WOOD DECAY PHYSIOLOGY OF T H E INKY CAP FUNGI
By
Jason P. Oliver
B.S. SUNY College of Environmental Science and Forestry, 2006
A THESIS
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Science
(in Ecology and Environmental Science)
The Graduate School
The University of Maine
December, 2008
Advisory Committee:
Jody Jellison, Professor of Biological Sciences, Advisor
Seanna Annis, Associate Professor of Biological Sciences
Bryan Dail, Research Scientist, Dept. of Plant, Soil, and Environmental Science
2008 Jason P. Oliver
All Rights Reserved
ii
W O O D D E C A Y P H Y S I O L O G Y O F T H E I N K Y CAP F U N G I
By: Jason P. Oliver
Thesis Advisor: Dr. Jody Jellison
A n Abstract of the Thesis Presented
in Partial Fulfillment of the Requirements for the Degree of
Master of Science
(in Ecology and Environmental Science)
December, 2008
Forest w o o d and litter decaying fungi are poorly understood despite their role in
forest ecosystem function. This is particularly true of the inky cap fungi, species originally in
the genus Coprinus. These fungi are c o m m o n on coarse woody debris and the litter layer of
forest soils however their degradative abilities haven't been adequately investigated.
T o characterize the w o o d decay potential of inky caps, ten isolates representing the
genera Coprinus, Coprinopsis, and Coprinellus were studied. Growth and colony characteristics
were measured on a variety of hard- and softwood substrates on agar and liquid medium and
in soil block jar decay cultures. C o m m o n wood-sugar monomers and hemicellulose extracts
were used as carbon substrates to investigate growth requirements and versatility of the
isolates. W o o d sugar composition of wood substrates before and following field and
laboratory decay was characterized by H P L C . Field and laboratory degraded hardwoods
were also used in decay tests to explore the role of inky caps as late stage decay fungi. In
addition to w o o d degradative physiology, tolerances to copper-based and polycyclic aromatic
hydrocarbon (PAH) compounds were measured to determine feasibility of fungal
remediation of treated wood. Results of all studies were analyzed for similarities and
differences both among and between species and genera.
Coprinellus species were typically the fastest growing, and were the only isolates to
decay w o o d blocks without pre-treatment. Poplar was the preferred substrate, with other
hardwood species decayed to a lesser extent. Preference for poplar is supported by the
resultant increase in biomass production when Coprinellus were cultured in media amended
with sugars abundant in hardwood hemicellulose, and failure to grow on media amended
with softwood hemicellulose sugars. Radial growth rates were not correlated with liquid
culture biomass production, however a stress reaction, a known strategy for late stage decay
fungi, is evident as cultures appeared abnormal with reduced biomass on softwood sugars
while growth rate increased.
All isolates produced higher weight losses following brown rot decay compared to
non-previously decayed controls. Coprinellus radians isolates produced the greatest weight
losses following four or more years of field decay and also had greater degradative
capabilities following white rot decay compared to untreated controls. Increased losses in
w o o d weight in the late stage decay experiments could not be attributed solely to w o o d sugar
levels and lignin chemistry and physical changes to the w o o d structure were likely involved.
N o n e of the tested isolates showed tolerance to copper or copper-based w o o d
preservatives. Observed tolerance to P A H preservatives was ambiguous, as some inky cap
fungi demonstrated ability to grow in the presence of these compounds in various Petri dish
cultures, but n o ability to decay P A H treated w o o d blocks.
This research suggests that some inky cap fungi, in particular Coprinellus isolates, are
effective at degrading hardwoods and that all may be capable of late stage decay in the
natural environment. Though further study of additional isolates is needed, this work
highlights distinct physiological differences between the genera and isolates of different
species.
ACKNOWLEDGEMENTS
I first want to thank the folks who introduced the world of fungi to me. In particular
this can be gratefully attributed to Alex Weir, June Wang, Tom Horton and Ed Setliff.
Without these inspiring folks I wouldn't have developed the skills or strong desire to attend
graduate school. They provided me the motivation to do so, and in particular, the interest
and excitement to explore this very intriguing group of litter fungi; the inky cap mushrooms.
I would like to thank my committee for their help, guidance and encouragement, especially
Jody who provided financial and infrastructural resources as well as the freedom to explore
all areas of interests as I learned to develop the skills of hypothesis development,
experimental design and organization. I would like to thank Caitlin Howell for her constant
friendship and advice, and for sharing the frustrations associated with being a young
scientist. Joan Perkins, thank you for endless edits and laughs and for keeping things in the
laboratory organized and functioning incredibly smoothly. I am indebted to my colleague
and friend Jonathan Schilling for his experience and support. Jessie Micales and Rita
Rentmeyer I would like to thank for their contribution of the fungal cultures that were used
in this research. Suping Lu, thank you for genetic identification of these cultures. Walter
Shorde should be thanked for both his wisdom and for the materials used in the wood decay
succession studies. I would like to thank Barry Goodell for his knowledge of wood decay
and preservation and for answering the many questions I had in these areas. Thanks to
Barbara Cole for answering of my many inquiries of wood chemistry. Sara Walton, thank
you for the access provided to wood sugar analytical equipment and the patience in
processing my many samples. Bill Halteman should be thanked for his expertise with
statistics and statistical software. Ban Weiping, thank you for supplying hemicellulose
extracts. Very special thanks must also be provided to the entire Jellison laboratory for
iii
coundess hours of assistance with the many tedious tasks that are required to produce
graduate research as well as for providing a both professional and comfortable atmosphere
to learn and work in. It was hard work but you all made the day very much a pleasure.
Thanks!
iv
TABLE OF CONTENTS
ACKNOWELEDGEMENTS
iii
LIST OF FIGURES
viii
LIST OF TABLES
xi
CHAPTER 1. INTRODUCTION & LITERATURE REVIEW
Introduction
1
Literature Review
3
Objectives
6
CHAPTER 2. BASIC PHYSIOLOGY & SUBSTRATE PREFERENCE
Abstract
7
Introduction
7
Materials and Methods
9
Cultural Conditions
9
Genetic Identification
10
Isolate Screening
10
Shake vs. Non-Shake Liquid Culture Technique
10
pH Preference
11
Wood Sugar Preference
11
Hemicellulose Extraction
12
Wood Species Preference
12
HPLC Wood Sugar Analysis
13
Ergosterol Analysis
13
Qualitative Enzyme Assays
14
Hemicellulase Quantitative Enzyme Assay
14
v
Statistics
14
Results
15
Discussion
28
Conclusions
38
CPiAPTER 3. SUCCESSIONAL DECAY STUDIES
Abstract
40
Introduction
40
Materials and Methods
41
USDA-FS Field Collected Forest Decayed Logs
41
Decay Test of Field Decayed Blocks
42
Brown Rot and White Rot Pretreatment
42
HPLC Wood Sugar Analysis
42
Analysis of Nitrogen
43
Results
43
Discussion
48
Conclusions
53
CHAPTER 4. TOLERANCE TO W O O D PRESERVATIVES
Abstract
54
Introduction
54
Materials and Methods
55
Copper Sulfate Tolerance — Petri Culture
Copper Sulfate Tolerance - Liquid Culture
Copper Sulfate Tolerance — Decay Tests
Tolerance to Ammonium Copper Quaternary - Petri Culture
vi
55
56
56
57
Tolerance to Ammonium Copper Quaternary — Liquid Culture
57
Tolerance to Ammonium Copper Quaternary — Decay Tests
57
PAH Tolerance - Petri Culture
57
PAH Tolerance — Decay Tests
58
Creosote Tolerance — Petri Culture
58
Creosote Tolerance - Decay Tests
58
Results
59
Copper Tolerance
59
PAH Tolerance
63
Discussion
67
Conclusion
70
CHAPTER 5. CONCLUSIONS
Objective 1 - Investigating the Ability of Inky Cap Fungi to Decay Wood
72
Objective 2 - Explore Inky Cap Fungi's Role as Late Stage Decay Fungi
74
Objective 3 -Prospect for Tolerance to Wood Treatment Chemicals
75
Objective 4 — Provide Substrate Preference and Physiological Data to Allow Interand Intra-species and Genus Comparisons
Remaining Work and Future Directions
77
78
BIBLIOGRAPHY
80
APPENDIX
87
BIOGRAPHY OF THE AUTHOR
88
vn
s«(S.
LIST O F FIGURES
Figure 2.1. O v e n dried biomass (mg) of inky cap cultures grown on CMM in shaken flasks
at a buffered p H ranging from 3-9
16
Figure 2.2. Average radial growth rate (mm/day) of inky cap isolates propagated on CMM
with glucose, CMM with hemicellulose sugars substituted for glucose, or with n o
added carbon
18
Figure 2.3. Comparison of radial growth on CMM Petri dish cultures solidified with agar
or the more highly purified agarose
19
Figure 2.4. Average radial growth rate (mm/day) of inky cap growth on CMM with glucose,
Avicel substitution or n o added carbon source
20
Figure 2.5. Oven dried biomass weight (g) produced by inky cap culture in CMM media with
glucose, other substituted hemicellulose sugars or n o carbon source
21
Figure 2.6. Average radial growth of inky cap isolates in Petri dish culture on CMM
amended with crude hemicellulose extract from hardwood and softwood chips
23
Figure 2.7. Average biomass production by inky cap isolates in liquid culture of CMM
amended with crude hemicellulose extract from hardwood and softwood chips
23
Figure 2.8. Average radial growth rates (mm/day) for inky cap isolates propagated on w o o d
powder amended CMM
25
Figure 2.9. Average percent weight losses in red spruce and poplar sapwood blocks decayed
by inky cap isolates in unmodified A W P A standard soil blocks jar assays
26
Figure 2.10. Average percent weight loss in sapwood blocks after 12 weeks of decay by inky
cap isolates
27
viii
Figure 3.1. Weight losses in long-term field decayed red maple sapwood blocks degraded by
inky caps. Sapwood blocks represent 0 to 10 years of field decay, collection years are
noted in the figure key
44
Figure 3.2. Inky cap mushroom decay of poplar wood blocks following an eight week
pretreatment by Gloeophyllum trabeum brown rot decay and Trametes versicolor'white
rot decay
45
Figure 3.3. Inky cap decay of red maple wood blocks following an 8 week pretreatment of
Gloeophyllum trabeum brown rot decay and Trametes versicolorwhite rot decay
46
Figure 3.4. Monosaccharide concentrations of control, G. trabeum and T. versicolor decayed
poplar blocks
47
Figure 3.5. Monosaccharide concentrations of control, G. trabeum and T. versicolor decayed
maple blocks
48
Figure 4.1. Average radial growth rate of inky cap isolates on CMM with increasing molar
copper concentrations of copper sulfate
59
Figure 4.2. C. radians ME-209 biomass and filtrate pH in liquid cultures of copper amended
CMM
60
Figure 4.3. C. radians ME-209 biomass and radial growth rate on Cu amended CMM
60
Figure 4.4. Average radial growth rate of inky caps on CMM amended with ACQ treatedSYP and untreated-SYP
61
Figure 4.5. 2 week dry weight biomass of inky cap isolates grown in shaking liquid culture
with either untreated southern yellow pine (SYP) or ACQ-treated SYP as the carbon
source
62
Figure 4.6. Average radial growth rate of inky cap isolates on CMM, Birch, Pine and Oak
amended CMM medias overlaid with the PAHs pyrene and phenanthrene
IX
64
Figure 4.7. Average radial growth on oak amended CMM and CMM amended with railroad
tie material removed from service
65
Figure 4.8. Average percent weight loss associated with 12 weeks of inky cap decay of oak
blocks, oak blocks treated with acetone and oak blocks treated with pyrene using an
acetone solvent
66
Figure 4.9. Inky cap average radial growth on CMM amended with oak wood powder and
railroad tie wood powder
67
x
LIST O F TABLES
Table A.l. Table of Isolates
87
XI
C H A P T E R 1: I N T R O D U C T I O N & L I T E R A T U R E R E V I E W
Introduction
Many species of basidiomycetous fungi are known to modify and degrade wood and
other cellulosic materials. Some of these wood decaying species, including Gloeophyllum
trabeum, Serpula lacrymans, Phanerochaete chrysosporium, Pleurotus ostreatus and Trametes versicolor,
have been thoroughly investigated generating insight into the mechanisms and chemical
changes of fungal decay (Goodell et al., 2003). With the exception of these model decay
species, biomodification and physiology studies of most w o o d inhabiting and w o o d decaying
fungi are limited. This is especially true of most forest litter and late-stage decaying species.
These fungi have not been used for biopulping, scaled-up bioremediation projects and are
not known to cause structural damage or failure of wood in service, and their lack in
economic importance has resulted in litde attention given towards their basic biology and
degradative abilities.
T h e inky cap mushrooms are one subset of litter decaying fungi that have recently
gained some research attention. Inky caps are a deliquescent group of basidiomycetous fungi
once known collectively as the genus Coprinus, part of the family Coprinaceae, but now
assigned to the four separate genera Coprinus sensu stricto in the family Agaricaceae, and the
genera Coprinopsis, Coprinellus and Parasola in the new family Psathyrellaceae (Redhead et al.,
2001). This group of fungi has recently been investigated as a model for laboratory-based
life history studies of mushroom-forming basidiomycetes based on their ease of culture and
short life cycle (Kiies, 2000). Inky caps have also attracted attention for their efficient
peroxidase production and the ability of these enzymes to detoxify phenolic compounds
from industrial effluents (Kauffmann et al., 1999). Despite these recent interests,
characteristics of inky cap substrate biomodification, wood decay and degradative physiology
have largely been ignored.
1
This thesis was intended to establish a basic understanding of the degradative
physiology of five inky cap species {atramentarius, cinereus, comatus, micaceus and radian),
representing three of the new genera (Coprinus, Coprinopsis and Coprinellus). The project relied
on adaptations of early coprinoid growth studies with the goal of applying new wood
biomodification and degradative physiological studies to produce a more complete
understanding of these fungi. Basic physiological analysis investigated preferences for
substrate, monitored production of degradative enzymes and followed changes to the
structure and chemistry of wood during and after decay. Owing to previous findings that the
affinity some inky caps have for certain hemicelluloses and pentose sugars (Fries, 1953), it
was hypothesized that these fungal isolates will preferentially decay hardwood substrates over
softwoods. It was recognized that some species of inky caps decay woods in late stages of
degradation (Peiris et al., 2008) and as a result it was also hypothesized that these fungi are
better able to successfully colonize and efficiendy decay highly modified woody substrates.
While few inky cap fungi have been shown to be of economic importance in the
decay and loss of treated wood products, their cultivability, enzymology, and potential
tolerance of wood preservatives may make them candidates for the bioremediation of human
generated wastes. Selected isolates were also evaluated for potential to remediate wood
preservatives, as the disposal of treated woods, construction debris and contaminated soils is
of great environmental concern. Tolerance and metabolism of organic treatment
compounds (creosote and other polyaromatic compounds) may be observed in some of the
isolates as peroxidase production has been detected in most members of this group;
peroxidases have previously been shown to be efficient agents of polyaromatic contaminant
bioremediation (Gadd, 2001).
2
Inter- and intra-species and genus differences were also investigated. It was
hypothesized that isolates of similar species will have similar substrate preferences and
physiological responses. It was also hypothesized that species within the same genera will
behave physiologically similarly and will be significantly different from isolates of other
genera. These analyses will be used to better understand the physiologic underpinnings to
the recent changes in the taxonomy of these fungi based upon DNA phylogenetic
differences.
