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This is to certify that the

dissertation entitled

The Involvement of Hydroxyl Radicals Derived From
Hydrogen Peroxide in Lignin Degradation By The
White-Rot Fungus Phanerochaete chrysosporium

presented by

Larry J. Forney

has been accepted towards fulfillment

of the requirements for
Microbiology and
Ph.D. degreein Public Health

Major profysror

Date 42/2‘3/ 87/
/ F

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

 

 

 

 

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Place in book drop to
remove this checkout from
your record. FINES will
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Stamped below.
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THE INVOLVEMENT OF HYDROXYL RADICALS DERIVED FROM
HYDROGEN PEROXIDE IN LIGNIN DEGRADATION BY THE WHITE-ROT FUNGUS
PHANEROCHAETE CHRYSOSPORIUM

 

By

Larry J. Forney

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1982

ABSTRACT
THE INVOLVEMENT OF HYDROXYL RADICALS DERIVED FROM HYDROGEN PEROXIDE

IN LIGNIN DEGRADATION BY THE WHITE-ROT FUNGUS
PHANEROCHAETE CHRYSOSPORIUM

By
Larry J. Forney

Accumulated data on the chemistry of lignin and its degradation by
white-rot fungi has recently led to the postulation that oxygen radicals
are involved in the extracellular transformations of the polymer ef-
fected by these organisms. In this study we investigated the possibili-
ty that the hydroxyl radical (-OH) derived from hydrogen peroxide (H202)
is involved in the degradation of lignin by Phanerochaete'chrysosporium,

a typical white-rot fungus. When 3, chrysosporium was grown in low N

 

medium (2.4 mM_N) an increase in the specific activity for H202 produc-
tion in cell extracts was observed to coincide with the appearance of lig-
ninolytic activity and both activities appeared after the culture en-
tered the stationary growth phase. Using cytochemical staining techni-
ques and 3,3'-diaminobenzidine (DAB) the H202 production activity in these
cells was shown to be localized in the periplasmic space of cells from
ligninolytic cultures. The intensity of the staining reaction (oxidized
DAB deposits) in cells of various ages was qualitatively related to the
levels of H202 production activity and in turn to the ligninolytic ca-
pacity of the cells. Similar oxidized DAB deposits were not observed in
cells from cultures grown in low N medium which had not entered the
stationary growth phase and therefore were not ligninolytic. The

production of -OH in ligninolytic cultures was demonstrated by

 

 

 

I-kEIO'l
peroxide
radic l
ated hy<
and by I
magneti:
oxide r
agents

nous ca
sence o
soecifi
results

gral r0

Larry J. Forney

a—keto-y-methiolbutyric acid dependent formation of ethylene. Hydrogen
peroxide dependent ~0H formation was also shown in cell extracts. The
radical species produced was demonstrated to be the -0H by the -OH medi-
ated hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid
and by using 5,5-dimethylpyrroline-N-oxide (DMPD) and electron para-
magnetic resonance spectrometry to detect the production of the nitr-
oxide radical of DMPD. These reactions were inhibited by ~DH scavenging
agents and were stimulated when azide was added to inhibit the endoge-
nous catalase. Lignin degradation was markedly suppressed in the pre-
sence of the .OH scavenging agents mannitol, benzoate and the non-
specific radical scavenging agent butylated-hydroxytoluene. The above
results clearly indicate that the -0H derived from H202 plays an inte-

gral role in lignin degradation by P, chrysosporium.

 

To Sue, Kelly and my parents

11'

ACKNOWLEDGMENTS

I would like to express my appreciation to Dr. C.A. Reddy for
his valued counsel during the course of this study. I am also grate-
ful to Drs. S.D. Aust. F. Dazzo, M.J. Klug, H.L. Sadoff and J.M.Tiedje
for serving on my guidance committee. The critical suggestions and
assistance of Dr. Ming Tien, H. Stuart Pankratz, Robert Kelly, Thomas
McFadden and numerous others have been very useful and are gratefully
acknowledged. Finally, I thank the Department of Microbiology and

Public Health for the financial assistance provided to me.

TABLE OF CONTENTS

 

 

Page
List of Tables ..........................
List of Figures .........................
Literature Review
Introduction ......................... I
Biosynthesis of Lignin .................... 1
Distribution of lignin in plant tissues ........... 11
Importance of lignin in the carbon cycle ........... 11
Role of lignin in the utilization of lignocellulosic
materials ......................... 12
Substrates Used in the Study of the Biological Degradation
of Lignin
Introduction ........................ 14
(14C-lignin) lignocellulose ................ 15
(14c) Synthetic lignins .................. 17
Polyguaiacol ........................ 19
Biological Degradation of Lignin
Introduction ........................ 20
Degradation of lignin by bacteria ............. 20
Degradation of lignin by white-rot fungi .......... 20
Summary of transformations effected in lignin by
white-rot fungi . .................... 23
Evidence for the involvement of enzymes in the '
initial steps in lignin degradation ........... 24
Low specificity of ligninolytic system ........... 25
Hydrogen peroxide production by white-rot fungi ...... 26
Degradation of lignin by brown-rot fungi .......... 27
Degradation of lignin by soft-rot fungi .......... 28
Degradation of lignin by other fungi ............ 29
Lignin Degradation by Phanerochaete chrysosporium
Introduction ........................ 29
Effect of nitrogen, carbon and sulfur ........... 30
Effects of oxygen partial pressure ............. 35
Effect of culture agitation ................ 36
Metabolism of Lignin Model Compound by P, chrysosporium
B-Guaiacyl ether linked dimeric lignin model compounds. . . 37
Regulation of metabolism of B-guaiacyl ether
linked model compounds .................. 37
Metabolism of model compounds containing phenolic ethers. . 38
Phenylcoumaran linked dimeric lignin model compounds. . . . 41
Literature Cited ....................... 48

iv

Chapter 1. Biological Studies on Lignin Degradation: A Simple
Procedure for the Synthesis from Ferulic Acid of
Coniferyl Alcohol, a Precursor of Synthetic Lignin

Abstract ........................... 58
Introduction ......................... 59
Materials and Methods .................... 60
Results ........................... 66
Discussion .......................... 69
Literature Cited ....................... 71

Chapter II. The Involvement of Hydroxyl Radicals Derived from
Hydrogen Peroxide in Lignin Degradation by the
White-Rot Fungus Phanerochaete chrysosporium

Summary ........................... 85
Introduction ......................... 85
Materials and Methods .................... 89
Results ........................... 96
Discussion .......................... 103
References .......................... 120

CHAPTER III. Ultrastructural Localization of Hydrogen Peroxide
Production in Ligninolytic Cells of Phanerochaete

 

 

chrysosporium
Abstract ........................... 124
Introduction ......................... 125
Materials and Methods .................... 127
Results ........................... 129
Discussion .......................... 132
Literature Cited ....................... 135

Appendices

Appendix 1. Speculation on the involvement of oxygen
radicals in lignin degradation by wood decomposing

fungi .......................... 141
Oxygen chemistry ...................... 143
The involvement of hydroxyl radical in lignin

degradation ....................... 144
Inhibition of lignin degradation by o-phtalate ....... 145
Cosubstrate requirement for lignin degradation ....... 145
Effect of oxygen partial pressure ......... . . . . 146
Literature Cited ...................... 148

Appenggx 2. Assay of Ligninolytic Activity (14C-lignin +
02) by Phanerochaete chrysosporium: Evaluation
of methods for the recovery and quantification of

14C02
Introduction ........................ 152
Evaluation of methods to trap 14C02 ............ 152
Effect of flushing time on the recovery of 14C02 ...... 156
Determination of quench correction curves ......... 157
Literature Cited ...................... 160

V

LIST OF TABLES

Literature Review

Table 1.

Chapter I

Table 1.

Chapter II

Table 1.

Table 2.

Table 3.

Table 4.

Chapter III

Table 1.

Appendix 2

Table 1.

Elemental and functional group analyses of lignin

following decay by white-rot fungi ........

Comparison of the elemental composition of the

synthetic lignin with that of other lignins. . . .

Effect of -OH radical scavenging agents on the
production of ethylene gas from KTBA by cultures

of P, chrysosporium ................

The effect of -DH scavenging agents and azide on
the hydroxylation of p-hydroxbenzoic acid to form
protocatechuic acid by cell extracts of

P, chrysosporium .................

 

Effect of .OH scavenging agents and azide on the
hydroxylation of p-hydroxybenzoic acid to form
protocatechuic acid in reactions containing

glucose oxidase ..................

' Effect of -OH scavenging agents on the lignino-
lytic activity and Phanerochaete chrysosporium . . .

 

Effect of culture age and nitrogen concentration
DAB reaction, hydrogen peroxide production
and 14C-synthetic lignin degradation by P.

chrysosporium ...................

 

IZEeCt of flushing time on the recovery of
0

vi

2 ooooooooooooooooooooooo

Page

22

74

110

111

112

113

138

Literature
Figure

Figure

Figure

Figure

Figure

Chapter I
Figure

Figure

Figure

Figure

LIST OF FIGURES

Review

1.

