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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 Un i v e r s i t y , 1961 M.Sc, Hebrew Un 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 of Forestry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1970 i i ABSTRACT A study into the d i s t r i b u t i o n of thermophilic and thermotolerant fungi i n a spruce-pine wood chip p i l e i n Prince George was c a r r i e d out. Five treatments c o n s i s t i n g of pine, spruce, incorporation of wood f i n e s into spruce, s t e r i l i z e d spruce and s t e r i l i z e d spruce inoculated with a Ptychogaster sp. were examined. Samples of wood chips buried at s i x d i f f e r e n t locations i n the chip p i l e were examined a f t e r 3, 6 and 12 months storage periods. From 100 randomly selected chips from each sample the fungi were i s o l a t e d on 2% malt, 0.5% malic acid and 2% agar at 25° and 45°C. Data on temperature during storage and a c i d i t y of wood chips, moisture content and weight loss at the time of sampling were recorded for the s i x posit i o n s i n the wood chip p i l e . Thermophilic fungi colonized the inner regions while thermotolerant fungi inhabited the outer regions of the wood chip p i l e . Among the ther-mophilic fungi, l i s t e d according to frequency of i s o l a t i o n were Byssochlamys emersonii Stolk-Apinis, 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 , Sporotrichum thermophile A p i n i s , Thermoascus aurantiacus Miehe and Humicola lanuginosa (Griffonand Maublanc) Bunce. The most common thermotolerant fungi were A s p e r g i l l u s fumigatus. Fresenius and Chrysosporium pruinosum (Gilman and Abbot) Comb. Nov. i i i Fungal d i s t r i b u t i o n was generally r e l a t e d to p o s i t i o n i n the wood chip p i l e . Of the associated f a c t o r s temperature (17°-45°C) was most strongly r e l a t e d to fungal d i s t r i b u t i o n , whereas a c i d i t y of wood chips and moisture content did not vary g r e a t l y between p o s i t i o n s . Incubation of wood samples on cultures demonstrated the a b i l i t y of a l l the common thermophilic and thermotolerant fungi to cause weight loss of lodge pole pine Pinus contorta Dougl. v a r l a t i f o l i a , ponderosa pine Pinus ponderosa Laws, and spruce Picea glanca (Moench) Voss sap wood samples. These weight losses v a r i e d from 0.65% to 25% a f t e r s i x weeks incubation. Temperature, medium and type of wood affected the a b i l i t y of the fungi to cause weight l o s s . No s y n e r g i s t i c or antagonistic e f f e c t s ' existed between the thermophilic fungi. Chemical a n a l y s i s of degraded wood indicated that the thermophilic fungi u t i l i z e d the arabinose f r a c t i o n of the hemicellulose p r e f e r e n t i a l l y . i v -In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the r e q u i r e -ments f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y 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 reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission Department of Forestry 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: August, 1970 V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS . v LIST OF TABLES i x LIST OF FIGURES x i ACKNOWLEDGEMENTS x i i I INTRODUCTION . 1 II FIELD STUDY OF FUNGAL DISTRIBUTION 6 A INTRODUCTION 6 B LITERATURE REVIEW 7 1. Advantages and disadvantages of outside wood chip storage 7 2. Types of wood chip p i l e s 9 3. Environmental conditions i n wood chip p i l e s 13 4. Fungi i s o l a t e d from wood chip p i l e s 18 5. Damage i n wood chip p i l e s 21 C MATERIALS AND METHODS 24 1. The experimental wood chip p i l e 24 2. Sampling p o s i t i o n s 27 3. Treatments 27 4. Preparation of wood chip samples 29 5. Measurement of environmental conditions 30 - v i Page a. Temperature i n the wood chip p i l e 30 b. Moisture content of wood chips 32 c. A c i d i t y of wood chips 32 6. I s o l a t i o n of fungi 32 a. Sampling procedure 32 b. I s o l a t i o n and grouping of fungi 33 7. Determination of wood chip weight loss 35 D RESULTS 36 1. L i s t of fungi i s o l a t e d 36 2. D i s t r i b u t i o n of common thermophilic and thermotolerant fungi 36 3. Environmental f a c t o r s 38 a. Temperature i n the wood chip p i l e 38 b. Moisture content of wood chips 43 c. A c i d i t y of wood chips 45 4. Weight loss of wood chips 46 5. Evaluation of treatments 47 6. Relationship between the v a r i a b l e s measured 51 E DISCUSSION 53 1. Fungi i n the wood chip p i l e 53 2. Temperature i n the wood chip p i l e 56 3. A c i d i t y of wood chips 58 4. Moisture i n the wood chip p i l e 58 5. Damage 59 - v i i -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 OF A METHOD FOR THE STUDY OF THE ABILITY OF THE FUNGI TO CAUSE WEIGHT LOSSES 69 1. E f f e c t of media 69 2. E f f e c t of wood sample s i z e 75 3. E f f e c t of methods of in o c u l a t i o n 78 4 . E f f e c t of duration of incubation 80 5. E f f e c t of temperature 82 6. Summary 85 E WEIGHT LOSSES CAUSED BY THE THERMOPHILIC AND THERMOTOLERANT FUNGI 85 1. Evaluation of 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 of lodgepole pine and white spruce wood to attack by several fungal i s o l a t e s 87 3. E f f e c t of mixed i s o l a t e s on weight losses 90 F DISCUSSION 92 IV CHEMICAL ANALYSIS OF DEGRADED WOOD 100 A INTRODUCTION 100 B LITERATURE REVIEW 100 C MATERIALS AND METHODS 101 D RESULTS 103 - v i i i -Page E DISCUSSION 104 V GENERAL DISCUSSION AND CONCLUSION 105 VI REFERENCES 111 VII APPENDIX 1 116 VIII APPENDIX 2 117 - i x -LIST OF TABLES Page Table 1. F i n a l moisture content of wood chips 44 Table 2. F i n a l a c i d i t y of wood chips 45 Table 3. F i n a l weight loss of samples i n percent 46 Table 4. Mean fungal 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 chips f o r three months 48 Table 5. Average number of fungi i s o l a t e d per p o s i t i o n a f t e r three months storage 49 Table 6. Fungal counts at d i f f e r e n t storage times at the d i f f e r e n t p o s i t i o n s 50 Table 7. Fungal counts at d i f f e r e n t storage times with d i f f e r e n t treatments. These are averages f o r s i x p o s i t i o n s . 50 Table 8. Summary of regression analysis of t o t a l temperature and weight loss on t o t a l fungal count 51 Table 9. Percentage weight losses caused to ponderosa pine sapwood on d i f f e r e n t media 70 Table 10. Moisture content of samples on each medium 71 Table 11. Percent weight losses obtained f o r A. t e r r e s t r i s and T_. aurantiacus growing on medium prepared from c e l l u l o s e , MgSO4-7H.20 and K2HPO4 with the absence of yeast or NH4NO3 or KH2PO4 75 Table 12. Percent weight loss and absolute weight loss of wood caused by thermophilic fungi growing on d i f f e r e n t s i z e s of ponderosa pine sapwood during s i x weeks incubation 77 Table 13. Average percentage weight loss of wood samples caused by thermophilic fungi using d i f f e r e n t methods of providing inoculum source 19 Table 14. Percent weight loss of ponderosa pine caused by some thermophilic and thermotolerant fungi at 45°C incubation 86 Percentage weight loss of lodgepole pine and spruce sapwood caused by some thermophilic and thermotolerant fungi at 45°C a f t e r 6 weeks incubation Percentage weight loss f or stained and unstained spruce inoculated with some thermophilic fungi 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. emersonii and thermophile on weight losses of ponderosa pine sapwood incubated at 45°C and 50°C Concentration of various chemical components of ponderosa pine sapwood a f t e r degradation by some thermo p h i l i c fungi. Percentages are based on degraded wood x i -LIST OF FIGURES Page Figure 1. East and West view of the chip p i l e showing l o c a t i o n ^ o f chip samples 1 to 6. 26 Figure 2. South view section of the chip p i l e 26 Figure 3. The four sections of the chip p i l e which were broken down a f t e r 3, 6, 12 and 24 months 28 Figure 4. R e t r i e v a l of the chip samples 28 Figure 5. The chip p i l e 34 Figure 6. Byssochlamys emersonii growing out of chips on 2% malt and 0.5% malic acid agar 34 Figure 7. Fungal count 37 Figure 8. Daily 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 r i t y - f o u r days of storage of chips 39 Figure 9. Temperature curves f o r the s i x p o s i t i o n s i n the chip p i l e during 80 weeks of storage of chips 41 Figure 10. Average monthly ambient temprature i n Prince George 42 Figure 11. The r e l a t i o n s h i p between t o t a l fungal count and moisture content of chips 52 Figure 12. The r e l a t i o n s h i p between t o t a l fungal count and t o t a l temperature 52 Figure 13. The r e l a t i o n s h i p between t o t a l fungal count and wood weight los s 52 Figure 14. A l l e s c h e r i a t e r r e s t r i s and Thermoascus aurantiacus growing on Abrams 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. aurantiacus 58 Figure 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 aurantiacus 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_. aurantiacus 73 Figure 16. Changes i n weight losses with duration of incubation 81 Figure 17. Changes i n weight losses with changes i n temperature 84 x i i ACKNOWLEDGEMENTS The project was introduced to me by Dr. R. W. Kennedy, Programme Manager, Forest Products Laboratory, Vancouver, B.C. F i n a n c i a l help for the study was provided by the Canada Department of F i s h e r i e s and Forestry. The management of the Laboratory has been generous i n i t s a s s i s -tance during the execution of the project. For t h i s , my sincere thanks go to the management of the Laboratory, e s p e c i a l l y to Dr. Kennedy. The author expresses h i s appreciation to Dr. R. S. Smith, Research S c i e n t i s t i n Wood Pathology at Forest Products Laboratory, for h i s f r i e n d l y guidance and encouragement throughout the study and preparation of the manuscript, and to Dr. J . V. Hatton, Research S c i e n t i s t i n Pulping Processes for h i s help i n providing him with some valuable data. Technical help when needed was kin d l y provided by Mr. E r i c Johnson, Mrs. C h r i s t i n e Sharman and Mrs. C. B. Johansen. I express my thanks to them. My appreciation goes to Dr. Apinis and Dr. Carmichael for t h e i r assistance i n i d e n t i f y i n g some of the c u l t u r e s . I thank also Dr. Bart van der Kamp, Ass i s t a n t Professor of Forest Pathology and my major advisor i n the Faculty of Forestry f or h i s kind guidance and worthwhile suggestions during the execution of t h i s work and i n the preparation of the manuscript. x i i i The author expresses his appreciation to a l l his student f r i e n d s who were a source of encouragement when things became d i f f i c u l t and f i n a l l y to h i s wife f o r her patience. I INTRODUCTION The storage of wood i n the form of wood chip p i l e s i s a p r a c t i c e widely adopted by pulp m i l l s around the world. The method has many advantages over roundwood storage such as reduced handling cost, reduced storage area, the a b i l i t y to mix various types of chips to any proportion e a s i l y , and the a b i l i t y to use sawmill residues for pulp economically. Storage of wood i n chip p i l e s also has c e r t a i n d i s -advantages such as the high temperatures commonly generated i n the p i l e with the attendant r i s k of f i r e and the degradation of the wood chips. I t has been estimated, f o r instance, that i n the i n t e r i o r of B r i t i s h Columbia, annual losses of $5,000,000 to $30,000,000 w i l l occur by 1987 due to these factors i f c o r r e c t i v e a c t i o n i s not taken (Hatton, Smith and Rogers, 1968). In the i n t e r i o r the main species used are lodgepole pine (Pinus  contorta Dougl. var l a t i f o l i a ) and spruce. For the purpose of t h i s thesis spruce r e f e r s to trees belonging to the complex c o n s i s t i n g of Picea glauca (Moench) Voss, Picea engelmanii Parry and the various hybrids formed between these two species as i t i s found around Prince George B.C. No attempt was made to d i s t i n g u i s h between the various forms. The wood i s ei t h e r chipped at the pulp m i l l from f r e s h l y cut logs or else i t a r r i v e s at the m i l l as chips produced from sawmill residue. In - 2 -contrast to the losses sustained by pulp m i l l s i n the i n t e r i o r , coastal pulp m i l l s i n B r i t i s h Columbia and the P a c i f i c North West, using d i f f e r e n t species of wood, report l i t t l e degradation of wood chips even a f t e r a year of storage (Wright, 1954). It follows that the experience gained on the coast cannot be applied d i r e c t l y to the new s i t u a t i o n i n the i n t e r i o r . Hence a large scale study of the problems associated with the storage of wood i n chip p i l e s i n the i n t e r i o r was i n i t i a t e d by the Forest Products Laboratory. The phenomenon of heat generation and degradation i s not r e s t r i c t e d to p i l e d wood chips, but i t occurs generally i n p i l e d organic matter such as hay, straw, manure, peat and grain (Miche, 1907; Isachenko and Mai'chevskaya, 1936; Waksman, Cordon and Hulpoi, 1939). In general two conditions must be met. F i r s t l y , there must be an adequate supply of water and secondly, the p i l e must be large enough so that heat d i s s i p a t i o n does not exceed heat generation u n t i l a high temperature i s reached. The maximum temperature reached depends on these two factors plus the nature of the material being stored and the e f f e c t s of various environmental conditions on the mechanisms of heat production. Various mechanisms for the production of heat have been proposed. These are r e s p i r a t i o n of the plant material, r e s p i r a t i o n of organisms, that u t i l i z e the stored material as a source of food, and auto-oxidation of organic substances i n the stored materials. The o r i g i n of heat production i n chip p i l e s i s not a subject of t h i s t h e s i s . - 3 -Isolati o n s from such n a t u r a l l y heated p i l e s of organic matter have y i e l d e d b a c t e r i a , fungi and actinomycetes, a l l with the p e c u l i a r a b i l i t y to be metabolically a c t i v e at high temperatures. A l l these classes of organisms have been i s o l a t e d from wood chip p i l e s . General experience with wood degradation has shown, however, that on wood fungi are much more important than e i t h e r b a c t e r i a or actinomycetes: Since, i t was impossible to sample a l l three groups adequately i n the experimental chip p i l e used i n the present study because of lack of time and space, i t was decided to l i m i t sampling to fungi. Most fungi i n contrast to b a c t e r i a are unable to grow at • temperatures above 35°C, although spores and various other r e s t i n g stages of these fungi such as s c l e r o t i a may oc c a s i o n a l l y survice exposure to higher temperatures. A r e l a t i v e l y small number of fungi can grow at temperatures up to 60°C. These have been divided into two groups c a l l e d thermophilic and thermotolerant fungi. For the purpose of th i s t h e s i s , thermophilic fungi are defined as those fungi able to grow on a 2% malt extract, 2% agar, and 0.5% malic a c i d medium at 45°C but not at 25°C. Thermotolerant fungi are those fungi which grow at both 25°C and 45°C on the above medium. Fungi which are able to grow at 25°C but not at 45°C are c a l l e d mesophilic f u n g i . In large commercial wood chip p i l e s , the temperature attained i n the i n t e r i o r region generally exceeds 45°C. I t appears therefore that apart from the outer layers of such a chip p i l e , fungal a c t i v i t y i s r e s t r i c t e d to thermophilic and thermotolerant fungi. For t h i s reason t h i s study deals only with thermophilic and thermotolerant fungi and does not concern i t s e l f with - l i -the a c t i v i t y of mesophilic fungi i n the outer, cooler regions of the p i l e . This thesis forms a part of the larger i n v e s t i g a t i o n mentioned above and deals with the r o l e of those fungi which inhabit the hotter parts of a wood chip p i l e . The c e n t r a l hypothesis i s that the fungi i n h a b i t i n g the hotter parts of the chip p i l e are d i r e c t l y responsible for the l o s s of wood substance and that t h i s l o s s leads to a decrease i n pulp production. To support t h i s hypothesis the following w i l l be necessary. F i r s t l y , i t must be v e r i f i e d that fungi are present i n the hotter parts of a chip p i l e and that weight losses are incurred i n these p o s i t i o n s . Secondly, i t must be demonstrated that the v a r i a t i o n i n weight losses observed between various 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 of f u n g i . Thirdly*/it must be shown that environmental conditions i n the p i l e due to such factors as temperature, moisture and a c i d i t y , i f they can be cor r e l a t e d with weight losses, cannot by themselves r e s u l t i n the observed weight losses, and that such c o r r e l a t i o n s can be explained by the e f f e c t of these environmental factors on the presence of fungi and t h e i r a b i l i t y to degrade wood chips. Fourthly, i t must be established that the fungi i s o l a t e d from the chip p i l e are capable of causing weight losses of wood chips under c a r e f u l l y c o n t r o l l e d environmental conditions. L a s t l y , i t must be shown that at l e a s t part of the weight loss of wood incurred during storage i s a t t r i b u t a b l e to the loss of components of the wood which are u t i l i z e d as pulp. - 5 -The thesis i s divided into three parts. The f i r s t part deals with the study of fungi, environmental conditions, and weight losses i n an experimental chip p i l e , the second i s concerned with the demonstration of the a b i l i t y of commonly i s o l a t e d fungi to cause weight losses and the l a s t part deals with the chemical analysis of degraded wood. - 6 -II FIELD STUDY OF FUNGAL DISTRIBUTION A INTRODUCTION This s e c t i o n of the thesis i s concerned with the demonstration that thermophilic and thermotolerant fungi are present and a c t i v e i n the hot regions of a chip p i l e , that weight losses of wood occur i n these regions, and that such weight losses are r e l a t e d to the d i s t r i b u t i o n of fungi. Furthermore, i t deals with the r e l a t i o n s h i p s between fungal d i s t r i b u t i o n , weight l o s s , and various environmental f a c t o r s . L a s t l y , an attempt i s made to show how fungal d i s t r i b u t i o n , weight losses and environmental conditions develop i n time. The experimental chip p i l e u t i l i z e d f o r t h i s study was designed by the Forest Products Laboratory. I t was not planned s o l e l y f o r the purpose of t h i s study. For instance samples inserted into the p i l e had to be large enough to allow experimental pulping studies of the stored chips. This made the accurate determination of weight losses rather d i f f i c u l t . The p i l e consisted of four sections, to be broken down a f t e r 3, 6, 12 and 24 months. This study contains the r e s u l t s of the f i r s t three periods only. I t was assumed that s i m i l a r conditions p r e v a i l e d at s i m i l a r p o s i t i o n throughout the whole chip p i l e . - 7 -Several treatments were incorporated into the chip p i l e . The f i r s t two consisted of bags f i l l e d with pure spruce and pure pine chips i n order to determine the e f f e c t of these species on the various f a c t o r s studied. A t h i r d treatment consisted of the incorporation of " f i n e s " into the sample bag. L a s t l y two treatments consisted of the incorporation of a "core" bag containing spruce chips inoculated with a species of Phytogaster, a thermotolerant basidiomycete i s o l a t e d from chip p i l e s i n eastern Canada into the sample bag, and a c o n t r o l c o n s i s t i n g of s t e r i l e spruce chips i n a core bag. B. LITERATURE REVIEW 1. Advantages and disadvantages of outside wood chip storage Compared to roundwood storage, the advantages of outside chip storage are many (Anon, 1961; Holekamp, 1962; Westaway, 1968). One of these advantages i s the lower cost i n handling and storage of the m a t e r i a l . It i s estimated that a medium sized m i l l can save up to $50,000 annually using outside wood chip storage instead of roundwood storage (Clark, 1963). Operational advantages (Holekamp, 1962; Clark, 1963) have been reported as follows: - 8 -1. Reduced f i b e r l o s s . 