Literature Review
Coprinus, a genus in the family Coprinaceae, is the original taxonomic grouping of the
litter decomposing basidiomycetous fungi commonly known as the inky caps. Hopple and
Vilgalys (1994) were the first to show that Coprinus was paraphyletic and they hypothesized
the genus to be polyphyletic. Coprinus, now recognized as polyphyletic, has since been
subdivided into the genera Coprinus sensu stricto, Coprinellus, Coprinopsis, and Parasola by
Redhead et al. (2001) based upon molecular phylogenetic research. There is little debate over
the phylogenetic separation of the genus Coprinus, but there is disagreement over the naming
of the new genera. Currendy. the type specimen of the old grouping C. comatus holds the
genus name Coprinus while the majority of the remaining species from the old grouping are
included in a new genus; Coprinopsis (Hawksworth, 2001). Some have argued that hasty
changes in nomenclature lead to confusion and presents problems in educating new
mycologists (Anonymous, 2001). Jorgensen et al. (2001) has proposed that Coprinus should
instead be typified by C. atramentarius and Coprinus comatus and Coprinus sterquilinus should be
renamed Coprinopsis instead of changing over a hundred species to Coprinopsis. As of this
writing, C. comatus remains the type species of Coprinus, and this thesis will use the new names
proposed by Redhead et al (2001).
3
Comprehensive investigations into the life history and developmental processes of
the model organism Coprinus (Coprinopsis)rinereushave been completed and reviewed by Kues
(2000). Despite these extensive studies, very little work has been done on other aspects of
inky cap physiology such as wood biomodification and degradative physiology. Early
nutritional and physiological investigations conducted by Rege (1927) focused on
decomposition of cellulosic material by Coprinus species. Coprinus (Coprinellus) radians-was
shown to rapidly decompose hemicellulose and cellulose (Waksman, 1931). Additional
investigations of nutritional physiology particular to Coprinus were carried out by Fries (1955).
She suggested that there were three types of Coprinus decomposers based on enzyme tests
and carbon source utilization studies, but found no correlation between ecological niche and
decomposing type: Type I- Capable of decomposing cellulose and lignin, Type II - Capable
of decomposing cellulose but not lignin, and Type III - Capable of lignin decomposition but
not cellulose degradation. The group as a whole aggressively metabolizes hemicelluloses and
pentose sugars according to Fries (1955), which supports previous work done by Rege (1927)
and Waksman (1931).
Fries also investigated the influence of pH, concentration of metals and temperature
on inky cap growth (1956), reporting an optimal incubation temperature of 25°C for most
isolates. The preferred pH range was approximately 5 to 7 with Coprinus (Coprinellus) micaceus
preferring a pH of 5, Coprinus comatus a pH of 6, and Coprinus (Coprinopsis) atramentarius a pH
of 7.
More current physiological studies have focused on enzyme production, particularly
peroxidases and their application in enzymatic systems designed to remove or detoxify
aromatic and phenolic compounds from wastewater. Class II fungal peroxidases with no
ligninolytic activities were isolates, purified and characterized from Coprinus (Coprinopsis)
rinereus in the 1980s (Shinmen et al, 1986, Morita et al, 1988). The Coprinus peroxidase (CiP)
4
is recognized to be a member of the structurally distinct class II peroxidase sharing 89% of
its amino-acid sequences with class II peroxidases produced by the white rot fungus
Phanerochaete chrysosporium. The substrate specificities of Coprinus peroxidase (CiP) however
are more similar to horseradish peroxidase than those obtained from other white rotting
species in spite of the fact that the horseradish peroxidase enzymes share just 18% of their
DNA sequences with the sequences coding for CiP in inky cap species. These structural
similarities but substrate differences of CiP compared to other class II white rot peroxidases
highlight the need for definition of a new family of peroxidases (Baunsgaard, 1993). CiP
production has subsequently been detected in cultures of Coprinus (Coprinopsis) friesii
(Heinzkill et al. 1998), Coprinus (Coprinopsis) lagopus (Ikehata and Buchanan, 2002), Coprinus
(Coprinopsis) echinospora, Coprinus (Coprinopsis) macrocephala (Ikehata et al., 2004), Coprinus
(Coprinellus) radians and Coprinus (Coprinellus) micaceus (Anh et al. 2007). Anh et al. (2007) also
demonstrated the potential for peroxidase activity in at least eleven other inky cap species.
Dritsa et al. (2007) investigating a culture of Coprinus (Coprinellus) xanthothrix did not detect
peroxidase activity, but manganese peroxidase activity was measured, a peroxidase not seen
in any other inky cap isolate.
Interest in Coprinus peroxidase is growing as it is easily produced and has high specific
activity and broad substrate specificity. Al-Kassim et al. (1994a, 1994b) have attempted to
optimize a bioreactor-scale phenol removal system using a Coprinus (Coprinopsis) macrorhi^us
peroxidase. Ikehata et al. (2003) have investigated the treatment of oil refinery waste water
with a crude extract of Coprinus (Coprinopsis) cinereus peroxidase. More recently, the use of
fungal peroxidases, including CiP, and laccases for waste water treatment were reviewed by
Ikehata et al 2004.
5
Some investigations into the production of laccases and hemicellulases by inky cap
species have also been published. Laccase activity has been measured in Coprinus (Coprinopsis)
psychromorbida by Inglis et al. (2000), in Coprinus (Coprinopsis) cinereus by Hoegger et al. (2006),
and in Coprinus (Coprinellus) xanthothrix by Dritsa et al. (2007). Xylanase, a class of enzymes
that degrade the hemicellulose xylan, has recently been detected in Coprinus (Coprinopsis)
psychromorbida (Inglis et al., 2000), one of the only publications addressing inky cap
hemicellulose production.
This research focused on the basic physiology and substrate requirements of the inky
cap fungi in the genera Coprinus sensu stricto, Coprinopsis, and Coprinellus, all formerly members
of the genus Coprinus, and the effect of substrate on their growth and enzyme production.
This investigation will improve our knowledge of the differences among these genera, the
basic ecology of this group and their role in fungal succession, as well as assess the potential
these fungi have as agents of bioremediation.
Objectives
1
- To investigate the ability of Coprinus, Coprinellus and Coprinopsis species of
inky cap fungi to decay wood and in particular, the characterization of their
growth responses on various woods and wood sugars.
2
- To explore inky cap fungi's role as late stage decay fungi through growth
responses in decay tests using field and laboratory pre-decayed woods.
3
- Prospect for potential tolerances to copper based and organic based wood
preservatives.
4
- To analyze inter- and intra-species and genera similarities and differences to
address physiological underpinnings of recent phylogenetic changes to the
classification of these fungi.
6
CHAPTER 2: BASIC PHYSIOLOGY & SUBSTRATE PREFERENCE
Abstract
Litde is known about the basic biology, physiology, and ecology of the inky cap
fungi. Their ability to grow on various wood sugars and wood species was investigated using
Petri dish-based and liquid culture-based studies as well as with laboratory wood decay
experimentation. All isolates seem to have high growth rates and large biomass production
on the wood sugars mannose, glucose and xylose. There was reduced growth on galactose
amended media and media with no added carbon (C) source. There was particularly poor
growth when the sugar arabinose was used as the C source. Successes on particular C
substrates were determined by measuring growth rates on Petri dish cultures and with dry
weight biomass measurements of liquid cultures. There was no correlation between these
two measurements as C sources that didn't provide sufficient nutrition, evident by changes in
culture characteristics and reductions in biomass production, typically caused increases in
radial growth rates. Only the Coprinellus isolates were capable of decaying wood and this
ability was limited to hardwoods, in particular the tested poplar species (IJriodendron tulipifera).
Preference for poplar could be attributed to preference for hardwood hemicellulose sugars
but might also be the result of low extractive levels, lignin structure, and physical properties
of the wood such as diffuse pores and thin cell walls (Cole, 2008). Further investigation is
needed to better understand Coprinellus species decay preferences.
Introduction
Our understanding of the basic biology and physiology of the inky cap mushrooms is
in its infancy. Known to be the commonest basidiomycetes on dung (Richardson, 2001),
and prevalent in productive soils, on forest litter and wood chips; inky caps have also been
observed fruiting in abundance on living and dead trees (Rayner and Boddy, 1988;
Heilmann-Clausen and Boddy, 2005; Peiris et al, 2008; & Setliff, 2008). Despite their
7
abundance on various woody and humified substrates, there is a dearth of published work
about their degradative ability (Guiraud, 1999). Lisbeth Fries publications are some of the
only work addressing inky cap decay, but her focus was primarily nutrition and not
degradation. The commonality of inky caps on woody substrates suggests they play a
significant role in the break down and humification of wood and forest litter to soil and
humic materials despite the lack of research attention
Some studies peripheral to addressing inky cap decay abilities have focused on
interactions with early stage decay fungi and with the production of wood degrading
enzymes. Competition assays addressing interaction responses in the presence of other
decay fungi have been completed in Petri dish culture by Heilmann-Clausen and Boddy
(2005) and by Peiris et al. (2008). Extracellular wood degrading enzyme activities of inky cap
basidiomycetes have been addressed with qualitative Petri plate assays by Fries (1955),
Saponsathien (1998), and Steffen et al. (2000). Quantitative measurements have also been
made, with particular focus upon peroxidase, its characterization, and its potential to degrade
PAHs to the exclusion of natural or model lignin substrates (Andersen et al., 1991; Kjalke et
al., 1992; Ikehata et al., 2004; and Anh et al, 2007). Guiraud et al. (1999) has addressed
degradation of lignin-like phenolic compounds, however his focus was more on
chloroaromatic compounds than lignin per se.
Beyond improving our understanding of the ecology of these coarse woody-debris
decaying fungi, wood degradation studies could aid the development of new technologies.
Commercial cultivation of some of the choice edible species in this group such as Coprinus
comatus or Shaggy Mane, which is coveted by wild collectors, could be accomplished with a
better understanding of preferred woody and wood sugar substrates. To optimize ethanol
production the cellulosic biofuels industry requires economical methods for the removal of
lignin and 5 carbon sugars from wood to increase accessibility to fermentable sugars.
8
Current hydrolysis techniques are energy intensive however and attention has recently
focused on biological pretreatments of cellulosic materials (Galbe and Zacchi, 2007;
Agbogbo and Coward-Kelly, 2008). With additional research, inky caps, recognized as
efficient degraders of hemicelluloses with abilities to attack lignin, could potentially be used
in these industrial applications.
Agar media, liquid culture, and soil block decay tests were used to study colonization
by Coprinus, Coprinopsis and Coprinellus species and to identify optimal pH and preferences for
specific wood sugars and wood types. Investigating growth and physiological responses to
substrate composition including decay of various wood types will highlight intra and inter
genus and species similarities and differences among these new phylogenetic groups, while
also increasing understanding of late successional wood and litter decay fungi.
Materials and Methods
Culture Conditions
Several isolates of inky cap fungi were initially obtained for the research. Rate of
growth and colony characteristics were monitored on potato dextrose agar (PDA) and malt
extract agar (MEA) (n=4) and isolates with poor reproducible growth rates were excluded
from future experiments. Ten isolates representing five species and three genera of
Coprinaceae were selected for this investigation and were maintained on Coprinus minimal
medium (CMM) slants (Stevens, R.B., 1981). All ten isolates were obtained from national
culture collections and are listed in the Appendix. One isolate of Coprinopsis atramentarius, two
isolates of Coprinus comatus, two isolates of Coprinellus micaceus, and two isolates of Coprinellus
radians were all obtained from the USDA-FS mycological culture collection maintained at the
Forest Products Laboratory in Madison, Wisconsin. Two isolates, one Coprinus comatus and
9
one Coprinellus micaceus, were purchased from the American Type Cultural Collection (ATCC)
Manassas, VA. Coprinopsis cinerea was obtained from the Fungal Genetics Stock Center at the
University of Missouri at Kansas City.
Genetic Identification
Identifications of cultures were checked by amplifying the noncoding rDNA region
of the inky caps using the primer pairs ITS r F and ITS4-B and PCR techniques. The
amplified regions were sequenced and the sequences were checked against sequences
previously uploaded in the NCBI-Genbank by BLAST searching.
Isolate Screening
Isolates Coprinopsis atramentarius DAOM 192254, Coprinopsis lagopus var. spaerospora
ME-271A-R and Coprinopsis quadrifidus FP-101909-T which were obtained from the Forest
Products Laboratory in Madison, WI were excluded from further experimentation due to
irreproducible and poor growth characteristics.
Shake vs. Non-Shake Uquid Culturing Techniques
75ml of malt extract or CMM liquid media were dispensed into 125ml Erlenmyer
flasks. To compare biomass production between shaken and non agitated liquid culture
growth 10 replicates for each inky cap isolate for each medium were inoculated by floating
two 0.5cm diameter plugs of 2 week old plate cultures onto the media. Five of the replicates
were placed in the dark at room temperature for incubation. The other 5 replicates were
incubated in the dark at room temperature and shaken at 100 rpm on an orbital shaker.
Cultures were collected by vacuum filtration through a Buchner funnel with a sterile, tarred
#1 Whatman filter paper. Dry biomass was determined following 24hrs of drying at 105°C.
10
pH Preference
pH preference was investigated using Clark and Lubs, National Bureau of Standards
0.1 M Potassium buffer solutions (Bower & Bates, 1955). Isolates were tested in solutions of
pH 3, 4, 5, 6, 7, 8, and 9. The buffers replaced deionized, distilled water in the Coprinus
minimal medium. Aliquots of this modified media (75 ml) were measured into 125 ml
Erlenmeyer flasks, adjusted for pH following sterilization aseptically using a sterile solution
of ION sodium hydroxide, inoculated and incubated at room temperature and 100 rpm
rotation for 2 weeks [n=4]. Cultures were collected by vacuum filtration as above.
The pH of the various wood species used later in this chapter in decay tests
were determined by homogenizing 10ml of 40-mesh wood flour with 10ml of deionized
distilled water by vortexing and then measuring the pH of the water fraction using a
accumet® Research AR-10 pH meter (Fisher Scientific) outfitted with a accumet® 13-620108 probe.
Wood Sugar Preference
Primary cultures were propagated for two weeks on Coprinus minimal medium Petri
dish plates. An inoculum plug 0.5 cm in diameter was taken from the margin of growth and
inoculated at the edge of the modified CMM Petri dish plates. The monomers arabinose,
galactose, glucose, mannose and xylose were used at a concentration of 2% (w/v) as the sole
carbon sources in the media along with a no sugar control. Growth was measured every
three days from the inoculum plug to the edge of growth on Parafilmed Petri dish cultures
incubated at 25°C to determine average daily growth rates [n=5].
Purified cellulose (Avicel ® PH-101, Fluka) [2% (w/v)] and crude hardwood and
softwood hemicellulose extracts [2% (v/v)], or [2% (w/v)] following lyophylization, were
also used as test sugar polymer substrates [n=5].
11
Water hemicellulose extractions are outlined below. Purified lignin (Organosolv, Aldrich)
[2% (w/v)] was also substituted as the carbon source in CMM for measurement of growth
rates.