The structures of (a) trans-p-coumaryl alcohol,
(b) trans-coniferyl alcohol, (c) trans-sinapyl
alcohol, the immediate precursors used in the

biosynthesis of lignin ............. .

Structures of the mesomeric forms of the free
radical derived from coniferyl alcohol. (a)
phenoxyl radical, (b) o-methylene quinonoid
radical, (c) p-methylene quinonoid radical,

(d) p-quinone methide radical ...........

Schematic representation of gymnosperm lignin

(from reference 57) ................

Frequencies of major interunit linkages in
lignins from spruce wood and birch wood (from

reference 86) ...................

Structures of dimeric lignin model compounds
and products of their metabolism (compounds I -

XXIV) .......................

Reaction sequence for the synthesis of coniferyl

alcohol from ferulic acid .............

Analysis by high performance liquid chromato-
graphy of coniferyl alcohol (a) standard and

(b) that synthesized by the methods described. . . .

here.

Mass spectra of (a) coniferyl alcohol synthesized
by the methods described here and (b) that of an

authentic coniferyl alcohol standard .......

Infra-red spectra of (a) synthetic lignin pre-
pared by the methods described here (———)

and (b) milled spruce wood lignin (----) .....

vii

Page

10

44

76

78

80

82

Figure 5.

Chapter II

Figure 1.

Figure 2.

Figure 3.

Chapter III

Figure 1.

Degradation of (2'—14C) synthetic lignin (0),
prepared in this study, and (U ring- 4C) synthetic
lignin of Kirk (O) by Phanerochaete

 

chrysosporium ....................

 

Relationship between the specific activity for
production in cell extracts (‘), the
mgtgbolism of [2'- 14C] synthetic lignin to

4C02] (C) and mycelial dry weight (U) .....

The production of -OH in ligninolytic cultures

of P. chrysosporium .................

 

Electron paramagnetic resonance spectra of
DMPO- 0H adduct formed in reactions containing

and Fe( (II) (Fenton' s reagent; A) or cell
eitFa

ct (B- K) ....................

Transmission electron micrographs of P. chr so-
sporium showing details of cell wall TCW,

periplasmic area and cytoplasmic membrane (CM) . . .

viii

Page

84

115

117

119

140

LITERATURE REVIEW

Biosynthesis, Distribution and Importance of Lignin

Introduction

Lignin is one of the most abundant and widely distributed organic
polymers in nature. It occurs as an integral cell wall component and in
the middle lamellae of all vascular plants (104). Within plants, lignin
imparts structural rigidity, resistance to impact, compression and bend-
ing (105), decreases water permeation across cell walls of xylem tissue
and protects plant tissues from invasion from pathogenic microorganisms
(47, 111). Interest in the mechanism of lignin degradation by micro-
organisms stems from its importance in the carbon cycle and because lig-
nocellulosic materials represent a vast renewable resource for the pro-

duction of fuels and chemicals (6).

Biosynthesis of lignin

Lignins are formed through the dehydrogenative polymerization of
primarily three cinnamyl alcohol derivatives: trans-p-coumaryl alcohol,
trans-coniferyl alcohol and trans-sinapyl alcohol (Figure 1). The ratio
of the three percursors varies in lignins of different plant species (1)
and within different tissues of the same plant species (111). On average,
gymnosperm lignin (softwood lignin) contains approximately 94% coniferyl
alcohol, 5% p-coumaryl alcohol and 1% sinapyl alcohol, whereas angio-
sperm lignin (hard wood lignin) contains roughly equal proportions of
coniferyl and sinapyl alcohols and a minor amount of p-coumaryl alcohol.

1

 

Figure 1.

The structures of (a) trans-p-coumaryl alcohol,
(b) trans-coniferyl alcohol, (c) trans-sinapyl
alcohol, the immediate precursors used in the
biosynthesis of lignin.

 

 

 

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During lignification in young plant tissues a higher proportion of guaiacyl
units (derived from coniferyl alcohol) are incorporated, whereas during
maturation, the proportion of syringyl units (derived from sinapyl alco-
hol) increases (36, 47, 111).

At the site of lignification, the phenolic hydroxyl groups of the
cinnamyl alcohol precursors are oxidized by a phenol oxidase enzyme,
usually peroxidase (EC 1.11.1.7, donor: hydrogen peroxide oxidoreductase),
to produce phenoxy radicals (47). Due to the extended irelectron sys-
temsoof the precursors the free radicals are stabilized through equili-
brium with several mesomeric forms (57). The resonance structures of
the radical produced from coniferyl alcohol are shown in Figure 2. The
stability of each mesomer determines its relative concentration and
therefore, its availability to participate in coupling reactions to pro-
duce covalent bonds. The phenolic hydroxyl groups of oligomeric pro-
ducts are also oxidized and the nonenzymatic polymerization proceeds pri-
marily through addition to the growing polymer (40). As a result of the
mechanism of polymerization, all asymmetric carbons in the polymer (C-a
and C98 of the monomer side chain) exist in both R and S stereoisomeric
”forms (106) .

The structural and chemical complexity of the polymer is clearly
evident in the schematic representation of gymnosperm lignin shown in
Figure 3 (39, 57). More than 24 different intermonomer bonding types
have been found to occur in lignin (1). The major intermonomer bonding
types and their frequencies in representative gymnosperm (spruce) and
angiosperm (birch) lignins are shown in Figure 4 (1, 86). The aryl
glycerol-ES-aryl ether linkage (Figure 4, substructure A) occurs most

frequently, with 48% and 60% of the monomers participating in this

 

Figure 2.

Structures of the mesomeric forms of the free radical
derived from coniferyl alcohol. (a) phenoxyl radical,
(b) o-methylene quinonoid radical, (c) p-methylene
quinonoid radical, (d) p-quinone methide radical.

 

 

 

H C
2' OH
CH
ll
HC

CONIFERYL ALCOHOL

ll

ll

H C|IOH

 

Figure 3. Schematic representation of gymnosperm lignin
(from reference 57).

 

 

 

 

 

 

 

 

Figure 4. Frequencies of major interunit linkages in lignins
from spruce wood and birch wood (from reference 86).

 

 

 

C

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Q

C

1

-C -C

O

C

l

C

l

C
C
I
. C)
l
C—O

(3'-(3 --(5

 

of C9 units participating

 

 

Substructure type spruce birch
A 48 60
8 9-12 6
C 9.5-11 4.5
D 6-8 6-8
E 7 8
F 3.5-4 6.5

 

11

type of linkage in gymnosperm and angiosperm lignins, respectively.
Most other intermonomer bonding types involve less than 10% of the lig-
nin monomers. The molecular weight of purified lignin varies from a
few thousand to more than 1 X 106 depending on the method used for iso-
lation (46). The elemental composition for lignin from spruce wood has

been shown to be C H D (OCH

9 7.49 2.53 (40)°

3)1.39
Distribution of lignin in plant tissues
Lignin is deposited within cell walls to form an interpenetrating
network with hemicelluloses (111). This matrix surrounds the cellulose
fibrils in a sheath-like manner to form a physically impenetrable barrier
such that polysaccharide degrading enzymes cannot gain access to a large
portion of the cellulose and hemicelluloses in fully lignified tissue.
Ultraviolet inicroscopycrf thin sections of plant tissues revealed that
72-81% of the total lignin in black spruce early wood is located within
the secondary cell wall layers, with the balance present in the middle
lamellae (35). The middle lamellae consists largely of lignin (approxi-
mately 72%). Since 45-50% of the secondary cell wall is polysaccharide,
the largest portion of lignin in plant tissues occurs in close physical
association with and is chemically bonded to cellulose and hemicellu-

loses (112).

Importance of lignin in the carbon cycle
Due to their abundance in nature, lignins play an important role
in the earth's carboncycle. An estimated 5 X 108 metric tons of lig-

12

nin are biosynthesized annually (114)and 1-3 X 10 metric tons of

carbon has been calculated to be present in the organic matter of soil

12

(114), primarily as peat and humus which in a large part are derived
from lignins (65).

