 50°C, which The  the wood samples  (0.2 x 1.9 x 4.4 cm) a r e p l a n t e d onto the c u l t u r e .  t e s t i s then i n c u b a t e d f o r 6 weeks a t the optimum temperature f o r the  growth o f the fungus.  E  after  T h i s may v a r y from 40° to 50°C.  WEIGHT LOSSES CAUSED BY THE THERMOPHILIC AND THERMOTOLERANT FUNGI  1.  E v a l u a t i o n o f the common i s o l a t e s  The f o l l o w i n g f u n g i , i s o l a t e d a t 45°C from wood c h i p s , b u t not  used i n p r e v i o u s experiments, were examined f o r t h e i r a b i l i t y  to cause  -  86  -  weight l o s s e s of ponderosa p i n e sapwood. because i t f a i l e d for  this  test.  to grow on the Abrams c e l l u l o s e medium which was used  The method f o r the t e s t was the same as o u t l i n e d i n  C-General methods.  T a b l e 14.  _H. l a n u g i n o s a was n o t examined  Each treatment was r e p l i c a t e d  twelve t i m e s .  P e r c e n t weight l o s s of ponderosa p i n e caused by some p h i l i c and t h e r m o t o l e r a n t f u n g i a t 45°C  Fungi  % Wt. l o s s  incubation.  S.D.  Control  0.12  0.12  S p o r o t r i c h u m B.  4.08  0.49  A s p e r g i l l u s fumigatus  3.62  0.42  Unknown ACIO  3.52  0.14  Unknown 61  3.40  0.24  Unknown 6214  3.35  0.77  Byssochlamys e m e r s o n i i 11  3.30  0.23  Byssochlamys  s p e c i e s 201  3.26  0.36  Byssochlamys  e m e r s o n i i 12  3.07  0.29  Unknown 618  3.01  0.43  Unknown 624  1.68  0.37  Unknown 6310  0.71  0.40  All p i n e sapwood  the f u n g i examined  thermo-  c o u l d cause weight l o s s e s o f ponderosa  (Table 14). S p o r o t r i c h u m B. caused a h i g h weight l o s s  compared  -  to the o t h e r f u n g i .  Two  87  -  f u n g i Unknown 624  and Unknown 6310  low weight l o s s e s , p r o b a b l y because of t h e i r poor growth on  caused  very  Abrams-cellulose  agar.  2.  S u s c e p t i b i l i t y of l o d g e p o l e p i n e and white  s p r u c e wood  to a t t a c k by s e v e r a l f u n g a l i s o l a t e s . ' In the e a r l i e r experiments of i t s a v a i l a b i l i t y  i n the l a b o r a t o r y and  s t u d i e s i n North America.  The  study have been i s o l a t e d was f o r e an i n v e s t i g a t i o n was l o d g e p o l e p i n e and outlined  spruce.  i n C-General  Each treatment  was  ponderosa p i n e sapwood was  c h i p p i l e from which the f u n g i used  made to study The  twelve  i n this  spruce.  There-  the a c t i o n of some of the f u n g i on  e x p e r i m e n t a l p r o c e d u r e was  methods except  because  i t s e x t e n s i v e use i n wood decay  made up of l o d g e p o l e p i n e and  replicated  used  f o r the d i f f e r e n t times.  The  the same as  tree species  used.  r e s u l t s a r e shown i n T a b l e  15, There were remarkable f u n g i on p i n e and after  spruce.  v i s u a l d i f f e r e n c e s i n the a t t a c k of  Most of the f u n g i c o m p l e t e l y  ten days of i n c u b a t i o n , but not s p r u c e .  i n c u b a t i o n p e r i o d d i d a l l f u n g i completely s t a i n i n g of s p r u c e was  noticed e a r l i e r .  Not  covered  u n t i l halfway  the  pine into  c o v e r s p r u c e , even though  the  -  T a b l e 15.  Percentage caused  88 -  weight l o s s of l o d g e p o l e p i n e and spruce sapwood  by some t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i a t 45°C  a f t e r s i x weeks i n c u b a t i o n .  Fungi  Spruce % Wt. l o s s  Control Allescheria  terrestris  Lodgepole p i n e % Wt. l o s s  1.53  1.96  3.06  9.17  Sporotrichum  thermophile  3.39  5.18  Sporotrichum  B.  5.02  6.69  2.03  3.74  Byssochlamys e m e r s o n i i 19  2.54  3.88  Byssochlamys e m e r s o n i i 12  2.65  3.63  S t e r i l e mycelium  2.85  3.72  Unknown 6214  3.60  5.65  2.51  3.91  2.58  . 3.95  Chrysosporium  Aspergillus  pruinosum  fumigatus  Thermoascus a u r a n t i a c u s  All and  ,.  spruce.  the f u n g i examined caused weight l o s s e s o f l o d g e p o l e p i n e Higher weight l o s s e s o c c u r r e d i n l o d g e p o l e p i n e than i n s p r u c e  as shown i n T a b l e 15.  -  89  -  A t the end o f the e x p e r i m e n t , some of the s p r u c e samples were s t a i n e d throughout and some had a s t r e a k of s t a i n i n the m i d d l e . was  o b s e r v e d f o r samples i n o c u l a t e d w i t h the f o l l o w i n g A.  Staining  fungi:  terrestris  S^. t h e r m o p h i l e Sporotrichum Unknown  B.  6214  When s t a i n e d and u n s t a i n e d samples were compared, the u n s t a i n e d samples had l o s t more w e i g h t t h a n the s t a i n e d samples ( T a b l e 1 6 ) .  T a b l e 16.  P e r c e n t a g e w e i g h t l o s s f o r s t a i n e d and u n s t a i n e d s p r u c e i n o c u l a t e d w i t h some t h e r m o p h i l i c f u n g i Fungus  Unstained % Wt. l o s s  Stained % Wt. l o s s  t  0-5  Unknown 6214  4.69  2.82  2.79*  S p o r o t r i c h u m B.  6.02  4.29  3.46*  Sporotrichum thermophile  3.77  3.01  2.62*  t  p.5 2.28  The d i f f e r e n c e s between the u n s t a i n e d and s t a i n e d s p r u c e samples were s i g n i f i c a n t f o r a l l the t h r e e f u n g i examined.  A comparison was  c a r r i e d out f o r A. t e r r e s t r i s , s i n c e t h e r e were n o t enough u n s t a i n e d  not samples.  -  3.  90  -  E f f e c t o f mixed i s o l a t e s on weight  From a s m a l l d i f f e r e n t species  losses  s t e r i l i z e d p i e c e of wood i t was p o s s i b l e to i s o l a t e  of thermophilic  fungi.  I n mixed c u l t u r e s o f these f u n g i on  malt agar, zones o f i n h i b i t i o n were n o t found. species  a l l the d i f f e r e n t  have d i f f e r e n t c a p a c i t i e s f o r a t t a c k i n g wood, i t was thought  some o f the f u n g i might depend on o t h e r s wood.  