Modified CMM liquid cultures (75 ml) shaken at 100 rpm were inoculated by floating
two 0.5 cm diameter fungal plugs transferred from primary plate CMM cultures. Cultures
were incubated with the individual C sources at room temperature, in the dark, at 100 rpm
for 14 days. Cultures were collected by vacuum filtration through a Buchner funnel with # 1
Whatman filter paper. Dry biomass was determined following 24hrs of drying at 105°C. In
some cases cultures were homogenized using a blender prior to filtrations and 2 ml of fungal
puree was removed and extracted into 3 ml of cold 100% methanol for ergosterol analysis.
Hemicellulose Extract
Southern yellow pine, loblolly pine (Pinus taeda), and a hard wood species, red maple
(Acer rubrum), were chipped and wood chips less than 10mm wide were extracted in
deionized water using 300ml cylindrical bomb digesters at 160°C for 70 minutes according to
methods outlined in Yoon et al. (2008). Extracts were added to CMM for Petri dish cultures
or buffered liquid cultures at 2% v/v. Remaining extracts were freeze dried using a VirTis
Freezemobile 25 SL Lyophilizer yielding dry crudely extracted hemicellulose powders also
used as the carbon source in CMM at 2% w/v.
Wood Species Preference
Wood species preference was determined in a manner similar to wood sugar
preference by measuring growth rates [n=5] and liquid culture biomass [n=4] on Coprinus
minimal medium amended with 40-mesh milled wood flour [2% (w/v)]. Wood species
tested were southern yellow pine (either Pinus echinata, palustris, taeda or elliottii), red spruce
(Picea rubens), eastern white cedar (Thuja occidentalis), red oak (Quercus rubra), yellow poplar
(JJriodendron tulipifera), yellow birch (Betula alleghaniensis), and red maple (Acer rubrum).
12
The different hardwood and softwood species were also used in the standard soil
block jar wood decay test method (AWPA, 2006). Weight losses and in some cases, HPLC
wood sugar analysis were used to characterize physical and chemical changes to the wood
after decay.
HPLC Wood Sugar Analysis
The various wood species blocks were analyzed for wood sugars using an Shimadzu
HPLC with a RID 10A refractive index detector (Marlborough, MA) fitted with a Bio-RAD
Aminex HPX-87P (lead ion) column (Hercules, CA) heated to 80°C, using deionized distilled
water as a mobile phase, a 20 [i.1 injection volume and a flow rate of 0.6 ml min"\ Prior to
HPLC analysis blocks were ground in a Wiley-Mill to pass a 40-mesh screen. Wood powders
were depolymerized to monomers with 4% H 2 S0 4 , followed by neutralization with calcium
hydroxide, and filtered through a Whatman G D / X 0.2[i.m PES filter.
ErgosterolAnalysis
Ergosterol, a component of fungal membranes was extracted from 2 week old liquid
cultures in cold 100% HPLC grade methanol and separated with HPLC using a method
developed by Schilling & Jellison (2005). Samples [n=4] were refluxed, saponified with 4%
KOH in ethanol, refluxed, and centrifuged twice. Supernatants were combined and mixed
with 1 ml deionized distilled water and 2 ml pentane. The pentane layer was removed after
15 minutes and allowed to evaporate over night. Methanol was used to re-dissolve the
precipitate and the solution was filtered through 0.2 ja.m PTFE into sampling vials.
The ergosterol was quantified using a Synergi Hydro-RP (Phenomenex, Torrance,
CA) 80A reverse-phase column (5[im, 250 x 4.6 mm I.D.) heated at 38°C, using methanol as
the mobile phase, a 20|j.l injection volume and a flow rate of 0.6 ml min \ Detection was at
285 nm and quantification was peak area. Elution times were compared to a 95 % pure
ergosterol (Aldrich) standard
13
Qualitative Enzyme Assays
Xylanase production was assayed by measuring radial growth of cultures side
inoculated onto CMM agarose medium Petri plates with birch wood xylan (Sigma) as the
soul carbon source (Luo et al., 2005). Radial growth was measured every three days until
hyphal growth crossed the plate.
Ligninolytic activity was assayed by the color change associated with the presence of
laccase or peroxidase produced on 2,21-azmo-bis-3-ethyl-benz-thiazoline-6-sulfonic acid
(ABTS) amended CMM media. Plates center inoculated were observed for 4 weeks for the
formation of green or green-blue zones under or around the cultures (Steffen et al., 2000).
Manganese peroxidase was assayed by monitoring center inoculated cultures of CMM
media with added MnCl 2 -4H 2 0 (200[i.M) for 6 weeks for flecks of oxidized Mn. All plates
for these assays were secured against desiccation using Parafilm, and incubated in the dark at
25°C. Five replicates were used for each assay.
Hemicellulase Quantitative Enzyme Assays
In addition to measurements of growth rate, biomass production, and decay,
hemicellulase production was measured using azo-labeled xylan, galactan, and galactomannan
purchased from Megazyme (Country Wicklow, Ireland). Changes in absorbance from
standard blanks, attributed to dye labeled sugar monomers liberated from sugar polymers,
were measured at 590 nm on a Spectronic® Genesys 2 spectrophotometer.
Statistics
The statistical program R 2.5.1 was used for the analysis of results. Analysis of
variance (ANOVA) was the primary analysis conducted, comparing controls to treatments.
The Tukey HSD post-hoc analysis was used for multiple comparisons of means. Substrate
utilization comparisons were made between Petri dish, liquid, and solid wood block studies
14
using correlation analysis. Inter and intra isolate differences were compared. All error bars
are standard deviation of the mean. Linear regression modeling was used to find the best
correlates of decay rate. A significance value of p=0.05 was used for all analyses.
Results
NCBI-Genbank blast search results from amplified ITS regions of each isolate
matched the culture identification with the exception of isolate Coprinellus micaceus ATCC
20122. Identifications of this isolate generated by the Blast search were in the same genera,
but differ by species as illustrated by the 97% congruency of the amplified ITS region from
isolate C micaceus ATCC 20122 with Coprinellusfloccuksus, 92% with Coprinellus radians and
only 91% shared base pairs with documented C. micaceus sequences.
Screening for optimal pH growth conditions were conducted in liquid culture as agar
has difficulty solidifying at low pHs (Hastrup, 2007). Growth by all isolates in non-agitated
liquid culture were compared to growth of isolates under similar cultural conditions except
shaken at 100 rpm on an orbital shaker. Agitated flask cultures resulted in significant
increases in biomass for all isolates on both malt extract and CMM and as a result. All liquid
culture experimentation used in this thesis were shaken at 1 OOrpm.
Growth in liquid culture by all Coprinopsis and most Coprinus cultures was poor
(Figure 2.1). Biomass measurements indicated that most Coprinopsis isolates grew most
vigorously at a pH of 9. Coprinopsis atramentarius FP-101910-T had the greatest average
biomass at pH 7, but growth at pH 5 and pH 9 were not significantly different. Isolate
Coprinopsis cinereus FGSC 9003 produced the most biomass at pH 9, but growth at pH 7 and
pH 8 were not statistically different. All Coprinus isolates had a statistically significant positive
growth response at pH 8 compared to biomass accumulated at all other pH levels. Coprinellus
micaceus isolates had a significant preference for pH 6 while the erroneously identified culture
of this species ATCC 20122 had greatest biomass production at pH 7, but this growth was
15
not significandy different from that measured at other pHs. Coprimllus radians isolates
seemed to grow equally as well in a pH range of 6-8 (Figure 2.1). The buffered pH media
showed no significant change in pH between initiation of the study and at biomass harvest in
replicates of all isolates.
700.000
600.000
Ia
D3
S4
Q5
06
400.000
Bio
P
500.000
*J
• 7
• 8
• 9
Wei
300.000
a
200.000
100.000
0.000
if
<*'
s y
i$>
J?
y
y
C?
^
^
/
rf*
>
y
Isolates
Figure 2.1. Oven dried biomass (mg) of inky cap cultures grown on CMM in shaken flasks
at a buffered pH ranging from 3-9.
Petri dish screening of Coprinopsis cinereus FGSC 9003 was unsuccessful due to failure
to grow on CMM, however, when grown on MEA solid media, the growth trends were
similar to diose observed of other isolates. All other isolates grew successfully on CMM.
Coprinellus isolates compared well under the cultural conditions used for Petri culture
assaying of hemicellulose preference; they typically had the greatest average growth rates with
particularly significant differences seen in the growth of C. radians isolates. In these
screenings, the greatest growth rates for all isolates were typically on CMM amended with
16
mannose, followed by growth on glucose then xylose amended plates. The Coprinopsis isolate
grew most vigorously on CMM with glucose and mannose as the C source, and the poorest
on arabinose amended CMM. The largest radial growth rates for all Coprinus isolates were on
glucose, mannose, and xylose, growing least impressively on the other sugars. Isolates
Coprinellus micaceus FP-101781-T and isolates ME-798, and with the exception of mannose,
isolate Coprinellus radians ME-352, all produced statistically greater biomass on CMM with no
C source followed by growths on mannose, glucose, and xylose successively. The probable
misidentified C micaceus ATCC 20122 also grew significandy fastest on mannose, glucose,
and xylose, but had poor growth on CMM with no added C source. Coprinellus radians isolate
ME-209 grew significandy greatest on media with mannose, followed by growth on CMM
with no additional C source, xylose then galactose. Growth rate on CMM without a sugar
addition was also significantly greater than glucose and xylose growth rates with isolate C
radians ME-209 (Figure 2.2).
17
s
• No Sugar
0 Arabinose
D Galactose
B Xylose
o
QDMannose
M Glucose
•o
<
J>
>'
<*'
&
cPv
<5»
cPN
^
>
&
.#
^
Isolates
Figure 2.2. Average radial growth rate (mm/day) of inky cap isolates propagated on CMM
with glucose, CMM with hemicellulose sugars substituted for glucose, or with no added
carbon.
Observed cultural characteristics for all isolates on xylose, mannose, and glucose
media were normal with matted aerial hyphae of average diameter which was usually
pigmented white to rusty-orange. Isolates grown on arabinose, galactose, and especially on
media with no added sugar typically had sparse, hyaline hyphae with smaller diameters as
compared to those grown on supplemented media and generally lacked aerial hyphae. When
more highly refined agarose was substituted for agar to address the possibility that cultures
were using the agar as a carbon source, growth rates were significantly reduced on the media
with agarose (Figure 2.3).
18
rJ*
Isolates
Figure 2.3. Comparison of radial growth on CMM Petri dish cultures solidified with agar
or the more highly purified agarose.
When growth on CMM media without a carbon source, unmodified CMM (glucose
carbon source) and supplemented with crystalline a-cellulose were compared, average radial
growth rate was significantly greatest for Coprinopsis and Coprinus isolates on unmodified
CMM with glucose (Figure 2.4). Coprinellus isolates with the exception of C. micaceus ATCC
20122 grew the slowest on unmodified CMM. The remaining C. micaceus isolates grew most
rapidly on media lacking an added carbon source though this growth was not significant. The
C. radians isolates grew fastest on Avicel amended media, with this growth and hyphal
extension on CMM media with no added carbon source statistically greater than growth on
unmodified CMM (Figure 2.4). Organosolv was also used as the sole carbon source in Petri
dish cultures, but no appreciable growth was produced by any isolate.
19
Coprinopsis
atramentarius
FP-101910-T
Coprinus
comatus FP101592-T
Coprinus
comatus FP101691-T
Coprinus
Coprinellus
Coprinellus
Coprinellus
comatus
micaceus FP- micaceus MEmicaceus
ATCC 12640
101781-T
798
ATCC 20122
Coprinellus
radians ME352
Coprinellus
radians ME209
Isolates
Figure 2.4. Average radial growth rate (mm/day) of inky cap growth on CMM with glucose,
Avicel substitution or no added carbon source.
Hemicellulose preference screening in liquid culture generated results that were not
consistent with growth rates achieved on solid media with the same C source (Figures 2.2 &
2.5). Biomass production in CMM media with no C source, or with arabinose or galactose as
a carbon source was miniscule; less than 0.07g after 2 weeks for Coprinopsis and Coprinus
cultures and less than 0.03g for Coprinellus cultures. Biomass production in Coprinopsis and
Coprinus cultures on the other sugars was also limited, < 0.1 g after 2 weeks, while Coprinellus
isolates grown on glucose and xylose achieved significantly greater growth during the study
period. Media amended with mannose stimulated the Coprinellus isolates to produce the
greatest biomass of any treatment source, with the exception of lagging growth by Coprinellus
micaceus ATCC 20122 (Figure 2.5).
20
Isolates
Figure 2.5. Oven dried biomass weight (g) produced by inky cap culture in CMM media with
glucose, other substituted hemicelluloses sugars or no carbon source. No (no carbon source
added), ara (arabinose), gal (galactose), xyl (xylose), man (mannose) and glu (glucose).
Measurement of hemicellulase levels in the filtrates of the liquid cultures suggested
that expression of some of these enzymes may be inducible in certain inky cap cultures.
While significandy higher endo-l,4-(3-xylanase levels were not measured in xylose amended
CMM for most the Coprinus or Coprinopsis isolates, a noticeable increase was detected in
cultures of C. comatus FP-101691-T. Significant increases in hemicellulase activities were
detected in xylose amended CMM by two C. micaceus isolates and isolate C. radians ME-209 as
well. An increase was observed in C. radians ME-352 but this was not significant. Xylanase
levels were typically between 50 and 500 mg/ml for most isolates and treatments. On
mannose amended CMM, endo-l,4-|3-mannanase levels were greater, in many cases
significandy, than those measured in CMM with other C sources, a significant difference for
21
C. comatus FP-101592-T and FP-101691-T. There were no observed increases of mannanase
expression with Coprinellus or Coprinopsis isolates. Mannanase levels did not exceed 50 mg/ml
and were typically around 5 mg/ml. No significant differences of endo-l,4-(3-galactanase
activities were measured on different treatments. Galactanase levels were typically between
100 and 300 mg/ml, but some measurements were as high as 2000mg/ml. No isolate
routinely expressed hemicellulases in excess of 500mg/ml.
When degrading enzymes were measured qualitatively in Petri culture, an ability to
grow and produce enzymes was observed on xylan by all isolates, particularly strong in the
case of the Coprinellus species. There was an observed color change in ABTS plates of all ten
isolates implying that either laccase or peroxidase was being produced. None of the isolates
were able to oxidize Mn suggesting no production of manganese peroxidase under the
conditions of this study.
Plate growth comparing inky cap average radial growth on crudely extracted
hemicellulose wood polymers revealed significandy greater growth rates on hardwood vs.
softwood extracts amended media by the isolate Coprinellus radians ME-352 (Figure 2.6).
When comparing isolate growth in liquid culture containing softwood or hardwood extracts,
no significant differences in biomass were observed except for isolate Coprinopsis atramentarius
FP-101910-T. C atramentarius FP-101910-T produced greater biomass on hardwood
hemicellulose amended media (Figure 2.7).
22
Isolates
Figure 2.6. Average radial growth of inky cap isolates in Petri dish culture on CMM
amended with crude hemicellulose extract from hardwood and softwood chips.
0.06
Isolates
Figure 2.7. Average biomass production by inky cap isolates in liquid culture of CMM
amended with crude hemicellulose extract from hardwood and softwood chips.