In soil, plant tissues are subject to degradation by microorgan-
isms. While the sugars and polysaccharide fractions are degraded com—
paratively rapidly, the lignin fraction is metabolized rather slowly
(94). The oxidized, partially condensed lignin polymer, in conjunc-
tion with various carbohydrates, proteinaceous material and other plant
and microbially produced phenolic compounds, iknmihumus (59, 93, 65).
The humus found in the upper layers of soils performs several impor-
tant functions (93). It influences the structure of soil increasing
its aeration and moisture holding capacity. It also serves as an ion
exchange resin and is able to sequester and release various ionically
charged organic molecules and inorganic ions, in general adding to
soil fertility. Humus is degraded very slowly with an estimated half-
life of 76 to 326 y depending on the soil type investigated (52). The
recalcitrance of humus is reflected in the observed occurrence of lignin
derived phenolic residues in lignite (61), bituminous coal (61) and in

8 y old (107).

silicified wood which was 2.8 X 10
Role of lignin in the utilization of lignocellulosic materials

In recent years, numerous investigators have been exploring the
potential of using "biomass" for the production of various fuels and
chemical feedstocks (6). The term biomass generally refers to the poly-
saccharides present in lignocellulosic materials, including agricultural
residues and crops grown expressly for use in such processes. It has

8

been estimated that approximately 8 X 10 tons of lignocellulosic by-

products and residues are generated annually (6). Obviously, these

13

materials represent a renewable resource of considerable magnitude.
Lignin, due to its close physical and chemical association with
plant polysaccharides, has been shown to present both a chemical and phy-
sical barrier to enzymes capable of hydrolyzing cellulose and hemicellu-
loses (15, 78). Thus, lignin is an important factor which impedes the
utilization of these polysaccharides in various industrial processes.
Therefore, any treatment which depolymerizes, solubilizes, or otherwise
removes lignin increases the availability and susceptibility of cellu-
lose and hemicelluloses to enzymatic or chemical attack (14, 15, 78).
Various physical and chemical pretreatments individually and in various
combinations have been shown to be effective (23, 25). However, most
suffer from one or more of the following disadvantages (25, 101): (a) are
energy intensive and are therefore not economical; (b) generate large
quantities of chemical waste which muSt be efficiently recycled or dis-
posed of, or (c) are not effective when used on an industrial scale.
The use of microorganisms to partially delignify lignocellulosic mater-
ials, in conjunction with the use of other chemical or physical pretreat-
ments, could potentially decrease the energy and chemicals required to
disrupt the lignin-polysaccharide association and therefore decrease
the cost of substrate pretreatment in processes which utilize ligno-
cellulosic materials (101) as sources of fuels, chemicals and feeds.
Lignin also has potential as a source of industrially useful
polymers and as a source of aromatic chemical feedstocks (6). Although
chemical procedures for the conversion of lignin into commercially valu-
able products have been developed, none are widely used and many suffer
from low product yield (6). A process using microorganisms may offer

greater specificity and therefore be more cost effective. Perhaps the

14

most efficacious approach would be to produce modified lignin polymers
for use as dispersants, emulsion stabilizers, complexing agents, coagu-
lants and antioxidants, controlled desorption agents, ion exchange resins,
or in thermosetting resins and rubber reinforcement (6). Furthermore,
it may be possible to use biological processes to generate aromatic
chemical feedstocks such as phenols, benzene, cresols and cresolyic
acids from lignin in yields that would be economically attractive (6).

Substrates used in the Study of the Biological Degradation of Lignin
Introduction

A survey of the literature reveals that lignin is transformed by

many organisms and in many environments (10, 17, 49, 93, 94). However,
study of the biological decomposition of lignin has been hindered due
to unavailability of sensitive assays. By far the most common assay
used in the past in studies on the biological degradation of lignocellu-
losic materials has been the Klason assay (26). In this procedure, the
carbohydrates are solubilized by hydrolysis with 72% H2504 and by re-
fluxing in dilute acid. The lignin remains insoluble and is quantified
gravimetrically. Using this assay, underestimation of the lignin pre-
sent in a sample may result from the increased solubility of lignin
through oxidation by microorganisms. Conversely, the lignin may
be overestimated if condensation reactions occur between lignin
and proteinaceous material in the sample during the assay proce-
dure (90). 11fi5.assay is usually indicative only of the amount of lignin
which remains polymeric and acid-insoluble, and no information is ob-
tained as to the extent to which the lignin is depolymerized or re-
spired as C02. Furthermore, specific transformations, such as demethy-

lation of methoxyl groups, cannot be assessed. Obviously, this assay

15

is of very limited use to biologists. In recent years, procedures for

the preparation of (14C-lignin) lignocelluloses and 14C-synthetic lignin

have been developed and are widely used in studies on the degradation
of lignin by microorganisms (20). These lignins have all the advantages

14

of other C-labelled substrates used in the study of microbial meta-

bolism including high sensitivity and the ability to specifically label

‘2'-14c side chain labelled, 14

portions of the polymer (i.e., C-ring
labelled or methoxyi group (ONCHB) labelled). However, these substrates
are primarily used to monitor 14C02 production which does not adequately

reflect the complex process of lignin degradation.

(l4C-Lignin) lignocelluloses
Crawford and Crawford (16) and Haider and Trojanowski (51) have

14

developed procedures for the preparation of C-lignin labelled plant~

tissue for use in studies of the microbial degradation of lignin. These
procedures employ radioactively labelled biosynthetic precursors of lig-
nin such as phenylalanine, cinnamic acid or ferulic acid which are either
injected into the cambial layer of the plant or a cut twig or stem is
placed in a solution containing the precursor (16, 51, 108). The pre-

14C-

cursor is then incorporated by the plant into lignin (63). The
lignocellulose is isolated by scraping the cambial layer from the
wood or the entire tissue is used. After milling to reduce the particle

14C-precursor, waxes, resins and oils are ex-

size, the unincorporated
tracted and the lignocellulose is dried.

The (14C-lignin) lignocellulose produced should be fully char-
acterized to determine the distribution of label within the tissue.

The analyses should include a Klason lignin assay and quantification of

16

the label incorporated into carbohydrates and protein, following hydro-
lysis of the wood polysaccharides and digestion with a protease, respec-
tively (20). When the Klason assay is performed on (14C-lignin) lig-
nocelluloses, the label present in the acid soluble and acid insoluble
fractions are determined by total combustion analysis and by liquid
scintillation counting. However, it has been shown that a certain frac-
tion of the lignin remains "acid-soluble". As a result, the amount of
label incorporated into non-lignin fractions cannot be accurately de-
termined. The amount of acid-soluble label found varies, depending on
the plant species examined, from approximately 50% (e.g. Sgartina
alterniflora, Picea excelas) to 2% (lypha latifola) with most species
being 25-35% (20). It can probably be assumed that if a large percen-
tage of the label is found in the "acid-soluble" fraction, that plant
components other than lignin have been labelled (e.g. phenolic compounds,

flavonoids, etc.).

If (14C)—phenylalanine is used as the precursor the determina-
tion of 14'C-protein present in 14’C—lignocellulose is especially
important. However, experimentation has shown that in the plant

species examined, little (less than 3%) of the label is incorporated

14

into protein. This problem is easily circumvented by using C-

14

ferulic acid or C-cinnamic acid where upon incorporation into protein

has been found to be less than 1%.

The incorporation of label into non-lignin fractions and less con-
densed, i.e., recently synthesized, lignin (which is probably degraded
more readily), constitutes the major disadvantage to the use of (14C-
lignin) lignocellulose. This is especially true if low levels of de-

gradation are observed.

17

For certain investigations, the use of (14C-lignin) lignocellulose
is desirable since the lignins are degraded while in their "natural"
states. Thus, if the question under study is whether or not an organism

(14

can degrade lignin in plant tissue, the use of C-lignin) lignocellu-

lose has obvious advantages.

(14C) Synthetic lignins

(14C)-synthetic lignins or unlabelled synthetic lignins are pre-
pared by the jfl_yitrg dehydrogenative polymerization of specifically
labelled lignin precursors, using a horseradish peroxidase and H202 (40).
Such model lignin polymers were used extensively by lignin chemists to
elucidate the structure of lignin (40, 57) and have been shown to ex-
hibit many of the structural and chemical features of naturally occuring
lignin (81, 103). Several significantly different protocols for the
synthesis of synthetic lignins have been published (40, 81). Despite
careful attention to experimental detail, these lignins frequently differ
in their average molecular weight (12, 31), susceptibility to degrada-
tion by microorganisms and solubility in various organic solvents (31,
L.J. Forney and C.A. Reddy, unpublished data). These differences occur
when the same procedure is used on different occasions. This obviously
leads to difficulty in rePVOdUClblllty of results in biodegradation
studies and comparison of results between different laboratories. Clearly
a thorough examination of the factors affecting the preparation of
these lignins is needed.

14C-Synthetic lignins are not commercially available and the

14C-labelled precursors of synthetic lignin, although available are

18

rather expensive. As a result, most investigators must undertake the
synthesis of the precursors themselves. The synthesis of the precursors
is somewhat involved and this has discouraged the use of synthetic
lignins in studies on the fate of lignin in pure culture and in the
environment.