2. Decreased handling cost. 3. Large manpower saving. 4. Twenty to twenty-five percent increase i n production from wood yard to wood room. 5. Reduced maintenance costs. 6. E l i m i n a t i o n of production los s because of wood room-wood yard breakdowns. 7. Increased bark y i e l d and reduced f u e l costs. 8. Improved uniformity i n the digester f u r n i s h . 9. Greater storage per acre. 10. Less housekeeping. 11. Smooth wood yard and wood room operation. 12. Better and more accurate species handling. This method, however, has i t s disadvantages which are as follows (A l l e n , 1968; Hatton, Smith and Rogers, 1968): 1. Risk of f i r e r e s u l t i n g from the tendency of p i l e s to heat up. 2. Losses i n pulp y i e l d due to d e t e r i o r a t i o n of chips during storage. 3. Reduction i n strength properties of the chemical and r e f i n e r -groundwood pulps. 4. Losses i n refiner-groundwood pulp brightness. 5. Increased requirements f o r cooking and bleaching chemicals. - 9 -6. Corrosion problems with chip reclaiming systems due to the low pH of severely deteriorated chips. 7. Production of off-grade chips due to contamination of chip p i l e s by a i r borne p a r t i c l e s , such as f l y ash and sand. Despite these disadvantages, the f a c t that a large number of m i l l s are using or contemplating using t h i s method might i n d i c a t e that the advantages outweigh the disadvantages. 2. Types of wood chip p i l e s There are as many p i l e forms as there are m i l l s . No two p i l e s are the same i n s i z e , l o c a t i o n or volume. However, two main shapes are it i n common use, the c o n i c a l p i l e s (Bjorkman and Haeger, 1963) and the it rectangular p i l e s (Bjorkman and Haeger, 1963; Shields and U n l i g i l , 1968). There i s also no standard design for a system of outside chip storage (Hajny, 1966). Recently, construction of p i l e s i n a r i n g form with a section of the r i n g p i l e removed so that the two r e s u l t i n g surfaces c o n s t i t u t e the beginning and end of the p i l e has been advocated (Croon, 1966). Chips are transported to the m i l l s by trucks, t r a i l e r s , r a i l , barge and ship (Bergman and Nilsson, 1966; Shields, 1967). In some instances round wood i s transported to the m i l l to be chipped there (Shields and U n l i g i l , 1968). There i s l i t t l e agreement as to what material to use for the base of the p i l e (Blackerby, 1958; Holekamp, 1959; ti Annergren, D i l l n e r , Haglund and Jagerud, 1965). The e a r l i e s t p i l e s were b u i l t d i r e c t l y on the ground, and packed clay and sand were used as the base. Under these conditions 2 to 3 feet of chips have been l e f t as the base of - 10 -a new p i l e . In some cases hogged f u e l or bark have been used i n place of t h i s layer of chips. A hard surfaced base such as black top or concrete i s generally considered to be the most s a t i s f a c t o r y , although the most expensive. An experimental p i l e was constructed on a polyethylene sheeting (Butcher and Howard, 1968). Chips are blown on to the area by pneumatic chip blowers and are then spread out by crawler type t r a c t o r s which compact them at the same time. Not a l l the p i l e s are mechanically compacted and i n such cases the chips are allowed to compact themselves. The volume and the f i n a l height of the chip p i l e are two important f a c t o r s determining the e f f i c i e n c y of the processes which take place i n the p i l e . Experimental p i l e s have generally been small and low (Butcher and Howard, 1968) varying from 9 feet to 145 f e e t . The experimental p i l e s i n Sweden were u s u a l l y large and t a l l (Annergren, D i l l e n it and Vardheim, 1964; Annergren, D i l l n e r , Haglund and Jagerud, 1965). The volumes of p i l e s i n Canada have averaged from 21,000 cords to a huge 100,000 cords (Robinson, 1968; Shields and U n l i g i l , 1968). These varying p h y s i c a 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 i n part for the tremendous v a r i a b i l i t y of the r e s u l t s from studies on chip p i l e s . The major species used i n chip p i l e s on the west coast of North America are Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco], western hemlock [Tsuga heterophylla (Ref.) Sarg] white f i r (Abies concolor Gord. - 11 -and Glend), western red cedar (Thuja p l i c a t a Don), pines (Pinus sp.), red alder (Alnus rubra Bong.) and cottonwood (Populus trichocarpa Torr. and Gray) (Wright, 1954; Blackerby, 1958; Hensel, 1958). In the southern U.S.A. many p i l e s are b u i l t of pine species. In eastern Canada, balsam f i r (Abies balsamea (L.) M i l l ) , spruce (Picea sp.), pine (Pinus sp.) and eastern hemlock (Tsuga canadensis (L.) Carr) have been stored as chips (Blackerby, 1958; Shields and U n l i g i l , 1968). Pulp m i l l s i n Scandinavian countries have experimented with b i r c h (Betula sp.) spruce and pine species (Annergren, D i l l e n and Vardheim, 1964; Annergren, D i l l n e r , Haglund and Jagerud, 1965; Bjorkman and Haeger, 1963; Bergman and Nilsson, 1966). Butcher and Howard (1968) have studied the behaviour of Pinus r a d i a t a D. Don. i n small p i l e s during winter and summer storage i n New Zealand. C o n f l i c t i n g accounts of the behaviour of wood i n outside storage are common, pos s i b l y because of the v a r i a t i o n i n resi s t a n c e to 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 of Douglas-fir on the west coast of North America do not seem to s u f f e r even a f t e r three years of outside storage while alder i s badly deteriorated a f t e r four months of storage. A mixture of alder and Douglas-fir could stand longer outside storage than alder alone (Wright, 1954). The length of time that chips can be kept i n storage v a r i e s with species, climate and pulping process (Hajny, 1966; Shields, 1967). In the P a c i f i c Northwest chips have been stored f o r 2 to 3 years or more with l i t t l e evidence of d e t e r i o r a t i o n (Blackerby, 1958; Burke, 1962). - 12 -It i s concluded that although many of the west coast species can be stored for lengthy periods without d e t e r i o r a t i o n , such a condition might not p r e v a i l with other commercial species and that t e s t i n g each species separately might be necessary. In the southern United States chips of southern pines d e t e r i o r a t e much more r a p i d l y than i n the P a c i f i c Northwest. This c o n d i t i o n i s r e f l e c t e d i n the storage time recommended by workers i n the southern United States. Holekamp (1959) recommends summer storage of three months, whilst Somsen (1962) gives 16 weeks as the maximum storage time. Rothrock, Smith and Lindgren (1961) stored slash pine chips f o r 5 months i n the southern U.S.A. and showed that outside summer storage of pine wood i s f e a s i b l e within t h i s l i m i t . In Nova Scotia, Robinson (1963) recommends two months storage. D e t e r i o r a t i o n of chips was found to be severe i n the lower t h i r d of a spruce and balsam f i r chip p i l e at approximately nine months, i n d i c a t -ing that complete u t i l i z a t i o n of a p i l e should occur before eight months at l e a s t i n order to minimize reductions i n chip q u a l i t y that could a f f e c t the pulp adversely (Shields and U n l i g i l , 1968). Storage of softwood chips for 15 or more months involves r i s k s of d e t e r i o r a t i o n to spruce and pine chips (Bjorkman and Haeger, 1963) and Forssblad (1965) suggests that chip p i l e s be mixed completely every few months to minimize t h i s r i s k . Experiments i n Georgia (Saucier and M i l l e r , 1961) i n d i c a t e that d e t e r i o r a t i o n of pine chips stored i n the winter i s one-third to one-half l e s s than that of summer stored chips. Many of the pine chips stored i n Sweden (Bergman and Nilsson, 1966) from the end of October to the following - 13 -May were found to be frozen during most of t h i s period at the bottom l e v e l of the p i l e . Where average storage temperature i s close to f r e e z i n g , i t i s found that s p e c i f i c g r a v i t y losses are very low i n comparison to losses i n samples stored i n the warmer areas of the p i l e . 3. Environmental conditions i n wood chip p i l e s A spontaneous r i s e i n temperature i n chip p i l e s occurs as a r e s u l t of processes which go on i n the p i l e . This heat generation a f f e c t s water vapour movement within the p i l e , causes changes i n wood ex t r a c t i v e s , and creates conditions for a d i f f e r e n t m i c r o f l o r a not normally associated with wood d e t e r i o r a t i o n . Several small experimental chip p i l e s have been b u i l t i n the southern United States from southern pine chips (Rothrock, Smith and Lindgren, 1961; Saucier and M i l l e r , 1961; Somsen, 1962; Davis, 1963). The sloping sides 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 these p i l e s showed a degree of uniformity. In the i n t e r i o r compacted portions of the p i l e s , a rapid i n i t i a l r i s e of temperature occurred. No mention was made of the ambient temperatures. Within the f i r s t two weeks the temperature rose to between 54° and 63°C. During the next two to four weeks the temperature dropped sharply to about 49°C followed by a gradual decline to about 38°C a f t e r about f i v e months storage. In the uncompacted portions of the p i l e s tempera-tures rose r a p i d l y at f i r s t , but only to 38-49°C, and soon thereafter f e l l to ambient temperature. - 14 -In Sweden, Bjorkman and Haeger (1963) b u i l t a softwood p i l e i n the shape of a cone with a base diameter of 15 meters and a height of 11 meters. In the deep i n t e r i o r of the p i l e the temperature remained above ambient f o r 15 months, the maximum d i f f e r e n c e being 20.6°C i n January. The temperature near the periphery followed the a i r temperature. In two studies on large p i l e s of 7400-8800 cubic 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 observations on temperature changes. Three of the p i l e s were of spruce and one of b i r c h . Temperatures i n the spruce p i l e s rose to about 55°C and remained at t h i s l e v e l f o r 3 to 4 months followed by a dec l i n e . In the b i r c h p i l e the temperature rose to 65°C i n the f i r s t two weeks and remained at t h i s l e v e l f o r 4 1/2 months at which time the p i l e was dismantled. Annergren, D i l l e n and Vardheim (1964) and Annergren, D i l l n e r , Haglund and Jagerud (1965) also confirmed the f i n d i n g that temperatures near the sides of the p i l e are lower than those near the centre of the p i l e . Ljungqvist (1965) made an extensive study of temperature v a r i a -t i o n i n several commercial p i l e s i n Sweden. P i l e s of b i r c h , pine and spruce (exact species not i d e n t i f i e d i n paper) b u i l t i n summer reached maximum temperature of 69°C, 63°C and 58°C r e s p e c t i v e l y . Although temperatures i n the i n t e r i o r of eastern Canadian chip p i l e s do r i s e very sharply a f t e r the f i r s t weeks of construction, with longer storage the temperatures drop to between 30°C and 43°C (Shields and U n l i g i l , 1968). The drop sometimes occurs very abruptly (Rothrock, - 15 -Smith and Lindgren,1961; Saucier and M i l l e r , 1961) but t h i s decline has also been shown to occur more gradually i n the l a r g e r , more compacted p i l e s i n Sweden (Annergren, D i l l e n and Vardheim, 1964). This d i f f e r e n c e i n the i n t e r n a l temperatures has been a t t r i b u t e d to the l a r g e r volume of chips i n the Swedish p i l e s which serves to i n s u l a t e the i n s i d e of the p i l e from environmental changes. The degree of compaction i s believed to be one of the factors responsible f or the i n t e r n a l temperature f l u c t u a t i o n s (Annergren, D i l l e n and Vardheim, 1964) . Greater compaction r e s u l t s i n higher average temperatures. Studies i n New Zealand on very small p i l e s c l e a r l y i l l u s t r a t e the e f f e c t of volume of chips and compaction on the temperature devleopment i n the p i l e . Butcher and Howard (1968) have shown that there are differences i n the temperature of winter p i l e s and summer p i l e s . The temperatures recorded i n a winter p i l e 9 feet on a 30 by 50 foot base, with self-compacted chips, were very e r r a t i c over the f i r s t seven weeks of storage. Reheating occurred, followed by several major f l u c t u a t i o n s i n temperature. The summer p i l e , which was 10 feet high on a 70 by 35 foot base and i n which the chips were compacted by crawler t r a c t o r , had temperatures which were not influenced by ambient conditions. The r i s e i n i n t e r n a l temperature was around 37°.C to 38 °C for most of the nine months of storage. It has been suggested that the r i s e i n temperature which occurs i n the outside stored chip p i l e s i s i n part due to the condensation of water vapour on the chips, the presence of f i n e s , the presence of metals and the - 16 -mi c r o b i a l a c t i v i t y within the p i l e (Shields, 1967; A l l e n , 1968; Chalk, 1968). No experiments have been done on any of these f a c t o r s . In large chip p i l e s a transfer of warm moist a i r from the lower regions of the p i l e to the upper cooler chips i s believed to occur. This trans f e r creates a chimney e f f e c t whereby the evaporation of moisture from the lower chips r e s u l t s i n cooler temperatures i n these regions while condensa-t i o n of water vapour on the chips i n the uppor portion of the p i l e s causes an increase i n temperature (Rothrock, Smith and Lindgren, 1961; Annergren, D i l l e n and Vardheim, 1964; Ljungqvist, 1965). Moisture d i s t r i b u t i o n i n chip p i l e s 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 moisture of the wood occurs during the storage period. Bjorkman and Haeger (1963) indicated that the r e l a t i v e humidity measured i n a p i l e stored f or 15 months averaged 98 to 100%. It i s generally accepted that the i n t e r i o r of a p i l e i s less moist than the outer regions owing to the high temperatures that are associated with the core of the p i l e (Rothrock, Smith and Lindgren, 1961; Annergren, D i l l e n and Vardheim, 1964; Bergman and Nilsson, 1966). Fungal a c t i v i t y may lead to an increase i n the moisture content of the chips to some extent. Increased fungal metabolism r e s u l t s i n the production of carbon dioxide and water (Lindgren and Eslyn, 1961) as end products of degradation of carbohydrates i n wood. P r e c i p i t a t i o n i s «an important source of moisture which can d r a s t i c a l l y a f f e c t the behaviour of the p i l e . Small p i l e s react to changes i n the r a i n f a l l (Butcher and Howard, 1968). The amount of moisture i n small - 17 -p i l e s i n the west coast 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 moisture content remained uniformly high during storage (Wright, 1954). The i n i t i a l moisture content of stored chips i s generally within the range of 45 to 50 percent, green weight b a s i s . With longer storage periods the moisture content becomes more or less uniform throughout the p i l e (Wright, 1954; Saucier and M i l l e r , 1961). A d d i t i o n a l moisture i n the surface of the p i l e i s contributed by r a i n f a l l or snow (Holekamp, 1958; Rothrock, Smith and Lindgren, 1961; Zak and Krauthauf, 1964). High temperatures and moisture are factors which contribute to brown chemical s t a i n i n g and are reported (Annergren, D i l l e n and Vardheim, 1964) to r e s u l t i n the deacetylation of hemicelluloses i n the wood. The pH of wood chips gradually decreases during storage. A c e t i c acid odour i s a very common phenomenom. This product i s found to r e s u l t from the deacetylation of the hemicelluloses i n the wood. Shields (1970) has shown that the pH of the wood dropped from 5.92 i n the fresh chips to 2.87 i n chips stored f o r 259 days. This pH affe c t e d the growth and type of microorganisms appearing i n the p i l e and the b a c t e r i a l i s o l a t i o n s dropped suddenly at pH 3.50 while the frequency of fungi i s o l a t i o n s was highest at thi s pH. Annergren, D i l l e n and Vardheim (1964) a t t r i b u t e the brown d i s -c o l o u r a t i o n of chips to the pH and the high temperatures. I t i s believed 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 responsible for the brown d i s -- 18 -colouration but that other chemical or enzymatic reactions are probably involved (Shields, 1970). 4. Fungi i s o l a t e d from wood chip p i l e s A wide v a r i e t y of fungi, belonging to a l l c l a s s e s , has been i s o l a t e d from chips. Some of these organisms cause wood decay while others s t a i n the wood. A large group of organisms has been i s o l a t e d whose behaviour on wood i s as yet unknown. The number of d i f f e r e n t fungi i s generally much larger i n chip storage than i n round wood storage per wood unit (Nilsson, 1965) and probably influences the rate and type of d e t e r i o -r a t i o n . Because of the high temperatures developed i n a chip p i l e a unique group of fungi i s found which i s able to survive at high temperatures. These fungi are c a l l e d thermophilic fungi. The l a r g e s t group of fungi i s o l a t e d belongs to the Fungi Imperfecti c l a s s and includes Trichoderma sp., Paecilomyces sp., Graphium sp.,Phialophora sp., Gliocladium sp., P e n i c i l l i u m sp., and A s p e r g i l l u s sp. (Lindgren and Eslyn, 1961; Bjorkman and Haeger, 1963; Nilsson, 1965; Bergman and Nilsson, 1966; Shields and U n l i g i l , 1968; Shields, 1970). Members of the genera Chrysosporium found i n chips during storage are among the most destr u c t i v e fungi. Chrysosporium lignorum, a thermotolerant fungus, was reported to cause 33 percent weight los s i n laboratory tests on pine sapwood a f t e r four months incubation at 25°C (Nilsson, 1965; Bergman and Nilsson, 1966). Another thermotolerant Fungus Imperfectus commonly i s o l a t e d i s - 19 -Asp e r g i l l u s fumigatus. Unlike Chrysosporium lignorum i t i s not highly d e s t r u c t i v e . The second commonest class of fungi found i n chip p i l e s i s the Ascomycetes, which includes Ceratocystis sp.causing s t a i n i n g of wood, Chaetomium •sp,. A l l e s c h e r i a sp,, Thermoascus sp. and Byssochlamys sp, (Nilsson, 1965; Bergman and Nilsson, 1966). Basidiomycetes are not very common i n outside chip storage except i n small p i l e s where the ambient conditions a f f e c t the behaviour of the p i l e , and i n p i l e s stored over long periods (Nilsson, 1965; Bergman and Nilsson, 1966). From two spruce p i l e s stored f o r 6 and 7 months r e s p e c t i v e l y , no Basidiomycetes were i s o l a t e d . From the p i l e s stored over a long period, 13 months, a number of Basidiomycetes were i s o l a t e d . Two of these were Fomes annosus and Odontia b i c o l o r (Nilsson, 1965). When the Basidiomycetes were present t h e i r frequency of i s o l a t i o n was very low. A heat tol e r a n t Basidiomycete, Ptychogaster species has been commonly i s o l a t e d from eastern Canadian chip p i l e s (Shields and U n l i g i l , 1968). It caused considerably more decay of pine sapwood when incubated at 37°C than at 27°C. The majority of the wood-decaying Basidiomycetes from wood chip p i l e s caused white rot i n laboratory tests (Nilsson, 1965). Peniophora gigantea and Polyporus species were the most commonly i d e n t i f i e d organisms i n t h i s group although other species such as Ptychogaster do cause white r o t . - 20 -Several of the Basidiomycetes have caused wood losses between 20-40% over 3 months (Nilsson-, 1965j Bergman and Nilsson, 1966). E s p e c i a l l y dangerous i n chip storage are those rot fungi which can t o l e r a t e high