23
CMM cultures using dried hemicellulose extract as the carbon source produced
different results than those obtained using hemicellulose extracts in solution. Only Coprinellus
and two of the Coprinus isolates produces measurable growth in Petri cultures and in every
case, radial growth on softwood was greatest, significandy for isolate C. radians ME-209. In
liquid culture using the dried hemicelluloses, there were no significant differences in biomass
production between the softwood and hardwood sources of the extract.
Average radial growth rates on CMM amended with wood powder were comparable
to the growth rates of the isolates on many of the hemicellulose amended CMM treatments
and the Coprinellus isolates again grew most rapidly. Growth rates on hardwood amended
CMM were greater than those on softwood amended CMM for all isolates. While birch
amended CMM supported the greatest growth in general, there were few growth rates that
differed significandy from rates measured on other hardwood amended media. Again, the
isolate Coprinellus micaceus ATCC 20122 behaved significandy different from other isolates of
the same species (Figure 2.8).
24
Figure 2.8. Average radial growth rates (mm/day) for inky cap isolates propagated on wood
powder amended CMM.
Standard AWPA (2006) soil block decay tests using red spruce and poplar blocks
decayed by inky cap isolates suggest an inability by all isolates to decay sapwood blocks cut
from softwood species. The hardwood blocks were undecayed by Coprinopsis and Coprinus
isolates, but significant weight losses were seen in the poplar blocks decayed by all of the
Coprinellus isolates (Figure 2.9). These weight losses are much smaller than what is typically
reported for most wood decaying species.
25
• Spruce 16wks
• Spruce 24wks
@ Poplar 8wks
• Poplar 12wks
y
N
C°
/
</ •
r
</ /
,<*•
-o?
if
.o<?
Isolates
Figure 2.9. Average percent weight losses in red spruce and poplar sapwood blocks decayed
by inky cap isolates in unmodified AWPA standard soil blocks jar assays.
Decay tests using a larger variety of wood species indicated the general inability of
most inky cap isolates to significanuy decay the selected species of wood. The softwood
species of wood were not decayed significanuy by any of the inky cap isolates. Coprinopsis
and Coprinus isolates did not significanuy decay any species of wood block. Coprinellus
isolates, with the exception of isolate Coprinellus micaceus ATCC 20122, measurably decayed
poplar blocks. C. micaceus FP-101781-T, ATCC 20122 as well as C. radians ME-209
significandy decayed red maple and oak, but weight losses were minimal; less than 1.5% and
2% respectively (Figure 2.10).
26
D Red Spruce
BSYP
B Cedar
• Poplar
B Birch
• Red Maple
• Red Oak
Isolates
Figure 2.10. Average percent weight loss in sapwood blocks after 12 weeks of decay by inky
cap isolates.
No correlations were observed between growdis of a particular culture in the three
different medias (Petri plate, liquid culture, wood block). No measured growth indicators
could accurately predict decay ability. For example: radial growth on xylose was not a
accurate predictor of biomass production on xylose and no biomass or radial growth
measurement on galactose could predict the extent of decay of a softwood block.
Ergosterol analysis conducted on samples harvested from Petri dish, liquid, and
decayed wood block samples yielded no usable results. Standard curves were reproducible
and clear in HPLC outputs, but sample data was variable and most often negligible. Because
original samples were hypothesized to be too dilute, subsequent samples were lyophilized.
Unfortunately, this concentration step yielded no measurable peaks on die chromatograms
and no usable data, probably owing to problems resulting from the extraction process.
27
Biomass removed from liquid culture was also extracted for ergosterol to ensure sufficient
material was being extracted and again no measurable peaks were produced. The filtration
step of the extraction seemed the likely point for loss of extracted ergosterol as syringe filters
with a PES membrane had been substituted for the PTFE filters in the early analyses, the
only change from the optimized extraction procedure.
Standard ergosterol samples were analyzed in triplicate by HPLC without filtration
and after filtration with PTFE and PES filters of the same pore size. All three filtrates
produced measurable peaks that accurately represented the concentrations of the standards.
When standards were prepared and processed following the same protocol used in the
exttaction procedure, the ergosterol was lost as evidenced by the lack of peaks on the HPLC
cbromatograms in the expected range for elution of standards. In some instances, residual
ergosterol was observed in test tubes in the final step of the extraction after overnight
evaporation of the pentane solvent. Attempts to recover ergosterol consistently at this step
were unsuccessful.
Discussion
Measurement of ergosterol extracted from Petri dish, liquid and soil block decay
cultures was not possible and as a result biomass from Petri dish and decay tests was not
quantified. Biomass harvested from liquid culture was quantified, but probable error exists
in dry weight biomass measurements due to hyphal production of extracellular mucilaginous
material, metabolic exudates previously having been shown to account for approximately
20% of the dry weight (Jellison et al., 1997). Ergosterol, a membrane bound sterol, can be
used as an estimate of fungal cells as extracellular mucilaginous material, typically produced
in greater quantities under stress conditions, is not measured by this analysis
28
(Vesentini et al., 2005). Discussion of biomass measurement hereafter refers to dry weight
biomass measurements obtained by vacuum Buchner filtration through a # 1 Whatman filter
paper subsequently dried for 24hrs at 105°C.
Of the isolates investigated in this study there was no wood decay ability shown by
any of the species in the genera Coprinopsis or Coprinus. Coprinellus isolates on the other hand,
had a distinct ability to decay hardwoods, in particular poplar. Though the weight losses
associated with Coprinellus decay were significant, the losses are less than those reported for
most decay fungi. Lacking ergosterol—based biomass production data on wood powder
amended media, a comparison between Petri and liquid culture could not be made. This was
a result of the weight of the wood powder creating too large an error to account for small
differences in liquid culture biomass and no dry weights of Petri culture biomass was
attempted. The Petri dish cultures amended with wood powder did however provided
observational data on biomass in the form of radial growth rates. Growth rates on
hardwood amended media for all Coprinellus isolates were greater than rates on media
amended with softwood. This held true for Coprinopsis and Coprinus isolates with the
exception of C. atramentarius and C. comatus FP-101592-T grown on poplar. Colony
characteristics on softwood were similar to those on galactose, arabinose, and with no sugar
source in Petri dish cultures. Although ergosterol analyses failed and there was no observed
correlation of statistical significance between radial growth rate and decay on different wood
species, observations of biomass on hardwood plates (in particular poplar) seems to be in
line with ability to cause decay.
It was interesting to note the large biomass production of Coprinopsis atramentarius in
liquid culture on these substrates, especially on hardwood. This was by far the greatest
growth by this isolates on any of the substrates tested.
29
Solid media screenings comparing radial growth on CMM amended with various
hemicelluloses showed that the greatest growth rates were typically on mannose, glucose, and
xylose, while growth on arabinose was typically the slowest. These higher growth rates were
significant for Coprinus isolates, and compared to arabinose and galactose they were
significantly greater for Coprinellus isolates on mannose and Coprinopsis growth on glucose and
mannose. Of the three genera, growth rates of Coprinellus species were typically the greatest,
with C. radians rates significantly greater than those of C. micaceus.
In many cases however, and in particularly with the Coprinellus isolates, hyphal
extension rates measured with no added sugar were not significandy different from the
extension rates measured for mannose, glucose, and xylose-amended CMM. The correcdy
identified Coprinellus micaceus isolates and C. radians ME-352 had significandy the greatest
growth rate on media with no additional C source. C. radian ME-209 also showed rapid
growth rates on CMM minus an additional C source, but these rates were not significandy as
great as rates on mannose amended CMM. Comparable growth rates were also noted on
galactose CMM but these rates were significandy different.
Dry weight biomass measurements, like radial growth rates, were significandy greater
for Coprinellus isolates, and were greatest on glucose, xylose, and mannose amended CMM.
In liquid culture however, biomass measurements in galactose or with no added sugar were
minimal and didn't represent the radial growth rates measured. Biomass on galactose or in
the absence of an additional C source were significandy less than biomass harvested from
cultures grown with glucose, xylose and mannose sugar treatments for most isolates.
Coprinus growth on galactose and no sugar was significandy different from growth on
mannose and both mannose and glucose respectively. Coprinopsis growth was significandy
stunted widiout additional C amendment. Arabinose addition however did not promote
radial growth nor boost liquid culture biomass and must be completely un-metabolizable.
30
Correlation analysis showed no positive or negative agreements between data
obtained using Petri culture radial growth rate and the dry weight of liquid culture biomass
for any treatment or isolate. Successful ergosterol analysis, correcting for error associated
with extracellular materials may have yielded similar results according to Guillen and Angela
(2008) as they also reported no correlation between radial growth and biomass for nine decay
fungi and a blue stain fungus.
Increased radial growth rates without production of biomass on galactose, and in the
absence of additional C sources, may indicate a nutritive stress reaction. Starvation may
trigger a fungal culture to search for new substrates by increasing hyphal extension at the
cost of biomass production, the later being a typical indicator of culture health. Reduced
biomass measurements in liquid culture correspond with observations of colony
characteristics in Petri dish culture, i.e., thin hyphal networks may quickly extend over solid
media, yet little biomass accrual occurred as compared to luxuriant growth and spore
production observed on media with additional C sources. Specifically, cultures grown on
galactose and sugarless solid media were atypical compared to normal growth seen on CMM,
MEA, or PDA with woolly, aerial mycelium lacking and reduced pigmentation, density and
diameter of the hyphae. Absorptive hyphae were reduced and small chords and exploratory
hyphae were more abundant. These characteristics were also seen with arabinose amended
cultures which produced the smallest biomass production though it also generated minimal
radial growth rates for all genera. This suggests that arabinose may be completely unmetabolizable by inky caps so any growth is the result of energy stored in the original
inoculum plug. Under nutrient-stressed conditions, inky cap fungi, particularly Coprinellus
isolates, may be capable of increasing growth rates of exploratory hyphae to seek out new,
more appropriate substrates.
31
These findings are contrary to most radial growth rate results of starved cultures, but
most of this work has centered on the Deuteromycete Aspergillus. The mold Aspergillus ory^ae,
for instance, had a significant reduction in growth rate in media free of glucose (Pollack et
al., 2008). Pollack et al.'s (2008) work is in agreement with the concept that specific growth
rate is a function of glucose concentration (Nielsen and Krabben, 1995; Agger et al., 1998;
Spohr et al., 1998). Perhaps the furanose sugar arabinose's affect on inky cap growth might
be in line with these findings.
The reductions in biomass and changes in morphology observed on CMM with
galactose, arabinose, and with no carbon source are in agreement with the models presented
by Bartnicki-Garcia et al.(1989) and updated in 2001 (Gierz and Bartnicki-Garcia). They
suggested that hyphal diameter will depend on material supplied (i.e. nutrients) to assemble
new cells. Agger et al. (1998) also noted a reduction in hyphal diameter under low nutrient
conditions. Muller et al. (2000) suggested that the relationship between hyphal diameter and
growth rate is related to a need of the fungal culture to maintain (per nucleus) a relatively
constant cytoplasmic volume. Therefore, in order for the inky cap isolates to increase
exploratory hyphal length to search for more suitable growth substrates upon nutrient
deficient media, hyphal growth must be reduced in other parts of the mycelium.
Boddy and Rayner (1983) suggested that late colonizers invade substrates in
conditions of low environmental stress and disturbance but that these decomposer
communities are products of primarily competitive interactions. Work by Peiris et al. (2008)
suggested that these competitions are reduced between middle and late stage as compared to
early and middle stage colonizers. Stated another way, late stage colonizers are less
aggressive competitors. Peiris et al. (2008) observed Coprinellus micaceus invading fallen wood
following 10-15 years of decomposition by means of yellow-brown mycelial cords. Others
have also observed inky caps to be extremely late stage decay fungi (Setliff, 2008). Perhaps a
32
rapid growth rate on a depleted media is a mechanism employed by inky caps for quickly
scavenging a substrate, metabolizing some of the freely available sugars that remain while
avoiding competition.
The preference for xylose, mannose and glucose and the stress induced on galactose
and arabinose may help explain inky cap's, in particular Coprinellus species', ability to decay
hardwoods and not softwoods. It is widely accepted that hardwood hemicelluloses are
primarily acetylated xylan with a minor unacetylated glucomannan hemicellulose fraction.
Softwood hemicellulose on the other hand is primarily acetylated glucomannan with
galactose groups 1-6 linked with a minor xylan fraction with acid and arabinose side chains
(Sjostrom, 1981). All hardwoods tested in this study had significantly greater xylose levels
than any of the softwoods. This was also seen in work comparing hemicelluloses in many
different plants including Vopulus and Pinus tree species and was summarized by Puis and
Schuseil (1993). Poplar glucose levels, though not significantly so, were greater than all other
wood types. Perhaps this partially explains the preference for poplar over all other wood
species in decay tests. Owing to high variability among assays, an increased sample size may
have made such differences significant.
Regarding other sugar contents in the wood decay analyses, there was no significance
in mannose observed, but arabinose and galactose levels were significantly greater in the
softwoods. Higher levels of arabinose and galactose in softwoods are expected as these
sugars are not present in hardwood hemicelluloses (Sjostrom, 1981) and were reported by
Puis and Schuseil (1993). The lack of biomass production on these sugars in pure cultures
grown on solid media implies an inability of inky caps to metabolize them. These sugars
therefore should be avoided in commercial cultivation substrates and could explain why
there was no appreciable weight losses in sound softwoods by inky caps.
33
The polymerized hemicelluloses of the crude extracts neither in Petri nor liquid
culture by inky caps supported the hypothesis that hardwood hemicelluloses are preferred
and more easily metabolized than softwood hemicelluloses. In both cases most of the
differences in growth were non-significant between hardwood and softwood hemicellulose.
In solid media cultures, only one isolate, Coprinellus radians ME-352, grew better on hardwood
hemicellulose amended CMM, and in liquid culture only isolate Coprinopsis atramentarius FP101910-T grew better on hardwood hemicellulose amended CMM. Similar lack of significant
difference were seen when the crude hemicellulose extracts were standardized by
lyophylization to dryness and added at a weight to volume concentration. Again, increasing
replicate size may better examine these materials as a growth media. In concert with better
characterization of the substrates, our understanding of hemicelluloses role in the
degradation of woods by inky caps would be gready improved.
Generally, it is recognized that appendages on backbone chains of hemicelluloses
impair the depolymerization action of hemicellulase enzymes. The side chains do however
increase aqueous solubility, increasing the chances that these enzymes will interact with
hemicellulose polymers (Puis and Schuseil, 1993). Yoon et al. (2008) found xylan-arabinose
side chains to be cleaved in softwood hemicellulose during acid hydrolysis but to still be
present following the hot water hemicellulose extraction used for this research. The
arabinose appendages likely reduced the effectiveness of the xylanase enzymes produced by
the inky caps. Galactose of softwood hemicellulose was found to remain attached to
glucomanan even following acid hydrolysis or water extraction and likely inhibits backbone
hemicellulose attack. It is quite possible then that limited growth on softwood hemicellulose
polymers was occurring due to side chain interference with enzymatic activity, but this was
not detected by the assays used in this study.
34
Perhaps the growth and biomass that was produced on the softwood hemicellulose
extract that was measurable can be attributed to the debranching and arabinofuranosidase
properties of the xylanases produced by the inky caps. Some types of xylanases described
have, in addition to the ability to break xylan into xylose oligosaccharides, xylobiose, and
xylose, the ability to cleave L-arabinosyl branch points (Reilly, 1981). Arabinases however,
were not measured in these studies. To date, there has been little characterization of inky
cap hemicellulases, in particular xylanases. There is need for the characterization of inky cap
hemicellulases.