For some investigations, the use of (14C)-synthetic lignin has
several advantages as compared to 14C-lignocelluloses. Synthetic lignins
can be recovered from microbial cultures without further (artificial)
modifications for use in various1analytical procedures. In contrast,
lignin in plant tissue is modified during the ball milling and extrac-
tion or solvolysis which is required to recover it (90). Synthetic
lignin is readily fractionated into various molecular weight ranges
(12, 31) and the average molecular weight of the lignin can be determined.
Therefore, changes in the molecular weight profile of synthetic.
lignin which occur during degradation can be determined. Such
determinations can not be made using lignocelluloses. Furthermore,
since synthetic lignin does not contain carbohydrates or other extrane-
ous materials, it can be used to determine if an organism is capable of
using lignin as a sole carbon source or for examining the ability of
various compounds to serve as cosubstrates for lignin degradation.
However, synthetic lignins are not associated with polysaccharides in
plant tissue and the extrapolation of conclusions made from experiments
using such an I'isolated" lignin to lignin in plant tissue may not be
possible or accurate. For example, organisms capable of degrading iso-
lated lignin may not attack lignin in plant tissue and vice versa.

Robinson and Crawford (102) found that Bacillus sp. could degrade

 

19

approximately 12% of (14C-lignin) lignocellulose but only 0.3% of the

14C-synthetic lignin to 14

C02 in 20 d. Conversely, Odier et. al (99)
isolated numerous strains of bacteria which could degrade more than
50% of acidolysis wheat straw lignin (an "isolated" lignin, albeit not

synthetic lignin) but were unable to degrade (14C-lignin) poplar wood.

Polyguaiacol

Recently, Crawford gt _1 (21) described a procedure for the syn-
thesis of (14C)-polyguaiacol which they proposed may serve as a lignin
model polymer. The (14C)-guaiacol is polymerized using a procedure analo-
gous to that used for the preparation of synthetic lignins. The poly-
mer is composed of o-o and p-p linked guaiacol moieties and infrequently
o-p biphenyl bonds and p-diphenoquinone structures. The polymer had an
approximate molecular formula of C6H2.300.3 Carbonyl (0H)0.7(0CH3)1.0
and an average molecular weight between 5,000 and 15,000. Experiments
with Phanerochaete chrysosporium suggested that the biodegradation of the
polymer was affected by the same culture parameters as lignin degradation

(see Lignin degradation by Phanerochaete chrysosporium). For example,

 

(140)-polyguaiacol was degraded in low N cultures (2.4 mM_N) but not in
the presence of high N (24 liN). Furthermore, the rate of degrada—

tion in an atmosphere of 80% 02 - 20% N2 was significantly greater than
in an atmosphere of 21% 02. Since the structure of polyguaiacol is less
complicated and more easily defined than that of synthetic lignin, it

may be easier to identify the events which occur during its degradation
by white-rot fungi. However, the potential andlimitations of this model

substrate have not yet been fully explored.

20

Biological Degradation of Lignin

Introduction

The range of microorganisms conclusively shown to degrade lignin
includes a wide variety of fungiiand bacteria (4, 19). In addition,
preliminary evidence suggests that certain yeasts can degrade lignin (70).
Wood-decomposing fungi have been classified into three main groups (77)
according to the effect they have on wood tissue: (a) white-rot fungi,
(b) brown-rot fungi, and (c) soft-rot fungi. The characteristics of wood

decomposition by these organisms will be discussed below.

Degradation of lignin by bacteria

Certain strains of Rhodococcus, Nocardia, Pseudomonas, Flavo-
bacterium, Aeromonas and Streptomyces have been shown to degrade
14C-synthetic lignin, 14C-lignocellulose, milled wood lignin, dioxane-
lignin, kraft lignin and various other industrial lignins (18, 28, 53,

72, 88, 110). The degradation of lignin by bacteria has recently been

reviewed by Crawford and Crawford (19).

Degradation of lignin by white-rot fungi

Several hundred species of Hymenomycetes and a few species of

 

Ascomycetes, collectively known as white-rot fungi, are capable of simul-

 

taneously effecting extensive degradation of all major components of
wood (4, 77, 85, 86). During the degradation of wood by white-rot fungi,
the lignin is concurrently depolymerized and condensed so that a por-
tion of the lignin becomes higher molecular weight and a portion be-

comes lower molecular weight (48, 66, 84). Chemical and spectrosc0pic

21

analyses of the residual white-rot decayed lignin suggest that, in gen-
eral, the degraded polymer is structurally more complex than the unde-
graded lignin (79, 80).

Previous investigators have shown lignin degradation by white-rot
fungi to be highly oxidative in nature. Several investigators (60, 68,
80) have compared the empirical formula, total hydroxyl, carboxyl and
a-carbonyl content for spruce wood lignin degraded by pure cultures of
white-rot fungi to that of undegraded lignin (Table 1). In general, a
progressive increase in oxygen content and a decrease in hydrogen con-
tent was observed which was reflected in an increase in oxygen containing

functional groups following white-rot decay. For example, the carboxylic

 

acid group content of spruce wood degraded by E. versicolor increased
from 0.02 per C9 to 0.106 per C9 after 180 d of incubation (60). The
oxidative nature of lignin degradation was also reflected in the obser-
vation that white-rot fungi did not degrade lignin in an atmosphere
which contained 5% 02 despite the fact that growth was normal (84).

14C-labelled synthetic lignins

In addition, experiments conducted using
have shown that lignin is not degraded in many anaerobic environments
49).

Kirk and Chang (80) have shown that white-rot fungi were able to
degrade the aromatic rings of the polymer while it is still in the ex-
tracellular environment. This was based upon the increase in a,B:un-
saturated carboxylic acid moieties which were'anderived from the
cinnamic acid residues (in the side-chains) of the polymer. Analogous
a,B-unsaturated carboxylic acids are known to be produced during
the oxidation of low molecular weight aromatic compounds by micro-

1

organisms (11). A decrease in the absorbance at 1515 cm' in the

22

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23

infrared spectrum relative to other absorption bands and a decreased
proportion of aromatic protons in the proton nuclear magnetic resonance
spectrum of the degraded lignin provided further evidence for cleavage
of the aromatic ring within the intact polymer.

In addition to aromatic ring cleavage, these fungi are apparently
able to oxidatively shorten the aliphatic side chains of the intact
polymer and to demethylate methoxyl groups in lignin (60, 68, 80, 109).
Hata (60) hydrolyzed spruce wood lignin which had been degraded by Pgria
subacida and intact milled wood lignin and determined the yield of C6-C1
compounds as compared to C6-C3 compounds. After incubation with the
fungus for six months, the yield of vanillic acid increased 5-fold
whereas the yield of coniferyl alcohol decreased 6-fold. These results
suggest that the terminal two carbons of the side chains of some sub-
structures in lignin are removed to produce C6-C1 units which can be
liberated by hydrolysis. Furthermore, there was approximately a 25%
decrease in the methoxyl group content of the residual lignin decayed by

_P. versicolor (Table 1). However, extensive demethylation of methoxyl

 

groups in lignin prior to its metabolism does not seem to occur since
the methoxyl group content of the residual lignin was similar after 28

and 180 d of incubation (60, 80).

Summary of transformations effected in lignin by white-rot fungi

The following conclusions can be made regarding the extra-
cellular transformation effected while lignin is still polymerized
(60, 68, 79, 80): (1) The reactions are primarily oxidative. The
degraded lignins show significant increases in oxygen containing func-

tional groups such as carboxyl and carbonyl groups and a decreased

24

hydrogen content; (b) the C-o carbon of the propyl side chains are oxié
dized to carbonyl groups; (c) the aromatic rings are oxidatively cleaved;
and (d) methoxyl groups are demethylated to yield hydroxyl groups. Iden-
tification and characterization of the low molecular weight products
formed during lignin degradation has provided data to support the above
conclusions and to suggest that hydroxylation at C-2 of some rings occurs
(86). This presumably allows for intradiol ring cleavage following
demethylation of the adjacent methoxyl groups. Thus, many of the
structural elements of the polymer appear to be subject to oxidation

during the course of lignin degradation by white—rot fungi.

Evidence for the involvement of enzymes in the initial steps in lignin
degradation

Since the reactions discussed above occur in the extracellular en-
vironment, one would expect the agents responsible for these trans-
formations to be found in the medium ofligninolytic cultures of white-
rot fungi. However, surprisingly low levels of extracellular enzymes
have been observed in these cultures (54). It has been suggested that
two extracellular enzymes, phenol oxidase (3, 4, 67, 74, 75, 98) and
aromatic alcohol oxidase (32, 64), are involved in the degradation of
the lignin polymer. Aromatic alcohol oxidase, shown to be elicited by
Coriolus versicolor and Fusarium solani, oxidizes a wide range of com-
pounds that possess a,B-unsaturated primary alcohols, including those
within the lignin polymer, to the corresponding aldehyde and hydrogen
peroxide (32, 64). Phenol oxidases, which are produced by many, but
not all lignin decomposing fungi (58) oxidize phenols to the corres-

ponding quinones (8). These enzymes are incapable of effecting all of

25

the structural changes which occur during lignin degradation (8, 37)

and their roles, if any, in lignin degradation are unclear. It is im-
portant to note that cell-free culture filtrates do not decompose lig-
nin (41, 42, 54, 69, 109) and conclusive evidence for the involvement

of any enzyme in lignin degradation is lacking.