temperatures. Among these are Polyporus species and an unknown Basidio-mycete both i s o l a t e d i n Sweden (Nilsson, 1965). Few Phycomycetes have been i s o l a t e d and the commonest from coniferous p i l e s i s Mucor (Bjorkman and Haeger, 1963; Nilsson, 1965; Bergman and Nilsson, 1966). The damage caused by the Phycomycetes i s as yet unknown. Rhizopus has been i s o l a t e d from a Pinus r a d i a t a p i l e (Butcher and Howard, 1968). A large number of the thermophilic fungi are Ascomycetes but a few are Fungi Imperfecti. The Fungi Imperfecti class includes: Sporotrichum  thermophile Apinis and Humicola lanuginosa ( G r i f f o n and Maublanc) Bunce (Nilsson, 1965). Among the thermophilic 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 emersonii, Chaetomium thermophile La Touche and Thermoascus aurantiacus Miehe. Most of the i s o l a t e s of these fungi have come from softwood chip p i l e s (Nilsson, 1965; Bergman and Nilsson, 1966; Shields and U n l i g i l , 1968). L i t t l e i s known about the nature of the thermophilic fungi's attack on wood. I t has been demonstrated that A. t e r r e s t r i s caused a s o f t rot of hardwoods (Bergman and N i l s s o n 1967). However, th i s may not be the case i n softwoods. The nature of attack i s very important i n e l u c i d a t i n g the function of thermophilic fungi i n chip p i l e s . - 21 -Sporotrichum thermophile, A_. terres t r i s and Humicola sp. can u t i l i z e c a l l u l o s e as a sole source of carbon w h i l s t T_. aurantiacus cannot (Fergus, 1969). No such study has been done f or v a r i e t i e s of Byssochlamys sp. i s o l a t e d from chip p i l e s . I t i s believed that these thermophilic fungi are p a r t l y responsible f o r the high temperatures usually recorded i n chip p i l e s . No r e s u l t s of heat production by fungi on wood are a v a i l a b l e . Results from heat generation experiments i n other organic substrates l i k e hay, straw, compost and grain i n d i c a t e d that thermophilic fungi are mainly responsible for 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 s t e r i l i z e d straw with A s p e r g i l l u s fumigatus, a very common fungus i n chip p i l e s , and demonstrated that the temperature could r i s e from 25°C to 55°C during 38 hours. Fenstenstein, Lacey, Skinner, Jenkins and Pepys (1965) have shown that a large number of thermophilic fungi can r a i s e the temperature of straw to about 60°C i n a very short time. 5. Damage i n wood chip p i l e s Chips i n storage are damaged by the a c t i v i t y of micro-organisms, such as fungi, which ei t h e r s t a i n or degrade the wood and also by conditions which develop i n the p i l e such as high temperatures. Damage by micro-organisms leads to a reduction of q u a l i t y and/or quantity of the wood. - 22 -A common damage to wood chips i s s t a i n i n g . L i t t l e d e t e r i o r a -t i o n of this type occurs i n chips stored on the west coast of North America (Blackerby, 1958; Hensel, 1958; Burke, 1962). Blackerby (1958) reported d i s c o l o u r a t i o n and rot i n softwood chips stored for one to three years at m i l l s i n B.C. and Oregon. Staining occurred early and was confined to the outer uncompacted parts of the p i l e . Studies i n Georgia (Holekamp, 1958) i n d i c a t e d that s t a i n i n g to a depth of about one foot from the surface of the p i l e occurred a f t e r four months storage. D e t e r i o r a t i o n due to s t a i n i n g may be very minor i n pine chips a f t e r 4 months storage (Blackerby, 1963). Most of the blue'gray fungal d i s c o l o u r a t i o n s i n the pine chips were sit u a t e d i n the outer one to two feet of uncompacted sides of one p i l e a f t e r one month's storage (Rothrock, Smith and Lindgren, 1961; Saucier and M i l l e r , 1961). A softwood p i l e became dark i n colour a f t e r 9 to 12 months storage i n Nova Scotia (Robinson, 1963) and did not regain i t s colour a f t e r drying. Shields and U n l i g i l (1968) found i n s i g n i f i c a n t d i s c o l o u r a t i o n i n spruce and balsam f i r chips stored i n New Brunswick a f t e r 4 to 8 months storage. A non-fungal yellowish brown or orange d i s c o l o u r a t i o n was found to occur i n a pine chip p i l e i n the southeastern U.S.A. (Lindgren and Eslyn* 1961^ Rothrock, Smith and Lindgren,1961; Saucier and M i l l e r , 1961) w i t h i n one month of storage. The d i s c o l o u r a t i o n was located p r i m a r i l y around r e s i n ducts i n the heartwood areas of pine chips. Brownish disco l o u r a t i o n s have been noticed i n the i n t e r i o r of several softwood p i l e s and have been a t t r i b u t e d to chemical changes i n the p i l e (Saucier and M i l l e r 1961; Shields and U n l i g i l 1968). The development - 23 -of the brown s t a i n i s progressive with increased storage and appears to be more widespread near the bottom than near the top of one p i l e (Young, 1961). Aft e r 4 months of summer storage and 5 months of winter storage, heavily stained pine chips were found to be s o f t and brashy (Lindgren and Eslyn, 1961; Rothrock, Smith and Lindgren, 1961; Saucier and M i l l e r , 1961). This condition has been a t t r i b u t e d to the a c t i v i t y of s o f t r o t fungi. In one study i t was found that the number of decay fungi i s o l a t e d from softwood chips i n the bottom h a l f of a large p i l e was greater than the number i s o l a t e d from other parts of the p i l e (Shields and U n l i g i l , 1968). Losses i n wood substance vary from one p i l e to another as work i n Sweden and southern U.S.A. i n d i c a t e s . Minor reductions i n s p e c i f i c gravity have been noticed i n pine chips stored f o r up to two months (Anon, 1961). The reduction i n the s p e c i f i c g r a v i t y corresponded to the lowering of temperature, to increase i n moisture content and perhaps to depletion of oxygen. A f t e r f i v e month's storage (Rothrock, Smith and Lindgren, 1961; Saucier and M i l l e r , 1961) there was an approximate average loss of 7% of wood. Losses i n the compacted centres of the p i l e s were low (about 4%). Even a f t e r 12 months storage, greater reductions (about 9%) occurred i n the outer three feet of the sloping sides of the p i l e where there was the l e a s t compaction and where v i s i b l e chip d e t e r i o r a t i o n r e s u l t i n g from growth of s t a i n and decay fungi was evident (Lindgren and Eslyn, 1961; Rothrock, Smith and Lindgren, 1961). - 24 -Losses i n Swedish spruce chip p i l e s (Annergren, D i l l e n and Vardheim, 1964; Nordin and Selleby, 1965) were reported to vary from two to f i v e percent for storage periods of four to f i v e months. At temperatures of 20°C to 30°C, losses of 5 to 10 percent occurred i n pine chips (Bergman and Nilsson, 1966) stored for seven months i n Sweden while l i t t l e or no loss occurred i n frozen chips during t h i s time. A loss of approximately 11 percent i n the s p e c i f i c g r a v i t y of brown and deteriorated balsam f i r and spruce chips was determined from samples which had been sotred for j u s t over nine months near the bottom of a p i l e i n New Brunswick (Shields and U n l i g i l , 1968). Further decreases i n the s p e c i f i c g r a v i t y of pine (Burks, 1962; Somsen, 1962; Selleby, 1965) spruce (Annergren, D i l l o n and Vardheim, 1964; Selleby, 1965) and b i r c h (Selleby, 1965) chips were s l i g h t beyond f i v e months. C MATERIALS AND METHODS 1. The experimental wood chip p i l e The experimental chip p i l e , used i n the study of the d i s t r i b u t i o n of thermophilic and thermotolerant fungi, was located on the grounds of the In t e r c o n t i n e n t a l Pulp Company Ltd., Prince George, B r i t i s h Columbia. I t was constructed on a l e v e l s i t e and oriented l o n g i t u d i n a l l y east-west. - 25 -The chip p i l e matrix consisted of white spruce and lodgepole pine into which the chip samples were ins e r t e d . The chips were from sawmill residues supplied by 15 sawmills. The p i l e contained 2,900 units of chips, each unit being 1,080 kg. of wood, chips. The r a t i o of spruce to pine was two to one. The b u i l d i n g of the p i l e took three weeks and data c o l l e c t i o n s t a r t e d on the second week of June, 1968. The south face of the p i l e was uniformly exposed to the weather throughout these studies so that 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 conditions on the e n t i r e front face would be constant. The chips were pneumatically delivered to the p i l e s i t e , then a c a t e r p i l l a r t r a c t o r was used to l e v e l the p i l e . This 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 samples and the compaction of the p i l e . The p i l e was 75ft (23.2m.) wide at bottom 28ft. (8.6m.) wide at the top and 25ft (7.7m.) high. The length of the p i l e was 400ft (124.m). Figures 1 and 2 give the dimensions of the p i l e i n the east and west and the south views. Samples of chips i n "flexmesh" p l a s t i c bags were incorporated into the p i l e during construction. For each treatment, sample bags were placed i n s i x d i f f e r e n t p o s i t i o n s within the p i l e as shown i n the east-west view of Figure 1. The spacing between the centres of any two sample bags was 8 f t . (2.4m.). The p i l e was constructed to comprise four i n d i v i d u a l sections, each se c t i o n containing a l l the treatments required for a study of one time period. - 2 6 -F i g . 1. East and West view of the chip p i l e showing locat ion of chip samples 1 to 6. F ig . 2. South view section of the chip p i l e . - 27 -The four time periods ( F i g . 3) were 3, 6, 12 and 24 months but only the f i r s t three were investigated i n t h i s study. The sample bags were r e t r i e v e d by c a r e f u l l y breaking up each se c t i o n of the p i l e at the required time i n t e r v a l ( Fig. 4). To reduce the p o s s i b i l i t y of environmental changes to the remaining sections, the samples i n each section were separated by 20ft. (6.2m.) long sections of matrix chips. When a s e c t i o n was removed from the p i l e , the p o s i t i o n and slope of the new face was adjusted to conform to those of the o l d . Samples removed from the p i l e were placed i n p l a s t i c bags and transported under r e f r i g e r a t i o n to the laboratory i n Vancouver where they were stored at 1°±0.1°C u n t i l used. 2. Sampling p o s i t i o n s Each of the four sections of the treatments placed i n s i x d i f f e r e n t middle, inner bottom, outer top, outer outer p o s i t i o n s were on the south face the wood chip p i l e ( F i g . 3) had a l l pos i t i o n s which were inner top, inner middle and outer bottom (Fig. 1). The of the p i l e . 3. Treatments The treatments were as follows: 1. Sample bags of pine chips 2. Sample bags of spruce chips 3. Sample bags of spruce chips containing a core bag of f i n e s Fig. 4. Retrieval of chip samples. - 29 - i 4. Sample bags of spruce chips containing a core bag of s t e r i l i z e d spruce chips 5. Sample bags of spruce chips containing a core bag of spruce chips inoculated with Ptychogaster sp. Each treatment was r e p l i c a t e d twice. 4. Preparation of wood chip samples White spruce and lodgepole pine were used i n preparing the samples. S u f f i c i e n t sound wood for the e n t i r e experiment was obtained from several logs i n the Prince George area. The wood was then chipped i n the m i l l . I n d i v i d u a l species were w e l l mixed to give homogeneous samples using the mixing f a c i l i t i e s of Rayonier Inc. i n Shelton, Washington. These samples were then transported back to Prince George i n a r e f r i g e r a t e d truck. The i n d i v i d u a l samples were?prepared on the s i t e by weighing 58 l b . (26.1 kg.) of chips into each flexmesh bag. Samples were taken from each bag, put i n a screw-capped b o t t l e , and weighed. These small samples were brought in t o the laboratory to determine the moisture content. A "core' sample bag" technique proposed by J.V.Hatton (1970) was used to prepare the majority of the samples. The technique consists of p l a c i n g a smaller "flexmesh" p l a s t i c bag containing a test sample into a bigger bag containing chips so that the small bag e s s e n t i a l l y forms the c e n t r a l core. Any influence due to the core must then pass outward and a f f e c t the sample chips. - 30 -The main sample hags weighed between 58 l b . (26.1kg) and 60 l b . (27.kg.) green weight of chips while the core samples weighed between 8 l b . (3.6kg.) arid 10 l b . (4.5kg.). The core bags f o r treatments 4 and 5 were prepared by autoclaving 10 l b . (4.5 kg.) spruce chips i n a wooden container at 15 p s i for 90 minutes. These were put a s e p t i c a l l y into "flexmesh" p l a s t i c bags which had been s t e r i l i z e d by d i p p l i n g them i n 5% phenol for a number of hours, followed by a r i n s e i n s t e r i l e water. The core bags i n treatment f i v e were inoculated with cultures of a Ptychogaster sp., growing on 2% malt agar, by c a r e f u l l y removing the colony from the p e t r i dish with a s t e r i l e spatula and putting i t i nto the p l a s t i c bag containing the s t e r i l e chips. The flexmesh p l a s t i c bags were placed i n s t e r i l e polyethylene bags and incubated f o r two and a hal f months at room temperature. They were then put i n another thick p l a s t i c bag a f t e r the incubation period and transported i n a r e f r i g e r a t e d truck to the s i t e of the p i l e . 5. Measurement of environmental conditions The environmental factors measured were temperature i n the wood chip p i l e , moisture content of wood chips and a c i d i t y of wood chips. a. Temperature i n the wood chip p i l e Thermocouples were inserted into the 2 year s e c t i o n of the p i l e to measure the temperatures i n every sample bag (60 bags). The thermal 1 gradient across the p i l e face to the depth of 96 i n . (2.4m) was also measured - 31 -on the south and north faces by plac i n g thermocouples at the surface and at depths of 3, 6, 12, 24, 48 and 96 i n (0.08, 0.15, 0.31, 0.61, 1.22 and 2.43m). In a l l , 74 thermocouples were used. Temperature readings were taken at d a i l y i n t e r v a l s f o r three months a f t e r which time twice weekly recordings were made. A l l 74 thermocouples were connected to a master c o n t r o l panel s i t u a t e d within an ins u l a t e d hut near the 2 year s e c t i o n of the p i l e . High temperatures are more favourable for the growth and reproduction of thermophilic fungi (Cooney and Emerson, 1964). Therefore the t o t a l number of fungi i s o l a t e d from a sampling bag a f t e r a given sampling period could, 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 determining a measure of the temperature regime corresponding to each bag, temperature values measured at a l l the bag p o s i t i o n s i n the 24 month se c t i o n of the chip p i l e were considered ap p l i c a b l e to the corresponding p o s i t i o n s i n the 3, 6 and 12 month sections of the chip p i l e ; also weekly average temperature values f.or each bag p o s i t i o n were summated f o r the period during which the respective bags were i n the p i l e and these summations c a l l e d the " t o t a l temperatures". These t o t a l temperatures were then c o r r e l a t e d with the t o t a l counts of fungi f o r each bag. - 32 -b. Moisture content of chips The change i n moisture content was determined by comparing the moisture content determination at the time t = 0 with those determined a f t e r 3, 6 and 12 months storage. To determine the moisture content the sample chips were f i r s t weighed and then oven-dried to constant weight. The d i f f e r e n c e i n weight represents the actual weight of water i n the chips. Using the formula M.C. % = Weight of water i n chips i n n . _^ . ^ ^ i i J — — s — r r ~ -7—, ——r~7^— 100 the percentage moisture content was c a l c u l a t e d . Oven dry weight of chips c. A c i d i t y of wood chips Five to lOg of chips were ground i n a Wiley M i l l (Model ED-5) to pass a 1-mm screen. One gram of wood meal (oven-dry basis) was s t i r r e d i n i t i a l l y f o r 15 seconds with 25-ml d i s t i l l e d water (of pH 6.0 - 6.5) and was determined with a Corning pH meter (Model 7). 6. I s o l a t i o n of fungi a. Sampling procedure A random sample of 100 chips was taken from each 60 l b . sample bag. These were surface s t e r i l i z e d i n d i v i d u a l l y by quickly passing them through a flame. A piece was removed from the centre of the chips using s t e r i l e bone forceps. This piece was s p l i t into two halves, and then surface s t e r i l i z e d again and plated on acid malt agar composed of 2% malt, 2% agar - 33 -and 0.5% malic a c i d . One piece on one plate was incubated at 45°C and the other piece on another plate at 25°C. Each p e t r i dish contained ten pieces of chips (Fig. 6). The plates were examined d a i l y and colonies growing on them were sub-cultured onto 2% malt 2%agar s l a n t s . The part of the colony that remained was removed and discarded. This process was repeated u n t i l drying of the agar prevented further i s o l a t i o n . b. I s o l a t i o n and grouping of fungi At the end of the sampling, a l l the sl a n t cultures were examined under the binocular microscope and were grouped into broad categories, mainly generic. The slants were then counted and recorded. A f t e r a thorough examination of a selected number of cultures from each group the remainder of the tubes were discarded. These representative cultures were then examined using a phase contrast microscope and i d e n t i f i e d wherever p o s s i b l e . The following categories were used. Group 1. This group was comprised of a s p e r g i l l u s type fungi which had no perfect stage. The culture was l i g h t green to green. The majority of the i s o l a t e s i n th i s group were A s p e r g i l l u s fumigatus. Group I I . This group was made up of a l l types of fungi which had p e n i c i l l a t e conidiophores. Some produced ascocarps while others did not. The most common fungus was Byssochlamys emersonii. - 34 -Figure 6. Byssochlamys emersonii growing out of chips on 2% malt and 0.5% malic acid agar. - 35 -Group I I I . This group consisted of a species with white thread-like mycelium. It had no asexual stage, grew slowly i n culture and had l i g h t brown ascocarps. The fungus i s s t i l l u n i d e n t i f i e d but resembles Byssochlamys. Group IV. This group was made up of a s i n g l e f a s t growing species which was i d e n t i f i e d as Chrysosporium pruinosum. The .culture was cream i n colour and powdery on top. Group V. The hyphae of the fungi i n t h i s group were hyaline and had no conidiopho res. The c l e i s t o t h e c i a which were produced i n large numbers were reddish brown and somewhat angular. This fungus grew very f a s t i n c u l t u r e . Group VI. This group was made up of fungi which had white to cream mycelium. They produced hyaline conidia which were pyriform to ova l . Some produced black c l e i s t o t h e c i a . I t contained both A l l e s c h e r i a and Sporotrichum. Group VII. This group contained the yeast l i k e organisms. They were infreque n t l y i s o l a t e d and were present mainly i n the samples stored f o r three months. 7. Determination of wood chip weight loss The weight losses were determined by comparing the ca l c u l a t e d oven-dry weight of chip samples at storage time t = 0 with those measured a f t e r 3, 6 and 12 months storage. The weight losses were expressed as percent of o r i g i n a l oven-dry weight of wood using the formula percent weight loss = ' i - Q o x 0 r i g i n a x ovendry weight of wood - ovendry weight of wood a f t e r test O r i g i n a l ovendry weight of wood. - 36 -D RESULTS 1. L i s t of fungi i s o l a t e d The following most commonly occurring fungi were i d e n t i f i e d : A s p e r g i l l u s fumigatus Fresenius A l l e s c h e r i a t e r r e s t r i s Apinis Cephaslosporium t e r r e s t r e Asexual state of A. t e r r e s t r i s . Byssochlamys emersonii S t o l k - A p i n i s . Chrysosporium pruinosum (Gilman and Abbot) Comb. Nov. Humicola lanuginosa ( G r i f f o n and Maublanc) Bunce. Sporotrichum thermophile A p i n i s . Thermoascus aurantiacus Miehe. In a d d i t i o n to the fungi l i s t e d above, 21 d i s t i n g u i s h a b l e fungi i s o l a t e d from the p i l e were not i d e n t i f i e d . 