Growth on the hardwoods may have been reduced due to lack of easily digestable
monosaccharides in the crude hemicellulose extract necessary to initiate growth and stimulate
production of energy expensive enzymes. Hemicellulose polysaccharide, despite its
solubility, cannot enter a fungal cell, the hemicellulose liquid culture experiments may have
contained no inducement for the synthesis of the hydrolytic enzymes. Xylanolytic systems
are known to be induced by xylobiose and xylotriose (Biely et al., 1980) as well as by xylose
alone (Yasui et al, 1984; Pou-Llinas and Driguez, 1987; Dobberstein and Emeis, 1989). The
initial enzymatic work done in this research supports the notion that xylanases are produced
by inky caps and that their production is induced by xylose in some of the isolates, Coprinellus
species, in particular. The same Coprinellus isolates that were successful degraders of wood
had increased endo-l,4-(3-xylanase levels on xylose amended CMM.
pH may be yet another factor affecting ability to decay a wood species. Coprinopsis
atramentarius and cinereus grew well across a pH range of 5-9 and 7-9 respectively. Fries's 1956
work with C. atramentarius suggested an optimum pH of 7 and documented a range of pH,
from 7.8 to 8.1 for tested substrates, from which specimens were collected. Work by
Soponsathien (1998) reported a pH preference between 6 and 7 for two isolates of C. cinereus
and the findings of Guiraud et al. (1999) who measured 7.0-7.5 as an optimum for C. cinereus,
35
both largely in agreement with the findings in this study. All Coprinus comatus isolates had
significandy greater growth at pH 8 in disagreement with Fries's previous work (1956) which
documented the greatest biomass production at pH 6. The pH of the C. comatus substrates
Fries tested ranged from 7.7 — 7.9 however; closer to the optimum found in this work.
Coprinellus micaceus and radians produced the greatest dry weight biomass at pH 6 and pH 6-8
respectively. Fries suggested pH 5-7 facilitated optimum growth and the pH of natural
substrates tested ranged from 6.6-7.6 (1956). Guiraud et al. (1999) found a pH preference of
5.0 — 6.5 for a C. micaceus isolate. Although Fries's work with C. comatus does not align
completely with current findings regarding optimal pH, all other species tested had similar
preference ranges to those previously documented. Substrate pH values were overlapped for
similar genera for all of the current species tested. The work of Fries and current work show
inky cap preferences for higher pHs than those of most wood degrading hymenomycetes
studies in liquid culture (Wolpert, 1924).
An alkaline preference is contrary to current work addressing preferred pH ranges of
well studied decay fungi. Significant reduction in pH by brown rot fungi have been
demonstrated in liquid culture (Jellison et al., 1997; Milagres, 2002; Muchuca, 2001) and in
Petri dish culture (Guillen and Angela, 2008). White rot fungi have also been shown to
lower the media pH in liquid culture (Milagres, 2002; Muchuca, 2001) and Petri dish culture,
but the change in pH was not significant with all species investigated (Guillen and Angela,
2008). Brown rot and white rot fungi have also been shown to significandy reduce pH in
decaying wood substrates. Jellison et al. (1992) found that after 4 months of decaying poplar
the white rot fungi T. versicolor and P. chrysosporium decreased the pH of wood from pH 5.9 to
5.2 and 4.4 respectively while the brown rot fungi G. trabeum and P. placenta reduced the pH
to 4.4 and 3.7 respectively. The more extreme reduction in pH by brown rot fungi is
thought to be a function of organic acid production, in particular oxalic acid. White rot fungi
36
also produce these acids but due to metabolic differences, they tend to accumulate them to a
lesser degree (Jellison et al., 1997; Machuca et al., 2001; Milagres et al., 2002). No effort has
been made to measure the production of these acids in inky cap fungi.
pH measurements of the wood species tested in the decay study did find poplar,
maple, and birch pHs to be significantly greater than oak, and the softwoods. Maple, the
least acidic, had a pH of 6.11 (±0.099) followed by poplar wood at pH 5.425 (±0.114).
These pHs are in the range of preference for the Coprinellus isolates and may partially explain
their ability to decay while the other genera can not. Personal observation as well as those of
others (Setliff, 2008) have observed inky caps in abundance on living and dead elm trees,
many of the isolates used in this work were originally collected from dying elm roots and
elm stumps. Elm pH was measured, but was in a range not significandy different from that
of the softwood species and disputes the significance of substrate pH. Inky caps likely
produce some organic acids with capacity to buffer pH external to the cell, though to date
they have not been investigated and no measured changes to liquid culture pH were
significant.
As neither wood sugar chemistry nor pH can clearly explain inky cap preference for
poplar wood, other possible explanations should be considered. Lignin, the other primary
polymer of wood could play a significant role. There are distinct differences in hardwood
and softwood lignins (Filley et al, 2000) and it is likely that minor differences exist from
species to species in addition to those between angiosperms and gymnosperms. Another
possible explanation is extractives; hardwoods tend to have lower levels of extractives and
poplar in particular is thought to be relatively low in extractive content (Cole, 2008). Cole
(2008) also mentioned that poplar is a diffuse porous tree species with thin cell walls
suggesting that physical, as well as chemical properties of these tree species are also playing a
37
role in subsequent colonization and decay. It is changes to both physical properties and the
w o o d chemistry which may dictate when late stage decaying inky caps invade w o o d in the
natural environment
At each level of organization (genus, species, and isolate) there were significant
difference among groups and cases of correlation among isolates of the same species and
species of the same genus. O n e exception was the isolate misidentified C. micaceus A T C C
20122 which was in many cases significantly different from the results of the other C. micaceus
isolates. These correlations and the many differences between genera outlined above lend
some support for the modern changes to phylogeny based on molecular techniques. These
new genera might also help explain Fries's inability to find correlation between ecological
character and decomposer type (Fries, 1955). There is some coherency between the
groupings of inky caps she made according to her findings and the recent reorganization of
the inky caps. This work suggests that genetic organization does somewhat match with
physiology and ecological niche. Coprinellus species seem to have rapid growth rates, an
ability to decay hardwoods supported by decay tests and observational data, xylose induced
xylanase, and a nutrient stress induced increase in hyphal extension rates. The isolates of the
other genera used in these assays did not express these same traits.
Conclusions
In conclusion, Coprinellus isolates have higher growth rates, produce more biomass
and are more capable of w o o d decay than the species nested in the other genera. All of these
isolates prefer p H in the neutral to alkaline range and do not tend to reduce extracellular or
media p H as observed with many white rot fungi. Nutrient-stressed conditions seem to
reduce biomass production but increase hyphal extension rates of Coprinellus isolates. As a
result, radial growth rate and biomass production were n o t correlated. This could be a
strategy employed by a late colonizing decayer to avoid competition by moving quickly.
38
Though Coprinellus isolates, in particular C. radians, could significandy decay hardwood
substrates compared to controls, the measured weight losses were small compared to
primary wood decaying fungi. Poplar was a preferred substrate. It appears that Coprinellus
are late colonizers of wood, have effective decaying machinery, and bridge the
transformation of wood and litter to soil humic matter.
39
CHAPTER 3: SUCCESSIONAL DECAY STUDIES
Abstract
To explore the effect of pre-decay treatments on inky cap fungal decay, field decayed
red maple sapwood blocks were exposed to inky cap fungi in pure culture decay tests. No
significant decay was produced by any Coprinopsis or Coprinus isolates. Great variation was
seen between replicates, but most Coprinellus isolates successfully degraded blocks subjected
to 4-10 years of field decay. The greatest decay rates were seen after the 10th year of field
decay. The brown rot G. trabeum and white rot T. versicolorwere used as pretreatments on red
maple and poplar blocks to explore the effect of laboratory decay pretreatments on
subsequent inky cap decay. Brown rot pretreatment significantly increased decay by most of
the inky cap isolates on both wood types. White rot pretreatments did not effect the
subsequent decay rates seen when the wood was exposured to Coprinopsis and Coprinus
isolates but did significandy increase decay by Coprinellus radians isolates. Both wood
chemistry and physical changes to the wood associated with prior attack by brown or white
rot fungi are believed to be responsible for increased inky cap decay.
Introduction
Late successional decay and the process of soil formation from coarse woody
residues are poorly understood. We depend on these processes for continued recycling of
plant debris and for contributions to soil formation and quality. An understanding of fungal
degradation is therefore crucial to inform resource managers to make the decisions that will
sustain and improve the health of human ecology and our natural environment.
Understanding these processes in detail may also facilitate development of innovative new
technologies for waste management, biofuels and energy production, and other industries.
Currently, only a minute number of the late stage litter and wood degrading fungi involved in
40
this process have been investigated (Steffen et al, 2007), with most of the research focused
on phylogeny/biodiversity, enzyme production, and with litde published work on one decay
group in particular, the inky cap fungi.
Most inky cap fungi have traditionally been considered soil or dung dwelling, hence
the genus 'Coprinu? (Coprophilus in Greek meaning 'dung-loving'). Field observation and
laboratory investigation however suggest some members of the genus are late successional
wood decayers growing on a range of substrates from sound wood to highly degraded
woods, humified lignin residues, and soils. Pekis et al. (2008) investigated the interaction of
early decay fungi with late stage decaying inky caps, but the work did not use natural wood
substrates. It is hypothesized that some of the species capable of decaying sound wood will
cause increased weight losses in woods that have undergone primary decay. To explore this
hypothesis, the role of inky cap fungi in wood degradation was investigated; characterized by
measuring growth and decay abilities on field degraded materials and on woods pre-decayed
in the laboratory by white and brown rots. In addition to enhancing understanding of inky
cap decay characterizing the degradative processes employed by inky caps lend physiological
and ecological credence to recent phylogenetic work that has moved the inky caps into the
family Psathyrellaceae, a litter and late stage decay family of fungi (Redhead et al. 2001).
Materials and Methods
USDA-FS Field Collected Forest Decayed Fogs
As part of a larger long-term study primarily conducted by Dr. Walter Shortle of the
United States Department of Agriculture-Forest Service investigating 'The Role of Fungi in
the Biotransformation and Nutrient Cycling in the Forest Ecosystem', approximately 3 inch
in diameter wood cookies of red maple boles decaying under the canopy in the Penobscot
41
Experimental Forest in Bradley, ME were collected every other year for the past 12 years.
These wood cookies were oven dried at temperatures greater than 90°C for 48 hours and
archived.
Decay Test of Field Decayed Blocks
One inch cubed sapwood blocks were cut from archived samples representing 0-10
years of field decay. Field degraded blocks were decayed in soil block jar decay tests (AWPA,
2006) incubated at 25°C for 8 weeks were used to assess inky cap decay post various
amounts of field decay (n = 3).
Brown Rot and White Rot Pretreatment
Soil block jar decay tests were prepared with red maple and poplar wood blocks
19mm and inoculated with either the brown rot fungus Gloeophyllum trabeum (American Type
Culture Collection isolate ATCC 11539) or the white rot fungus Trametes versicolor (USDA
Forest Products Laboratory isolate M-697). The blocks were steam sterilized in an autoclave
at 121 °C for 30 min and subsequently placed into soil block jars inoculated with inky cap
fungi to simulate successional decay after brown rot or white rot pretreatments (n = 4).
After 8 weeks of decay the blocks were harvested, dried, and weight loss calculated.
HPLC Wood Sugar Analysis
The field decayed blocks and brown rot/white rot pretreatment blocks were analyzed
for wood sugars using an Shimadzu HPLC with a RID 10A refractive index detector
(Marlborough, MA) fitted with a Bio-RAD Aminex HPX-87P (lead ion) column (Hercules,
CA) heated to 80°C, using deionized distilled water as a mobile phase, a 20 p.1 injection
volume and a flow rate of 0.6 ml min"1. Prior to HPLC analysis, blocks were ground in a
Wiley-Mill through a 40-mesh screen. Wood powders were depolymerized to monomers
with 4% H 2 S0 4 , neutralized with calcium hydroxide, and filtered through a Whatman G D / X
0.2[xm PES filter.
42
Analysis ofNitrogen
Nitrogen content was measured in field decayed blocks by dry combustion at 10501350°C in an oxygen atmosphere. Released gases carried by He gas are cleaned of oxygen via
passage through steel wool then a copper catalyst and moisture is removed using a
condenser. The copper catalyst converts nitrous oxide to N 2 gas. C 0 2 is scrubbed and N 2 is
measured by thermal conductivity and reported based on sample weight, atmospheric
pressure, and calibrated standards. Analyses were performed by the soil analytical lab at the
University of Maine in Orono, Maine.
Results
Coprinus and Coprinopsis isolates did not decay the archived Forest Service red maple
blocks regardless of the length of field decay pretreatment (Figure 3.1). Appreciable decay
by Coprinellus isolates was achieved with the exception of decay by C. micaceus isolate ATCC
20122. Weight losses following Coprinellus decay typically increased with the length of field
decay. Degradation by the C. radians isolates was significantly greater after 4 years of field
decay compared to time zero field decayed blocks and increased significantly with time with
the greatest weight losses in 10 year field-decayed blocks (Figure 3.1).
43
Isolates
Figure 3.1. Weight losses in long-term field decayed red maple sapwood blocks degraded by
inky caps. Sapwood blocks represent 0 to 10 years of field decay, collection years are noted
in the figure key. Stars (•&) denote significant weight loss as compared to time zero blocks.
Percentage weight loss for G. trabeum at 8 weeks for field decayed red maple and
poplar were 36.14 (±6.45) and 41.12 (±6.13) respectively. T. versicolor weight losses for red
maple and poplar were 16.12 (±4.17) and 17.64 (±5.68) respectively.
In laboratory studies, all fungal isolates caused greater average weight losses on
poplar wood following the G. trabeum brown rot decay pretreatment, than in the absence of
pretreatment, with many of these increases being significant. Significandy greater weight
losses following T. versicolor white rot decay pretreatment were only observed on poplar in C.
radians isolates (Figure 3.2).
44
o
J3
<
;
.^
^
&
Isolates
Figure 3.2. Inky cap mushroom decay of poplar wood blocks following an eight week
pretteatment by Gloeophyllum trabeum brown rot decay and Trametes versicolor white rot decay.
A star (•&) denotes significance from the control at p = 0.05.
Weight losses resulting from inky cap decay were also greater following G. trabeum
pretteatment than in the controls for red maple blocks. With the exception of the
misidentified C. micaceus, all these differences were significant. C. radians isolates had
significandy greater weight losses post T. versicolor decay as was also observed in the poplar
blocks (Figure 3.3).
45
Isolates
Figure 3.3. Inky cap decay of red maple wood blocks following an 8 week pretreatment of
Gloeophyllum trabeum brown rot decay and Trametes versicolorwhite rot decay. A star (•&)
denotes significant difference from the control at p = 0.05.
Wood sugar analysis of the field decayed control blocks found extractable glucose
and xylose levels to be ~3 mg/ml and ~1.5 mg/ml respectively with no significant change in
sugar concentrations over years of decay. Other free wood sugars were not detected by the
analysis. Total percent nitrogen of wood samples was also measured and although the values
increased in later stages of decay from 0.063(±0.003) at time zero field decay to
0.109(+0.054) at ten years of decay, no significance was observed despite years of decay.