Since  to o b t a i n  that  t h e i r n u t r i e n t s from the  I f t h i s were s o , then i t might be p o s s i b l e to o b t a i n h i g h e r  losses i f  more than one fungus were used to i n o c u l a t e the wood. An  experiment to study the e f f e c t o f mixed c u l t u r e s o f t h e r m o p h i l i c  f u n g i on weight l o s s e s o f ponderosa p i n e thermophilic in  f u n g i which c o u l d  sapwood was performed.  grow t o g e t h e r  The f o l l o w i n g  i n c u l t u r e and were v e r y  common  the p i l e were examined: A.  terrestris  13. e m e r s o n i i thermophile Two i n c u b a t i o n  temperatures o f 45°C and 50°C were used because  the maximum a c t i v i t y o f the t h r e e temperatures.  f u n g i was found to o c c u r a t d i f f e r e n t  P l a t e s were i n o c u l a t e d w i t h more than one fungus by p l a c i n g  s i d e by s i d e 2mm d i s k s from malt agar c u l t u r e s .  Each treatment was r e p l i c a t e d  6 times. The  mixed c u l t u r e s d i d n o t g e n e r a l l y cause h i g h e r  weight  losses  than t h e s i n g l e c u l t u r e s except f o r t h e c u l t u r e o f _B. e m e r s o n i i and S^. thermophile incubated incubated  a t 45°C and the mixed c u l t u r e s of a l l t h r e e  a t 50°C (Table 1 7 ) .  fungi  -  T a b l e 17.  Effect and  91  of i n t e r a c t i o n  S. t h e r m o p h i l e on  at 45°C and  -  between A.  terrestris,  weight l o s s e s of  B. e m e r s o n i i  ponderosa p i n e i n c u b a t e d  50°C.  Fungus  45°C loss  50°C % Wt. l o s s  % Wt.  Control  0.49  0.42  terrestris  2.99  3.39  Byssochlamys  emersonii  3.22  3.72  Sporotrichum  thermophile  3.50  0.93  Allescheria  A.  terrestris  & B.  emersonii  3.32  3.81  A.  terrestris  & S.  thermophile  3.24  2.71  4.14  3.59  3.22  4.07  B. e m e r s o n i i & S. A.  thermophile  t e r r e s t r i s & B. e m e r s o n i i & S. t h e r m o p h i l e  Dependence of the f u n g i on temperature than the i n t e r a c t i o n f u n g i was by  between the f u n g i .  Although  g e n e r a l l y n o t complementary, no obvious  the o t h e r o c c u r r e d .  was  more  significant  the a c t i v i t y inhibition  of the t h r e e  of one  fungus  F  DISCUSSION  Medium, u n l i k e temperature, has not been s t u d i e d i n the i n v e s t i g a t i o n o f the e f f e c t s o f t h e r m o p h i l i c a b i l i t y of thermophilic which i t i s grown.  studies of this subject,  a t t e n t i o n has been p a i d  1966, failed be  The  These d i f f e r e n c e s have been ,shown i n the p r e s e n t  l o s s e s caused by the t o t a l spectrum of t h e r m o p h i l i c  oversimplify  f u n g i on wood.  f u n g i to degrade wood depends on the medium on  i n which no s i n g l e medium was found to be the b e s t  previous  extensively  o n l y one medium  to the f a c t  study,  f o r the study o f weight fungi.  I n a l l the  has been used, and no  t h a t t h e use o f a s i n g l e medium can  the weight l o s s r e s u l t s ( N i l s s o n , 1965; Bergman and N i l s s o n ,  1967, 1968; S h i e l d s and U n l i g i l ,  1968).  F o r t h i s r e a s o n these a u t h o r s  to show t h a t a l a r g e number o f t h e r m o p h i l i c  f u n g i from wood c h i p s can  destructive. Some t h e r m o p h i l i c  f u n g i a r e h i g h l y s e n s i t i v e to changes i n the  c o m p o s i t i o n o f the medium i n which they grow, w h i l e o t h e r s changes i n the medium a f f e c t e d the a b i l i t y o f A. l o s s e s more than they a f f e c t e d T_. a u r a n t i a c u s . medium, w h i l e d e p r e s s i n g  terrestris  Savory  Drastic  to cause weight  The p r e s e n c e o f y e a s t  the weight l o s s e s caused by A. t e r r e s t r i s ,  those caused by T_. a u r a n t i a c u s .  in a increased  (1954) showed t h a t f o r a s o f t r o t  fungus, i m p r e g n a t i o n o f the wood w i t h ammonium s u l p h a t e losses  are not.  increased  the weight  to the wood caused by C^. globosum w h i l e the i m p r e g n a t i o n w i t h magnesium  sulphate different  l e d to n e g l i g i b l e l o s s e s . "preferences"  The two examples i n d i c a t e t h a t f u n g i have  f o r d i f f e r e n t c o m p o s i t i o n o f media.  -  93 -  A l t h o u g h i m p r e g n a t i o n o f wood w i t h s a l t s and s o l u t i o n l i k e Abrams has been found u s e f u l f o r the study o f weight l o s s e s caused by microfungi  (Savory, 1954; E s l y n , 1969) i t may n o t be a r e l i a b l e method,  since i t could  lead  to a h y d r o l y s i s o f some c h e m i c a l c o n s t i t u e n t s  wood which w i l l g i v e m i s l e a d i n g Autoclaving  might be o b t a i n e d The  r e s u l t s when f u n g i a r e grown on the wood.  mono and o l i g o s a c c h a r i d e s  original structure  (Ball,  of the  with s a l t s leads  1953; B r e t z l o f f , 1954).  to a change i n the  High weight  losses  which might n o t be due to the a c t i v i t y o f the fungus  aim o f t h i s study was to o b t a i n a medium t h a t would a l l o w  alone.  the fungus  to cause h i g h weight i o s s e s o f wood w i t h o u t p r e v i o u s l y a l t e r i n g  the c o m p o s i t i o n  of the wood. Some of t h e t h e r m o p h i l i c  f u n g i have a tendency to accumulate  m o i s t u r e i n the wood samples, depending upon the s u b s t r a t e grow. of  on which they  The h i g h m o i s t u r e c o n t e n t o f the sample might reduce the a c t i v i t y  the f u n g i .  The t h e r m o p h i l i c  f u n g i were h i g h l y v a r i a b l e and the same  medium which was e f f i c i e n t a t one time c o u l d g i v e a poor r e s u l t a t another time. Unligil  T h i s i s p r o b a b l y the r e a s o n f o r the statement by S h i e l d s and (1968) t h a t i t i s d i f f i c u l t  f u n g i i n the d e t e r i o r a t i o n o f wood  to a s s e s s the r o l e of the t h e r m o p h i l i c chips.  There i s no s i n g l e medium which i s s u i t a b l e f o r s t u d i e s e f f e c t of thermophilic  f u n g i i n wood.Based on the p r e s e n t  of the  r e s u l t s Abrams-  c e l l u l o s e medium was chosen as g i v i n g r e a s o n a b l e weight l o s s e s f o r the f u n g i tested.  -  The  I t is  and Ascomycetes i n the c h i p p i l e  to the abundance (Bergman and N i l s s o n ,  not p r o b a b l e t h a t the c h i p s i z e p e r se i n c r e a s e s  of wood l o s s are  -  s i z e o f the wood c h i p s may c o n t r i b u t e  of F u n g i I m p e r f e c t i 1966) .  94  the weight  How c h i p s i z e a f f e c t s weight l o s s e s depends on how the l o s s e s  calculated. The  absolute  directly proportional greater  weight l o s s caused by t h e r m o p h i l i c to the s i z e of the wood; the b i g g e r  the weight l o s s .  the sample the  The p e r c e n t a g e weight l o s s i s i n v e r s e l y p r o p o r t i o n a l  to the s i z e o f the sample thermophilic  fungi i s  ( F i n d l a y , 1953).  T h i s was n o t the case w i t h the  f u n g i examined, s i n c e the p e r c e n t a g e weight l o s s e s o f the  medium s i z e d samples were g r e a t e r  than those o f the s m a l l s i z e d samples.  Two d i f f e r e n t c l a s s e s o f f u n g i were used i n the two s t u d i e s and t h i s may have brought about the d i f f e r e n c e s . limited  i n the q u a n t i t y  proportion  c e l l u l o s e or l i g n i n .  c e l l u l o s e o r c e l l u l o s e and l i g n i n w i l l o f the wood to a t t a c k .  reduced sample w i l l ,  thermophilic  undoubtedly have a l a r g e r  I n t h i s s i t u a t i o n the a t t a c k o f wood by to volume.  fungus.  fungi.  i s known about the n a t u r e o f a t t a c k o f wood by  I t has been shown t h a t A. t e r r e s t r i s  of hardwoods and t h e r e f o r e w i l l These a u t h o r s a l s o p o i n t e d not  area  on the o t h e r hand, l i m i t what i s a v a i l a b l e to the  However, l i t t l e thermophilic  piece of  The B a s i d i o m y c e t e s i n  B a s i d i o m y c e t e s might depend more upon the r a t i o o f s u r f a c e The  f u n g i may be  o f n u t r i e n t s a v a i l a b l e to them i n a g i v e n  wood i f they do not a t t a c k attacking  The t h e r m o p h i l i c  attack  causes s o f t r o t  c e l l u l o s e (Bergman and N i l s s o n , 1967) .  out t h a t the moulds i s o l a t e d from c h i p p i l e s do  a t t a c k l i g n i n but attack  carbohydrates.  -  96 -  I f weight l o s s e s a r e to be c a l c u l a t e d i n p e r c e n t a g e s , small  sample p i e c e s  should  be chosen f o r the s t u d y .  losses are c a l c u l a t e d i n absolute  then  However, i f weight  terms, l a r g e r sample p i e c e s  a r e much more  appropriate. Thermophilic  fungi w i l l  a t t a c k wood so l o n g as the m o i s t u r e i n  the wood i s adequate and the temperature i s s u i t a b l e . unless  the c o n d i t i o n e d  that  wood had absorbed s u f f i c i e n t m o i s t u r e i t was n o t  p o s s i b l e to g e t the f u n g i to a t t a c k  the wood.  I n o c u l a t i n g wood w i t h t h e r m o p h i l i c the d i f f i c u l t i e s  I t was n o t i c e d  f u n g i d i d not present  associated with s o f t r o t fungi.  Savory  any o f  (1954) c o u l d n o t  s u c c e s s f u l l y i n o c u l a t e wood w i t h s o f t r o t f u n g i u n t i l he p l a n t e d  unsterilized  wood samples on Abrams medium and seeded them w i t h a s p o r e s u s p e n s i o n o f C^. globosum which l e d to the i n o c u l a t i o n o f the wood. P l a n t i n g the samples on weak o l d c u l t u r e s o f t h e r m o p h i l i c might have g i v e n h i g h e r  weight l o s s e s because the f u n g i would p r o b a b l y have  had  enough time to grow and produce a d a p t i v e  for  a t t a c k i n g wood.  enzymes which a r e n e c e s s a r y  In many s t u d i e s on the e f f e c t s o f t h e r m o p h i l i c samples have been i n c u b a t e d and  1968).  I t appears from the p r e s e n t  study t h a t i n c u b a t i o n beyond s i x weeks does n o t s e r v e the t h e r m o p h i l i c  which a r e u s u a l l y i n c u b a t e d  f u n g i on wood,  f o r two to three months ( N i l s s o n , 1965; Bergman,  N i l s s o n , 1966; S h i e l d s and U n l i g i l ,  In t h i s r e s p e c t  fungi  f u n g i may be s i m i l a r  any u s e f u l purpose. to the s o f t r o t f u n g i  f o r s i x weeks (Savory, 1954).  -  9J6 -  A s h o r t p e r i o d o f i n c u b a t i o n has c e r t a i n advantages the c o n d i t i o n s i n which e a r l i e r and  these t e s t s a r e c a r r i e d o u t .  considering  Results are obtained  the d r y i n g problem a t h i g h temperatures i s c o n s i d e r a b l y  reduced. Most of the wood d e g r a d a t i o n i n the l a b o r a t o r y first  takes p l a c e i n the  few weeks o f i n c u b a t i o n , and the r e s t of the i n c u b a t i o n time does not  c o n t r i b u t e a g r e a t d e a l to the t o t a l l o s s e s . but the f o l l o w i n g h y p o t h e s i s may may  on which  probably r e s u l t i n g  m e t a b o l i c b y - p r o d u c t s which may  t h i s happens i s not  e x p l a i n the phenomenon.  of the t h e r m o p h i l i c f u n g i the f u n g i depend,  Why  lead  be  The r a p i d  clear,  growth  to the r a p i d d e p l e t i o n of the n u t r i e n t s i n the a c c u m u l a t i o n of  inhibitory.  The p e r c e n t a g e weight l o s s o f wood caused by a t h e r m o p h i l i c  fungus  i n c r e a s e s w i t h temperature u n t i l optimum temperature i s reached, a t which point i t starts where a c t i v i t y  to d e c r e a s e u n t i l i s terminated.  by A. t e r r e s t r i s temperature  the maximum temperature i s r e a c h e d ,  S t u d i e s i n which  increased eleven f o l d  (Bergman and N i l s s o n , 1967)  the weight l o s s e s caused  from the minimum to the optimum support t h i s  conclusion.  