2. D i s t r i b u t i o n of common thermophilic and thermotolerant fungi The most common thermophilic fungus occurring i n the p i l e was B_. emersonii ( F i g . 7), which occurred mainly i n the i n t e r i o r region of the p i l e where temperatures were high. The numbers of i s o l a t e s of t h i s fungus increased with time of storage. - n — i . to to o o c rs rt T J 0 (J) H 0 z m CO CO c+ TO o X* TO O — N J C O K O l O N I O O O — o o o o o o o o o g F U N G A L C O U N T / l O O C H I P S ~ o o c F U N G A L C O U N T / l O O C H I P S •^4 0 0 0 0 0 0 o o o g ^ o F U N G A L C O U N T / l O O C H I P S - 38 -A. t e r r e s t r i s and S_. thermophile (Fig. 7) were commonly i s o l a t e d from the inner region of the p i l e and were most common i n the samples stored for three months. Thereafter, t h e i r numbers decreased with time of storage. _T. aurantiacus (Appendix 1) did not occur as frequently as the other fungi but would grow f a s t out of the wood i f i t were present. I t was e r r a t i c a l l y i s o l a t e d from both inner and outer regions of the p i l e . A. fumigatus (Fig. 7) was confined to the outer region of the p i l e and appeared i n large numbers. I t frequently occurred i n the outer top p o s i t i o n . Its numbers remained stable throughout the period of storage of the chips. (Z. pruinosum (Appendix 1) was more commonly i s o l a t e d from spruce than from pine chips. The highest number of i s o l a t i o n s came from outer top and outer middle p o s i t i o n s of the p i l e . I t was o c c a s i o n a l l y i s o l a t e d from the outer bottom p o s i t i o n . The numbers of i s o l a t e s of C^. pruinosum remained stable throughout the duration of storage of the chips. 3. Environmental factors a. Temperature i n the wood chip p i l e Changes i n temperature i n the s i x posit i o n s i n the p i l e f o r 34 days and 80 weeks are shown i n Figures 8 and 9 r e s p e c t i v e l y . ° F 140.0-130.0-120.0-110.0-100.0-90.0-UJ DC D h < CE UJ Q. 80.0-70.0-UJ 60.0-h 50.0- 1 40.0- 4.4 30.0-20.0-10.0-1.1 -6.7 •12.2 INNER BOTTOM POSITION OUTER BOTTOM POSITION INNER MIDDLE POSITION OUTER MIDDLE POSITION INNER TOP POSITION OUTER TOP POSITION Fig. 8. Daily temperature curves f o r the s i x positions in 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 chips. j i ~T~ 6 —J— 22 I 10 14 18 D A Y S 26 I 30 I 34 - 40 -The inner region of the p i l e had higher temperatures than the outer region. In the inner region, the highest temperatures were at the top followed by the middle and the bottom. The r i s e i n temperature at the top was very rapid and attained the maximum temperature of 60.6°C i n 19 days. Two weeks l a t e r the temperature had dropped to 49.4°C. The temperature trend was the same for the inner middle p o s i t i o n as the inner top p o s i t i o n but temperatures were never as high. The maximum temperature was 54.4°C and was attained t h i r t e e n days a f t e r the b u r i a l of the bags. The drop i n temperature was gradual, going from 54.4°C to 46.1°C i n two weeks. Low temperatures were recorded f o r the inner bottom p o s i t i o n of the p i l e compared to the other i n t e r i o r regions. A longer time was required to a t t a i n a maximum temperature of 42.5°C. Temperatures were stable i n t h i s part of the p i l e . In the outer regions of the p i l e temperatures fl u c t u a t e d a great deal, and except f or the outer top p o s i t i o n s , temperatures never rose above the i n i t i a l temperatures of the p i l e . The highest temperature recorded i n the outer region was 29.4°C at the outer top p o s i t i o n . Temperatures were r e l a t i v e l y high f o r the f i r s t three months a f t e r which there was a rapid drop. Temperatures were low during the l a t e f a l l , winter and early spring but started to r i s e again i n the summer months, though not a t t a i n i n g the values of the previous summer. In the winter months ambient temperature af f e c t e d the inner top p o s i t i o n more than the other op o C 140.0-130.0-120.0-110.0-100.0-90.0-80.0-LU CC D h < DC 70.0-LU CL >^ 60.0-LU h 50.0-40.0-30.0-20.0-10.0-INNER BOTTOM POSITION OUTER BOTTOM POSITION INNER MIDDLE POSITION OUTER MIDDLE POSITION INNER TOP POSITION OUTER TOP POSITION Temperature curves for the s ix posit ions in the chip p i l e during 80 weeks of storage of chips. o.o 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 \ W E E K T T" 50.0 55.0 60.0 65.0 70.0 75.0 80.0 Fig. 10. Average monthly ambient temperature in Prince George. - 43 -p o s i t i o n and the inner middle temperatures were sometimes higher than those of the inner top p o s i t i o n . Temperatures i n the outer regio followed the ambient temperature for most of the time (Fig. 9, 10). Freezing temperatures were common. Temperature r i s e was noticed i n the summer but was only marked i n the outer top p o s i t i o n of the p i l e ( F i g . 9). b. Moisture content of wood chips No d e f i n i t e pattern of moisture content existed for a l l the treatments or for the p i l e as a whole. The moisture content of the samples was highly v a r i a b l e (Table 1). Spruce always had a higher moisture content than pine. The chips i n the outer p o s i t i o n s had higher moisture content than the chips i n the inner p o s i t i o n u n t i l the "wet lens " e f f e c t developed at the inner top p o s i t i o n , thus b i a s i n g the moisture content values of the chips i n the -inner p o s i t i o n s i n the l a t e r time periods. Moisture, generally, must have been adequate f o r fungal development. - 44 -Table 1. F i n a l moisture content of wood chips. Treatment 3 months % M. C. 6 months % M. C. 12 months % M. C. Pine (1) Outer samples, 1, 3, 5 42.6 Inner samples 2, 4, 6 33.6 Spruce (2) Outer samples 1, 3, 5 64.7 Inner samples 2, 4, 6 58.6 S t e r i l i z e d spruce (4) Outer samples 1, 3, 5 70.8 Inner samples 2, 4, 6 65.6 Inoculated spruce (5) Outer samples 1, 3, 5 64.9 Inner samples 2, 4, 6 61.3 39.2 38.1 66.2 56.4 63.2 67.5 61.6 76.7 46.7 39.7 65.6 72.0 63.9 72.9 66.4 74.4 - 45 -c. A c i d i t y of wood chips Measurements of a c i d i t y of wood chips were taken only f o r the spruce and pine treatments except for samples i n the outer psotions of the f i r s t sampling period (Table 2). Table 2. F i n a l a c i d i t y of wood chips Storage periods Pine Spruce Months Outer 1, 3, 5 Inner 2, 4, 6 P o s i t i o n P o s i t i o n Outer 1, 3, 5 Inner 2, 4, 6 P o s i t i o n P o s i t i o n 0 4.80 4.80 5.40 5.40 3 4.60 5.00 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 very marked but generally followed the i n i t i a l pH of spruce and pine which indi c a t e d that the pH of spruce was higher than pine. The pH of the inner samples was lower than that of the outer samples. The change i n pH with duration of storage was e r r a t i c . pH generally went down a f t e r s t o r i n g the wood for three months but rose again a f t e r s i x months and continued to increase. The behaviour of the inner samples of - 46 -spruce did not follow the general pattern. pH decreased up to the s i x months' storage and started to increase only at twelve months. 4. Weight losses of wood chips The weight losses of wood chips are summarized i n Table 3. Weight losses were lower i n the outer region of the p i l e than i n the inner region. This d i f f e r e n c e was s i g n i f i c a n t at 5%. There was no s i g n i f i c a n t d i f f e r e n c e between treatments. Weight losses increased from three to s i x months, the d i f f e r e n c e being s i g n i f i c a n t at 5%. Although weight losses at twelve months were lower than weight losses at s i x months, the d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t . Table 3. F i n a l weight loss of samples i n percent P o s i t i o n Percent weight loss 3 months 6 months 12 months Outer: 1 , 3 , 5 1.1 2.0 1.9 Inner: 2 , 4 , 5 2.4 4.0 3.2 - 47 -5. Evaluation of treatments The treatments were analyzed on the t o t a l count of thermo-p h i l i c and thermotolerant fungi i s o l a t e d at 45°C following, incubation. The t o t a l count of fungi i s the number of fungi i s o l a t e d from one hundred chips. The treatments (Tables 4, 7) generally had no e f f e c t on the d i s t r i b u t i o n of fungi i n the p i l e . I t was only at the s i x months sampling period that d i f f e r e n c e s existed between treatments. P r e - i n o c u l a t i n g spruce with Ptychogaster sp had no e f f e c t on the d i s t r i b u t i o n of the fungi. There were also no differences between pine and spruce. The length of storage of chips (Tables 6,7) had a s i g n i f i c a n t e f f e c t on the d i s t r i b u t i o n of the fungi. Fungal population increased from the three months period to the s i x months period and remained constant between the s i x months and the twelve months periods. The p o s i t i o n of the samples i n the p i l e (Table 5) played a dominant r o l e i n the changes i n numbers of the fungal population. Most of the fungi were i s o l a t e d from the inner bottom and outer top po s i t i o n s of the p i l e i n the f i r s t three months. Examination of the chips a f t e r s i x months storage showed a large increase i n the t o t a l fungal population i n the inner upper and the inner middle p o s i t i o n s although increases occurred at a l l p o s i t i o n s . Changes i n t o t a l fungal population did not occur a f t e r twelve months storage. - 48 -The evaluation of the treatments was c a r r i e d out i n two steps using the analysis of variance. The analysis of the three months samples was done separately because a treatment which was not examined i n the subsequent time periods was included i n th i s a n a l y s i s . The second analysis was f o r the four treatments at the three time periods. Table 4. Mean fungal 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 chips f o r three months. Treatments Fungal count Pine 44 Spruce 55 Fines incorporated into spruce 65 S t e r i l i z e d spruce 53 Inoculated spruce 43 In the three months storage (Table 4) diff e r e n c e s between treatments were not s i g n i f i c a n t but diff e r e n c e s between po s i t i o n s (Table 5) were s i g n i f i c a n t at -5%. No s i g n i f i c a n t i n t e r a c t i o n existed between t r e a t -ments and p o s i t i o n s . A Duncan's multiple range test (Table 5) c a r r i e d out on the mean fungal counts f o r po s i t i o n s showed s i g n i f i c a n t d ifferences at 5% between fungal counts from inner bottom, outer top po s i t i o n s and a l l the other - 49 -p o s i t i o n s . The highest count of fungi was from inner bottom p o s i t i o n followed by the outer top p o s i t i o n . Table 5. Average number of fungi i s o l a t e d per p o s i t i o n a f t e r three months storage. * P o s i t i o n Mean Fungal Count Inner top Outer bottom Inner Middle Outer middle Outer top Inner bottom 24 28 39 45 79 99 * Any two p o s i t i o n s means sidescored by the same l i n e are not s i g n i f i c a n t l y d i f f e r e n t at 5% l e v e l . In the second analysis s i g n i f i c a n t d i f f e r e n c e at 5% existed between po s i t i o n s and time but not between treatments. There were s i g n i f i c a n t i n t e r a c t i o n s between a l l combinations of these factors except time and treatments (Table 6). - 50 -Table 6. Fungal counts at d i f f e r e n t storage times at the d i f f e r e n t p o s i t i o n s . P ositions Storage period i n months 3 6 12 Outer bottom 32 67 • ' 73 Inner bottom 99 119 108 Outer middle 48 68 73 Inner middle 41 112 100 Outer top 79 113 105 Inner top 25 118 100 The population of fungi changed with time of storage but not with the treatments. The s i g n i f i c a n t d i f f e r e n c e s existed between the treatments stored f o r 6 months but not for 3 or 12 months (Table 7). Table 7. Fungal counts at d i f f e r e n t storage times with d i f f e r e n t treatments. These are average f o r 6 p o s i t i o n s . Treatments Storage period i n months 3 6 12 Pine 44 82 92 Spruce 55 107 93 S t e r i l i z e d spruce 65 116 96 Inoculated spruce 53 94 92 - 51 -6. Relationships between va r i a b l e s measured Figure 11 shows the r e l a t i o n s h i p between t o t a l fungal count and moisture content. A simple regression analysis i n d i c a t e d that there was no s i g n i f i c a n t c o r r e l a t i o n between these v a r i a b l e s ( r = 0.014). Figure 12 shows the r e l a t i o n s h i p between t o t a l fungal count and t o t a l temperature. Figure 13 shows the r e l a t i o n s h i p between t o t a l fungal count and weight l o s s . A mult i p l e regression analysis on the data showed s i g n i f i c a n t c o r r e l a t i o n (R 2 = 0.34) between fungal count, t o t a l temperature and weight loss (Table 8). Table 8. Summary of regression analysis of t o t a l temperature and weight loss on t o t a l fungal count v a r i a b l e R-' TQ ^ temperature and weight loss 0.34 17.8 * temperature 0.29 28.9 * The f i n a l pH of the wood chips (Table 2) was not r e l a t e d to any of the other factors measured. pH varied from 4.74 to 5.57. It i s concluded that temperature, weight loss and fungal count are r e l a t e d . I t i s not cl e a r which of these f a c t o r s i s the independent v a r i a b l e . - 52 -162.9 • UJO^ 130.3 -DC h 3 Z 97.7 H ill 65.1 • o-z * u 32.5 -18 33 48 63 78 93 108 123 138 153 168 183 T O T A L F U N G A L C O U N T 11. The relationship between total fungal count and moisture content of chips. < h 0 h lil cc D < cc LU Q. h 4533 -3782 • 3031 2280 1530 17 31 44 58 71 86 99 113 127 141 155 168 T O T A L F U N G A L C O U N T 12. The relatinship between total fungal count and total temperature. 7.7 6.2 -4.7 3.2 1.8 • 17 31 44 58 72 86 99 113 127 141 155 168 T O T A L F U N G A L C O U N T 13. The relationship between total fungal count and wood weight loss. - 53 -E DISCUSSION 1. Fungi i n the chip wood p i l e This part of the study i n d i c a t e s that thermophilic and thermotolerant fungi occupy d i f f e r e n t regions of the chip p i l e ( F i g . 7). The thermophilic fungi mainly inhabit the inner regions w h i l s t the thermotolerant fungi are found i n the outer regions of the p i l e . Any d i s t r i b u t i o n a l and successional changes of fungi i n the p i l e r e s u l t from tenperature "preferences" of the d i f f e r e n t fungi. Bergman and Nilsson (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 between fungi and the temperatures i n the p i l e . This was not a s p e c i f i c c o r r e l a t i o n based on any fungal group. In the present study a c o r r e l a t i o n (0.29) i s shown between fungal i s o l a t i o n s at 45°C and temperatures i n the chip p i l e (Table 8). The t o t a l number of fungi does not decrease with length of storage of chips even a f t e r storage of the chips f o r twelve months. The low unfavourable temperatures from the s i x months' storage period to the twelve months period did not a f f e c t the population of the fungi. The reduction i n the numbers of fungi from the s i x months' sampling period to the twelve months sampling period was not s i g n i f i c a n t . This f i n d i n g i s contrary to that of Shields (1970), who showed that the numbers of fungi decreased sharply - 54 -with stonage time and even f e l l to n e g l i g i b l e values a f t e r seven and one-half months' storage of the chips. No explanation i s offered f o r this sharp de c l i n e , but i t might imply that the chips were s t e r i l i z e d a f t e r being at the high temperatures for that length of time. A l t e r n a t i v e l y , i t could s i g n i f y that the low i s o l a t i o n temperatures which Shields used were responsible for h i s f a i l u r e to i s o l a t e the thermophilic fungi which would have been present i n the chips even a f t e r that period of storage. The population of fungi i n the hotter region of the p i l e i s i n i t i a l l y low and comprised of A. t e r r e s t r i s , S_. thermophile and B. emersonii. With storage beyond three months, the population of fungi increases. The e n t i r e increase is accounted for by increase i n _B. emersonii population. The population of A. t e r r e s t r i s and S_. thermophile decreases, and probably they are replaced by B^ . emersonii. In succession, therefore, B_. emersonii follows A. t e r r e s t r i s and J3. thermophile. The high frequency of occurrence of the three fungi i s confirmed by Bergman and Ni l s s o n (1966) but no mention i s made of t h e i r succession. A. fumigatus i s an early c o l o n i z e r of the outer regions of the p i l e and i t may not be replaced by other fungi. In t h i s study the high i n i t i a l population of t h i s fungus remained at the same l e v e l throughout the three sampling periods. No successional pattern i s shown f o r <Z. pruinosum except that i t occurs i n the outer top and middle p o s i t i o n s . Although i n comparison to A. fumigatus i t s numbers were low, i t i s stable i n those areas where i t appeared. The chips i n these areas, however, were not heavily degraded, and unlike the findings of Bergman and N i l s s o n (1966), the decaying a c t i v i t y of t h i s organism must have been low i n the spruce-pine - 55 -chip p i l e at Prince George. The cause of th i s low decaying a c t i v i t y i s not cle a r but may be due to competition from fungi l i k e A. fumigatus. The high wood substance loss i n the inner region of the chip p i l e i s a t t r i b u t e d p a r t l y to the a c t i v i t y of the thermophilic fungi i n the p i l e . 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 fungal counts and weight losses of wood (Fig. 13 Table 8) although t h i s c o r r e l a t i o n was not as strong as the c o r r e l a t i o n between fungal counts and temperature (Fig. 12 Table 8) i n the p i l e . The thermophilic and thermotolerant fungi through t h e i r metabolic a c t i v i t y must be p a r t l y responsible f o r thermogenesis i n the chip p i l e s . Thermogenesis through fungal a c t i v i t y has not been shown i n wood, but thermogenetic c a p a b i l i t i e s have been shown for A. fumigatus i n other plant materials ( C a r l y l e and Norman, 1941). The inner bottom region probably had the highest fungal counts because the temperature was mild and s t a b l e . Because of the mildness and s t a b i l i t y t h i s area of the p i l e can harbour both thermophilic and thermotol-erant fungi. A s i m i l a r observation was made by Shields and U n l i g i l (1968) but they thought that the foundation of o l d chips contributed to the greater numbers of fungi i n that region. Ptychogaster sp. does not spread out of the inoculum bag into the chips. I t was not i s o l a t e d from the chip samples. Shields and U n l i g i l (1968) i s o l a t e d Ptychogaster sp. from balsam f i r and spruce chip samples i n the upper and middle layers of the p i l e . This fungus has not been i s o l a t e d from other p i l e s (Shields, 1967). I t i s pos s i b l e that Ptychogaster - 56 -sp. i s not able to compete with the thermophilic and thermotolerant species of fungi. Its i n a b i l i t y to compete i s i n f e r r e d by Shields and U n l i g i l (1968) who wrote that the fungus cannot stand temperatures above 50°C. 2. Temperature i n the wood chip p i l e A sharp r i s e i n temperature occurs s h o r t l y a f t e r the construction of most chip p i l e s . This generally takes two weeks to a t t a i n maximum temperature, and, depending upon the species of wood which i s p i l e d , the volume of the p i l e and the extent of compaction, the temperature w i l l vary from 37°C to 69°C (Bjorkman and Haeger, 1963; Annergren, D i l l o n and Vardheim, 1964; Ljungqvist, 1965; Shields and U n l i g i l , 1968; Butcher and Howard, 1968). A s i m i l a r sharp r i s e occurred i n the spruce-pine chip p i l e i n Prince George. In t h i s study, however, i t was noticed that the temperature r i s e was not the same for a l l the po s i t i o n s i n the inner regions of the p i l e , nor d i d i t take the same time f o r a l l the po 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 va r i e d from two to three weeks. The pattern of the decline i n temperature i s the same as given by other workers (Bjorkman and Haeger, 1963; Annergren, D i l l e n and Vardheim, 1964; Shields and U n l i g i l , 1968). Although di f f e r e n c e s i n temperature occur between p i l e s composed of d i f f e r e n t wood species, no differences were noticed between samples of spruce and pine. Ljungqvist (1965) found that as much as 5°C d i f f e r e n c e can - 57 -e x i s t between pine and spruce p i l e s . There are, however, species such as Douglas-fir (Hensel, 1958) where no r i s e i n temperature occurs during storage. This may be due to high e x t r a c t i v e content of the wood which i n h i b i t s the growth of fungi. Temperatures i n the outer region of the Prince George wood chip p i l e were much lower than those reported for southern pine species (Rothrock, Smith and Lindgren, 1961; Saucier and M i l l e r , 1961; Davies, 1963) although the compaction i n a l l these cases must have been the same. Such a d i f f e r e n c e can be accounted f o r by the differences i n the ambient temperatures of the d i f f e r e n t l o c a t i o n s . Comparison of the ambient c l i m a t i c conditions (Fig. 10) and the temperature changes ( F i g . 