Low specificity of ligninolytic system
Recent work with model compounds (see Model compound metabolism

by P, chrysosporium) has provided evidence that some agents involved in

 

the decomposition of lignin are relatively nonstereospecific (96). Methyl-

dehydroconiferyl alcohol is a dilignol which contains a phenylcoumaran

linkage and also has an a,B-unsaturated propyl side chain. When meta-

bolized by E, chrysosporium the a,e-unsaturated side chain is converted

 

to a glycerol moiety by nonsteroselective epoxidation followed by non-
enzymatic hydrolysis of the epoxide to yield both erythro and threo
isomers of the product. It was thought that an enzyme-mediated nonstereo-
selective epoxidation was unlikely. A

Other, more general, observations suggest that at least certain
components of the ligninolytic system have relatively low specificity.
Lignin has been shown to be extensively degraded (>90%) upon prolonged
incubation in pure cultures of white-rot fungi (13). This occurs des-
pite the variety of intermonomer linkages that are known to occur and
the racemic carbons in the polymer. Furthermore, lignin decomposing
fungi are capable of degrading structurally different lignins including
lignins in various species of plants (e.g. guaiacyl, syringyl and grass
lignins) within different tissues of the same plant (e.g. early, late

and compression wood) as well as industrially modified lignins

26

(e.g. kraft lignins and lignosulfonates) (7, 29, 77, 92). Thus, the
ligninolytic system is capable of recognizing structurally different
polymers as substrates. Furthermore, Keyser et. al. (73) suggested

that the relatively low rates of lignin degradation by basidiomycetes,
and the apparent inability of lignin to serve as a growth substrate for
fungi, may indicate low activity of the ligninolytic system and, in-
directly, that the system possesses low specificity. The data discussed
above suggest that the agents responsible for lignin degradation in the
extracellular environment are relatively nonspecific in their mode of

attack.

Hydrogen peroxide production by white-rot fungi

Koenigs (87) found that 29 of 32 strains (representing 23 species)
of white-rot fungi examined produced hydrogen peroxide (H202) which
could be detected extracellularly. Koenigs (87) suggested the follow-

'hu;role for H202 in the decomposition of wood by white-rot fungi:

The H202 molecule, because of its small size and the accom-
panying lack of restricted substrate range inherent to

an enzyme system would be much freer than is poly-

phenol oxidase, for example, to attack a polymer such

as lignin. Formation by H202 of organic peroxides or free
radical in lignin might lead to its subsequent depolymeri-
zation or furnish a "substrate" on which oxidation of lignin
can occur.

However, later studies on the production of H202 by wood-decomposing
fungi have been concerned with the involvement of oxygen radicals de-
rived from H202 in cellulose decomposition by brown-rot fungi (88; also
see Lignin degradation by brown -rot fungi). To my knowledge, studies on
the role of H202 in cellulose or lignin degradation by white-rot fungi

have heretofore not been conducted.

27

Degradation of lignin by brown-rot fungi

During wood decomposition by brown-rot fungi, the carbohydrates
are extensively depleted but the lignin residue is only partially de-
graded (4, 13, 77). The decay is characterized by extensive depoly-
merization of the cellulose prior to its metabolism by the fungus. This
is somewhat surprising since these fungi apparently do not produce
8-1,4-endoglucanase (62, 97). These fungi have been shown to produce
and excrete hydrogen peroxide (87) and it has been postulated that hy-
droxyl radicals derived from hydrogen peroxide in a Fenton type reaction
may serve to depolymerize the cellulose in lignocellulose during wood
decomposition by brown-rot fungi (87, 88, 89). Althoughihlliwell has
shown that hydroxyl radicals effectively disrupt the crystalline structure
cfl’cellulose and reduce the degree of polymerization (56). Conclusive
data to support this proposed mechanism of cellulose decomposition by
brown-rot fungi is lacking.

Kirk and Adler (76) found that Poria monticola and Lenzites trabea

 

extensively demethylate methoxyl groups in lignin. The demethylation

of methoxyl groups in either units with free phenolic hydroxyl groups

or in units with etherified phenolic groups, coupled with the hydro-
xylation of the aromatic ring at C-2, was found to result in the forma-
tion of o-diphenolic moieties (catechol) in lignin during brown-rot decay
(76, 82). These moieties were thought to undergo autooxidation to pro-
duce quinone-type chromophores which give brown-rotted wood its character-
istic color. Aromatic ring cleavage was minimal however and some 0-
diphenolic substructures were found to persist. There were twice as

many a-carbonyl and carboxyl groups in lignin decayed by L, 329922.35

compared to sound lignin. In contrast, to white-rot fungi, brown-rot

28

fungi appear unable to oxidatively shorten the alkyl side chains of
lignin.

In many respects, the structural alteration and decomposition of
lignin by brown-rot fungi resembles that of white-rot fungi. Kirk (82)
has suggested that the differing abilities of these two taxonomically re-
lated groups may be due to the inability of brown-rot fungi to metabo-

lize aromatic rings or the aliphatic products of aromatic ring cleavage.

Degradation of lignin by soft-rot fungi

The ability of soft-rot fungi to degrade lignin has not been ex-
tensively investigated. These fungi, which include certain ascomycetes
and fungi imperfecti, primarily metabolize the hemicelluloses and cellu-
loses in wood (77). However, they have also been shown to demethylate
methoxyl groups within lignin and to metabolize the side chains and
aromatic ring elements within the polymer (51). Analyses on wood de-
cayed by soft-rot fungi indicated the lignin component was partially
degraded and modified by these fungi in such a way that a larger portion
becomes acid soluble (50, 91). This is probably due to partial oxida-
tion and a reduction in the molecular weight of the polymer effected by
the fungi. Elsyn (30) found that four of six species studied depleted
the carbohydrates in wood faster than lignin whereas the other two spe-

cies depleted lignin faster than the carbohydrates.

29

Degradation of lignin by other fungi

Several fungi, which are not classified into the groups discussed
above, have also been shown to decompose lignin. Strains of Fusarium
oxysporus, Fusarium moniliforme, Fusarium solani and Fusarium s2. have
been shown to decompose synthetic lignin (64). Size exclusion chroma-
tography of lignins degraded by these fungi and degradation experiments
conducted using synthetic lignins of various molecular weight ranges
showed that the low molecular weight fractions were preferentially metabo-
lized.

Drew and Kadam (24) found that when 1% soluble starch was provided
as a cosubstrate, more than 50% of the added 14C-labelled kraft lignin

14

was converted to CO2 by Aspergillus fumigatus in 16 d. This was roughly

 

seven times greater than the rate observed for the white-rot fungus

.Q. versicolor when grown under similar conditions. The ability of cell-

 

free culture filtrates to degrade or modify lignin were rather limited
although cellobiose-quinone oxidoreductase and phenol oxidase activities
were demonstrated in these filtrates. Based on these studies, it would
appear that certain strains of A, fumigatus and possibly other sapro-
phytic fungi are able to rapidly and extensively degrade lignin.

Lignin Degradation by Phanerochaete chrysosporium

Introduction

Phanerochaete chrysgsporium Burds, causes a typical white-rot

 

 

type of decay and was isolated from beech (Fagus grandifolia) wood chips
collected in Waterville, Maine in 1964 by W. E. Elsyn (strain ME 446,
Center for Forest Mycology Research, Forest Products Laboratory, USDA,
Madison, WI or ATCC 34541, American Type Culture Collection, Rockville,

MD). The morphology and taxonomic criteria have been described by

3O

Burdsall and Elsyn (9). The fungus is readily cultivated on either com-
plex or chemically defined media, produces conidia in culture, has an
optimum pH for growth of 4.5-5.0 and a temperature optimum of 40°C.

The low level of phenol oxidase activity expressed by the fungus (73)
minimizes the undesirable oxidation and polymerization of phenols during
studies of lignin degradation. 3, chrysosporium (originally called
Peniophora “6“) is synonomous with Sporotrichum pulverulentum, Chrysos—
porium lignorum and g, pruinosum and has been the subject of numerous
investigations on the biological degradation of lignin (85, 86). Many
of the cultural and nutritional parameters which affect lignin degrada-
tion have been established (5, 71, 73, 83, 84, 100) and methodologies

for replica plating and mutant isolation have been described (43, 44).

Effect of nitrogen,gcarbon and sulfur

“Lignin degradation by E. chrysosporium has been shown to be de-
repressed following the cessation of primary growth due to either N,
C or S starvation (71, 73, 84, 100). The effect of N concentration on
lignin degradation has been investigated in detail. In cultures of E.

chrysosporium which contained 56 mfl_glucose and 2.4 mM_N, the extra-

 

cellular N was depleted after 2 d of incubation (73). The culture
appeared to enter stationary phase on day 3 and ligninolytic activity

first appeared after day 4. P, chrysosporium was shown to sequester N

 

intracellularly as arginine during the first three days of incubation,
during which time it accounted for 27-35% of the total amino acid N
(33). During the transition period between the depletion of N in the
medium and the onset of ligninolytic activity, the arginine pool de-

creased 5-fold. Concurrently, there was a rapid increase in

31

intercellular protein turnover which occurred at maximal rates of 5-7%
per hour.