The r i s e i n the a c t i v i t y of the fungus as the temperature  increases  to the optimum i s slower than the d e c l i n e i n the a c t i v i t y as the temperature i n c r e a s e s from optimum to the maximum. The optimum temperature f o r the a c t i v i t y of a t h e r m o p h i l i c i s a f f e c t e d by the s u b s t r a t e on which i t grows.  fungus  The optimum temperature f o r  -  A. t e r r e s t r i s  97 -  f o r r a d i a l growth on malt agar i s 45°C, but on b i r c h  the  optimum temperature a s s o c i a t e d w i t h the h i g h e s t weight l o s s e s i s 50°C (Bergman and N i l s s o n , 1967). a t which A. t e r r e s t r i s  In the p r e s e n t study the optimum  temperature  caused the h i g h e s t weight l o s s e s i n p i n e was  T h i s f i n d i n g agrees w i t h the work of Henningsson's  (1967).  He found  f o r s e v e r a l f u n g i from b i r c h and aspen the temperature f o r optimum growth  i s h i g h e r than t h a t f o r o p t i m a l decay The a b i l i t y  may  extend beyond  60°C i n the p i l e may e m e r s o n i i was  that  radial  activity.  to cause weight l o s s e s by some t h e r m o p h i l i c  60°C.  40°C.  fungi  I t has been p o i n t e d out t h a t temperatures above  completely s t e r i l i z e  the c h i p s  ( N i l s s o n , 1965) .  v e r y a c t i v e when the temperature o f i n c u b a t i o n was  13.  60°C f o r  f o u r weeks.  I t i s p o s s i b l e t h a t above 60°C i n c u b a t i o n J3. e m e r s o n i i may  cause weight  losses.  U n l i k e the o p t i m a l temperature, the s u b s t r a t e d i d n o t a f f e c t minimum temperature a t which  the a c t i v i t y of the fungus began.  temperature f o r r a d i a l growth o f _T. a u r a n t i a c u s on m a l t i s 30°C and N i l s s o n , 1966)  and 30°C was  the  The minimum (Bergman  the temperature a t which i t began to cause  weight l o s s e s i n p i n e as observed i n t h i s s t u d y . The c a p a c i t y of t h e r m o p h i l i c f u n g i to a t t a c k d i f f e r e n t  species of  c o n i f e r o u s wood i s v a r i a b l e , a l t h o u g h n e a r l y a l l the f u n g i examined degrade p i n e and s p r u c e . little  will  The observed d e g r a d a t i o n of l o d g e p o l e p i n e i s a  g r e a t e r than s p r u c e .  S i m i l a r r e s u l t s were o b t a i n e d by the Swedish  -  98 -  workers e x c e p t t h a t some o f the f u n g i they t e s t e d c o u l d a t a l l (Bergman in  and N i l s s o n , 1966).  not a t t a c k s p r u c e  The e x t e n t of a t t a c k was much lower  the f u n g i which Bergman and N i l s s o n  (1966) i n v e s t i g a t e d .  be expected because o f d i f f e r e n c e s i n s p e c i e s  T h i s might  o f wood, media and v a r i a t i o n  i n the f u n g i themselves. The cause of s t a i n i n g i n the s p r u c e samples i s unknown.  Staining  may be dependent upon the p o s i t i o n i n the t r e e from which the samples came, s i n c e some heartwood might have been i n a d v e r t e n t l y samples due t o d i f f i c u l t y zones i n s p r u c e . sometimes  included  i n the  i n d i f f e r e n t i a t i n g between heartwood and sapwood  However, the n a t u r e o f the o b s e r v e d s t a i n i n g , which may  form a s t r e a k  i n the m i d d l e o f a sample, does n o t s u p p o r t  this  contention. Stained  s p r u c e l o s t l e s s weight than u n s t a i n e d s p r u c e .  Oberg and F r e n c h (1957), found a l s o t h a t d i s c o l o u r e d  Hossfeld,  aspen a s s o c i a t e d  with  k n o t s , N e c t r i a canker and wet wood was more r e s i s t a n t to decay than the s a p wood c o n t r o l s .  I t was suggested by H o s s f e l d ,  the d i s c o l o u r e d  aspen wood c o n t a i n  toxic  Oberg and F r e n c h (1957)  e x t r a c t i v e components  that  some o f which were  towards wood d e c a y i n g f u n g i . The t h e r m o p h i l i c  f u n g i examined were n e i t h e r a n t a g o n i s t i c nor  synergistic i n their interaction.  Each fungus p r o b a b l y o c c u p i e s i t s own  zone i n the wood w i t h o u t depending upon the o t h e r f u n g i p r e s e n t . i n t e r d e p e n d e n c e between  f u n g i t h a t may take p l a c e w i l l  rely  Any  to a l a r g e  99  extent  on temperature.  Trichoderma l i g n o r u m was found to i n h i b i t the  a b i l i t y o f C_. l i g n o r u m to degrade wood but o n l y a t low temperatures (Bergman and N i l s s o n , 1967). of 1?. l i g n o r u m  As the temperature i n c r e a s e d  the e f f e c t  disappeared.  In summary, i t has been shown t h a t i n c o n t r o l l e d experiments, t h e r m o p h i l i c  and t h e r m o t o l e r a n t  laboratory  f u n g i can cause wood weight  l o s s e s and t h a t these l o s s e s a r e a f f e c t e d by medium, d u r a t i o n o f tion,  temperature o f i n c u b a t i o n ,  oculation.  the t r e e s p e c i e s  used and method o f i n -  A l t h o u g h sample s i z e and i n t e r a c t i o n of f u n g i were  they were found to have l i t t l e  incuba-  i n f l u e n c e on the e v e n t u a l  considered,  p e r c e n t a g e weight  l o s s e s o f the wood samples. In c o n c l u s i o n ,  these r e s u l t s suggest t h a t the o p t i m a l  conditions  f o r e v a l u a t i n g weight l o s s o f wood caused by these f u n g i would i n v o l v e  using  A b r a m - c e l l u l o s e medium, growing the f u n g i on t h i s medium f o r one week, p l a n t i n g wood samples incubating  (0.2 x 1.9 x 4.4 cm.) on the one week o l d c u l t u r e s and  the t e s t f o r 6 weeks a t 45°C f o r t h e r m o p h i l i c  thermotolerant  fungi.  f u n g i and 40°C f o r  T h i s method developed f o r the e v a l u a t i o n o f weight  l o s s of wood caused by these f u n g i i s an e f f i c i e n t one and the r e s u l t s obtained  show l e s s v a r i a b i l i t y  than those o f p r e v i o u s  workers.  -  IV  A  100  -  CHEMICAL ANALYSIS OF DEGRADED WOOD  INTRODUCTION  Thermophilic  f u n g i have been i s o l a t e d from c h i p s  a l s o been shown to degrade wood i n l a b o r a t o r y N i l s s o n , 1 9 6 6 , 1 9 6 8 ; S h i e l d s and U n l i g i l , information activity  1968).  (Bergman and  Relatively  i s a v a i l a b l e on the n a t u r e o f t h e i r s p e c i f i c  i n wood.  information  studies  The p r e s e n t  and have  little  chemical  study was undertaken t o p r o v i d e  some  on the s p e c i f i c c h e m i c a l a c t i v i t y o f some t h e r m o p h i l i c  fungi  i n wood. The  method o f a n a l y s i s f o l l o w e d was the h y d r o l y s i s o f both  c e l l u l o s e and h e m i c e l l u l o s e  to monosaccharides, the r e d u c t i o n  o f the  monosaccharides to sugar a l c o h o l s and the a c e t y l a t i o n o f t h e sugar a l c o h o l s -. These a c e t y l a t e d compounds were then i n j e c t e d i n t o a gas chromatograph.  B  LITERATURE REVIEW  Bergman and N i l s s o n fungi, including thermophilic not  ( 1 9 6 8 ) noted t h a t a number o f s o f t r o t f u n g i , i s o l a t e d from a b i r c h c h i p p i l e d i d  cause any l i g n i n l o s s e s and thus the s o f t r o t f u n g i m a i n l y degraded the  c a r b o h y d r a t e s o f the wood.  Chang ( 1 9 6 7 ) found t h a t b o t h h e m i c e l l u l o s e and  -  101  -  c e l l u l o s e were q u i t e s u s c e p t i b l e to m i c r o b i a l a t t a c k d u r i n g s e l f of  removed.  Henssen ( 1 9 5 7 )  thermophile (1969)  C  i n 60 days of composting  straw and  and  about 7 0 . 7 %  Chang ( 1 9 6 7 )  MATERIALS  AND  and  t h e r m o p h i l e would degrade  e m e r s o n i i and  1 2 weeks were employed.  was  taken f o r the  S_. t h e r m o p h i l e .  Carbohydrate  The  f u n g i used were A.  I n c u b a t i o n p e r i o d s of 2 ,  through  f o r each  a 6 0 mesh s i e v e .  6  treatment  From t h i s  1  analysis.  (Tappi s t a n d a r d T 1 3 0 5  lignin  Fergus  cellulose.  S i x of the wood samples  L i g n i n c o n t e n t s were determined  lignin.  and  S^  f o r the p r e p a r a t i o n of the wood samples f o r t h i s  were ground t o g e t h e r to pass gm.  Chang ( 1 9 6 7 )  pectin.  a n a l y s i s i s the same as o u t l i n e d i n P a r t I I I . I},  was  METHODS  The method used  terrestris,  of the c e l l u l o s e  demonstrated the a b i l i t y of  to decompose h e m i c e l l u l o s e and  have shown t h a t S^  heating  -  54,  1954)  by  the s t a n d a r d K l a s o n method  and were not c o r r e c t e d f o r a c i d - s o l u b l e  a n a l y s e s were made on the f i l t r a t e  from  the  Klason  determination. One  gram of ground wood to which 2 5 0 mg. of i n o s i t o l was  as an i n t e r n a l s t a n d a r d , was The m i x t u r e was  added  h y d r o l y s e d w i t h 1 5 ml. of 72% sumphuric  d i l u t e d w i t h d i s t i l l e d water and heated  i n an  acid.  autoclave  -  at by  212°F  f o r four hours.  102. -  The l i g n i n was removed by f i l t r a t i o n and e s t i m a t e d  the s t a n d a r d K l a s o n method.  To c o n v e r t the sugars to a l d i t o l s a n  a l i q u o t of the K l a s o n l i g n i n f i l t r a t e was brought of s a t u r a t e d barium h y d r o x i d e s o l u t i o n . then removed by c e n t r i f u g a t i o n . sodium b o r o h y d r i d e and l e f t  to a pH o f 4 by a d d i t i o n  The r e s u l t i n g p r e c i p i t a t e was  The c e n t r i f u g a t e was t r e a t e d w i t h 50mg.  over n i g h t .  The r e d u c i n g s o l u t i o n was a c i d i f i e d  w i t h 0.1ml g l a c i a l a c e t i c a c i d and taken to dryness under vacuum u s i n g a f l a s h e v a p o r a t o r i n a water b a t h a t temperature a c i d was removed as methyl b o r a t e (Wolfrom e v a p o r a t i o n s under vacuum w i t h methanol. volume o f methanol and t r a n s f e r r e d  o f 35° to 40°C.  The b o r i c  and Thompson, 1963) by f o u r The r e s i d u e was taken up i n a s m a l l  to a v o l u m e t r i c tube where the methanol  was a g a i n removed by e v a p o r a t i o n under vacuum.  The a d d i t i v e m i x t u r e was  a c e t y l a t e d by adding 1 l/2ml a c e t i c a n h y d r i d e and h e a t i n g the m i x t u r e f o r t h r e e hours a t 120°C. The excess a c e t i c a n h y d r i d e was removed by e v a p o r a t i o n and the r e s i d u e taken up i n 2ml methylene d i c h l o r i d e .  T h i s s o l u t i o n was then  injected  i n t o a gas chromatograph f o r a n a l y s i s . The  gas chromatograph used was S e r i e s 1520 V a r i a n - A e r o g r a p h  flame i o n i z a t i o n d e t e c t o r , i n i t i a l minute f o r 25 minutes,  oven temperature  i n j e c t i o n temperature  w i t h n i t r o g e n as c a r r i e r g a s .  The copper  3% ECNSS-M on Gas Chrom Q (Sawardeker, 1967).  with  140°C, h e a t i n g r a t e 1/2°/  250°C, d e t e c t o r temperature  column (1/8" x 2.5') was packed  S l o n e k e r and Jeanes,  with  1965; Oades,  The peak a r e a s were measured w i t h a Model 476 V a r i a n - A e r o g r a p h  i n t e g r a t o r w i t h i n o s i t o l as the i n t e r n a l s t a n d a r d .  225°C  digital  -  D  103 -  RESULTS  In t h i s e x p l o r a t o r y t e s t , c o n s i d e r i n g the e x p e r i m e n t a l t i o n i n the c h e m i c a l method  varia-  ( a p p r o x i m a t e l y ± 2%, p e r s o n a l communication,  Dr. K. Hunt) and a l s o the e x p e r i m e n t a l v a r i a t i o n to be expected  i n the  m y c o l o g i c a l method, any changes i n the r e s u l t s f o r g l u c o s e , mannose, x y l o s e and l i g n i n appear to be i n c o n c l u s i v e . a r a b i n o s e i n d i c a t e a d e c r e a s e of t h i s branch  However, the r e s u l t s f o r residue with increasing  time o f  a t t a c k by a l l of the f u n g i .  T a b l e 18.  C o n c e n t r a t i o n o f v a r i o u s c h e m i c a l components o f ponderosa p i n e sapwood a f t e r d e g r a d a t i o n by some t h e r m o p h i l i c f u n g i . a r e based  Fungi  Time i n wks.  on degraded wood.