9) i n the wood chip p i l e studied shows that the ambient conditions had a strong e f f e c t on the changes i n temperature. Seasonal changes aff e c t e d the p i l e i n Prince George more than the other studies i n d i c a t e . Generally, the outer regions freeze during the winter months, but even when the inner temperatures drop they do not approach ambient (Rothrock, Smith and Lindgren, 1961; Saucier and M i l l e r , 1961; II Bjorkman and Haeger, 1963; Shields and U n l i g i l , 1968). In the cold weather of Prince George, the inner regions of the p i l e s had temperatures near f r e e z i n g . The second r i s e i n temperature (Fig. 9) a f t e r the decline from the maximum i s not common with most p i l e s . This happens i n smaller p i l e s i n summer months (Butcher and Howard, 1968). This second r i s e i n temperature - 58 -i s probably due i n i t i a l l y to increasing external temperature. This perhaps w i l l , i n time, bring temperatures to l e v e l s which w i l l allow the resumption of thermophilic fungal a c t i v i t y . This i s shown by Figure 9 where the second r i s e i n temperature began with.the warm whether. 3. A c i d i t y of wood chips , The r e s u l t s on the pH of wood chips are at variance with findings of several authors who reported a r e c t i l i n e a r decrease of pH during storage (Annergren, D i l l e n and Vardheim, 1964; Shields, 1970). No attempt at c o r r e l a t i n g pH with time of storage was p o s s i b l e i n th i s study since no set pattern existed 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 production of a c e t i c a c i d as a r e s u l t of deacetylation of the hemicelluloses i n the wood (Annergren, D i l l o n and Vardheim, 1964; Shields, 1970). Although some deacetylation of hemicelluloses must have occurred i n the p i l e under study, the low wood substance l o s s could not have l e d to large a c e t i c a c i d production. Changes i n pH i n th i s p i l e w i l l not a f f e c t the a c t i v i t y and succession of the thermophilic and thermotolerant fungi. 4. Moisture i n the wood chip p i l e The extreme v a r i a t i o n i n moisture of chips during storage i s sai d to be responsible f o r the d i s t r i b u t i o n of fungi, and the r i s e i n temperature i n the inner top p o s i t i o n of the chip p i l e (Rothrock, Smith - 59 -and Lindgren, 1961; Saucier and M i l l e r , 1961; Bjorkman and Haeger, 1963; Annergren, D i l l e n and Vardheim, 1964; Nilsson, 1965; Bergman and Nilsson, 1966). Although the moisture content of the chips i n t h i s study was highly v a r i a b l e , e s p e c i a l l y between inner and outer samples, i t had a minimal e f f e c t on the d i s t r i b u t i o n of fungi. Moisture i n the wood must have been adequate for the support of fungal a c t i v i t y . The average moisture content of the chips was above 50% i n a l l samples. However, moisture content above 100% was common i n the samples from the inner top region, when they were stored for 12 months. This i s s i m i l a r to the findings of Bjorkman and Haeger (1963). The high moisture content of the wood i n the inner top of the p i l e i s due to the melting of the i c e and snow a f t e r the winter and to r a i n f a l l . Changes i n r a i n f a l l have a f f e c t e d the moisture content of chips i n small p i l e s (Butcher and Howard, 1968). The edges of the p i l e dry out quicker than the top, consequently the inner top i s wetter than the outer s i d e s . 5. Damage No serious damage as diagnosed by v i s u a l i n s p e c t i o n was done to the chips during the outside storage for a period of 12 months at Prince George. Staining of the chips was minimal and weight losses were low. Moderate s t a i n i n g of the chips occurred i n the outer chips stored for twelve months. These chips were mouldy, with very l i t t l e blue s t a i n i n g present. - 60 -Staining of balsam f i r and spruce has been reported a f t e r s i x to twelve months' storage, with l i t t l e blue s t a i n i n g occurring (Shields and U n l i g i l , 1968). The two findings are i n agreement. Although chips from the inner regions of the p i l e were l i g h t brown, they were not discoloured to any extent. In t h i s respect the spruce-pine p i l e at Prince George behaved d i f f e r e n t l y from p i l e s i n Sweden and eastern Canada (Annergren, D i l l e n and Vardheim, 1964; Shields, 1970) where i t was found that owing to the low pH of the chips, they were brown and discoloured. The weight losses of pine and spruce samples were low and did not increase from the s i x to twelve months' sampling period. The weight losses may not have increased from the second sampling period to the t h i r d sampling period because of the inclement weather conditions which could have a f f e c t e d fungal a c t i v i t y . Bergman and N i l s s o n (1966) showed that pine chips stored i n the warmer parts of the p i l e l o s t 1% per month during storage. High weight losses occur i n the inner, hotter regions of the p i l e . Saucier and M i l l e r (1961) found lower wood losses i n the centre of southern pine p i l e than i n the sides during both summer and winter storage. Bergman and N i l s s o n (1966) found lower wood losses i n the centre of a summer stored chip p i l e while i n winter storage the inner samples l o s t more. The spruce-pine p i l e i n Prince George behaved l i k e the winter p i l e which Bergman and N i l s s o n (1966) studied. - 61 -The conclusion from these findings i s that the weight losses are a f f e c t e d by external conditions. This i s possible i n a small p i l e where the i n s i d e i s a f f e c t e d by external conditions (Butcher and Howard, 1968). However, i n a b i g p i l e 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 not aff e c t e d by external conditions (Bjorkman and Haeger, 1963). Of the facto tested only temperature and d i s t r i b u t i o n of micro organisms are r e l a t e d and therefore could be responsible f o r weight l o s s e s . - 62 -III LABORATORY INVESTIGATION OF WOOD DEGRADATION CAUSED BY THERMOPHILIC AND THERMOTOLERANT FUNGI A INTRODUCTION Recent accounts (Bergman and Nilsson, 1966) of fungi i s o l a t e d from wood chips during storage have indicated that the thermophilic and thermotolerant fungi might play a r o l e i n wood chip 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 chip p i l e where high temperatures e x i s t . In the f i r s t part of t h i s study i t was shown that the thermophilic and thermotolerant fungi were common i n the inner region of the spruce-pine chip p i l e , where the highest weight losses of wood also.occurred. There was a r e l a t i o n s h i p between the wood substance los s and the number of these fungi. R e l a t i v e l y few laboratory studies have been made into the a b i l i t y of the thermophilic and thermotolerant fungi to degrade wood and the f a c t o r s a f f e c t i n g t h i s phenomenon. These fungi caused low weight losses and the r e s u l t s of the experiments performed using these fungi were also v a r i a b l e (Nilsson, 1965; Bergman and Nilsson, 1966; Shields and U n l i g i l , 1968). The reason for the v a r i a t i o n i n the r e s u l t s of the previous studies could be that the basic factors governing the a b i l i t y of these fungi to degrade wood have not been thoroughly investigated. The methods that have been used i n the e a r l i e r work were borrowed from those methods designed f o r the i n v e s t i g a t i o n of wood decay caused by Basidiomycetes and s o f t rot of wood caused by Ascomycetes and Fungi Imperfecti. - 63 -This section i s concerned with the demonstration that the common thermophilic and thermotolerant fungi i s o l a t e d from the wood chip p i l e are capable of causing weight losses of wood. In order to accomplish t h i s i t was f i r s t necessary to develop a method to test for t h i s a b i l i t y . This section of the thesis i s therefore subdivided into two parts. The f i r s t deals with the development of a method of t e s t i n g , the second deals with the demonstration of the a b i l i t y of the common i s o l a t e s to cause weight losses. The factors which were considered i n the development of a method f o r t e s t i n g the a b i l i t y of thermophilic and thermotolerant fungi to cause weight losses of wood were the following: 1. The e f f e c t of the culture 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 duration of incubation 5. The e f f e c t of temperature B LITERATURE REVIEW The e f f e c t of medium on weight losses of wood caused by thermophilic and thermotolerant fungi has been l i t t l e studied. In Sweden, Nilsson (1965) and Bergman and Nilsson (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 , while i n Canada, Shields and U n l i g i l (1968) used the s o i l block b u r i a l test to study the a c t i v i t y of thermophilic fungi on wood. Cooney and Emerson (1964) have pointed out the d i f f i c u l t y of e s t a b l i s h i n g , thermophilic fungi on laboratory media. Of the media tested they found these four to be u s e f u l : 1. Yeast starch agar 2. Yeast glucose agar 3. Oatmeal agar 4. Czapek agar Yeast starch agar and yeast glucose agar were p a r t i c u l a r l y favourable for the growth of thermophilic fungi. It has been pointed out that large numbers of microfungi are i s o l a t e d from chips because of the chip s i z e (Bergman and Nilsson, 1966). The small wood chips have large surface area to volume r a t i o and t h i s may be c r i t i c a l i n a f f e c t i n g the rate at which they are deteriorated. Findlay (1953), i n laboratory studies of natural d u r a b i l i t y of wood, found that the amount of decay increased as the volume of the t e s t species was reduced. In the previous studies of thermophilic fungi on wood, incubation periods ranged from two months to three months (Bergman and Nilsson, 1966; Shields and U n l i g i l , 1968). Bergman and Nilsson (1967) incubated C^. lignorum on pine sapwood at 40°C and showed that the weight losses s t a r t i n g from the f i r s t month of incubation continued to increase every month t i l l the f i f t h month when the experiment was terminated. In studies of weight losses caused by s o f t r o t fungi the usual incubation period i s s i x weeks (Duncan, 1953; - 65 -Savory, 1954). Bergman and N i l s s o n (1966, 1968) and Shields and U n l i g i l (1968) have tested some thermophilic and thermotolerant fungi and have shown that they could attack and cause weight losses i n wood samples i n the laboratory. The weight losses of coniferous species were lower than the weight losses of hardwood species and most of the fungi would not attack spruce. The thermophilic fungi caused considerably higher weight loss i n aspen and b i r c h samples. Work on s o f t rot of wood has shown that s o f t rot fungi extensively attack hardwoods, wh i l s t causing l i t t l e or no rot i n softwoods. Chaetomium  globosum caused extensive damage to hardwoods but did not cause a weight loss i n softwoods (Savory, 1954). The a b i l i t y of the s o f t rot fungi to cause weight losses i n wood i s a f f e c t e d by 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 nitrogen, i n the wood. It was shown that impregnation of the test wood with Abram's s o l u t i o n increased the weight losses s u b s t a n t i a l l y (Savory, 1954; Eslyn, 1969). Addition of n u t r i e n t s a l t s increased the rate of decay by C_. globosum, and the extent of decay appeared to be proportional to the amount of nutrients a v a i l a b l e to the fungus. Temperature i s a very 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 d i s t r i b u t i o n of many fungi. For a thermophilic fungus, temperature may be the most c r i t i c a l f a c t o r . Increasing the temperature up to the optimum generally increased the rate of growth and decay of wood by fungi. Bergman and Nilsson (1967) showed that the weight losses i n b i r c h , aspen, spruce - 66 -and pine wood caused by C_. lignorum increased r a p i d l y with increasing temperature and were highest at the optimum temperature for growth, which was 40°C. A. t e r r e s t r i s behaved s i m i l a r l y . Nilsson (1965) demonstrated that S^  thermophile at i t s optimum temperature of 45°C caused up to 7% weight loss to wood during two months incubation. Unlike the thermophilic fungi most Basidiomycetes are a c t i v e at lower temperatures. Henningsson (1967) found that only three Basidiomycetes of the many i s o l a t e d from b i r c h and aspen pulpwood had optimal growth and decay a c t i v i t y above 30°C. He also showed that low temperature fungi have a comparatively low decay a c t i v i t y and that many fungi with higher temperature optima were more a c t i v e at low temperatures than the low temperature fungi. C GENERAL METHODS Test wood pieces (1/16 x 3/4 x 1 3/4 in.) 0.2 x 1.9 x 4.4 cm. were cut from ponderosa pine sapwood so that the long axis was p a r a l l e l to the grain of the wood. The pieces were quickly impregnated with water and then conditioned at a constant temperature of 22.2°C and r e l a t i v e humidity of 50 ± 2% for seven days. The i n i t i a l conditioned weights then were measured. The te s t pieces were eit h e r s t e r i l i z e d i n ethylene oxide or autoclaved. They were then placed on "S" shaped glass rods on the fungal cultures growing i n p e t r i dishes on a medium. The wood had no d i r e c t contact with the medium. Three pieces were put i n each 9 cm. p e t r i dish. - 67 -The samples were incubated at 45°C for s i x weeks. To prevent the cultures from dessicating the r e l a t i v e humidity i n the incubator was kept high. A f t e r incubation the surfaces of the wood samples were cleaned of a l l mycelium and were weighed while wet. The treated wood was again conditioned at the same temperature and r e l a t i v e humidity f o r seven days when f i n a l weights were taken. Except f o r one experiment where d i f f e r e n t media were studied, Abrams c e l l u l o s e medium was used i n a l l studies. Media were autoclaved f o r t h i r t y minutes at 121°C. Sixty-mi portions were dispensed into p e t r i dishes and a f t e r cooling were inoculated with disks of inocula. The plates were incubated at 45°C for about four days to e s t a b l i s h the fungus. The general design was the randomized complete design. The analysis of variance was used to analyse the r e s u l t s . The fungi used i n these experiments had previously been i s o l a t e d from the spruce-pine chips from the Prince George experimental p i l e . Much at t e n t i o n was paid to the following because of t h e i r abundance i n the p i l e : 1. 15. emersonii 2. A. fumigatus 3. A. t e r r e s t r i s 4. S^ . thermophile 5. C_. pruinosum I igure 14. A l l e s c h e r l a t e r r e s t r l s and Thermoascus  aurantiacus growing on Abrams 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. auran t i a c u s . - 69 -D 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 of media Standard methods are a v a i l a b l e f o r t e s t i n g the a b i l i t y of f u n g i , e s p e c i a l l y the Basidiomycetes, to 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 the c a r e f u l study of media which serve as the s u b s t r a t e f o r the growth of the f u n g i . No study of t h i s nature i s a v a i l a b l e f o r the t h e r m o p h i l i c f u n g i . This study i s intended.to evaluate the e f f e c t of growth media on the a b i l i t y of t h e r m o p h i l i c f u n g i to cause weight l o s s e s of wood. The f o l l o w i n g media, the composition of which appear i n Appendix- 2, were used: 1. Abrams medium 2. 2% malt agar 3. Y e a s t - s t a r c h medium YpSS 4. Abrams-cellulose medium 5. 2% m a l t - c e l l u l o s e medium 6. Y e a s t - c e l l u l o s e medium Y nSc Table 9. Percentage weight l o s s e s caused to ponderosa pine sapwood on d i f f e r e n t media. Medium ,, Abrams Fungus C o n t r o l Thermoascus  aurantiacus Humicola sp. Sporotrichum  thermophile A l l e s c h e r i a t e r r e s t r i s % Wt. l o s s S.D. 1.42 0.24 3.46 0.40 3.10 0.41 3.52 0.64 3.64 0.80 Abrams c e l l u l o s e % Wt. l o s s S.D. 0.93 0.24 -3.41 0.28 3.44 0.51 3.82 0.45 4.39 0.46 2% Malt % Wt. l o s s S.D. 1.15 0.15 1.61 0.16 2.82 0.22 1.21 0.92 3.51 1.10 2% Malt 1% c e l l u l o s e % Wt. l o s s S.D. 0.85 0.52 1.64 0.43 3.21 0.57 2.50 0.69 3.31 0.89 YpSs % Wt. l o s s S.D. 0.50 0.35 2.63 0.83 3.80 0.57 2.69 2.84 3.90 0.88 YpSc. % Wt. l o s s S.D. 0.97 0.52 4.08 0.33 3.72 0.42 3.67 1.01 3.71 0.81 Table 10. Moisture content of samples on each medium A b r m g Abrams 2% . 2 % malt c e l l u l o s e malt 1% c e l l u l o s e YpSs YpSc. /o /a /a /a /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. C o n t r o l 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 36.4 5.0 33.5 3.8 65.0 14.2 53.2 13.4 91.3 32.7 37.5 6.0 aurantiacus Humicola sp. 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 35.6 7.2 33.0 7.4 178.1 9.9 187.6 15.7 96.7 34.7 46.7 16.7 thermophile A l l e s c h e r l a 31.5 6.6 41.3 7.4 103.4 34.7 140.5 59.3 55.5 20,1 49.5 13.3 t e r r e s t r i s - 72 -The f i r s t three media normally are used for the study of fungal decay and the other three were made by adding 1% cellulose to the f i r s t two media and replacing starch with cellulose in the third medium. The following fungi were used: A. terrestris, Humicola sp., thermophile and T. aurantiacus. Each treatment was replicated twelve times. Media had an affect on the a b i l i t y of the fungi to cause weight losses of ponderosa pine sapwood (Table 9). There were significant differences between media and between fungi. No single medium was the best for a l l the fungi and there was significant interaction between fungi and media. The two media resulting in the greatest wood weight losses were Abrams-cellulose and yeast-cellulose media. The addition of cellulose 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 in 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 effect of media on the weight losses caused by thermophilic fungi, i t was shown that fungi did not behave independently of medium. There were "preferences" for media and the most pronounced was the "preference" of A. terrestris for Abrams-cellulose medium and T. aurantiacus for yeast-cellulose medium. - 73 -Figure 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  aurantiacus 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. aurantiacus. - 7ft -The differences between these two media were the absence of yeast from Abrams-cellulose and ammonium n i t r a t e and potassium d i b a s i c phosphate from y e a s t - c e l l u l o s e medium. Since the search was for a medium which would be s u i t a b l e f o r a l l the fungi, i t was f e l t that a combination of these components might give the best medium. An experiment was set up to test the behaviour of the two fungi A. t e r r e s t r i s and T_. aurantiacus to the three constituents and t h e i r combinations. Eight media were tested. Each medium had a basal composition of magnesium sulphate, dipotassium monobasic phosphate, c e l l u l o s e and agar. The composition of the media appear i n the appendix. 