HPLC analyses of poplar wood extractable sugars showed an increase in xylose and
significant increases in glucose and mannose post white rot decay by T. versicolor. Brown rot
decay by G. trabeum reduced levels of extractable xylose and mannose and significandy
46
•ip"
reduced glucose levels of extractable sugar compared to the control (Figure 3.4). A similar
trend was seen in analyses of the red maple wood blocks except significance was seen in all
sugars and pretreatment fungi except the difference between mannose control and brown rot
decay (Figure 3.5).
Glucose
Xylose
Mannose
Treatment
Figure 3.4. Monosaccharide concentrations of control, G. trabeum and T. versicolor decayed
poplar blocks.
47
Glucose
Xylose
Mannose
Treatment
Figure 3.5. Monosaccharide concentrations of control, G. trabeum and T. versicolor decayed
maple blocks.
Discussion
Decay of the field materials suggests that Coprinopsis and Coprinus isolates are unable
to decay red maple despite the years of prior biological exposure in the field. Coprinellus
isolates on the other hand have some ability to decay field-degraded red maple with the
exception of C. micaceus isolate ATCC 20122, the only isolate whose identification didn't
match GenBank blast search results of the amplified ITS regions. C. radians isolates
produced significantly greater weight loss in blocks subjected to four years of field decay.
Weight losses were usually significantly greater with every additional increment of field decay
leading to approximately 20% weight losses on blocks collected after 10 years of field decay.
This result coupled with the fact that C. radians isolates failed to achieve 20% weight losses in
other decay tests conducted in this thesis investigation supports the hypothesis that these
48
fungi have evolved and thrive as very late stage w o o d decayers. Fungi associated with early
succession must increase availability of cell wall components of a decaying tree before
Coprinellus can produce weight loss.
H P L C analysis of acid extractable wood monosaccharides from the field decayed red
maples blocks showed n o significant changes over time. With n o difference between time 0
blocks and w o o d harvested after years of field decay; w o o d monosaccharides do not appear
to influence degradability of red maple by Coprinoid fungi. It was curious not to see
predicted changes as in a competitive environment like the forest floor, these free sugars
would be aggressively consumed by a consortium of microbes. Other modifications to the
w o o d of possible relevance might include changes in oligomer concentrations, cellulose
crystallinity, loss of extractives, and lignin degradation and the formation of degradation
products. Physical changes resulting from decay such as cell wall penetration and the general
opening of the w o o d matrix may also facilitate inky cap growth.
Field decay is highly variable as it is caused by a consortium of natural decay fungi
and is also shaped by bacterial, animal and abiotic activities. Possible changes to the wood
during field decay are numerous. In the laboratory, red maple and poplar blocks predecayed
with brown and white rot fungi were used to control for these changes to the decaying
wood. G. trabeum weight losses at 8 weeks for red maple (36.14 (±6.45)) and poplar (41.12
(+6.13)) compared reasonably well to losses reported previously. For instance, Shi et al.
(2007) reported 68.5% (±7.8%) average weight loss at 12 weeks on poplar by a different G.
trabeum isolate while Richter et al. (2005) working with the same G. trabeum isolate (ATCC
11539) measured an average weight loss of 3 8 . 1 % (±6.8%) at 12 weeks on southern yellow
pine sapwood blocks. T. versicolor weight losses were less than expected however. Work by
49
Celimene et al. (1999) on red maple and Schirp and Wolcott (2005) on poplar using the same
T. versicolor isolate (M-697) measured 55.94% (±5.59%) average weight loss at 12 weeks and
50.9% (±2.2%) average weight loss after just 3 weeks respectively.
As seen in the decay tests described earlier in Chapter 2 and the above testing of field
collected red maple, Coprinopsis and Coprinus are unable to achieve appreciable decay. This
trend is seen again in the minimal decay of the red maple and poplar control blocks with
some exceptions following brown rot decay. The isolates C. atramentarius FP-101910-T, C.
cinereus FGSC 9003 and C. comatus ATCC 12640 all produced significantly greater weight
losses in red maple blocks following a G. trabeum brown rot pretreatment. Coprinellus isolates
decayed red maple blocks minimally with no pretreatment but produced significandy greater
weight losses post brown rot pretreatment compared to no pretreatment. The single
exception was the weight loss produced by the likely misidentified isolate ATCC 20122.
Coprinellus radians isolates all incurred significandy greater weight loss following the white rot
pretreatment of both wood types, the only isolates to do so. A similar trend was observed
with poplar wood, except for isolates C. comatus FP-101592-T, C. micaceus FP-101781-T, or
the misidentified isolate C. micaceus ATCC 20122.
It is possible that brown rot pretreatments better enabled inky cap decay than white
rot pretreatments due simply to their higher measured weight losses and thus higher degree
of penetration and depolymerization of the wood blocks. It is also possible that the differing
decay mechanisms played a role.
Trametes versicolor is a simultaneous white rot fungus known to degrade all cell wall
components. Colonization of wood by T. versicolor occurs via rays and penetration between
wood cells through pits and bore holes. Decay starts in the cell lumen and moves erosively
toward the middle lamella adjacent to hyphal growth. Degradation is primarily via enzymatic
systems. Lignin undergoes side chain cleavage, demethoxylation, demethylation, and
50
mineralization via laccase and peroxidase oxidation. As the lignin matrix is degraded the
holocellulose is metabolized primarily by exoenzyme. Liberated monosaccharides regulate
wood polysaccharide enzyme expression such that holocellulose is repeatedly depolymerized
to meet metabolic demands.
Brown rot decay by G. trabeum on the other hand is typically associated with
hemicellulose and cellulose degradation with minimal demethylation and oxidation of the
lignin matrix following similar entry to the lumen (Highley, 1987). Hemicellulose
glucomannans are removed preferentially to xylan and xylan preferentially to cellulose
(Highley, 1987), but all of the holocellulose is depolymerized faster than degradation
products are utilized (Cowling, 1961). The free radical mechanisms employed by G. trabeum
allow for wood degradation at distances away from the invaded hyphae where liberated
sugars can not be absorbed (Daniel, 2003). For these reasons it was hypothesized that
brown rot decay would liberate more free oligomers of cellulose and cellobiose, and more
glucose monomers than white rot decay. These simple sugars are preferred by inky caps
according to Fries (1955) and this preference may help explain the greater decay rates of the
inky caps following brown rot pretreatment.
Wood sugar analysis of both wood types however, suggests an increase in glucose,
mannose and xylose monosaccharides after decay by T. versicolor and decreases in glucose and
xylose sugars following decay by G. trabeum; which is contrary to what was hypothesized. A
limitation with this analysis is the hydrolysis process. The 4% H 2 S0 4 is not strong enough to
cleave bonds between uronic acids and the wood sugars; thus these sugars are undetected by
the analysis. Stronger levels of acidity convert the sugars to breakdown products such as
hydroxyl-methyl-furfural from glucose and furfural from xylose. It is also possible that T.
versicolor enzymes cleaved these dilute acid-stable bonds increasing sugar detection while not
increasing the actual level of monosaccharides. G. trabeum may have liberated larger levels of
51
sugars, but the free radical mechanisms employed may not cleave the uronic acids and as a
result these sugars are not detected. G. trabeum's free radical mechanisms might also cleave
bonds causing these sugars to be converted by the hydrolysis to furfural like compounds
which are also undetected by the column used for analysis. More robust characterization of
the wood is needed.
If the sugar levels were significantly lowered by G. trabeum decay, it might be implied
that inky caps are very efficient at scavenging sugars late in decay. It might also imply that
inky caps can utilize the modified but not depleted lignin residue as a source of carbon. Only
a simple Petri culture screen was used to explore lignin metabolism and its uses as a carbon
source. These cultures produced insignificant growth rates and biomass for analysis. Lignin
structure and metabolism therefore can not be ruled out. The only certainty is that there is a
need to further investigate lignin's role in inky cap degradative physiology.
If utilization of lignin isn't directly supported and no monosaccharide changes were
correlated with inky cap decay in either field or laboratory pretreatments another possibility
is that physical modifications are actually more important than chemical ones. Perhaps
simply the greater percentage of decay by G. trabeum was correlated with more fungal
invasion of rays and increased boring through cell walls enabling subsequent inky cap
invasion and decay. Brown rot, unlike white rot, increases wood porosity during degradation
which would better enabling a latter colonizing fungus to decay by enabling better
penetration of enzymes and hyphae. Brown rot decay in particular opens up hardwood S2
cell wall layers to a greater extant than softwood S2 layers (Daniel, 2003).
We had re-
sterilized the blocks between fungal treatments, so if Gloeophyllum had secured regions of the
wood block, they were not there to compete with the late stage colonizing inky caps and
prevent them from rapidly invading those regions of the wood. Heilmann-Clausen and
Boddy (2005) suggest that late stage decay fungi would not be able to compete with primary
52
colonizers. This might explain the time it takes in the field for these fungi to colonize a
substrate and why significant losses were not seen with the field decayed woods until 4 or
more years. It is of interest to see if brown rot and white rot or field decayed pretreatments
will enable decay by inky caps of softwoods. These woods are sometimes observed as
substrates in the forest, yet there was no successful decay by any of the isolates in the
laboratory. More wood species and decay fungi pretreatments need to be explored in
association with more thorough analysis of chemical and physical changes occurring to the
wood.
Conclusions
This preliminary work suggests inky caps can be efficient late stage decay fungi. On
red maple, weight losses resulting from Coprinellus decay were significandy increased as length
of field decay increased and with G. trabeum brown rot pretreatment. Only the C. radians
isolates caused significandy greater weight loss following a white rot pre-treatment with T.
versicolor. The same increases in weight loss were seen on poplar for most isolates post brown
rot decay and for the C. radians isolates following white rot decay. No significant changes
were seen in monosaccharide levels in any of the field or laboratory decayed materials.
Increases in glucose and xylose subunits by T. versicolor and decreases in the sugars by G.
trabeum were measured. Results show sugar analysis does not support the greater growth and
weight losses seen post brown rot decay. It is likely that physical opening of the wood
matrix may be at least partially responsible for successful inky cap decay. Better
characterization of changes to wood physiology and chemistry are needed to clearly elucidate
the required changes essential to enable decay by inky cap isolates. This work clearly outlines
the importance of early decay to the successful degradation of wood by some of the inky cap
fungi.
53
CHAPTER 4: TOLERANCE TO WOOD PRESERVATIVES
Abstract
Inky cap peroxidases have been used previously in some bioremediation schemes for
treatment of petroleum and polycyclic aromatic hydrocarbons (PAHs). PAH compounds are
also contained in oil based wood preservatives such as creosote. To date no one has
investigated the ability of inky caps to tolerate or degrade such wood preservatives. A
literature review shows minimal investigation of the inky caps' ability to tolerate copper
either, a primary component of most water based wood preservatives. In this work, the inky
cap isolates were tested against copper, creosote, and creosote constituents to investigate
their bioremediation potential for wood preservatives. There were varying degrees of PAH
tolerance observed in some of the Coprinellus isolates. There were no significant tolerances to
copper observed. Inky caps were not able to decay woods treated with any of the wood
preservatives tested. The isolates showing tolerance to PAHs should be further explored for
their potential utilization in the bioremediation of preservatives.
Introduction
Currently used wood preservatives contain toxic metals (Humar et al., 2004) and or
organic chemicals which have toxic and carcinogenic properties (Cerniglia, 1993). The wood
protective industry is striving to develop environmentally safe chemical wood treatments
with reduced disposal concerns that still provide long-term resistance to microbial
degradation. To date, this ambitious goal has not been met and treated woods removed
from service (estimated in the millions of m3 yr"1 for copper chromium arsenate (CCA)
treated wood alone (Cooper, 1993)) are continuing to be a major waste disposal issue. The
persistent environmental pollutants used to treat woods are also ubiquitous contaminants of
soil and water as a result of widespread use, poor management of treatment facilities, and
improper disposal. Landfilling is considered the most undesirable disposal of treated wood
54
removed from service (Humar et al, 2004) due to limited capacity at specialized dumps and
the lack of public approval for the construction of new material reclamation sites (Stephan et
al., 1996). Biological methods of treated wood recycling are accepted as a promising
alternative to landfilling.
Some species of fungi have been shown to efficiently break down organic wood
preservative chemicals such as Pleurotus ostreatus (Bezalel et al., 1996a,b,c, 1997; Andersson et
al., 2003) while others can tolerate and accumulate copper such as Phanerochaete chtysosporium
(Falih, 1997; Sing and Yu, 1998). The need of better waste disposal methods, the novel
bioremediation abilities of some fungi and the lack of thorough bioprospecting for tolerance
and degradative ability towards treated materials by a wider array of fungi, highlights the need
for additional studies.
Coprinus peroxidases have already gained minor attention for their use in
bioremediation strategies for toxic phenolic compounds like polycyclic aromatic
hydrocarbons (PAHs). PAHs are a major component of creosote and other oil based wood
preservatives. Inky cap tolerance to PAHs has only been explored minimally however and
the potential of inky cap fungi to degrade copper containing water based wood preservatives
has not been explored. The physiology research for this thesis focused on copper, pyrene
and phenanthrene tolerance and the ability of the inky caps to degrade woods treated with
these compounds as well as ammonium copper quaternary (ACQ) and the organic creosote.
Materials and Methods
Copper Sulfate Tolerance — Petri Culture
Tolerance to treatment chemicals was measured using Petri dish and liquid cultures,
and soil block jar decay tests. Copper tolerance was measured in Petri dish culture by adding
copper(II) sulfate pentahydrate to CMM at 0.1, 0.01, 0.001, 0.0001 molar copper
concentrations with a control at 0 copper. The pH of the CMM was aseptically adjusted to
55
7.0 with sterile ION NaOH before pouring plates. Cooled Petri dishes were side inoculated
with a plug of inoculum 0.5 cm in diameter taken from the margin of growth of a 2-week old
culture. Plates were parafilmed and incubated at 25°C. Radial growth rates were measured,
in millimeters, from the inoculum point to the furthest margin of growth every 3 days until
cultures covered the entire plate (n=3).
Copper Sulfate Tolerance — Uquid Culture
Tolerance to copper sulfate was tested in liquid culture using the same treatment
concentrations and control used in Petri dish screens. 75 ml of CMM with copper and
adjusted to pH 7.0 was aliquoted into 125 ml Erlenmeyer flasks. Each flask was inoculated
by floating two 0.5cm in diameter plugs of fungal inoculum taken from 2-week old Petri
cultures. Each isolate was inoculated into five replicates per treatment. Flasks were
incubated in the dark, at room temperature, for 14 days on an orbital shaker at 100 rpm
rotation. Biomass was harvested by vacuum Buchner filtration through # 1 Whatman filter
papers, with a predetermined dry weight, which were subsequently dried for a second 24hrs
at 105°C to determine post harvest weight.
Copper Sulfate Tolerance — Decay Tests
Southern yellow pine (SYP) blocks were pressure treated in the laboratory by
submersion in aqueous copper sulfate solutions of 0.1, 0.5, and 1.0 molar copper
concentration with two processing cycles at 30 inches Hg vacuum for 30 minutes. Blocks
were subsequently air dried and retention calculated by measuring the gain in mass and via
copper analysis by inductively coupled plasma emission (ICP). Mass gain post treatment can
be attributed to materials accumulated in the wood from pressure treatment. Mass gain was
calculated by drying blocks for 48 hrs at 95°C before and after the treatment and comparing
the weights. ICP-AES analysis was performed on 40-mesh milled wood powders digested
using concentrated nitric acid. Treated blocks were used in AWPA soil block jars inoculated
56
with inky cap fungi to test the isolates aggressiveness toward copper treated wood. Wood
blocks were incubated at 25°C for 12 weeks. Weight losses were determined for decayed
blocks after harvest and drying at 95°C for 48 hours.