*  Lignin  Carbohydrate % & L i g n i n % Glucose Mannose Xylose  Arabinose  Control  2 12  24.7 25.1  40.0 41.3  8.0 12.3  5.0 5.6  1.5 1.6  Allescheria terrestris  25.2 25.2 26.3 25.5 25.1 25.8 25.5 26.1  38.0 46.0 46.2 41.7 46.0 43.0 48.7 41.6  10.0 9.5 11.0 10.4 11.0 13.6  Sporotrichum  2 6 12 2 6 12 2 6  10.1  5.1 5.6 5.6 4.5 5.0 4.8 4.0 5.3  1.5 0.0 0.0 0.8 0.2 0.1 0,4 0.2  thermophile  12  25.7  47.0  13.3  5.3  0.2  Byssochlamys emersonii  *  Percentages  11.0  S i n c e p r e v i o u s t e s t s showed t h a t these 3 f u n g i would g i v e no more  about 4% weight l o s s , c a l c u l a t i o n o f c h e m i c a l r e s u l t s c o u l d be based  than on e i t h e r  sound o r degraded wood w i t h v e r y l i t t l e  d i f f e r e n c e i n the r e s u l t i n g v a l u e s .  In t h i s  used.  t e s t degraded wood weights  were  -  E  104  -  DISCUSSION  The t h e r m o p h i l i c f u n g i i n v e s t i g a t e d do n o t a t t a c k l i g n i n and do n o t appear t o a t t a c k any o f the c a r b o h y d r a t e c o n s t i t u e n t s e x c e p t arabinose.  Bergman and N i l s s o n (1968) have i n d i c a t e d t h a t s o f t r o t f u n g i  from a b i r c h c h i p p i l e , w h i c h i n c l u d e d some t h e r m o p h i l i c f u n g i , d i d n o t a t t a c k l i g n i n but attacked carbohydrates.  No s p e c i f i c component o f t h e  c a r b o h y d r a t e was i n d i c a t e d as b e i n g a t t a c k e d . (1967) showed t h a t S_. t h e r m o p h i l e From t h i s l i m i t e d study  Henssen (1957) and Chang  would decompose h e m i c e l l u l o s e s and p e c t i n .  i t i s p o s s i b l e to hypothesize  t h a t some o f t h e sugar  c o n s t i t u e n t s o f the x y l a n s i n t h e wood, e s p e c i a l l y a r a b i n o s e by  the t h e r m o p h i l i c f u n g i .  may be a t t a c k e d  -  V  105  -  GENERAL DISCUSSION AND  CONCLUSIONS  A study of the d i s t r i b u t i o n of f u n g i i n a c h i p p i l e  should  i n c l u d e a l l types of f u n g i which might i n h a b i t t h i s e c o l o g i c a l l o c a t i o n . However, much of the e a r l i e r s t u d i e s fungi  (Bergman and  Unligil, The  1968;  S h i e l d s , 1970)  1967,  1968;  a t much h i g h e r  temperatures.  f u n g i were i s o l a t e d , u s i n g  I t was  soon e v i d e n t  m a i n l y on  E s l y n , 1967;  wood c h i p s  two  At  the b e g i n n i n g of  isolation  be p a i d  these f u n g i .  Eslyn  (1967) and  study.  this  capable  study,  temperatures  to the i s o l a t e s from 45°C than p r e v i o u s  Actinomycetes and  Shields  i t appeared t h a t  B a c t e r i a w i l l be  45°C.  occurred.  greater workers  common i n the c h i p  (1970) have i s o l a t e d b a c t e r i a and  Eslyn  were not  had piles.  (1967) has  these organisms were not  i m p o r t a n t i n q u a n t i t a t i v e wood d e g r a d a t i o n and  present  fungi  temperatures, 25°C and  Therefore  i s o l a t e d Actinomycetes from c h i p p i l e s , but to be  temperatures.  i n the a r e a s of the p i l e which produced l a r g e numbers  of i s o l a t e s a t 45°C l o s t more w e i g h t .  given  and  t h a t many more f u n g i were i s o l a t e d a t 45°C than a t 25°C,  stored  a t t e n t i o n should  Shields  f o r an emphasis on  e s p e c i a l l y from the a r e a s of the c h i p p i l e where h i g h e r The  mesophilic  which o c c u r commonly at o r d i n a r y  p e c u l i a r e c o l o g y of a c h i p p i l e c a l l e d  of l i v i n g all  N i l s s o n , 1966,  concentrated  considered  treated i n  the  -  The  succession  106  -  of f u n g i i n a c h i p p i l e w i l l depend upon many  e n v i r o n m e n t a l f a c t o r s such as  temperature, the a b i l i t y of the f u n g i to  wood as a n u t r i e n t s o u r c e , the a v a i l a b i l i t y  of m o i s t u r e , the pH  use  of the wood  and  a e r a t i o n i n the c h i p p i l e .  Aeration  i n the c h i p p i l e a t P r i n c e  George  was  not  been suggested t h a t a c c u m u l a t i o n of  carbon  i n v e s t i g a t e d but  i t has  d i o x i d e or d e p l e t i o n of oxygen does not J o r g e n s e n and were not  Ferrigan,  found to be  1967;  occur i n chip p i l e s  Bergman and  limiting  N i l s s o n , 1968).  (Hajny, M o i s t u r e and  f a c t o r s i n the development of f u n g i i n  P r i n c e George p i l e w h i l e a l l the f u n g i examined c o u l d use wood as source. present  The  major f a c t o r which determined the s u c c e s s i o n  study was  temperature.  to t h e i r  P r i n c e George c h i p p i l e .  This p a t t e r n w i l l vary  nutrient  of f u n g i i n  the  temperature t o l e r a n c e s from one  i s given  for  the  a r e a of the p i l e  to  other. The  initial  of growing a t o r d i n a r y Gliocladium, in  the  Thus a s u c c e s s i o n a l p a t t e r n based on groups o f  fungi delimited according  the  pH  this  c o l o n i z e r s of  the c h i p p i l e were f u n g i  temperatures, l i k e v a r i o u s  C e r a t o c y s t i s and  study.  Emerson, 1964)  fungal  species  of  l i k e A.  the t h e r m o p h i l i c  fungi  fumigatus and (Cooney and  C^. pruinosum.  Emerson, 1964)  e s p e c i a l l y when temperatures r o s e above 22°C. j5. t h e r m o p h i l e and  A.  Trichoderma,  P e n i c i l l i u m , which were i s o l a t e d  Together w i t h these f u n g i were t h e r m o t o l e r a n t  terrestris.  capable  but fungi  With r i s i n g  not  (Cooney  and  temperatures,  began to grow i n the  These i n c l u d e d  included  organisms  Above 30°C, IS. e m e r s o n i i and _T.  pile like  aurantiacus  -  started  107  -  to grow w h i l e some o f the m e s o p h i l i c f u n g i s t a r t e d  the c h i p p i l e .  from  Most of the m e s o p h i l i c f u n g i i n the p i l e might have d i e d above  40°C when the a c t i v i t y o f both optimum.  