2. The two fungi behaved d i f f e r e n t l y to the d i f f e r e n t media (Table 11). Differences between fungi and between media were s i g n i f i c a n t . 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 medium. Exclusion of yeast increased the weight losses caused by A. t e r r e s t r i s while decreasing that caused by _T. aurantiacus. When potassium d i b a s i c phosphate was removed the a c t i v i t y of A. t e r r e s t r i s was depressed while having no e f f e c t on the performance of T_. aurantiacus, The r e a c t i o n of the fungi to the exclusion of ammonium n i t r a t e was s i m i l a r to the exclusion of yeast. The removal of a l l three 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. aurantiacus. - 75 -Table 11. Percent weight losses obtained f o r A. t e r r e s t r i s and T_. aurantiacus growing on medium prepared from c e l l u l o s e , MgSO^ 7H 0 and K HPO. with the absence of yeast or NH NO or KH PO Composition A. t e r r e s t r i s % Wt. loss T. aurantiacus % Wt. loss Complete medium 3.89 4.46 Without yeast 4.42 3.85 Without KHoP0. 2 4 3.25 4.31 Without NH.NO. 4 3 4.32 4.03 Without yeast & KH^O^ 3.78 3.94 Without yeast & NH 4N0 3 2.16 3.71 Without KHoP0. & NH.N0o 2 4 4 3 3.91 4.17 None 1.92 3.60 jT. aurantiacus was generally not as s e n s i t i v e to changes i n the nutr i e n t composition of the medium as A. t e r r e s t r i s and benefited most from the incorporation of a l l three components into the medium. Af t e r these experiments, Abrams-cellulose medium was selected f o r the r e s t of the study. 2. E f f e c t of wood sample s i z e Two sample si z e s selected f o r t h i s study were s i m i l a r i n s i z e to chips and the t h i r d s i z e was chosen to be the same as that i n the ASTM - 7tf -D1413-61 t e s t . The influence of sample s i z e on weight losses of wood caused by thermophilic fungi was investigated. The s i z e s of samples were 0.2 x 1.9 x 4.4 cm. (1/16 x 3/4 x 1 3/4 i n s . ) , 0.4 x 2.5 x 5.1 cm. (1/8 x 1 x 2 in.) and 1.9 x 1.9 x 1.9 cm (3/4 x 3/4 x 3/4 i n ) , and were cut so that the long axis was p a r a l l e l to the grain of the wood. The s i z e s w i l l be r e f e r r e d to as small, medium and large hereafter. They were a l l cut from ponderosa pine sapwood. The tangential surface was placed on the c u l t u r e . The fungi used i n t h i s experiment were A. t e r r e s t r i s , 13. emersonii and S_. thermophile. Each t r e a t -ment was r e p l i c a t e d s i x times. Sample s i z e (Table 12) influenced the absolute weight loss of wood and the percentage weight loss of wood. There were s i g n i f i c a n t d i f f e r e n c e s between the percentage weight loss i n the d i f f e r e n t s i z e s , The percent weight loss values f o r the small and medium size d pieces were greater than those for the large pieces,- except for S_. thermophile where higher percent losses occurred i n the medium sized samples than i n the small or large samples. The re a c t i o n of the fungi was independent of the s i z e of wood. Sample s i z e (Table 12) aff e c t e d the absolute weight loss of wood i n such a way that the small pieces l o s t l e s s weight than the medium and large pieces. The d i f f e r e n c e i n weight los s between d i f f e r e n t s i z e s of blocks was s i g n i f i c a n t . 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 dependent on the sample, since a s i g n i f i c a n t i n t e r a c t i o n existed between fungi and s i z e of sample. The r e s u l t s of t h i s experiment did not lead to a change i n the s i z e of samples and i n a l l subsequent experiments the small s i z e pieces were s t i l l used. Table 12. Percent weight loss and absolute weight loss of wood caused by thermophilic fungi growing on d i f f e r e n t s i z e s of ponderosa pine sapwood samples during s i x weeks incubation. Percent weight loss Absolute weight loss i n grams (0.2 x 1.9 x 4.4) (0.4 x 2.5 x 5.1) (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) Small Medium Large Small Medium Large Control A l l e s c h e r i a  t e r r e s t r i s Byssochlamys emersonii Sporotrichum  thermophile 0.25 3.61 3.58 3.08 0.51 3.67 3.56 3.65 0.31 3.21 2.82 2.98 0.001 0.019 0.019 0.017 0.009 0.063 0.061 0.061 0.009 0.090 0.078 0.084 - 7§ -3. E f f e c t of methods of i n o c u l a t i o n This study was designed to inve s t i g a t e the e f f e c t of d i f f e r e n t methods of providing the inoculum source on the weight losses of wood caused by thermophilic fungi. The usual method of providing inoculum i n wood decay studies consists of growing the fungus on the basal medium f o r a period of time before the te s t blocks are-planted. This method i s unsuitable f o r s o f t r ot studies (Duncan, 1953; Savory, 1954). In s o f t r o t studies the wood, planted on Abrams medium, i s d i r e c t l y inoculated with a spore suspension. Although these two methods have been extensively used i n studies of microfungi from wood chips, no information i s a v a i l a b l e on the e f f e c t of the d i f f e r e n t methods of providing inoculum source on the weight lo s s e s . The following methods were tested: 1. Inoculation of wood samples. 2. Inoculation of medium, with wood samples subsequently planted on the medium on the same day. 3. Inoculation of medium, with wood samples subsequently planted on the cul t u r e a f t e r one week. Two fungi, A. t e r r e s t r i s and jB. emersonii were used and the method was the same as outlined under C-General methods. Each treatment was r e p l i c a t e d s i x times. The r e s u l t s are shown i n Table 13. - 79 -Table 13. Average percentage weight loss of wood samples caused by thermophilic fungi using d i f f e r e n t methods of providing the inoculum source. A. t e r r e s t r i s J3. emersonii Methods of providing inoculum % Wt. loss % Wt_. loss D i r e c t on wood 2.87 2.91 Inoculate medium-wood planted same day 2.65 2.91 Inoculate medium-wood planted a f t e r one week 3.24 3.03 The method of providing the inoculum source had an e f f e c t on the weight losses caused to the wood. Differences between these methods were s i g n i f i c a n t . The expression of these differences was li n k e d to the fungus used i n the t e s t . Both fungi caused higher weight losses i f the medium was inocu-l a t e d a week before the wood samples were planted. With both fungi there 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 loss i f the wood was inoculated d i r e c t l y or i f the medium was inoculated and the wood samples were planted on i t on the same day. Inoculating the medium a week before p l a n t i n g the wood samples was considered the best method f or i n o c u l a t i n g the wood samples and was used i n a l l subsequent experiments. - 8Q -4. E f f e c t of duration of incubation Limited information i s a v a i l a b l e on the e f f e c t s of duration of incubation on the weight losses caused by thermophilic and thermotolerant fungi. The incubation periods f o r previous studies were a r b i t r a r i l y chosen. This i n v e s t i g a t i o n was conducted to determine how duration of incubation af f e c t e d weight losses caused by the thermophilic and thermotolerant fungi. The fungi used i n the study were A. t e r r e s t r i s , ]3. emersonii, (3. pruinosum and £>\ thermophile. Six incubation periods were used varying from two to twelve weeks. Every two weeks, a whole set of treatments was taken out, conditioned and weighed. Each treatment was r e p l i c a t e d s i x times. The r e s t of the method has been ou t l i n e d before under C-General methods. The weight losses were p l o t t e d against period of incubation and the r e s u l t s appear i n Figure 16. A l l the graphs have been drawn from corrected data by deducting the weight losses i n the c o n t r o l samples from the treated samples. Rapid weight losses were caused by a l l of the fungi during the f i r s t s i x weeks with the exception of _C. pruinosum, which produced low weight losses i n the f i r s t four weeks followed by a rapid increase from the fourth week to the s i x t h week. Generally, no appreciable increases i n weight losses occurred a f t e r the s i x t h week except f or A. t e r r e s t r i s which attained a stable condition a f t e r the eighth week. Weight losses went up s l i g h t l y f o r three of the fungi i n the twelfth week, but t h i s could be the r e s u l t of a general drop i n moisture content of the wood since the samples s t a r t e d to dry out. Byssochlamys emersonii Chrysosporium pruinosum D U R A T I O N O F I N C U B A T I O N I N W E E K S - 82 -Fluctuations i n weight losses were common for samples inoculated with J3. thermophile. These f l u c t u a t i o n s were opposite to changes i n moisture content of the samples. Since the a c t i v i t y of most of the fungi had s t a b i l i z e d a f t e r s i x weeks incubation, t h i s time was chosen as the incubation period f o r the r e s t of the experiments. 5. E f f e c t of temperature. It was shown i n the f i r s t part of this study that temperature af f e c t e d the d i s t r i b u t i o n of thermophilic and thermotolerant fungi i n the chip p i l e . Wood substance loss was also higher i n the inner region of the p i l e where temperatures were high. This experiment was conducted to inv e s t i g a t e the e f f e c t of temperature on the weight losses caused by thermophilic and thermotolerant fungi. Temperatures ranging from 25°C to 60°C were used f o r incubating the fungi. The temperature i n t e r v a l was 5°C. In a few cases 35°C incubation was not used. A l l cultures were incubated at 45°C before the s t a r t of the experiment. The incubation period was four weeks and s i x samples were, used i n each examination. - 8 3 -The following fungi were tested. A. t e r r e s t r i s A. fumigatus _B. emersonii (]. pruinosum S^. thermophile _T. aurantiacus The r e s u l t s appear i n Figure 17. A l l the graphs have been drawn from corrected data by deducting the weight losses i n the co n t r o l samples from the treated weight l o s s e s . For a l l the fungi, increases i n temperature l e d to increases i n weight losses caused by them u n t i l the optimum temperature was reached and then weight losses decreased. Except for J3. emersonii and _T. aurantiacus, the highest weight losses occurred at 40°C. These two fungi caused t h e i r highest weight loss at 50°C. A l l the fungi were a c t i v e at 25°C except for j3. emersonii and _T. aurantiacus, which showed no a c t i v i t y at 25°C but sta r t e d at 30°C. The temperature at which the a c t i v i t y of the organisms stopped, va r i e d f o r d i f f e r e n t organisms. C^ . pruinosum a c t i v i t y ended at the maximum temperature 50°C, w h i l s t that of JJ. emersonii and T_. aurantiacus ended at or a l i t t l e above 60°C. The other three fungi had the same l i m i t a t i o n of a c t i v i t y maximum at a temperature of 55°C. The increase i n a c t i v i t y from the minimum to the optimum was rapid but the decline i n a c t i v i t y from optimum to the maximum was gradual . - 84 -25 30 35 40 45 50 55 60 Thermoascus aurantiacus 0 5-4-3-2-1 - / V \ 25 30 35 40 45 50 55 60 Byssochlamys emersonii 6r 5 4 3 2 1 6-5-4-3-2-1 -0 — ^ , , , , \ , _ l I I I I I \ I 25 30 35 40 45 50 55 60 Sporotrichum thermophile / _L 25 30 35 40 45 50 55 60 A. t e r r e s t r i s 6 r 5 -4 -3 -2 -1 -J L 25 30 35 40 45 50 55 60 Aspergillus fumigatus 25 30 35 40 45 50 55 60 Chrysosporium pruinosum ' T E M P E R A T U R E ( ° C ) F ig . 17. Changes in weight losses with changes in temperature. - 85 -except f o r C^. pruinosum and _T. aurantiacus. The production of ascocarps was greatly reduced at temperatures above 45°C and stopped completely at temperatures beyond 55°C. Mycelia production was sparse a f t e r 55°C except f o r J3. emersonii and T_. aurantiacus. Samples, generally, showed a moisture d e f i c i t and charring a f t e r four weeks incubation at 60°C. 6. Summary It appears from these r e s u l t s that the most s u i t a b l e method f or evaluating the a b i l i t y of thermophilic and thermotolerant fungi to cause weight losses of wood w i l l be by growing the tes t fungus on Abrams c e l l u l o s e medium f o r one week at temperatures between 40° and 50°C, a f t e r which the wood samples (0.2 x 1.9 x 4.4 cm) are planted onto the c u l t u r e . The t e s t i s then incubated f o r 6 weeks at the optimum temperature f o r the growth of the fungus. This may vary from 40° to 50°C. E WEIGHT LOSSES CAUSED BY THE THERMOPHILIC AND THERMOTOLERANT FUNGI 1. Evaluation of the common i s o l a t e s The following fungi, i s o l a t e d at 45°C from wood chips, but not used i n previous experiments, were examined f or t h e i r a b i l i t y to cause - 8 6 -weight losses of ponderosa pine sapwood. _H. lanuginosa was not examined because i t f a i l e d to grow on the Abrams c e l l u l o s e medium which was used for t h i s t e s t . The method for the test was the same as out l i n e d i n C-General methods. Each treatment was r e p l i c a t e d twelve times. Table 14. Percent weight loss of ponderosa pine caused by some thermo-p h i l i c and thermotolerant fungi at 45°C incubation. Fungi % Wt. loss S.D. Control 0.12 0.12 Sporotrichum B. 4.08 0.49 As 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 emersonii 11 3.30 0.23 Byssochlamys species 201 3.26 0.36 Byssochlamys emersonii 12 3.07 0.29 Unknown 618 3.01 0.43 Unknown 624 1.68 0.37 Unknown 6310 0.71 0.40 A l l the fungi examined could cause weight losses of ponderosa pine sapwood (Table 14). Sporotrichum B. caused a high weight loss compared - 87 -to the other fungi. Two fungi Unknown 624 and Unknown 6310 caused very low weight losses, probably because of t h e i r poor growth on Abrams-cellulose agar. 2. S u s c e p t i b i l i t y of lodgepole pine and white spruce wood  to attack by several fungal i s o l a t e s . ' In the e a r l i e r experiments ponderosa pine sapwood was used because of i t s a v a i l a b i l i t y i n the laboratory and i t s extensive use i n wood decay studies i n North America. The chip p i l e from which the fungi used i n t h i s study have been i s o l a t e d was made up of lodgepole pine and spruce. There-fore an i n v e s t i g a t i o n was made to study the a c t i o n of some of the fungi on lodgepole pine and spruce. The experimental procedure was the same as out l i n e d i n C-General methods except for the d i f f e r e n t tree species used. Each treatment was r e p l i c a t e d twelve times. The r e s u l t s are shown i n Table 15, There were remarkable v i s u a l differences i n the attack of the fungi on pine and spruce. Most of the fungi completely covered pine a f t e r ten days of incubation, but not spruce. Not u n t i l halfway into the incubation period did a l l fungi completely cover spruce, even though s t a i n i n g of spruce was noticed e a r l i e r . - 88 -Table 15. Percentage weight loss of lodgepole pine and spruce sapwood caused by some thermophilic and thermotolerant fungi at 45°C a f t e r s i x weeks incubation. Fungi Spruce Lodgepole pine % Wt. loss ,. % Wt. loss Control 1.53 1.96 A l l e s c h e r i a t e r r e s t r i s 3.06 9.17 Sporotrichum thermophile 3.39 5.18 Sporotrichum B. 5.02 6.69 Chrysosporium pruinosum 2.03 3.74 Byssochlamys emersonii 19 2.54 3.88 Byssochlamys emersonii 12 2.65 3.63 S t e r i l e mycelium 2.85 3.72 Unknown 6214 3.60 5.65 A s p e r g i l l u s fumigatus 2.51 3.91 Thermoascus aurantiacus 2.58 . 3.95 A l l the fungi examined caused weight losses of lodgepole pine and spruce. Higher weight losses occurred i n lodgepole pine than i n spruce as shown i n Table 15. - 89 -At the end of the experiment, some of the spruce 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 middle. S t a i n i n g was observed 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 f u n g i : A. t e r r e s t r i s S^ . thermophile Sporotrichum B. Unknown 6214 When s t a i n e d and unstained samples were compared, the unstained samples had l o s t more weight than the s t a i n e d samples (Table 16). Table 16. Percentage weight l o s s f o r s t a i n e d and unstained spruce 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 Stained % Wt. l o s s % Wt. l o s s t0-5 tp.5 Unknown 6214 4.69 2.82 2.79* 2.28 Sporotrichum B. 6.02 4.29 3.46* Sporotrichum thermophile 3.77 3.01 2.62* The d i f f e r e n c e s between the unstained and s t a i n e d spruce samples were s i g n i f i c a n t f o r a l l the three f u n g i examined. A comparison was not 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 there were not enough unstained samples. - 90 -3. E f f e c t of mixed i s o l a t e s on weight losses From a small s t e r i l i z e d piece of wood i t was possible to i s o l a t e d i f f e r e n t species of thermophilic fungi. In mixed cultures of these fungi on malt agar, zones of i n h i b i t i o n were not found. Since a l l the d i f f e r e n t species have d i f f e r e n t c a p a c i t i e s f o r attacking wood, i t was thought that some of the fungi might depend on others to obtain t h e i r nutrients from the wood. If th i s were so, then i t might be poss i b l e to obtain higher losses i f more than one fungus were used to inoculate the wood. An experiment to study the e f f e c t of mixed cultures of thermophilic fungi on weight losses of ponderosa pine sapwood was performed. The following thermophilic fungi which could grow together i n cult u r e and were very common i n the p i l e were examined: A. t e r r e s t r i s 13. emersonii  thermophile Two incubation temperatures of 45°C and 50°C were used because the maximum a c t i v i t y of the three fungi was found to occur at d i f f e r e n t temperatures. Plates were inoculated with more than one fungus by placing side by side 2mm disks 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 cultures did not generally cause higher weight losses than the s i n g l e cultures except f o r the culture of _B. emersonii and S^. thermophile incubated at 45°C and the mixed cultures of a l l three fungi incubated at 50°C (Table 17). - 91 -Table 17. 