Further studies showed that ligninolytic activity was repressed
by nitrogen (73). If 24 umoles NH4+ nfi-l was added to the cultures on
day three, the appearance of ligninolytic activity was delayed (73).
Furthermore, when 2.4 umoles NHafiLml"1 was added to 6 day'old, actively
ligninolytic cultures, lignin degradation was not affected for 16 h, but
then was transiently repressed for 40-50 h (73). All of the fourteen
nitrogenous compounds examined repressed ligninolytic activity in a
manner similar to NH4+ when added at equimolar N concentrations, but
with varying degrees of effectiveness (33). Glutamate, glutamine and
histidine were the most effective and repressed ligninolytic activity
by 83, 76, and 76% respectively, as compared to cultures to which no
N was added. Ammonium chloride repressed ligninolytic. activity approxi-
mately 50%. Protein synthesis increased when these various N sources were
added- However,the stimulation was not correlated to the relative abili-
ties to serve as N sources for growth or to repress ligninolytic
activity. Therefore, the repressive effects of theses compounds
does not seem to be due to a resumption of primary growth of the
fungus.

The intracellular pools of arginine and glutamine increased sharply
following the addition of NH4+, but not glutamate, to nitrogen starved
cultures (34). Thus high levels of these amino acids did not appear
to be essential for the initiation or maintainence of repression. Glu-
tamate pools increased immediately following the addition of either NH4+
or glutamate to ligninolytic cultures. However; the pool size returned

to that found in control cells by 18 h after glutamate addition, but

32

the repression of ligninolytic activity persisted. Thus it would

appear that neither arginine, glutamate or glutamine is directly in-
volved in the repression of ligninolytic activity but rather the effect
of N appears to be a more general one. This conclusion is further sup-
ported by the inhibition of the synthesis of the secondary metabolite
(3,4-dimethoxy) benzyl alcohol (veratryl alcohol) observed upon the addi-
tion of N to nitrogen starved cultures.

Cycloheximide, a protein synthesis inhibitor, prevented the appear-
ance of ligninolytic activity when added to nitrogen limited cultures
3-5 d after inoculation (73). These results indicate that the lignino-
lytic system, or at least an essential component, is formed gg.ggy9.
following the depletion of N in the culture. When added to actively lig-
ninolytic cultures, cycloheximide suppressed ligninolytic activity in
a manner similar to NH4+ and glutamate. It would appear that certain
nitrogenous compounds repress key enzyme(s) involved in lignin degrada-

tion by_P. chrysosporium. Repression of ligninolytic activity, as op-

 

posed to inhibition or competition, by N is supported by the following:
(a) Repression but not inhibition or competition would be expected to

take several hours for maximum development, (b) the kinetics of repression
by cycloheximide, NH4+ and glutamate were found to be nearly identical,
and (c) ligninolytic activity was found to be derepressed in response to
carbon limitation in the presence of nitrogen at concentrations which
would be suppressive in nitrogen starved cultures (discussed below).

The repression does not appear to be mediated by suppression of central
carbon metabolism, since both glutamate and NH4+ stimulated the

oxidation of glucose and succinate but repressed the production of

14 14

C02 from C-lignin (34).

33

Kirk et al (83) have clearly demonstrated the inability of lignin
to serve as a sole carbon source for growth of P, chrysosporium. There-
fore, it is somewhat surprising that ligninolytic activity is derepressed
in carbohydrate limited medium following the depletion of carbohydrate
(71). The extent of (14C)-synthetic lignin degradation observed in car-
bohydrate limited cultures was positively correlated to the amount of
carbohydrate added and therefore to the amount of mycelium formed in the
culture. Mycelial dry weight was found to decrease immediately after
the carbohydrate was exhausted and the appearance of ligninolytic activity
*was associated with this event. Theinycelial dry weight decreased
throughout the period in which lignin was degraded and no further de-
crease was observed when lignin degradation stopped (73, 100). ‘P.

chrysosporium has been shown to produce copious amounts of extracellular

 

polysaccharide and to have intracellular carbon reserves (L. Forney,

S. Pankratz and C. A. Reddy, unpublished). Therefore, it seems likely
that upon exhaustion of glucose, the necessary energy to support lignin
degradation is provided by extracellular or intracellular reserves.

This is consistent with the observation that lignin degradation does

not occur in the absence of a cosubstrate and that the extent of degrada-
tion observed is dependent on the amount of substrate available (71,

83).

The derepression of ligninolytic activity in carbohydrate limited
cultures following the depletion of carbohydrate, despite the presence
of high levels of nitrogen in the medium, is somewhat surprising in view
of the established ability of N to inhibit lignin degradation. In an
attempt to clarify this, Jeffries et al (71) examined the effect of

NH4+, glutamate and a-ketoglutarate addition on lignin degradation in

34
carbohydrate limited cultures. When glutamate was added to carbohydrate

limited cultures, lignin degradation decreased approximately 50% as com-
pared to control cultures. The inhibition was not due to the presence of
additional nitrogen in the medium since the addition of an equimolar a-
mount of NH4+ had no effect on lignin degradation. Nor was the effect
due to added carbon as an equimolar addition of a-ketoglutarate stimulated
lignin degradation by approximately 30%. The effect of adding glutamate
and a-ketoglutarate was the same as adding glutamate only. a-Ketoglutar-
ate and NH4+ stimulated lignin degradation to a degree equal to that ob-
served upon addition of a-ketoglutarate only. Therefore, in carbohydrate
limited cultures, glutamate pg§_§g inhibits lignin degradation, however,
the mechanistic basis for this has not been established. The repressive
species produced upon the addition of N to nitrogen starved ligninolytic

cultures of E, chrysosporium is apparently not produced by the fungus un-

 

der carbohydrate limited conditions, despite the presence of high levels

of N.
Lignin degradation was also found to be derepressed in cultures

which initially contained limiting levels of $04= (20 ufl) and excess car-
bohydrate and nitrogen after 7 d of incubation (71). The sulfur concen-
tration in the medium at that point was not determined; When the initial
504: concentration in this medium was 200141, ligninolytic activity

did not appear. Dual limitation of $04: and nitrogen did not result in
more rapid or more extensive degradation than when only nitrogen was
limiting. Unfortunately, the effect of these culture conditions on

mycelial dry weight was not examined. The basis of the observed effect

of sulfur on lignin degradation by P, chrysosporium is not known.

 

In summary, ligninolytic activity in cultures of P, chrysosporium

 

was derepressed following the cessation of growth due to N, C or S

starvation and developed as a part of idiophasic growth. The

35

complex regulation of the process is not well understood, however, it
would appear to be associated with the shift to secondary metabolism in

the cells.

Effects of oxygen partial pressure

Oxygen partial pressure has been shown to have a profound effect
on the rate and extent of lignin degradation by E, chrysosporium (73).
When cultures limited in nitrogen were incubated in 0.2 atm of 02,
lignin degradation appeared on day 4 followed by a linear rate of de-
gradation from day 8 through day 28, at which time roughly 40% of the
lignin had been degraded. When the cultures were incubated in 1 atm of
02, the onset of lignin degradation was unaffected, however, the rate of
degradation was significantly greater. There was a very rapid evolution

of 14

C02 from day 4 to day 12 during which 40% of the lignin was de-
graded. Lignin was not degraded during a 35 d incubation period in 0.05
atm of 02. The effects of 02 partial pressure on lignin degradation were
also reflected in the molecular weight profile of the residual lignins
from these cultures. Lignin from cultures grown in 0.8 atm and 1 atm

02 was extensively depolymerized whereas little depolymerization of
lignin was seen in cultures grown in 0.05 atm of 02. Thus, lignin de-
gradation by E, chrysospgrium in 1 atm of 02 is roughly 2.5 times greater
than in 0.2 atm of 02, and is not degraded at all by the fungus under

low p02 (0.05 atm).

The oxygen partial pressure does not appear to affect fungal growth,

or the level of glucose metabolic enzymes. Bar-lev and Kirk (5) grew

.3. chrysosporium in 0.2 atm of 02 for 2.5 days and then shifted to either

 

0.2 atm or 0.8 atm of 02 for 5 days and determined the rate at

36

14C-synthetic

which glucose and lignin were metabolized. The rate of
lignin oxidation to 14CO2 was 5-fold greater in cultures which were in-
cubatedir10.8 atm of 02, whereas the rate of glucose metabolism was the
same under both atmospheres. Based on these results, the authors
suggested that immediately preceding the appearance of the lignin de-
grading system the partial pressure of 02 determines the amount of lig-
ninolytic activity that develops in cultures. To determine the effect

of 02 partial pressure on glucose metabolism and the activity of the lig-
ninolytic system after its appearance,cultures were grown in 0.2 atm 02
for 2.5 days and then maintained under 0.8 atm of 02 for an additional 4
days at which time the cultures were actively ligninolytic. The rates

of glucose and lignin oxidation in these 6.6 day old cultures were then
determined under different 02 atmospheres. Oxidation of both substrates
was enhanced by increasing 02 partial pressure. The oxidation of both
glucose and lignin was maximal at 0.4 atm 02 but was not significantly
lower at 0.8 atm 02. These results indicate that both glucose and lig-
nin metabolism were stimulated by high partial pressure of 02; however,

a satisfactory explanation for these observations is lacking.