to d i s a p p e a r  the t h e r m o p h i l i c and t h e r m o t o l e r a n t f u n g i was  Some o f the t h e r m o t o l e r a n t f u n g i p r o b a b l y d i e d a t temperatures  above  50°C when 15. e m e r s o n i i and T. a u r a n t i a c u s were most a c t i v e . With temperatures  above 55°C few f u n g a l s p e c i e s w i l l  among which were Ti. e m e r s o n i i and T. a u r a n t i a c u s . by  remain a c t i v e  T h i s p a t t e r n i s supported  the d i s t r i b u t i o n of f u n g i w i t h s t o r a g e time and the b e h a v i o u r  these organisms on wood w i t h changing  temperatures.  J3. e m e r s o n i i may be the most important the P r i n c e George a r e a . i n c r e a s e d temperature  o f some o f  fungus i n c h i p s t o r a g e i n  I t s development c o u l d r e s u l t i n l o s s o f wood  and e v e n t u a l s e r i o u s d e g r a d a t i o n o f wood.  substance,  In chip p i l e s  i n Sweden ( N i l s s o n , 1965; Bergman and N i l s s o n , 1966, 1967, 1968) C^. l i g n o r u m has been found was found  to be most d e s t r u c t i v e .  I n t h i s study, however,  to be a r a r e c o l o n i z e r o f t h e c h i p p i l e and these r e s u l t s  t h a t i t s h o u l d n o t pose a problem i n c h i p p i l e s I t i s suggested  here  suggest  i n and around P r i n c e George.  t h a t the f i n d i n g s on a c h i p p i l e from one p a r t o f the  w o r l d may n o t be t r u e f o r another may be.  Chrysosporium  p a r t o f the w o r l d , however s i m i l a r c o n d i t i o n s  No t h e r m o p h i l i c o r t h e r m o t o l e r a n t B a s i d i o m y c e t e s  were i s o l a t e d  from  the s p r u c e - p i n e c h i p p i l e i n P r i n c e George. Chips w i l l  come onto  of the p r e s e n t methods used old p i l e ,  the p i l e h e a v i l y contaminated  i n handling chips.  f u r t h e r inoculum w i l l  come from  by f u n g i because  When a new p i l e i s near  the o l d p i l e .  to an  I n a new a r e a where no  -  p i l e s have been b u i l t b e f o r e , postulated  that s u f f i c i e n t  spores to i n i t i a t e t h a t new  chips  108  -  where w i l l  the  inoculum w i l l  come from the s o i l  chip p i l e degradation.  should  not be put  f u n g i i n the l a b o r a t o r y were h i g h e r  to be do not  caused by  and  thermotolerant  In c h i p p i l e s ,  ( N i l s s o n , 1965;  c h i p p i l e where h i g h thermophilic  study t h a t no  the  thermophilic  Basidiomycetes  These f a c t o r s i n d i c a t e t h a t  T h i s view i s s u p p o r t e d by  f u r t h e r weight l o s s e s o c c u r r e d  increases  from the s i x months' to the  a l s o shown i n the l a b o r a t o r y  by  the frequency of i s o l a t i o n of  In a r e a s o f  temperatures o c c u r most of the damage w i l l be  a l s o been shown t h a t no  which  thermotolerant  studies  the o b s e r v a t i o n  losses the done by from  from the s i x months' to  this  the  temperatures i n the p i l e would  p r o b a b l y have a f f e c t e d the m e t a b o l i c a c t i v i t y of  occurred  conditions  are  and  S h i e l d s , 1970).  fungi.  obtained  f u n g i are compared to decay l o s s e s of wood  twelve months' sampling p e r i o d when the low  has  thermotolerant  When the weight l o s s e s of wood caused  i n the c h i p p i l e s are g e n e r a l l y caused by m i c r o f u n g i .  the  and  l o s s e s of wood i n the l a b o r a t o r y  B a s i d i o m y c e t e s , the l o s s e s by  f u n g i are low. low  thermophilic  than l o s s e s of wood s u b s t a n c e  higher  p r e v a i l i n the c h i p ; p i l e .  air-borne  !  expected s i n c e weight l o s s e s are s t u d i e d under o p t i m a l  thermophilic  is  The  from  on a base of o l d c h i p p i l e s which must  Weight l o s s e s of wood caused by  the P r i n c e George p i l e .  and  It is  I t i s , however, suggested h e r e  a c t as a l a r g e r e s e r v o i r of i n f e c t i o n .  in  inoculum come from?  the  i n thermophilic  thermophilic and  fungi.  thermotolerant  twelve months' sampling p e r i o d . t h a t a l l the  thermophilic  and  It  It fungi was  thermotolerant  -  109  -  f u n g i would cause h i g h weight l o s s e s of wood at temperatures above 40°C. T h i s study has  provided  A chip p i l e  a method f o r t e s t i n g t h e r m o p h i l i c f u n g i on wood.  i s o b v i o u s l y not b u i l t  i n c l u d e some heartwood. be l e s s  than 10%  shown i n these storage.  although  However, the p e r c e n t a g e of heartwood i s low  (Keays, 1970).  studies w i l l  Both l o d g e p o l e  t h e r m o p h i l i c f u n g i may  o n l y of sapwood, but w i l l  A fungus' a b i l i t y  reflect  p i n e and  its ability  spruce  a t t a c k spruce  and  Studies thermotolerant  fungi, especially  Bergman and  nature  e s p e c i a l l y J3. e m e r s o n i i .B. e m e r s o n i i  i n these p i l e s .  thermophilic  chip  necessary.  should  thermotolerant  fungi,  t h i s w i l l not  nullify  be r e s p o n s i b l e f o r thermogenesis i n the  pile. Bergman and  the i n s i d e of the p i l e piles,  and  I t might be shown c o n c l u s i v e l y t h a t  does not a t t a c k c e l l u l o s e i n the wood, but  the c o n t r o l of the fungus s i n c e i t may  pine.  of the a t t a c k , a r e  of t h e r m o p h i l i c and  storage  N i l s s o n (1967)  I t i s suggested t h a t c o n t r o l of c h i p d e t e r i o r a t i o n i n P r i n c e George i n v o l v e the p r e v e n t i o n of the spread  the  sapwood of  of a t t a c k of wood c h i p s , by the c h e m i c a l  may  during  p i n e heartwood d u r i n g  would a t t a c k b o t h the heartwood and  i n t o the n a t u r e  chips  a r e non-durable s p e c i e s and  not as s t r o n g l y as the sapwood i s a t t a c k e d .  