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. emersonii and S. thermophile on weight losses of ponderosa pine incubated at 45°C and 50°C. Fungus % 45°C Wt. los s % 50°C Wt. los s Control 0.49 0.42 A l l e s c h e r i a t e r r e s t r i s 2.99 3.39 Byssochlamys emersonii 3.22 3.72 Sporotrichum thermophile 3.50 0.93 A. t e r r e s t r i s & B. emersonii 3.32 3.81 A. t e r r e s t r i s & S. thermophile 3.24 2.71 B. emersonii & S. thermophile 4.14 3.59 A. t e r r e s t r i s & B. emersonii & S. thermophile 3.22 4.07 Dependence of the fungi on temperature was more s i g n i f i c a n t than the i n t e r a c t i o n between the fungi. Although the a c t i v i t y of the three fungi was generally not complementary, no obvious i n h i b i t i o n of one fungus by the other occurred. F DISCUSSION Medium, unlike temperature, has not been studied extensively i n the i n v e s t i g a t i o n of the e f f e c t s of thermophilic fungi on wood. The a b i l i t y of thermophilic fungi to degrade wood depends on the medium on which i t i s grown. These differences have been ,shown i n the present study, i n which no s i n g l e medium was found to be the best f o r the study of weight losses caused by the t o t a l spectrum of thermophilic fungi. In a l l the previous studies of t h i s subject, only one medium has been used, and no at t e n t i o n has been paid to the f a c t that the use of a s i n g l e medium can oversimplify the weight los s r e s u l t s (Nilsson, 1965; Bergman and Nil s s o n , 1966, 1967, 1968; Shields and U n l i g i l , 1968). For th i s reason these authors f a i l e d to show that a large number of thermophilic fungi from wood chips can be d e s t r u c t i v e . Some thermophilic fungi are hig h l y s e n s i t i v e to changes i n the composition of the medium i n which they grow, while others are not. Dra s t i c changes i n the medium af f e c t e d the a b i l i t y of A. t e r r e s t r i s to cause weight losses more than they af f e c t e d T_. aurantiacus. The presence of yeast i n a medium, while depressing the weight losses caused by A. t e r r e s t r i s , increased those caused by T_. aurantiacus. Savory (1954) showed that f o r a s o f t r ot fungus, impregnation of the wood with ammonium sulphate increased the weight losses to the wood caused by C^ . globosum while the impregnation with magnesium sulphate l e d to n e g l i g i b l e l o s s e s . The two examples i n d i c a t e that fungi have d i f f e r e n t "preferences" f o r d i f f e r e n t composition of media. - 93 -Although impregnation of wood with s a l t s and s o l u t i o n l i k e Abrams has been found useful f o r the study of weight losses caused by microfungi (Savory, 1954; Eslyn, 1969) i t may not be a r e l i a b l e method, since i t could lead to a hydro l y s i s of some chemical constituents of the wood which w i l l give misleading r e s u l t s when fungi are grown on the wood. Autoclaving mono and oligosaccharides with s a l t s leads to a change i n the o r i g i n a l s tructure ( B a l l , 1953; B r e t z l o f f , 1954). High weight losses might be obtained which might not be due to the a c t i v i t y of the fungus alone. The aim of this study was to obtain a medium that would allow the fungus to cause high weight iosses of wood without previously a l t e r i n g the composition of the wood. Some of the thermophilic fungi have a tendency to accumulate moisture i n the wood samples, depending upon the substrate on which they grow. The high moisture content of the sample might reduce the a c t i v i t y of the fu n g i . The thermophilic fungi were highly v a r i a b l e and the same medium which was e f f i c i e n t at one time could give a poor r e s u l t at another time. This i s probably the reason f o r the statement by Shields and U n l i g i l (1968) that i t i s d i f f i c u l t to assess the r o l e of the thermophilic fungi i n the d e t e r i o r a t i o n of wood 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 studies of the e f f e c t of thermophilic fungi i n wood.Based on the present r e s u l t s Abrams-c e l l u l o s e medium was chosen as giving reasonable weight losses f o r the fungi tested. - 94 -The s i z e of the wood chips may contribute to the abundance of Fungi Imperfecti and Ascomycetes i n the chip p i l e (Bergman and Nilsson, 1966) . I t is not probable that the chip s i z e per se increases the weight of wood l o s s - How chip s i z e a f f e c t s weight losses depends on how the losses are c a l c u l a t e d . The absolute weight loss caused by thermophilic fungi i s d i r e c t l y proportional to the s i z e of the wood; the bigger the sample the greater the weight l o s s . The percentage weight loss i s i n v e r s e l y proportional to the s i z e of the sample (Findlay, 1953). This was not the case with the thermophilic fungi examined, since the percentage weight losses of the medium size d samples were greater than those of the small s i z e d samples. Two d i f f e r e n t classes of fungi were used i n the two studies and thi s may have brought about the d i f f e r e n c e s . The thermophilic fungi may be l i m i t e d i n the quantity of nutrients a v a i l a b l e to them i n a given piece of wood i f they do not attack c e l l u l o s e or l i g n i n . The Basidiomycetes i n attacking c e l l u l o s e or c e l l u l o s e and l i g n i n w i l l undoubtedly have a la r g e r proportion of the wood to attack. In th i s s i t u a t i o n the attack of wood by Basidiomycetes might depend more upon the r a t i o of surface area to volume. The reduced sample w i l l , on the other hand, l i m i t what i s a v a i l a b l e to the thermophilic fungus. However, l i t t l e i s known about the nature of attack of wood by thermophilic fungi. It has been shown that A. t e r r e s t r i s causes s o f t r ot of hardwoods and therefore w i l l attack c e l l u l o s e (Bergman and Nilsson, 1967) . These authors also pointed out that the moulds i s o l a t e d from chip p i l e s do not attack l i g n i n but attack carbohydrates. - 96 -If weight losses are to be calculated i n percentages, then small sample pieces should be chosen f o r the study. However, i f weight losses are calculated i n absolute terms, larger sample pieces are much more appropriate. Thermophilic fungi w i l l attack wood so long as the moisture i n the wood i s adequate and the temperature i s s u i t a b l e . I t was noticed that unless the conditioned wood had absorbed s u f f i c i e n t moisture i t was not possible to get the fungi to attack the wood. Inoculating wood with thermophilic fungi did not present any of the d i f f i c u l t i e s associated with s o f t r o t fungi. Savory (1954) could not s u c c e s s f u l l y inoculate wood with s o f t r ot fungi u n t i l he planted u n s t e r i l i z e d wood samples on Abrams medium and seeded them with a spore suspension of C^ . globosum which led to the i n o c u l a t i o n of the wood. Planti n g the samples on weak old cultures of thermophilic fungi might have given higher weight losses because the fungi would probably have had enough time to grow and produce adaptive enzymes which are necessary for attacking wood. In many studies on the e f f e c t s of thermophilic fungi on wood, samples have been incubated for two to three months (Nilsson, 1965; Bergman, and Nilsson, 1966; Shields and U n l i g i l , 1968). It appears from the present study that incubation beyond s i x weeks does not serve any us e f u l purpose. In t h i s respect the thermophilic fungi may be s i m i l a r to the s o f t r ot fungi which are usually incubated for s i x weeks (Savory, 1954). - 9J6 -A short period of incubation has c e r t a i n advantages considering the conditions i n which these tests are c a r r i e d out. Results are obtained e a r l i e r and the drying problem at high temperatures i s considerably reduced. Most of the wood degradation i n the laboratory takes place i n the f i r s t few weeks of incubation, and the r e s t of the incubation time does not contribute a great deal to the t o t a l l o s s e s . Why t h i s happens i s not c l e a r , but the following hypothesis may explain the phenomenon. The rapid growth of the thermophilic fungi may lead to the rapid depletion of the nutrients on which the fungi depend, probably r e s u l t i n g i n the accumulation of metabolic by-products which may be i n h i b i t o r y . The percentage weight loss of wood caused by a thermophilic fungus increases with temperature u n t i l optimum temperature i s reached, at which point i t s t a r t s to decrease u n t i l the maximum temperature i s reached, where a c t i v i t y i s terminated. Studies i n which the weight losses caused by A. t e r r e s t r i s increased eleven f o l d from the minimum to the optimum temperature (Bergman and Nilsson, 1967) 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 decline i n the a c t i v i t y as the temperature increases from optimum to the maximum. The optimum temperature for the a c t i v i t y of a thermophilic fungus i s a f f e c t e d by the substrate on which i t grows. The optimum temperature for - 97 -A. t e r r e s t r i s 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 associated with the highest weight losses i s 50°C (Bergman and Nilsson, 1967). In the present study the optimum temperature at which A. t e r r e s t r i s caused the highest weight losses i n pine was 40°C. This f i n d i n g agrees with the work of Henningsson's (1967). He found that for several fungi from b i r c h and aspen the temperature for optimum r a d i a l growth i s higher than that f or optimal decay a c t i v i t y . The a b i l i t y to cause weight losses by some thermophilic fungi may extend beyond 60°C. I t has been pointed out that temperatures above 60°C i n the p i l e may completely s t e r i l i z e the chips (Nilsson, 1965) . 13. emersonii was very a c t i v e when the temperature of incubation was 60°C f o r four weeks. I t i s po s s i b l e that above 60°C incubation J3. emersonii may cause weight l o s s e s . Unlike the optimal temperature, the substrate did not a f f e c t the minimum temperature at which the a c t i v i t y of the fungus began. The minimum temperature for r a d i a l growth of _T. aurantiacus on malt i s 30°C (Bergman and Nilsson, 1966) and 30°C was the temperature at which i t began to cause weight losses i n pine as observed i n t h i s study. The capacity of thermophilic fungi to attack d i f f e r e n t species of coniferous wood i s v a r i a b l e , although nearly a l l the fungi examined w i l l degrade pine and spruce. The observed degradation of lodgepole pine i s a l i t t l e greater than spruce. Similar r e s u l t s were obtained by the Swedish - 98 -workers except that some of the fungi they tested could not attack spruce at a l l (Bergman and Nilsson, 1966). The extent of attack was much lower i n the fungi which Bergman and Nilsson (1966) inv e s t i g a t e d . This might be expected because of diff e r e n c e s i n species of wood, media and v a r i a t i o n i n the fungi themselves. The cause of s t a i n i n g i n the spruce samples i s unknown. Staining may be dependent upon the p o s i t i o n i n the tree from which the samples came, since some heartwood might have been inadvertently included i n the samples due to d i f f i c u l t y i n d i f f e r e n t i a t i n g between heartwood and sapwood zones i n spruce. However, the nature of the observed s t a i n i n g , which may sometimes form a streak i n the middle of a sample, does not support t h i s contention. Stained spruce l o s t l e s s weight than unstained spruce. Hossfeld, Oberg and French (1957), found also that discoloured aspen associated with knots, 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 sap-wood c o n t r o l s . I t was suggested by Hossfeld, Oberg and French (1957) that the discoloured aspen wood contain e x t r a c t i v e components some of which were toxic towards wood decaying fungi. The thermophilic fungi examined were neither antagonistic nor s y n e r g i s t i c i n t h e i r i n t e r a c t i o n . Each fungus probably occupies i t s own zone i n the wood without depending upon the other fungi present. Any interdependence between fungi that may take place w i l l r e l y to a large 99 extent on temperature. Trichoderma lignorum was found to i n h i b i t the a b i l i t y of C_. lignorum to degrade wood but only at low temperatures (Bergman and Nilsson, 1967). As the temperature increased the e f f e c t of 1?. lignorum disappeared. In summary, i t has been shown that i n c o n t r o l l e d laboratory experiments, thermophilic and thermotolerant fungi can cause wood weight losses and that these losses are af f e c t e d by medium, duration of incuba-t i o n , temperature of incubation, the tree species used and method of i n -oc u l a t i o n . Although sample s i z e and i n t e r a c t i o n of fungi were considered, they were found to have l i t t l e influence on the eventual percentage weight losses of the wood samples. In conclusion, these r e s u l t s suggest that the optimal conditions f o r evaluating weight loss of wood caused by these fungi would involve using Abram-cellulose medium, growing the fungi on th i s medium for one week, pl a n t -ing wood samples (0.2 x 1.9 x 4.4 cm.) on the one week old cultures and incubating the te s t f o r 6 weeks at 45°C for thermophilic fungi and 40°C f o r thermotolerant fungi. This method developed f o r the evaluation of weight loss of wood caused by these fungi 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 of previous workers. - 1 0 0 -IV CHEMICAL ANALYSIS OF DEGRADED WOOD A INTRODUCTION Thermophilic fungi have been i s o l a t e d from chips and have also been shown to degrade wood i n laboratory studies (Bergman and Nils s o n , 1 9 6 6 , 1 9 6 8 ; Shields and U n l i g i l , 1 9 6 8 ) . R e l a t i v e l y l i t t l e information i s a v a i l a b l e on the nature of t h e i r s p e c i f i c chemical a c t i v i t y i n wood. The present study was undertaken to provide some information on the s p e c i f i c chemical a c t i v i t y of some thermophilic fungi i n wood. The method of analysis followed was the hydro l y s i s of both c e l l u l o s e and hemicellulose to monosaccharides, the reduction of the monosaccharides to sugar alcohols and the a c e t y l a t i o n of the sugar alcohols -. These acetylated compounds were then i n j e c t e d into a gas chromatograph. B LITERATURE REVIEW Bergman and Nils s o n ( 1 9 6 8 ) noted that a number of s o f t r o t fungi, i n c l u d i n g thermophilic fungi, i s o l a t e d from a b i r c h chip p i l e did not cause any l i g n i n losses and thus the s o f t r ot fungi mainly degraded the carbohydrates of the wood. Chang ( 1 9 6 7 ) found that both hemicellulose and - 1 0 1 -c e l l u l o s e were quite susceptible to m i c r o b i a l attack during s e l f heating of straw and i n 60 days of composting about 70 .7% of the c e l l u l o s e was removed. Henssen ( 1 9 5 7 ) and Chang ( 1 9 6 7 ) demonstrated the a b i l i t y of S^  thermophile to decompose hemicellulose and p e c t i n . Chang ( 1 9 6 7 ) and Fergus ( 1 9 6 9 ) have shown that S^  thermophile would degrade c e l l u l o s e . C MATERIALS AND METHODS The method used for the preparation of the wood samples f o r t h i s a n alysis i s the same as o u t l i n e d i n Part I I I . The fungi used were A. t e r r e s t r i s , I}, emersonii and S_. thermophile. Incubation periods of 2 , 6 and 1 2 weeks were employed. Six of the wood samples f o r each treatment were ground together to pass through a 60 mesh sieve. From t h i s 1 gm. was taken for the a n a l y s i s . L i g n i n contents were determined by the standard Klason method (Tappi standard T 1 3 05 - 5 4 , 1 9 5 4 ) and were not corrected f o r a c i d - s o l u b l e l i g n i n . Carbohydrate analyses were made on the f i l t r a t e from the Klason l i g n i n determination. One gram of ground wood to which 2 5 0 mg. of i n o s i t o l was added as an i n t e r n a l standard, was hydrolysed with 15 ml. of 72% sumphuric a c i d . The mixture was d i l u t e d with d i s t i l l e d water and heated i n an autoclave - 102. -at 212°F f o r four hours. The l i g n i n was removed by f i l t r a t i o n and estimated by the standard Klason method. To convert the sugars to a l d i t o l s a n a l i q u o t of the Klason l i g n i n f i l t r a t e was brought to a pH of 4 by ad d i t i o n of saturated barium hydroxide s o l u 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 then removed by c e n t r i f u g a t i o n . The centrifugate was treated with 50mg. sodium borohydride and l e f t over night. The reducing s o l u t i o n was a c i d i f i e d with 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 using a f l a s h evaporator i n a water bath at temperature of 35° to 40°C. The b o r i c acid was removed as methyl borate (Wolfrom and Thompson, 1963) by four evaporations under vacuum with methanol. The residue was taken up i n a small volume of methanol and transferred to a volumetric tube where the methanol was again removed by evaporation under vacuum. The a d d i t i v e mixture was acetylated by adding 1 l/2ml a c e t i c anhydride and heating the mixture f o r three hours at 120°C. The excess a c e t i c anhydride was removed by evaporation and the residue taken up i n 2ml methylene d i c h l o r i d e . This s o l u t i o n was then i n j e c t e d into a gas chromatograph f o r a n a l y s i s . The gas chromatograph used was Series 1520 Varian-Aerograph with flame i o n i z a t i o n detector, i n i t i a l oven temperature 140°C, heating rate 1/2°/ minute for 25 minutes, i n j e c t i o n temperature 250°C, detector temperature 225°C with nitrogen as c a r r i e r gas. The copper column (1/8" x 2.5') was packed with 3% ECNSS-M on Gas Chrom Q (Sawardeker, Sloneker and Jeanes, 1965; Oades, 1967). The peak areas were measured with a Model 476 Varian-Aerograph d i g i t a l i n t e g r a t o r with i n o s i t o l as the i n t e r n a l standard. - 103 -D RESULTS In t h i s exploratory t e s t , considering the experimental v a r i a -t i o n i n the chemical method (approximately ± 2%, personal communication, Dr. K. Hunt) and also the experimental v a r i a t i o n to be expected i n the mycological method, any changes i n the r e s u l t s f o r glucose, mannose, xylose and l i g n i n appear to be inconclusive. However, the r e s u l t s f o r arabinose i n d i c a t e a decrease of t h i s branch residue with increasing time of attack by a l l of the fungi. Table 18. Concentration of various chemical components of ponderosa pine sapwood a f t e r degradation by some thermophilic fungi. Percentages are based on degraded wood.* Fungi Time Carbohydrate % & L i g n i n % i n wks. L i g n i n Glucose Mannose Xylose Arabinose Control 2 24.7 40.0 8.0 5.0 1.5 12 25.1 41.3 12.3 5.6 1.6 A l l e s c h e r i a 2 25.2 38.0 10.0 5.1 1.5 t e r r e s t r i s 6 25.2 46.0 9.5 5.6 0.0 12 26.3 46.2 11.0 5.6 0.0 2 25.5 41.7 10.4 4.5 0.8 Byssochlamys 6 25.1 46.0 11.0 5.0 0.2 emersonii 12 25.8 43.0 13.6 4.8 0.1 2 25.5 48.7 11.0 4.0 0,4 Sporotrichum 6 26.1 41.6 10.1 5.3 0.2 thermophile 12 25.7 47.0 13.3 5.3 0.2 * Since previous tests showed that these 3 fungi would give no more than about 4% weight l o s s , c a l c u l a t i o n of chemical r e s u l t s could be based on e i t h e r sound or degraded wood with very 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 values. In t h i s test degraded wood weights were used. - 104 -E 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 not a t t a c k l i g n i n and do not appear to a t t a c k any of the carbohydrate c o n s t i t u e n t s except arabinose. Bergman and N i l s s o n (1968) have i n d i c a t e d that s o f t r o t f u n g i from a b i r c h chip p i l e , which 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 not 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 of the carbohydrate was i n d i c a t e d as being attacked. Henssen (1957) and Chang (1967) showed that S_. thermophile would decompose h e m i c e l l u l o s e s and p e c t i n . From t h i s l i m i t e d study i t i s p o s s i b l e to hypothesize that some of the sugar c o n s t i t u e n t s of the xylans i n the wood, e s p e c i a l l y arabinose may be attacked by the t h e r m o p h i l i c f u n g i . - 105 -V GENERAL DISCUSSION AND CONCLUSIONS A study of the d i s t r i b u t i o n of fungi i n a chip p i l e should include a l l types of fungi which might inhabit 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 studies concentrated mainly on mesophilic fungi (Bergman and Nilsson, 1966, 1967, 1968; Eslyn, 1967; Shields and U n l i g i l , 1968; Shields, 1970) which occur commonly at ordinary temperatures. The p e c u l i a r ecology of a chip p i l e c a l l e d f or an emphasis on fungi capable of l i v i n g at much higher temperatures. At the beginning of t h i s study, a l l fungi were i s o l a t e d , using two i s o l a t i o n temperatures, 25°C and 45°C. It was soon evident that many more fungi were i s o l a t e d at 45°C than at 25°C, e s p e c i a l l y from the areas of the chip p i l e where higher temperatures occurred. The wood chips stored i n the areas of the p i l e which produced large numbers of i s o l a t e s at 45°C l o s t more weight. Therefore i t appeared that greater a t t e n t i o n should be paid to the i s o l a t e s from 45°C than previous workers had given these fungi. Actinomycetes and Bacteria w i l l be common i n the chip p i l e s . Eslyn (1967) and Shields (1970) have i s o l a t e d b a c t e r i a and Eslyn (1967) has i s o l a t e d Actinomycetes from chip p i l e s , but these organisms were not considered to be important i n q u a n t i t a t i v e wood degradation and were not treated i n the present study. - 106 -The succession of fungi i n a chip p i l e w i l l depend upon many environmental factors such as temperature, the a b i l i t y of the fungi to use wood as a n u t r i e n t source, the a v a i l a b i l i t y of moisture, the pH of the wood and aeration i n the chip p i l e . Aeration i n the chip p i l e at Prince George was not investigated