Tolerance to Ammonium Copper Quaternary — Petri Culture
To investigate tolerance to copper-treated materials, ACQ treated SYP and untreated
SYP wood flours, ground in a Wiley Mill through a 40 mesh screen, were added to CMM in
place of glucose at a 2% (w/v) concentration. ACQ was selected for screening as it is
quickly replacing CCA as an arsenate free preservative treatment. Culture conditions were
maintained as outlined above with the exception of pH adjustment (n=3) and radial growth
was recorded at similar intervals until the fungi covered the entire surface of each plate.
Tolerance to Ammonium Copper Quaternary — Uquid Culture
SYP and ACQ-SYP 40-mesh milled wood powders were added to liquid CMM
cultures at a 2% (w/v) concentration. 75ml of liquid culture was aliquoted into 125 ml
Erlynmeyer flasks. Four flasks of each treatment, per isolate were inoculated by floating 2
0.5 cm diameter plugs of inoculum taken from 2 week old Petri dish cultures. The
inoculated flasks were incubated in the dark at room temperature on 100 rpm orbital
rotation. Dry biomass was collected and measured as outlined in chapter 2.
Tolerance to Ammonium Copper Quaternary — Decay Tests
Copper tolerance was also investigated in AWPA soil block decay tests as above
with SYP blocks and commercially treated ACQ-SYP blocks.
PAH Tolerance — Petri Culture
To investigate the tolerance of inky cap fungi to organic compounds, model PAH
compounds pyrene (98+%) (Acros Organics, N.V.) and phenanthrene (98+%) (Acros
Organics, N.V.) each dissolved in 100% acetone were added as an overlay to CMM agar
57
plates and 2% (w/v) wood powder amended CMM plates to achieve coverage of 0.02g of
PAH/plate. Oak, SYP, and birch wood powder amended CMM were used. Plates were side
inoculated, incubated and measured for growth rates as above.
PAH Tolerance — Decay Tests
Oak wood blocks were pressure treated with PAH components by submerging the
blocks in a 2% solution of pyrene or phenanthrene in acetone and exposing the blocks to 30
inches Hg vacuum for 2x 30 minutes. Blocks were allowed to air dry and blocks were placed
in soil block jars inoculated with fungi and incubated 12 weeks.
Creosote Tolerance — Petri Culture
In Petri dish culture, creosote tolerance was measured by amending CMM and oak
amended CMM with liquid creosote (2% v/v) or with 40-mesh wood flour from creosote
treated oak railroad ties removed from service (2% w/v). Treatments were added aseptically
to sterile media. As noted previously, radial growth was measured every 3 days and the same
culture conditions were employed.
Creosote Tolerance — Decay Tests
Creosote tolerance was also investigated using modified soil block jars. Fresh and
railroad tie material (oak treated with creosote) removed from service was cut into 1" square
blocks with initial dry block weights recorded following a 48 hr equilibration period at 21 °C
and 65% relative humidity. Blocks were surface sterilized with 70% EtOH and aseptically
added to previously inoculated jars and incubated at room temperature for 8 weeks. After
harvest, blocks were similarly conditioned for 48 hours at 21 °C and 65% relative humidity
before weighing.
58
Results
Copper Tolerance
At molar concentration 0.1 Cu, average radial growth rate and biomass production in
liquid culture were significandy reduced for all isolates and completely inhibited in most.
Growth was also inhibited in both mediums at Cu levels greater than 0.001 molar for most
isolates (Figures 4.1 & 4.2). pH, which was measured in liquid culture, was also significandy
reduced at these higher molar concentrations but remained within a range tolerated by the
fungi (Figure 4.2). Biomass and radial growth rate were positively correlated on CMM
amended with copper sulfate (Figure 4.3).
Isolate
Figure 4.1. Average radial growth rate of inky cap isolates on CMM with increasing molar
copper concentrations of copper sulfate.
59
Figure 4.2. C. radians ME-209 biomass and filtrate pH in liquid cultures of copper amended
CMM.
0.5
0.4
f
0.3
&
a
0.01 M
0.001 M
0.0001 M
Molar Copper Concentration
Figure 4.3. C. radians ME-209 biomass and radial growth rate on Cu amended CMM.
60
There was no difference in Coprinopsifs average radial growth rate on SYP amended
and ACQ-SYP amended CMM, but it's important to note the isolates' minimal growth on
both media. The Coprinus isolates all grew significandy faster on CMM amended with SYP
wood powder amended CMM than CMM amended with ACQ-SYP suggesting an inhibitory
affect of ACQ on hyphal extension of these isolates. In contrast, most Coprinellus isolates
showed a significant increase in average radial growth rate on ACQ-SYP wood powder
amended medium suggesting ACQ may stimulate hyphal extension. Of the Coprinellus
isolates, the only exception was the misidentified isolate C micaceus ATCC 20122 that grew
significantly slower in the presence of the ACQ treated SYP wood powder (Figure 4.4).
/
J?
/
/
/
/
/
/
/
-
;
Isolates
Figure 4.4. Average radial growth rate of inky caps on CMM amended with ACQ treated
SYP and untreated-SYP.
Biomass production was greater on CMM amended with untreated SYP than on
SYP-ACQ amended CMM for all isolates with the exception of CoprinopsistinereusFGSC
9003. Significandy, greater biomass production on untreated SYP amended agar than ACQ-
61
SYP amended CMM was found for all the isolates except for both the Coprinellus radians
isolates and the misidentified C. micaceus ATCC 20122. Biomass measurements for the C.
radians isolates, which have typically been greater than those of the other isolates in this work,
were reduced in both the treatments and the controls. ACQ had overall significant inhibiting
effects on biomass production in liquid culture (Figure 4.5).
Isolates
Figure 4.5. 2 week dry weight biomass of inky cap isolates grown in shaking liquid culture
with either untreated southern yellow pine (SYP) or ACQ-treated SYP as the carbon source.
Soil block jars set up to test laboratory treated copper sulfate found no significant
decay by tested isolates. There was no obvious decay or growth on the treated blocks. All
measurable weight losses in decay tests could be attributed to "kick-back" or leaching of
treatment chemicals. Even in the lowest treatment concentration,(0.1 mol Cu), where
leaching was minimal, there was no significant weight loss in any of the inoculated soil block
jars. The post incubation wood, treated with the 0.1, 0.5 and 1.0 molar Cu concentrations
had retained copper levels of approximately 6885, 19990, and 28138 respectively.
62
Soil block jar assays yielded greater weight losses for untreated SYP than for any of
the treated blocks for all of the isolates, but these differences were not significant. In fact,
after 12 weeks of incubation, weight loss for all blocks including untreated SYP was less than
1 %, a result not significandy different from non-inoculated controls.
PAH Tolerance
Average radial growth rate in the presence of PAHs was dependent on the medium
type. On un-amended CMM, no significant tolerance to PAHs was measured with only
Coprinellus radians isolates producing any measureable growth in the presence of pyrene.
Phenanthrene inhibited the growth of all isolates. On pine wood amended medium all
isolates produced greater growth without PAHs although there was appreciable growth by
many of the isolates in the presence of pyrene, and by Coprinopsis atramentarius FP-101910-T
in the presence of both pyrene and phenanthrene. On birch wood powder amended CMM,
most of the isolates had measurable growth in the presence of either one or both of the
PAHs. Only Coprinopsis cinereus FGSC 9003, Coprinus comatus ATCC 12640 and Coprinellus
micaceus ATCC 20122 were unable to grow on the pine or birch wood in the presence of
PAHs. There was no significant difference between growth on medium with the pyrene
overlay and on the control agar for isolate C. radians ME-209, and between the phenanthrene
medium and the control for isolates C. radians ME-352 and ME-209. On red oak media,
minimal growth in the presence of PAHs was achieved by isolates C. micaceus FP-101781-T,
and the two C. radians isolates. No growth comparable to that on the control was seen in the
PAH overlay on oak amended CMM. Growth on medium overlaid with either of the model
PAH compounds never equaled the growth on the untreated control. Growth rates for any
of the isolates on the controls were lower than the growth rates measured in chapter two
which explored wood species preference (Figure 4.6).
63
a)
b)
4t
u
o
e
c)
^
A
3.5
3
4
2.5
2
1.5
1
0.5
O
Figure 4.6. Average radial growth rate of inky cap isolates on CMM (a), Birch (b), Pine (c),
and Oak (d) amended CMM medias overlaid with the PAHs pyrene and phenanthrene.
64
There was no measurable growth of any isolate on CMM and oak amended CMM
media when they were amended with creosote. Growth was seen in the presence of creosote
treated oak railroad-tie amended CMM. This growth was always significandy less than
growth on untreated oak amended CMM for all isolates (Figure 4.7).
Isolates
Figure 4.7. Average radial growth rate on oak amended CMM and CMM amended with
rail road tie material removed from service.
In soil block jar decay tests, no significant weight loss was seen in control blocks or
pyrene treated red oak sapwood blocks (Figure 4.8). Acetone treated blocks were used as a
control to test the affect of the solvent the PAH was dissolved in. Weight losses of acetone
treated blocks were great by Coprinellus micaceus ME-798, but this was quite variable and was
not significant. No appreciable weight losses were measured after 8 weeks of incubation on
creosote treated rail road tie materials by any of the isolates (data not shown).
65
&
*r
Isolates
Figure 4.8. Average percent weight loss associated with 12 weeks of inky cap decay of oak
blocks, oak blocks treated with acetone and oak blocks treated with pyrene using an acetone
solvent.
Growth of all isolates was completely inhibited on CMM and oak CMM Petri dish
cultures containing creosote. There was measurable growth, particularly of Coprinellus
isolates, on CMM amended with wood powder of a rail road tie removed from service. This
growth was significandy reduced compared to that of the untreated oak control (Figure 4.9).
Appearance of the cultures was also typical of the stressed cultures described previously.
66
Isolates
Figure 4.9. Inky cap average radial growth on CMM amended with oak wood powder
and railroad tie wood powder.
Discussion
At a molar copper concentration of 0.1, radial growth and biomass production of all
isolates were significandy inhibited with almost no growth. Radial growth and biomass were
significandy reduced at concentrations greater than 0.001 moles Cu. Biomass measurements
were positively correlated with radial growth rates in copper tolerance tests.
Higher molar copper concentrations (0.1 and 0.01) significandy lowered media pH to
approximately 6.2 from 7.0. This change may have affected the outcome of the liquid
culture screens although a pH of 6.2 should not inhibit growth of these isolates. The pH
preference experiments showed acceptable growth at this pH and some isolates such as C.
micaceus isolates preferred a pH in this range.
67
Though weight loss measurements in decay tests using copper sulfate treated SYP
blocks may have been affected by leaching of the treatment compound, blocks still had
measurable levels of copper and no evidence of decay. Humar et al. (2004) noted
approximately 32% leaching of copper sulfate from vacuum treated spruce block as the
result of four days of intensive leaching testing.
The least concentrated treatment solution used, 0.1 mole Cu, did provided
comparable concentrations of copper in the treated block to those currently accepted in
commercial treatments, even post kick-back. The AWPA standard suggests retentions of
0.24 pounds cubic foot"1 of copper for copper azole B (CA-B), and 0.53 pounds cubic foot"1
of CuO for ammoniacal copper quat B (ACQ-B) (AWPA, Tl-06). Goodell et al. (2007)
measured 0.349 pounds cubic foot"1 Cu in commercially available CA-B treated SYP and
0.429 pounds cubic foot"1 CuO in ACQ-B treated SYP. Using the retention standards for
CA-B and ACQ-B and assuming a cubic foot of southern yellow pine weighs approximately
38 pounds (Bepler, 1890), the standard treatment calls for approximately 6316 mg and
13947mg of copper/kg of wood respectively. ICP analysis measured an average retention of
6885.6 (±355.5) mg copper/kg for SYP blocks treated in the laboratory with the 0.1 molar
solution. These copper levels are greater than those in CA-B and less than those of ACQ-B.
Future work should be done with further conditioning of blocks to help reduce leaching or
with alternative treatment chemicals.
Coprinus isolates were inhibited by ACQ in both Petri and liquid cultures. In contrast,
Coprinellus isolates showed a significant increase in average radial growth rate on ACQ-SYP
wood powder amended media suggesting that ACQ may stimulate a stressed hyphal
extension reaction. The story for Coprinellus radians isolates may be different however as their
biomass production was not significantly reduced by ACQ. These isolates usually
outperform all other isolates in biomass production, which is not seen here in the untreated
68
SYP control and can possibly account for the lack of significant difference among the
control and treatments. This lack of significance might also suggest that these isolates have
some minimal tolerance to ACQ.
Soil block jar assays using commercially ACQ treated SYP blocks do not provide
evidence for ACQ tolerance by any isolate. It may be that a minimal tolerance is not enough
to decay new commercially treated woods, but possibly C. radians isolates could attack such a
substrate following failure of the ACQ due to decay as previous finding suggest their role
later in the timeline of decay.
Overall, the poor growth of all of these isolates on softwoods, the wood typically
treated with copper containing treatment chemicals, such as CCA and ACQ, limits their use
in bioremediation schemes for these materials. Even isolates such as C. radians ME-352
which had high growth rates on pine amended CMM, had colony characteristics typical of
the stressed cultures discussed in chapter 2. An inability to grow on softwoods along with a
lack of copper tolerance makes it unlikely that these fungi have potential in the waste
management of CCA and ACQ treated materials.
PAH tolerance Petri dish testing highlights some tolerance of certain inky cap isolates
to PAHs, but this is highly media dependent. On CMM medium all of the Coprinellus
isolates, except C. radians ME-209, produced radial growth rates in the presence of pyrene
that were not significandy different from the controls. Tolerance to both pyrene and
phenanthrene was also found on birch amended medium for both C. radians isolates. No
PAH tolerances were measured on other wood powder amended mediums such as oak,
which is the species of wood typically treated with creosote. The differences on various
wood powder amended media may be due to the stimulated production of a phenolic
degrading system such as peroxidases in the presence of certain substrates. Elisashvili et al.
(2008) have shown that production of cellulytic and ligninolytic enzymes depends on the
69
fungal species and lignocellulosic growth substrate. Peroxidase production by the isolates
was not compared on the different mediums or in the presence/absence of PAHs.
Unpublished real-time PCR work by Suping Liu suggests that expression of peroxidases are
increased in the presence of low levels of pyrene.
Creosote added to CMM and oak amended CMM media completely inhibited the
growth of all isolates. When creosote treated oak rail road tie material removed from service
was ground and added to CMM, there was measurable growth by many of the isolates
however, particularly Coprinellus. However, the cultures lacked the pigmentation, size, and
hyphal thickness typified by the growth on un-amended CMM and the radial growth rates
were in all cases significantly less than those on untreated oak amended media.