showed t h a t C^. l i g n o r u m  and  to a t t a c k sapwood as  to d e s t r o y  lodgepole  also  N i l s s o n (1968) have suggested t h a t s m a l l e r p i l e s , i s near to f r e e z i n g , be b u i l t  i n the w i n t e r  and  where the i n s i d e of the p i l e w i l l be a t or about 60°C be b u i l t  summer.  This suggestion  i s the r e s u l t of l a r g e r l o s s e s i n w i n t e r  where larger  i n the  piles  in  -  Sweden when the smaller  temperature was  about 40°C i n the i n t e r i o r  l o s s e s i n summer p i l e s when the  interior. like  110  I t must be p o i n t e d  temperature was  of  the p i l e  about 60°C i n  that i f l a r g e r p i l e s  are b u i l t d u r i n g  summer i n P r i n c e George, l o s s e s can o n l y be p r e v e n t e d by k e e p i n g the temperature of the p i l e above 60°C f o r most of the time. s i n c e l a r g e r volumes of c h i p s  can  spontaneous combustion of the  chips.  a r e i n h a b i t e d by of the p i l e . and  i t has  f u n g i and  start  T h i s may  a c h a i n r e a c t i o n which may  t h a t weight l o s s e s a r e  Controlled laboratory  i n c u r r e d i n these  conditions. indicated may  be  chip  lead  pile  weight  fungi  fungi  losses  c o n t r o l s m a i n t a i n e d under the same e x p e r i m e n t a l  the x y l a n s  I t i s concluded that thermophilic  responsible  to  regions  A c h e m i c a l a n a l y s i s of wood exposed to common t h e r m o p h i l i c  destroyed.  pile.  difficult  experiments have shown t h a t the  t h a t some of the sugar c o n s t i t u e n t s of  are d i r e c t l y  be  p o s i t i v e l y c o r r e l a t e d with both  commonly i s o l a t e d from the c h i p p i l e were c a p a b l e o f c a u s i n g when compared to u n i n o c u l a t e d  the  inside  been shown t h a t the h o t t e r p a r t s o f the  Furthermore, weight l o s s was  temperature.  the  out here t h a t the p i l e at P r i n c e George behaved  the w i n t e r p i l e i n Sweden and  In c o n c l u s i o n ,  and  and  especially  thermotolerant  fungi arabinose fungi  f o r the weight l o s s e s i n c u r r e d i n the e x p e r i m e n t a l wood  -  I l l -  REFERENCES  A l l e n , D.  1968. 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Outside storage of southern pine c h i p s .  Tappi  45(8):  623-628. Tappi Standard.  1954.  L i g n i n i n wood.  T 13 os-54.  Waksman, S. A., T. C. Cordon and N. H u l p o i . 1939. I n f l u e n c e of temperature upon the m i c r o b i a l p o p u l a t i o n and d e c o m p o s i t i o n p r o c e s s e s i n composts of s t a b l e manure. S o i l Sc., 47:83-113. Westaway, A. G.  Paper. Wolfrom, M.  1968.  Guidelines f o r outside chip handling.  E.  and  L. and A. Thompson. 1963. Reduction Products. (20) Reduction w i t h sodium b o r o h y d r i d e . In Methods i n Carbohydrate Chemistry V o l . 2. E d i t e d by w h i s t l e r , R. L. and M. L. Wolfrom. Academic  P r e s s , New York. Wright,  Pulp  42(36): 24,30.  p. 65-67.  1954. A p r e l i m i n a r y study of the d e t e r i o r a t i o n of a l d e r and D o u g l a s - f i r i n outdoor p i l e s . U.S. F o r e s t S e r v i c e . P a c i f i c Northwest F o r e s t and Range Expt. S t a . Res. Note No. 99.  Young, C. E. 1961. The economics of c h i p s from m i l l r e s i d u a l s - s u p p l y and demand and mutual b e n e f i t s . Southern Pulp and Paper M a n u f a c t u r e r .  24(2): 55-56, 58, 60. Zak,  H.,  and E. K r a u t h a u f . 1964. Changes i n s u l p h i t e p u l p q u a l i t y owing open a i r s t o r a g e of hardwood and softwood c h i p s . Das P a p i e r  18(11): 691-699.  to  -  116 -  Appendix 1  T o t a l count/100 c h i p s o f T_. a u r a n t i a c u s , Byssochlamys s p . and C. pruinosum a f t e r s t o r i n g c h i p s f o r 12 months  Thermoascus aurantiacus  Byssochlamys s p .  Chrysosporium pruinosum  Position i n Pile  3  DURATION OF STORAGE OF CHIPS IN MONTHS 6 12 12 3 6 6 3  Outer bottom  0  0  1  2  0  0  1  2  1  Inner bottom  0  0  0  1  0  00  1  0  '0  Outer m i d d l e  0  1  1  0  1  0  21  18  15  Inner m i d d l e  2  0  0  0  0  0  1  4  2  Outer top  0  0  0  1  11  0  6  6  5  Inner top  0  0  0  0  0  0  1  0  0  12  -  117  -  Appendix 2 Composition o f media used  Abrams medium NH NO-  3.0 g.  K HP0  4  2.0 g.  KH P0  4  2.5 g.  4  3  2  2  MgS0 *7H 0 4  2.0 g.  2  Agar  20.Og.  D i s t i l l e d water  1000.0 c c .  A b r a m s - c e l l u l o s e medium Same as Abrams w i t h 10.0 g. o f c r y s t a l l i n e c e l l u l o s e YpSs:  Y e a s t - S t a r c h agar (Emerson, 1941) D i f c o powdered K HP0 2  yeast extract  1.0 g.  4  MgS0 :.7H 0 4  0.5 g.  2  Soluble starch  15.0 g.  Agar  20.0 g.  Water YpCs:  4.0 g.  (1/4 t a p , 3/4 d i s t i l l e d ) 1 0 0 0 . 0 g.  Y e a s t - c e l l u l o s e agar D i f c o powdered K„HP0. 2 4  4.0 g. 2.0 g.  MgS0 .:7H 0 4  yeast extract  2  Cellulose D i s t i l l e d water  2.0 g. 10. g. 1000.0 c c .  added.  -  113  M a l t agar D i f c o malt  20.0 g.  Agar  20.0 g.  Water Malt-cellulose  1000.0 c c .  (distilled) agar  D i f c o malt  20.0 g.  Cellulose  10.0 g.  Agar  20.0 g.  Distilled  1000.0 c c  water  Combinations of NaNO , KH P0 2  4'  Difco yeast extract  Medium 1  2  3  4  5  6  7  8  NH.N0„ 4 3  3.0 g. -  +  +  -  -  -  +  +  K HE0  4  2.0 g. +  +  +  +  +  +  +  +  KH P0  4  2.5 g. -  +  -  +  -  +  2.0 g. +  +  +  +  +  +  +  Components  2  2  MgS0 -7H 0 4  2  Difco yeast  extract  4.0 g. -  +  +  +  +  -  +  +  Cellulose  10.§.g.+  +  +  +  +  +  +  +  Agar  20.0 g.  +  +  +  +  +  +  +  +  +  +  +  +  +  +  Distilled  water  + = Component i s p r e s e n t - = Component i s absent  1000.0 c c .  -  Medium f o r i s o l a t i n g  119  fungi  D i f c o malt e x t r a c t  20.0 g  D i f c o agar  20.0 g  Malic acid  5.0 g  D i s t i l l e d water Medium f o r growing  1000.0 c  fungi  D i f c o malt e x t r a c t  20.0 g  D i f c o agar  20.0 g  D i s t i l l e d water  1000.0 c  

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