but i t has been suggested that accumulation of carbon dioxide or depletion of oxygen does not occur i n chip p i l e s (Hajny, Jorgensen and Ferrigan, 1967; Bergman and N i l s s o n , 1968). Moisture and pH were not found to be l i m i t i n g f a c t o rs i n the development of fungi i n the Prince George p i l e while a l l the fungi examined could use wood as n u t r i e n t source. The major f a c t o r which determined the succession of fungi i n the present study was temperature. Thus a successional pattern based on groups of fungi delimited according to t h e i r temperature tolerances i s given for the Prince George chip p i l e . This pattern w i l l vary from one area of the p i l e to the other. The i n i t i a l fungal colonizers of the chip p i l e were fungi capable of growing at ordinary temperatures, l i k e various species of Trichoderma,  Gliocladium, Ceratocystis and P e n i c i l l i u m , which were i s o l a t e d but not included i n t h i s study. Together with these fungi were thermotolerant fungi (Cooney and Emerson, 1964) l i k e A. fumigatus and C^ . pruinosum. With r i s i n g temperatures, the thermophilic fungi (Cooney and Emerson, 1964) began to grow i n the p i l e e s p e c i a l l y when temperatures rose above 22°C. These included organisms l i k e j5. thermophile and A. t e r r e s t r i s . Above 30°C, IS. emersonii and _T. aurantiacus - 107 -started to grow while some of the mesophilic fungi started to disappear from the chip p i l e . Most of the mesophilic fungi i n the p i l e might have died above 40°C when the a c t i v i t y of both the thermophilic and thermotolerant fungi was optimum. Some of the thermotolerant fungi probably died at temperatures above 50°C when 15. emersonii and T. aurantiacus were most a c t i v e . With temperatures above 55°C few fungal species w i l l remain a c t i v e among which were Ti. emersonii and T. aurantiacus. This pattern i s supported by the d i s t r i b u t i o n of fungi with storage time and the behaviour of some of these organisms on wood with changing temperatures. J3. emersonii may be the most important fungus i n chip storage i n the Prince George area. I t s development could r e s u l t i n loss of wood substance, increased temperature and eventual serious degradation of wood. In chip p i l e s i n Sweden (Nilsson, 1965; Bergman and Nilsson, 1966, 1967, 1968) C^ . lignorum has been found to be most d e s t r u c t i v e . In t h i s study, however, Chrysosporium was found to be a rare co l o n i z e r of the chip p i l e and these r e s u l t s suggest that i t should not pose a problem i n chip p i l e s i n and around Prince George. I t i s suggested here that the findings on a chip p i l e from one part of the world may not be true f o r another part of the world, however s i m i l a r conditions may be. No thermophilic or thermotolerant Basidiomycetes were i s o l a t e d from the spruce-pine chip p i l e i n Prince George. Chips w i l l come onto the p i l e heavily contaminated by fungi because of the present methods used i n handling chips. When a new p i l e i s near to an old p i l e , further inoculum w i l l come from the old p i l e . In a new area where no - 108 -p i l e s have been b u i l t before, where w i l l the inoculum come from? I t i s postulated that s u f f i c i e n t inoculum w i l l come from the s o i l and from air-borne spores to i n i t i a t e chip p i l e degradation. I t i s , however, suggested here that new chips should not be put on a base of old chip p i l e s which must act as a large r e s e r v o i r of i n f e c t i o n . ! Weight losses of wood caused by thermophilic and thermotolerant fungi i n the laboratory were higher than losses of wood substance obtained i n the Prince George p i l e . The higher losses of wood i n the laboratory are to be expected since weight losses are studied under optimal conditions which do not p r e v a i l i n the c h i p ; p i l e . When the weight losses of wood caused by thermophilic and thermotolerant fungi are compared to decay losses of wood caused by Basidiomycetes, the losses by the thermophilic and thermotolerant fungi are low. In chip p i l e s , the frequency of i s o l a t i o n of Basidiomycetes i s low (Nilsson, 1965; Shields, 1970). These f a c t o r s i n d i c a t e that losses i n the chip p i l e s are generally caused by microfungi. In areas of the chip p i l e where high temperatures occur most of the damage w i l l be done by the thermophilic fungi. This view i s supported by the observation from t h i s study that no further weight losses occurred from the s i x months' to the twelve months' sampling period when the low temperatures i n the p i l e would probably have a f f e c t e d the metabolic a c t i v i t y of the thermophilic fungi. I t has also been shown that no increases i n thermophilic and thermotolerant fungi occurred from the s i x months' to the twelve months' sampling period. I t was also shown i n the laboratory studies that a l l the thermophilic and thermotolerant - 109 -fungi would cause high weight losses of wood at temperatures above 40°C. This study has provided a method for t e s t i n g thermophilic fungi on wood. A chip p i l e i s obviously not b u i l t only of sapwood, but w i l l also include some heartwood. However, the percentage of heartwood i s low and may be l e s s than 10% (Keays, 1970). A fungus' a b i l i t y to attack sapwood as shown i n these studies w i l l r e f l e c t i t s a b i l i t y to destroy chips during storage. Both lodgepole pine and spruce are non-durable species and the thermophilic fungi may attack spruce and lodgepole pine heartwood during storage although not as strongly as the sapwood i s attacked. Bergman and N i l s s o n (1967) showed that C^ . lignorum would attack both the heartwood and sapwood of pine. Studies into the nature of attack of wood chips, by thermophilic and thermotolerant fungi, e s p e c i a l l y the chemical nature of the attack, are necessary. I t i s suggested that c o n t r o l of chip d e t e r i o r a t i o n i n Prince George should involve the prevention of the spread of thermophilic and thermotolerant fungi, e s p e c i a l l y J3. emersonii i n these p i l e s . I t might be shown con c l u s i v e l y that .B. emersonii does not attack c e l l u l o s e i n the wood, but t h i s w i l l not n u l l i f y the c o n t r o l of the fungus since i t may be responsible f o r thermogenesis i n the chip p i l e . Bergman and N i l s s o n (1968) have suggested that smaller p i l e s , where the i n s i d e of the p i l e i s near to f r e e z i n g , be b u i l t i n the winter and l a r g e r p i l e s , where the i n s i d e of the p i l e w i l l be at or about 60°C be b u i l t i n the summer. This suggestion i s the r e s u l t of l a r g e r losses i n winter p i l e s i n - 110 Sweden when the temperature was about 40°C i n the i n t e r i o r of the p i l e and smaller losses i n summer p i l e s when the temperature was about 60°C i n the i n t e r i o r . I t must be pointed out here that the p i l e at Prince George behaved l i k e the winter p i l e i n Sweden and that i f larger p i l e s are b u i l t during the summer i n Prince George, losses can only be prevented by keeping the i n s i d e temperature of the p i l e above 60°C for most of the time. This may be d i f f i c u l t since larger volumes of chips can s t a r t a chain r e a c t i o n which may lead to spontaneous combustion of the chips. In conclusion, i t has been shown that the hotter parts of the p i l e are inhabited by fungi and that weight losses are incurred i n these regions of the p i l e . Furthermore, weight loss was p o s i t i v e l y c o r r e l a t e d with both fungi and temperature. Controlled laboratory experiments have shown that the fungi commonly i s o l a t e d from the chip p i l e were capable of causing weight losses when compared to uninoculated controls maintained under the same experimental conditions. A chemical analysis of wood exposed to common thermophilic fungi in d i c a t e d that some of the sugar constituents of the xylans e s p e c i a l l y arabinose may be destroyed. I t i s concluded that thermophilic and thermotolerant fungi are d i r e c t l y responsible f o r the weight losses incurred i n the experimental wood chip p i l e . - I l l -REFERENCES A l l e n , D. 1968. F i r e i n chip p i l e s ! What to do. Pulp Paper 42 (27): 33-34. Annergren, G., S. D i l l e n and S. Vardheim. 1964. On Outside storage of spruce wood c h i p s . Svensk P a p e r s t i d n . 67: 125-245. Annergren, G., B. D i l l n e r , B. Haglund and G. Jagerud. 1965. Outside chip storage experiment. Svensk P a p e r s t i d n . 68: 309-326. American S o c i e t y f o r t e s t i n g m a t e r i a l s . 1961. Standard method of t e s t i n g wood p r e s e r v a t i v e s by l a b o r a t o r y s o i l - b l o c k c u l t u r e s . ASTM D1413-61. Anon, 1961. Chip p i l e s grow higher i n South as round wood handling d e c l i n e s . Pulp Paper 35(21): 79-81. B a l l , E. 1953. H y d r o l y s i s of sucrose by a u t o c l a v i n g media, a neglected aspect i n the technique of c u l t u r e of p l a n t t i s s u e . B u l l . Torey Botan. Club 80: 409-411. Bergman, 0. and T. N i l s s o n . 1966. On o u t s i d e storage of pine chips a t Lovholmen's Paper M i l l . Res. Notes. R53. Dept. of Forest Prod., Royal C o l l e g e of F o r e s t r y , Stockholm. Bergman, 0. and T. N i l s s o n . 1967. On o u t s i d e storage of aspen chips a t Hornefor's S u l p h i t e M i l l . Res. Notes R55. Dept. of Forest Prod., Royal College of F o r e s t r y , Stockholm. Bergman, 0. and T. N i l s s o n . 1968. On o u t s i d e storage of b i r c h chips a t Morum's Sulphate M i l l . Res. Notes R60. Dept. of Forest Prod., Royal College of F o r e s t r y , Stockholm. Bjorkman, E. and C. E. Haeger. 1963. Outdoor storage of chips and damage by microorganisms. Tappi 46: 757-776. Blackerby, L. H. 1958. Outside chip storage. Pulp Paper 32: 129-133. Blackerby, L. K. 1963. Separating chips lowers f i r e r i s k . Pulp Paper 37: 89-90. B r e t z l o f f , C. W. J r . 1954. The Growth and f r u i t i n g of S o r d a r i a f i m i c o l a . Am. J . Botany 41: 58-67. - 112 -B r i t i s h Standard I n s t i t u t i o n . 1961.. Methods of t e s t f o r t o x i c i t y of wood p r e s e r v a t i v e s to f u n g i . BS 838. Burke, M. 1962. Outside storage and handling of wood chips i n the west coast. Tappi 45(7): 164A-167A. Butcher, J . A. and M. Howard. 1968. Outside storage of Pinus r a d i a t a wood chips i n New Zealand. Tappi 51(4): 117A-122A. C a r l y l e , R. E. and A. E. Norman. 1941. M i c r o b i a l thermogenesis i n decomposition of p l a n t m a t e r i a l s . P a r t I I . Factors i n v o l v e d . J . of B a c t e r i o l . 41(6): 699-724. Chalk, E. 1968. Wood d e t e r i o r a t i o n during o u t s i d e chip storage. Pulp Paper Mag. Can. 69: 75-85. Chang, Y. 1967. The Fungi of wheat straw compost I I . Biochemical and p h y s i o l o g i c a l s t u d i e s . Trans. Br. Mycol. Soc. 50(4): 667-677. C l a r k , J . H. 1963. Economies w i l l accrue to any m i l l . Inherent advantages of O.C.S. make i t a good investment. Pulp Paper 37(10): 127-129. Cooney, D. C. and R. Emerson. 1964. Thermophilic Fungi. Freeman and Company, Lond. 188 p. Croon, I . 1966. A new continuous system f o r storage of c h i p s . P a p e r t e r i e 88: 1142-1143. Davis, V. J r . 1963. Outside storage of pine wood chips i n the South. South Pulp Paper Mfr. 26(1): 62, 66-67, 109. Duncan, C. G. 1953. S o i l b l o c k and agar b l o c k technique f o r e v a l u a t i o n of o i l type p r e s e r v a t i v e s creosote, copper naphthanate and pentachlorophenol. B e l t s v i l l e , Md. U.S. Dept. of A g r i c . D i v i s i o n of Forest Pathology. E s l y n , W. E. 1967. Outside storage of hardwood chips i n the Northeast. 2. M i c r o b i a l e f f e c t s . Tappi 50(6): 297-303. E s l y n , W. E. 1969. A new method f o r a p p r a i s i n g decay c a p a b i l i t i e s of microorganisms from wood chip p i l e s . U.S.D.A. For e s t S e r v i c e Research Paper F.P.L. 107' 8 p. Fe n s t e n s t e i n , G. N., J . Lacey, F. A. Skinner, P. A. Jenkins and J . Pepys. 1965. S e l f h e a t i n g of hay and g r a i n i n Dewar F l a s k s and the development of farmer's lung a n t i g e n . J . Gen. M i c r o b i o l . 41:383-407. - 113 -Fergus, C. L. 1969. The c e l l u l o l y t i c a c t i v i t y of t h e r m o p h i l i c f u n g i Actinomycetes. Mycologia. 60(1): 120-129. F i n d l a y , W. P. K. 1953. Infl u e n c e of sample s i z e on decay r a t e of wood i n c u l t u r e . Timb. Technol. and Mach.Woodwork. 61:160-162. F o r s s b l a d , L. H. 1965. Outside chip storage. P a p e r i Puu. 47(8): 455-462. Hajny, G. J . 1966. Outside storage of wood c h i p s . Tappi 49(10): 97A. Hajny, G. J . , R. N. Jorgensen, and J . J . F e r r i g a n . 1967. Storage of hardwood chips i n the Northeast. Tappi 50(2): 92-96. Hatton, J . V., R. S. Smith and I . H. Rogers. 1968. Outside chip storage: i t s e f f e c t s on pulp y i e l d and pulp q u a l i t y . Pulp and Paper Mag. Can. 69(15): 33-36. Hatton, J . V. 1970. P r e c i s e s t u d i e s on the e f f e c t s of o u t s i d e chip storage on f i b e r y i e l d . White spruce and lodgepole p i n e . Tappi 53(4): 627-638. Henningsson, B. 1967. Physiology of f u n g i a t t a c k i n g b i r c h and aspen p u l p -wood. S t u d i a F o r e s t a l i a Suecica. No. 52. Royal C o l l e g e of F o r e s t r y . Hensel, J . S. 1958. Storage of chips i n o u t s i d e p i l e s . Paper Trade J o u r n a l 142(12): 40-42. Henssen, A. 1957. Uber d i e Bedeutung der thermophilen Mikroorganismen f u r d i e Zesetzung des S t a l l m i s t e s . Arch. M i k r o b i o l . 27, 63-81. Holekamp, J . A. 1958. Outside storage of southern pine c h i p s . Paper Trade J . 142(49): 36-38. Holekamp, J . A. 1959. Further progress on the o u t s i d e storage of southern pine c h i p s . Paper Trade. J . 143(48): 34-36. Holekamp, J . A. 1962. Outside storage and handling of wood chips i n the South. Amer. Pulpwood A s s o c i a t i o n . Tech. Release No. 62R-11. H o s s f e l d , R. L., J . C. Oberg and D. W. French. 1957. The appearance and decay r e s i s t a n c e of d i s c o l o u r e d aspen. Forest Products J o u r n a l . 7(10):378-382. Hunt, K. 1970. Personal communication. - 114 -Isachenko, B. L. and N. N. Mai'chevskaya. 1936. Biogenic spontaneous heating of peat. Dokl. Akad, Nauk SSSR, 13:377-380. (In En g l i s h ) . Keays, J . L. 1970. Personal communication. Lindgren, R. M.and W. E. Eslyn. 1961. B i o l o g i c a l d e t e r i o r a t i o n of pulpwood and pulp chips during storage. Tappi 44(6):419-429. L i n d t , W. 1886. Mitteilungen uber einige neue pathogene Schimmelpilze. Arch. exp. Path. Pharmakol. 21:269-298. Ljungqvist, K. J . 1965. Temperature and moisture v a r i a t i o n i n a number of chip p i l e s . Svensk Papperstidn. 68(16): 527-533. Miehe, H. 1907. Die Selbsterhitzung des Heus. Eine b i o l o g i s c h e Studie. Gustav Fischer, Jena. 1-127 p. Nilsson, T. 1965. Mickroorganismer i f l i s s t a c k a r . Svensk Papperstidn. 68(15): 495-499. (Microorganisms i n chip p i l e s ) . Nordin, B., and L. Selleby. 1965. The e f f e c t of outside chip storage on t a l l o i l y i e l d . Svensk Papperstidn. 68, 1.6. Oades, J . M. 1967. G a s - l i q u i d chromatography of a l d i t o l acetates and i t s a p p l i c a t i o n to the analysis of sugars i n complex hydrolyzates J . Chromatogr. 28(2): 246-252. Robinson, G. W. 1963. Sawmill chip p i l e storage at Bowaters Mersey. Paper presented at annual meeting of the A t l a n t i c Branch, Technical Section, Canadian Pulp and Paper Association, Dalhousie, New Brunswick, Sept. 6, Mimeo. 15 p. Robinson, G. W. 1968. Prevention of wood chip d i s c o l o u r a t i o n at Bowaters Mersey. Pulp and Paper Mag. Can. 96(3): 50-54. Rothrock, C. W. J r . , W. R. Smith, and R. M. Lindgren. 1961. The e f f e c t s of outside storage on sla s h pine chips i n the south. Tappi 44(1): 65-73. Saucier, J . R. and R. L. M i l l e r . 1961. D e t e r i o r a t i o n of southern pine chips during summer and winter storage. Forest Products Jour. 11(8): 371-379. Savory, J . G. 1954. Breakdown of timber by Ascomycetes and Fungi Imperfecti. The Annal of appl. B i o l . 41(2): 336-347. - 115 -Sawardeker, J . S., J . M. Sloneker, and A. Jeanes. 1965. Quantitative determination of monosaccharides and t h e i r a l d i t o l acetates by g a s - l i q u i d chromatography. Anal. Chem. 37(12): 1602-1604. Selleby, L. 1965. Vednedbrytning och massakvalitet v i d f l i s l a r g r i n g . Svensk Papperstidn. 68(14): 477-481. (English Summary). Shields, J . K. 1967. M i c r o b i o l o g i c a l d e t e r i o r a t i o n i n the wood chip p i l e . Dept. Pub. No. 1191. Canada Forestry Branch. Shields, J . K. and H. H. U n l i g i l . 1968. D e t e r i o r a t i o n of softwood chips owing to outside storage i n New Brunswick. Pulp and Paper Mag. Can. 69(21): 62-67. Shields, J . K. 1970. Brown-stain development i n stored chips of spruce and balsam f i r . Tappi 53(3): 455-457. Somsen, R. A. 1962. Outside storage of southern pine chips. 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. Hulpoi. 1939. Influence of temperature upon the m i c r o b i a l population and decomposition processes i n composts of stable manure. S o i l Sc., 47:83-113. Westaway, A. G. 1968. Guidelines for outside chip handling. Pulp and Paper. 42(36): 24,30. Wolfrom, M. L. and A. Thompson. 1963. Reduction Products. (20) Reduction with sodium borohydride. In Methods i n Carbohydrate Chemistry V o l . 2. Edited by w h i s t l e r , R. L. and M. L. Wolfrom. Academic Press, New York. p. 65-67. Wright, E. 1954. A preliminary study of the d e t e r i o r a t i o n of alder and Douglas-fir i n outdoor p i l e s . U.S. Forest Service. P a c i f i c Northwest Forest and Range Expt. Sta. Res. Note No. 99. Young, C. E. 1961. The economics of chips from m i l l residuals-supply and demand and mutual b e n e f i t s . Southern Pulp and Paper Manufacturer. 24(2): 55-56, 58, 60. Zak, H., and E. Krauthauf. 1964. Changes i n s u l p h i t e pulp q u a l i t y owing to open a i r storage of hardwood and softwood chips. Das Papier 18(11): 691-699. - 116 -Appendix 1 To t a l count/100 chips of T_. aurantiacus, Byssochlamys sp. and C. pruinosum a f t e r s t o r i n g chips f o r 12 months Thermoascus Chrysosporium  aurantiacus Byssochlamys sp. pruinosum DURATION OF STORAGE OF CHIPS IN MONTHS P o s i t i o n i n P i l e 3 6 12 3 6 12 3 6 12 Outer bottom 0 0 1 2 0 0 1 2 1 Inner bottom 0 0 0 1 0 0 0 1 0 '0 Outer middle 0 1 1 0 1 0 21 18 15 Inner middle 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 - 117 -Appendix 2 Composition of media used Abrams medium NH4NO-3 3.0 g. K 2HP0 4 2.0 g. KH 2P0 4 2.5 g. MgS04*7H 20 2.0 g. Agar 20.Og. D i s t i l l e d water 1000.0 cc. Abrams-cellulose medium Same as Abrams with 10.0 g. of c r y s t a l l i n e c e l l u l o s e added. YpSs: Yeast-Starch agar (Emerson, 1941) Difco powdered yeast extract 4.0 g. K 2HP0 4 1.0 g. MgS04:.7H20 0.5 g. Soluble starch 15.0 g. Agar 20.0 g. Water (1/4 tap, 3/4 d i s t i l l e d ) 1 0 0 0 . 0 g. YpCs: Y e a s t - c e l l u l o s e agar Difco powdered yeast extract 4.0 g. K„HP0. 2.0 g. 2 4 MgS04.:7H20 2.0 g. Ce l l u l o s e 10. g. D i s t i l l e d water 1000.0 cc. - 113 Malt agar Difco malt Agar Water ( d i s t i l l e d ) M a l t - c e l l u l o s e agar Difco malt C e l l u l o s e Agar D i s t i l l e d water 20.0 g. 20.0 g. 1000.0 cc. 20.0 g. 10.0 g. 20.0 g. 1000.0 cc Combinations of NaNO , KH2P0 4' Difco yeast extract Components Medium 1 2 3 4 5 6 7 8 NH.N0„ 4 3 3.0 g. - + + - - - + + K 2HE0 4 2.0 g. + + + + + + + + KH 2P0 4 2.5 g. - + - + - + + MgS0 4-7H 20 2.0 g. + + + + + + + Difco yeast extract 4.0 g. - + + + - + + C e l l u l o s e 10.§.g.+ + + + + + + + Agar 20.0 g. + + + + + + + D i s t i l l e d water 1000.0 cc. + + + + + + + + = Component i s present - = Component i s absent - 119 Medium for i s o l a t i n g fungi Difco malt extract 20.0 g Difco agar 20.0 g Malic acid 5.0 g D i s t i l l e d water 1000.0 c Medium for growing fungi Difco malt extract 20.0 g Difco agar 20.0 g D i s t i l l e d water 1000.0 c 

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