Decay tests found the inky cap fungi were able to degrade pyrene treated oak wood
blocks or creosote treated railroad ties. These tests focused on high density oak wood as this
wood species is still preferred for rail road ties which are one of the only commercial
applications where treatment with creosote is still allowed.
Despite extensive study of the usefulness of Coprinus peroxidase in the treatment of
petroleum based aromatics, this work shows limited tolerance to PAHs and creosote by inky
caps and an inability to decay oak treated with these materials. As the Coprinellus isolates
were more prolific decayers following other fungal decay pretreatments, perhaps there are
still potential applications of these fungi in the recycling of such treated materials; following a
primary biological pretreatment for instance. The ability of these fungi to decay treated
woods following various decay pretreatments should be investigated.
Conclusions
The inky cap fungi investigated in this work showed no significant tolerance to
copper based wood preservatives or any ability to decay wood treated with them. There was
limited tolerance to pyrene and phenanthrene by Coprinellus radians isolates in birch wood
70
powder amended CMM and no tolerance on oak or SYP amended media. No ability to
decay oak treated with PAHs or creosote was reported. Despite this, the tests imply that
while there may not be degradation of PAHs, some isolates were at least tolerant to certain
PAHs, but this was highly isolate and substrate dependant. As these compounds are
ubiquitous contaminants of many various terrestrial and aquatic systems, the inky cap fungi
still might be useful in some specific applications of PAH clean up. Further investigations
exploring more PAH compounds, their break down products and more diverse substrates
for growth might find useful application for certain inky cap fungi.
71
CHAPTER 5 - CONCLUSIONS
Objective 1 - Investigate the Ability of Inky Cap Fungi to Decay Wood
Inky cap fungi have traditionally been thought of as copriphilic fungi and as a result
their role in the recycling of wood and woody forest litter has not been investigated. Soil
block jar decay testing was utilized to determine which wood species, if any, the inky cap
fungi can degrade. Inky cap fungi in the genus Coprimllus, including the species micaceus and
radians, were the only fungi tested to yield significant weight losses. Other isolates used in
the study, representing the genera Coprinus and Coprinopsis, exhibited no ability to decay
wood. Of the wood species tested only hardwood species were appreciably decayed by C.
micaceus and C. radians. Poplar was decayed to the greatest extent by the Coprinellus isolates,
but all weight losses were reduced compared to typical rates of standard decay fungi
(Celimene et al, 1999; Richter et al., 2007; Schirp and Wolcott, 2005). These decay results
were not predicted by complimentary experimentation using Petri dish or liquid cultures with
Wiley-milled wood powders used as the sole carbon source.
Inky cap fungi can be efficient degraders of certain hemicellulose sugars (Fries, 1955).
These hemicelluloses were investigated in an effort to explain wood species preferences. All
isolates grew faster and produced greater biomass on hardwood hemicelulloses and typically
produced litde growth on the softwood hemicelluloses arabinose and galactose. While
growth rates were increased on galactose and with no sugar compared to unmodified CMM
containing glucose, biomass measurements were minute. Arabinose seemed to limit both
growth rates and biomass production. The inability of inky caps to metabolize arabinose
warrants further examination. Despite the apparent preference for hardwood monomer
hemicelluloses, this was not reflected in experiments where crudely extracted hemicelluloses
were used as the carbon source. Nor was HPLC analysis of different wood species able to
explain fungal preference, but with more replication, increased glucose levels observed in
72
poplar compared to other hardwood and softwood species may have been more important.
Hemicellulose structure likely plays a role in the ability of inky caps to decay wood and cause
appreciable weight loss, but better characterization of the hemicelluloses is needed to
accurately elucidate this relationship.
pH data suggests the inky cap fungi prefer more neutral to alkaline pHs, results that
agree with previous findings (Fries, 1956) but pH cannot explain the hardwood preference
shown by the inky cap isolates used in this work. Physical differences working in concert
with the chemical conditions are likely responsible for preventing or enabling inky cap decay.
Physical differences of the wood among tree species have not been explored in this work,
but it is known that poplar is diffuse porus and has a thin cell wall (Cole, 2008). Wood
extractives might also inhibit inky cap growth and poplar (LJriodendron tulipiferd) is a species
known to have relatively low levels of extractives. There is need for better characterization
of the wood species both physically and chemically to better understand the role inky caps
potentially may have in wood and woody debris degradation.
An interesting observation made during the wood degradation work is the apparent
increase in radial growth in response to poorly metabolizable sugars which littie growth and
biomass are produced on. This work suggests that many of the inky caps, in particular
Coprinellus spp., have an ability to limit growth of absorptive and combative hyphae while
increasing exploratory hyphal elongation in response to nutrient deprivation. This could
function as an effective late stage colonizer strategy enabling quick scavenging of sugars
while avoiding competition. Peiris et al. (2008) suggests that late stage decay fungi are poor
competitors, so such a strategy would prove most effective in avoiding competition.
73
This work has demonstrated the ability of some of the inky caps to decay selected
wood species. Though this work couldn't ascertain why hardwoods, in particular poplar,
were preferred, or why only the isolates Coprinellus were efficient at decay, it does
demonstrate the need for further investigation of the degradative physiology of this
mushroom group and their role in late stage decay and humification.
Objective 2 - Explore Inky Cap Fungi's Role as Late Stage Decay Fungi
In the natural environment inky cap fungi are typically observed fruiting and growing
on highly modified wood and humifying materials. In addition to observational findings
(Personal Observations; Setliff, 2008) the current work confirms the proposed role of inky
caps as late stage decayers (Peiris et al., 2008). To explore this role, inky cap fungi were
exposed to wood pre-decayed either in the field or in the laboratory by a white rot or a
brown rot primary decay fungus. Field decay did not affect the growth of most isolates, but
weight losses from Coprinellus radians decay were significantly greater with wood samples
decayed in the field for greater than four years and weight losses increased with additional
years of field decay to a maximum weight loss in 10 year field decayed blocks. The weight
losses of the blocks that were field decayed for a decade were the largest seen in any of the
inky cap decay tests outlined in this thesis. This might be related to inky caps evolution as a
late stage decayer, with poor competitive ability but capable of thriving on nutrient depleted
woods, Boddy and Rayner (1983) suggested that mid stage decayers needed an ability to
compete with primary colonizers while the late stage colonizers could invade the woody
substrates in lower competitive environments but must be tolerant of low nutrient availability
and conditions of stress. Peiris et al. (2008) didn't observe Coprinellus micaceus invasion of oak
in the field until 10-15 years of forest floor decomposition had occurred.
74
Only Coprinellus radians isolates' decay rates of both red maple and poplar were
significandy affected by a pretreatment with the white rot fungus Trametes versicolor.
Pretreatment with the brown rot Gloeophyllum trabeum however, significandy increased decay
by all isolates, except the erroneously identified C. micaceus ATCC 20122, on red maple and
most of the other isolates on poplar. The brown rot pre-treatment allowed decay to occur
by Coprinus and Coprinopsis isolates that could not decay sound wood. Differences in wood
sugar chemistry were contrary to those hypothesized and cannot fully explain why only the
brown rot pretreatment enabled inky cap decay. There are well characterized differences in
the degradative mechanisms of brown rot and white rot decay fungi, so it is likely that
different chemical and physical changes occurred during the pretreatment. Brown rot, unlike
white rot, increases wood porosity during degradation better enabling a latter colonizer to
decay by increasing the penetration of wood by enzymes and hyphae. Brown rot decay in
particular opens up hardwood S2 cell wall layers to a greater extant than softwood S2 layers.
No softwood species were investigated in the successional studies, but it would be
interesting to see if a brown rot pretreatment facilitates softwood decay by the inky cap
fungi. Other wood species and fungal pretreatments must also be explored to enhance
understanding of the fungal succession that enables inky caps to decay wood. Incorporation
of other field collected materials into a similar experimental design would also be of interest.
Objective 3 - Prospect for Tolerance to Wood Treatment Chemicals
Innovative waste treatments are being developed by employing decay fungi to reduce
waste volumes and to detoxify debris. The inky caps tested showed no tolerance to copper
at any concentration above 0.001 M and no tolerance to the copper-based wood preservative
ACQ. None of the isolates decayed copper or ACQ treated SYP. The poor ability to decay
sound softwoods and the lack of tolerance to copper suggest that it is unlikely that these
fungi could decay copper or ACQ treated wood removed from service. Goodell et al. (2007)
75
reported that several species of brown rot fungi could efficiendy degrade copper treated
wood. It might be of interest to test inky caps ability to degrade copper treated wood
following degradation by these brown rotting species as the inky caps seem to have increased
decay abilities following previous brown rot decay according to results from chapter 3.
Polycyclic aromatic hydrocarbons (PAHs), ubiquitous in creosote, a preservative used
in railroad ties, as well as oil based pollutants, are of increasing environmental concern.
Some white rot fungi have shown the ability to degrade some of the PAHs of wood
preservation and oil compounds (Field et al., 1992; Cerniglia, 1997; Tortella et al, 2005).
Enzymes of some inky cap species have been shown to be effective at removal of PAHs
from effluents (Al-Kassim et al., 1994a,b; Ikehata et al., 2003, 2004) Tolerances to the model
PAHs measured in this work suggested a dependence on the growth substrate. There was
no significant tolerance to pyrene or phenanthrene when they were overlaid on pine and oak
containing media. On birch wood powder amended CMM overlaid with the model PAH
compounds however, the growth of Coprinellus radians isolates was not significandy different
than in the controls. Decay of PAH treated wood was not successful. There was no
tolerance to creosote found for any of the isolates. Although C. radians isolates did not decay
wood treated with PAHs, their tolerance to PAHs in media suggest a need for further
investigation. Perhaps their peroxidases, like those of Coprinopsis cinerea (Ikehata et al., 2003,
2004) have some ability to degrade such compounds. Further analysis could address this
through detection of breakdown products and physiological responses. Additional isolates
and PAHs must be tested with a wider range of carbon sources before conclusion of
investigations into inky caps' abilities to tolerate and degrade PAHs is complete.
76
Objective 4 — Provide Substrate Preference and Physiological Data to Allow Interand Intra-species and Genus Comparisons
To properly group and organize the Kingdom Fungi, modern changes based on
molecular phylogenetics must be supported by physiological investigations. The unique
individual characteristics of the inky cap species being taxonomicaUy reorganized warranted
characterization of their physiology to support or dispute proposed taxonomic changes
(Hopple and Vilgalys, 1999). Throughout the investigations of this thesis there has been
clear similarity between isolates of the same species and species of the same genus following
the genera names proposed by Redhead et al., (2001). The erroneously identified C. micaceus
ATCC 20122 isolate almost never behaved similarly to the other C. micaceus isolates.
Different genera typically were significantly different from one another in growth and decay
rates on the variety of the media tested.
With the exception of experiments utilizing the brown rot pre-treated wood, only
Coprinellus isolates significantly decayed the wood. These fungal isolates were also typified by
rapid growth rates, inducible xylanase production and a preference for pH around 7.
Coprinopsis isolates had the lowest growth rates and a preference for pH 8. Coprinus isolates
had growth rates and a pH preference between those of the other genera, and most of the
isolates had inducible mannanase.
Physiological results from this work were sometime variable due to isolate
differences. For example, C. radians ME-209 caused greater weight loss in decay tests than
those seen for C. radians ME-352. However, both these isolates caused significantly greater
decay than C. micaceus isolates. The small number of isolates investigated also limited the
strength of these analyses. For example, with only three isolates of C. micaceus, although the
erroneously identified isolate typically behaved differentiy from the others, it is possible that
this is coincidental and simply reflects normal variation among the species. In the cases of
77
Coprinopsis atramentarius, there was only one isolate representing an entire species. Small
replicate numbers created large standard deviations possibly hiding trends and significant
differences that may or may not have supported the proposed genera differences and
phylogenetic reorganization. Despite these limitations there is some support for the new
ordering of the inky caps and these findings encourage further work in physiology and
analysis of their support for phylogenetic changes.
Remaining Work and Future Directions
This work demonstrates an abihty of some inky caps to decay wood. A most
interesting finding, beyond the apparent genus-specific ability to decay wood, is the concept
of decay succession and the effects of the different decay pretreatments. Currently, decay
tests are being set up to investigate the effects of wood extractives and other pretreatment
factors on the ability of inky cap fungi to decay wood. The pretreatment factors being
explored are the extent of brown rot decay required to be an effective pretreatment, the use
of more white and brown rot species as pretreatments and their affects on both softwood
and hardwood degradation. It would also be of interest to utilize other field decayed
archived materials to bridge the laboratory investigations of this group of fungi back into the
natural environment.
Another major improvement can be made by simply increasing the number of
isolates and species being investigated to more completely represent the inky cap group of
fungi and their ability to decay wood. Investigating more isolates would also better
illuminate inter- and intra-species and genus differences and similarities. Though the ten
isolates represented three of the newly proposed genera, only five species were examined and
in some cases only one isolate represented a species.
78
With improved characterization of the changes to the woody substrates, a solid
understanding of late stage decay of forest litter and course woody debris degradation could
be built. This knowledge would greatly improve our management of forest resources, our
respect for small brown mushrooms, and could help us more towards greener futures both
ecologically and technologically.
79
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86
«Ssfe
APPENDIX
Original Genus
Coprinus
Coprinus
Coprinus
Coprinus
Coprinus
Coprinus
Coprinus
Coprinus
Coprinus
Genus
Coprinopsis
Coprinopsis
Coprinus
Coprinus
Coprinus
Coprinellus
Coprinellus
CoprineOus
Coprinellus
Species
atramentarius
cinereus
comatus
comatus
comatus
micaceus
micaceus
micaceus
radians
Isolate
FP-101910
FGSC 9003
FP-101592-T
FP-101691-T
ATCC 12640
FP-101781-T
ME-798
ATCC 20122
ME-352
Source
FPL
FGSC
FPL
FPL
ATCC
FPL
FPL
ATCC
FPL
Substrate of Isolation
Ulmus stump
Unknown
Lawn
Quercus
Grassy lawn
Decaying U/mus roots
Quercus alba timber
Soil
Populus pulp log
Coprinus
Coprinellus
radians
ME-209
FPL
Populus pulp log
Table A.l. Table of Isolates
Abbreviations for isolate sources:
-FPL (USDA-FS Forest Products Laboratory, Madison,WI)
-FGSC (Fungal Genetics Stock Center, University of Missouri)
-ATCC (American Type Cultural Collection, Manassas, VA)
87
BIOGRAPHY OF T H E A U T H O R
Jason P. Oliver was born in Auburn, N e w York on the 5 th of May in 1984. All of his
pre-college education was at the Union Springs Central School District in Cayuga County,
N e w York where he graduated from the High School with a Regents Diploma in May of
2002. H e continued on to earn a Bachelors of Science at the State University of N e w York,
College of Environmental Science and Forestry in Syracuse, N e w York in May of 2006 in the
Environmental and Forest Biology program with a focus of Forest Pathology, Microbiology,
and Mycology. H e began his Masters of Science work in August of 2006, advised by Dr.
Jody Jellison, at the University of Maine in O r o n o , Maine exploring the fields of w o o d decay
science, fungal physiology and ecology. The topic of his thesis is the w o o d decay physiology
of the inky cap fungal genera Coprinus, Coprinellus and Coprinopsis with attention on decay
succession and bioremediation potential. Jason is a candidate for the Master of Science
degree in Ecology and Environmental Science from the University of Maine in December
2008.
88