Effect of culture agitation

Culture agitation has also been shown to have a marked yet rather
anomalous effect on lignin degradation by E, chrysosporium (84).
Lignin degradation was rapid and extensive in stationary cultures incu-
bated under 1 atm 02, but not degraded in cultures which were
shaken from the time of inoculation. Similarly, only 4% of the added
lignin was degraded in agitated cultures incubated under 0.2 atm 02

for 34 d as compared to the 16% degradedin stationary cultures

37

during the same period. Kirk et al (84) suggested that the "pellet"

form of growth that results upon agitation entraps the lignin within the
pellet wherein the effective 02 concentration was too low to allow for
the efficient degradation of lignin. This explanation was supported by
the fact that pelleted cultures, when incubated without agitation, formed
mycelial mats and decomposed lignin at a rate and extent proportional to
the amount of lignin which had not previously associated with the pellets.
In other experiments, cultures with different partial pressures of 02
were incubated without agitation for 9 d to allow for the formation of
mycelial mats and then agitated under 0.2 atm 02. Lignin degradation
was unaffected by agitation in these cultures. However, in contrast

to those results, lignin degradation was dramatically suppressed in
cultures under 1.0 atm 02 upon agitation of the culture. The basis

for the differential effect of agitation on these cultures is not clear

at this time.

Metabolism of Lignin Model Compound by E, chrysosporium

 

B-Guaiacyl ether linked dimeric lignin model compounds.
The metabolism of dimeric lignin model compounds with B-ether

linkages by E, chrysgsporium has been extensively investigated (27,

 

28, 45, 96, 113). The e-ether linkage constitutes a major intermonomer
linkage in lignin and hence the study of the metabolism of these com-
pounds may be useful to elucidate the metabolism of these substructures

in the lignin polymer.

Regulation of metabolism ofge-guaiacyl ether linked model compounds.
The metabolism of some of these model compounds has been found

to be regulated in a manner similar to that of the lignin polymer.

38

Weinstein et al (113) examined the effect of culture parameters on the
metabolism of 14C-labelled guaiacylglycerol-B-guaiacylether (I) and
veratrylglycerol-e-guaiacyl ether (II) (Figure 5) by E, chrysosporium.
In nitrogen limited (1.2 mM N) cultures these compounds were found to be
metabolized only after the culture entered the stationary phase of growth
and in response to N starvation. In cultures grown with 12.0 mM_N, the
14C02 released from I and II was roughly 10-15% of that observed in ni-
trogen limited cultures. Furthermore, the evolution of 14CO2 from I and
II in nitrogen limited cultures was repressed upon the addition of NH4+
to the culture and was stimulated 2—3 fold in cultures incubated under
1.0 atm 02 as compared to those incubated in an atmosphere of air (0.2
atm 02). After correcting for differences in cell yield the metabolism
of I_and II in agitated cultures was 12.5% and 7.5%, respectively, of
that observed in stationary cultures. Thus, the effect of nitrogen con-
centration, 02 partial pressure and culture agitation on the metabolism
of these model compounds is analogous to their effect on ligninolytic
activity (5, 73, 84) and it may be possible to extrapolate conclusions

from these studies to lignin degradation by E, chrysosporium.

Metabolism of model compounds containinggphenolic ethers.

It is generally thought that lignin model compounds containing
phenolic ethers are useful models of "internal" substructures in lignin.
Whereas compounds with free phenolic hydroxyl groups more closely re-
flect peripheral substructures in lignin.

Goldsby et al (45) studied the metabolsim by E, chrysosporium
of guaiacylglycerol~B-guaiacyl ether (I), which has a free phenolic

hydroxyl group. 3-Hydroxy-2-(o-methoxyphenoxy)propionic acid (III) and

39

2-(o-methoxyphenoxy)1,3-propanediol (IV) were isolated as degradation
products, suggesting that cleavage of the alkyl-phenyl bond had occurred.
Cleavage of the alkylphenyl bond also occurred during the metabolism of
a-deoxyguaiacylglycol-B-guaiacyl ether (V) to 2-(o-methoxyphenoxy)
ethanol (VI). The latter compound was also observed as a product of

the metabolism of the a-hydroxy analog, guaiacylglycol-e-guaiacyl ether
(VII). Similar results were obtained using a-deoxyguaiacylglycerol-e
-guaiacyl ether (VIII). The nature of the Y-carbon of the side chain
does not appear to be critical since (IV), which lacks a Y-carbon appears
to be metabolized by a mechanism similar to (V). These results suggest
that cleavage of the alkyl-phenyl bond proceeds via an a-hydroxylated
intermediate.

Interestingly, the a-deoxy model compounds (V, VIII) were only
metabolized in nitrogen-limited stationary cultures whereas the a-‘
hydroxylated model compounds (VII, I), which were the products of the
first reaction in the metabolism of V and VIII, respectively, were
metabolized in nitrogen sufficient, agitated cultures. Similarly, VI,
IX and XI were metabolized in nitrogen sufficient, agitated cultures.
This strongly suggests that regulation of the metabolism of certain lig-
nin model compounds and lignin by nitrogen is at the step of hydroxyla-
tion of the arcarbon of the side chains.

4-Ethoxy-3-methoxyphenylglycerol-3-guaiacyl ether (XIII), which
contains a phenolic ether, was metabolized by E. chrysosporium only in
nitrogen limited cultures (1.2 mM N) and not in nitrogen sufficient
media (12mM_N) (27, 28). The e-ether linkage of XIII was cleaved to
produce 4-ethoxy-3-methoxyphenyl glycerol (XVI) and guaiacol (XVII).

The a-deoxy and Y-deoxy analogs (XIV and XV, respectively) were

4O

metabolized at rates comparable to XIII and resulted in the formation
of analogous products. The product of the metabolism of the a-deoxy
analog, 4-ethoxy-3-methoxyphenyl-2,3-dihydroxypropane (XVIII), was
metabolized through two pathways. In one pathway the a-carbon of
XVIII is hydroxylated to form 4-ethoxy-3-methoxyphenyl glycerol (XVI)
which is then cleaved between the a and B carbons to form 4-ethoxy-3-
methoxybenzyl alcohol (XIX). Alternatively. 8,1 cleavage of the side
chain of XVIII occurred to form 4-ethoxy-3-methoxyphenyl~2-hydroxy
ethane (XX). In analogous reactions, guaiacol and 4-ethoxy-3-methoxy-
phenyl—l,2-dihydropropane (XXI) were the products of the metabolism
of the y-deoxy analog (XV). The side chain of XXI was then cleaved between
the a and B carbons to form (XIX). The triol was metabolized by both
cleavage of the age bond and the 8,7 bond.

These results indicate that the catalyst which initiates the metabo-
lism of B-guaiacyl ether linked model compounds containing phenolic ethers
is quite nonspecific and requires neither the a nor 7 hydroxyl group of
the side chain. Previous studies have shown that the cleavage of the
B-ether bond in other model compounds is catalyzed by phenol oxidases
and by cell-free culture filtrates. The reactions were shown to pro-
ceed by oxidation (radicalization) of the free phenolic hydroxyl group
(75). The mechanism by which XIII, XIV and XV were metabolized was
clearly different since cleavage of the phenolic ether to generate a
free phenolic hydroxyl groups was not a prerequisite for B-ether cleavage.
The agents which catalyze the cleavage of the side chains of XVIII and
XXI were also shown to exhibit low specificity as both the 6,8 dihy-
droxy and the B,y dihydroxy homologs were metabolized.

These studies on the metabolism of B-guaiacyl ether linked dimeric

lignin model compounds suggest that model compounds and analygous

 

41

substructures in lignin which have free phenolic hydroxyl groups are met-
abolized via cleavage of the 6,8 bond of the side chain. In contrast
when the structure has a phenolic ether present, the B-ether linkage

of the side chain is cleaved.

Phenylcoumaran linked dimeric lignin model compounds.

The phenylcoumaran substructure accounts for 6% and 9-12% of the
intermonomer linkages in birch and spruce wood lignins, respectively
(1). The initial reactions in the metabolism of methyldihydroconiferyl
alcohol (XXII), a model compound with a phenylcoumaran linkage, by

P, chrysosporium were elucidated by Nakatsubo et al (96).. The first

 

step in the conversion of XXII was the formation of XXIII via hydro-
xylation of the cinnamyl alcohol moiety. Both erythro and threo forms
were produced suggesting that the oxidation of the double bond was not
stereoselective or that subsequent reactions cause recemization. To
rationalize these results Nakatsubo et al (96) suggested that the in-
sertion of oxygen at the a and B carbons is probably not enzymatically
controlled. The reaction probably proceeds by nonstereoselective expoxi-
dation of Ca - CB followed by nonenzymatic hydrolysis of the epoxide.
The side chain is then cleaved between the a and e carbons in a reaction
analogous to that observed for e ether compounds by Weinstein et al (113)
and Enoki et al (27, 28) and the a carbon is oxidized to a carboxylic
acid (XXIV).

The nonstereoselective oxidation of the cinnamyl alcohol moiety
is extremely interesting. It is consistent with the widely held belief

that the ligninolytic system of white-rot fungi must be nonspecific and

42

supports the suggestion (2, 55, 95) that oxygen radicals are involved

in lignin degradation.

43

Figure 5. Structures of dimeric lignin model compounds and
products of their metabolism (compounds I - XXIV).

 

44

CH OH
1 2 I a, . a, - 4w utmigimi-u-umyi m
H? - a, ll 01 - ‘05 I: - 4|! bratty! glycerol-Ionic”! other
HCR '11! I! - 4w I2 - on owiuylglml-O-guiacyl other
2 OCH3
: OCH
OR 3
l
71 i
HT - 0 Q m 5 ' m NW) manic am
CH OH OCH ' u T‘ . 3 "‘°"m¥l-I.Wioi
’- a
V II I 0“
H C - 0 Q m 'm'“""m'°fi-Iniacyl other
:CR ‘1 . a "'m""“"’*'0¢rl cum
1 OCH
© 3
OCH3

 

45

VI 5‘ ' '05" bio-DWI m1

R
' I ll I1 - m 24M) back at“
H2c '0 ©

OCH3

R :1 Il - oeuzul I2 - ~00, nutty! alcohol

IV” I! t 0H .2 o .a. ”1”}

©...

3
OCIIIIZCH3

‘ . .0120... a2 = .011 Cathay-Jdnthmnhunyllglycml-roguhcyl M

m l‘ - outta I, - ~01 i-(O'WJ'Wll-z-(rml-

W

IV I! 0 a, I2 0 4w “'ootbly-J'flthxmnyl1-2-(2'fitM-ymlol-

hydro typropone

46

R
'2 XVI R1 = -OH R2 = -CH20H 4-ethoxy-3-meth0xyphenyl glycol
HCOH XVIII R‘ = -H R2 =- -CH20H Q-othoxy—J-methoxyphenyl-2.3-dihydroxypropane
CIR XXI R1 I -OH R2 - - CH3 4-ethoxy-3—methoxyphenyl-l.Z-dihydroxypropane
l
OCH:5
OCH CH
2 3
R x” R} ' 4112011 4'0thy-3-Ilothoxybenzyl alcohol
1 n “1 a ““2050" “'"Mlyd'lflmupmi‘lyl-2-hydroxyethano
OCH3
OCH CH
2 3
HZCOH XXII methyldehydroconiferyl alcohol
I
CH
I
HC
H COH
21
HC
I OCH:5
OCH:5
OCH

47

H COH
HCG‘I
HOCH
HZCG-I

HC—O

COD...

OCH

COW
H cm I!"

HC
HC ——0

OCH
OCH

IO.

11.

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Ishihara, T. 1980. The role of laccase in lignin biodegradation,
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Ishikawa, H., W.J. Schubert and F.F. Nord. 1963. Investigations
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Jeffries, T.W., s. Choi and T.K. Kirk. 1981. Nutritional regula-
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Kawakami, H. 1976. Bacterial degradation of lignin. I. Degrada-
tion of MWL by Pseudomonas ovalis. Mokuzai Gakkaishi 22:
252-257.

 

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system of Phanerchaete chrysosporium: synthesized in the
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Kirk, T.K. and Kelman. 1965. Lignin degradation as related to
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Biochemistry of microbial degradation, Marcel Dekker, New
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Lignins: occurence formation, structure and reactions,
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Americang2§§(1):34-44.

CHAPTER I -

Biological Studies on Lignin Degradation: A Simple Procedure
for the Synthesis from Ferulic Acid of Coniferyl Alcohol,

a Precursor of Synthetic Lignin

A relatively simple procedure for the conversion of ferulic acid to
coniferyl alcohol, a precursor of synthetic lignin, is described. In
this procedure, unlabeled or (2'-]4C) ferulic acid was reacted with N,O-
bis-(trimethyl-silyl) acetamide in dioxane-pyridine (l0:l) to form the
trimethylsilyl ester of ferulic acid which was then reduced to coniferyl
alcohol with LiAlH4. Excess LiAlH4 was destroyed with NaOH, the organic
solvent layer was evaporated and the precipitated aluminum salts were
removed by filtration. The coniferyl alcohol in the filtrate was
extracted into ethyl ether, which was washed with water and dried with
Na2504. The yield of coniferyl alcohol was approximately 47%. The
purity and identity of the coniferyl alcohol was confirmed by gas
chromatography, high pressure liquid chromatography and mass spectrometry.
The unlabeled or (2'-14C) coniferyl alcohol synthesized was polymerized to

14C) synthetic lignin, respectively. The

produce unlabeled or (2'-
synthetic lignin was similar to synthetic lignins described in
the literature in elemental composition, gross structural characteristics

and in its susceptibility to degradation by Phanerochaete chrysosporium.

INTRODUCTION

In recent years, investigators have used synthetic lignin (a
dehydrogenated polymer of coniferyl alcohol), which contains inter-
monomer linkages similar to those in naturally occurring lignins (4,10),
as a model substrate for studies on the biological degradation of lignin
(2,6,8,l3,l4,l6). Although unlabeled or 14C-coniferyl alcohol, the
common precursors for synthetic lignin, are available commercially, they
are relatively expensive. Hence, there is a need for developing a
relatively simple and inexpensive procedure for the synthesis of

unlabeled or 14

C-coniferyl alcohol.

Existing methods for the synthesis of coniferyl alcohol are
relatively complex (l,3,5,7,l0,13). In contrast, the reduction of
ferulic acid (4-hydroxy-3-methoxycinnamic acid) to coniferyl alcohol (4-
hydroxy-3-methoxycinnamyl alcohol) has several advantages: (l) Only a
few steps are required for the conversion of ferulic acid to coniferyl

alcohol; (2) Ferulic acid is relatively inexpensive and is readily

available commercially; and (3) An established simple procedure (l5)

exists for synthesizing (2'-14C) ferulic acid which could be used as a

14C) synthetic lignin.

precursor for synthesizing(2'-
In this paper we describe a simple method for the synthesiscfi’coni-

feryl alcohol from ferulic acid which was then utilized in the

preparation of synthetic lignins for use in studies of lignin bio-

degradation.

59

MATERIALS AND METHODS
synthesis of (2'-]4C) ferulic Acid. (2'-‘4C) ferulic acid was
synthesized using the procedure of Pearl and Beyer (15). In this

procedure vanillin and (2'-14

C) malonic acid were added to pyridine
which contained a small amount of piperidine and stirred in the dark.
After 3 weeks, the reaction was acidified with HCl and the precipitated
ferulic acid was filtered from the solution, washed with distilled water
and dried under vacuum in an oven. The yield of ferulic acid was 78%
(based on the initial amount of vanillin added).

Synthesis of coniferyl alcohol. The trimethylsilyl derivative of
ferulic acid was synthesized by adding 2.50 g ferulic acid (Aldrich
Chemical Co., Milwaukee, WI), 50 ml dioxane-pyridine (l0:l) and 40 ml
N,0-bis-(trimethylsilyl) acetamide (BSA; Sigma Chemical Co., St. Louis,
M0) to a 500 ml round bottom flask which was closed with a drying tube
containing silica gel. The reaction mixture was stirred for 30 min at
room temperature, cooled in a dry ice-ethanol bath, and l.5 g LiAlH4
(Aldrich Chemical Co.) was gradually added to reduce the trimethylsilyl
ester to coniferyl alcohol. After evolution of H2 had subsided, the
reaction mixture was refrigerated for l2 h. The residual LiAlH4 was
destroyed by slowly adding 30 ml of l M NaOH to the reaction mixture
held in an ice bath. After evaporation of the organic layer under a
stream of air, the aluminum salts were removed by filtration on a
Whatman GF/C filter (Whatman Inc., Clifton, NJ), washed with 70 ml of

l M NaOH and the washings were added back to the alkaline reaction
mixture. The pH of the reaction mixture was adjusted to 7.0 with

l M HCl and extracted four times with 500 ml portions of ethyl ether.

60

61

'Hueethyl ether extracts were then washed with 125 ml distilled water,
pooled and dried with anhydrous Na2S04. The volume of the ethyl ether
extracts was reduced from 2 1 to 500 ml by evaporation under a stream
of air.

14C-coniferyl alcohol was synthesized exactly as described above

except that 2-14

C ferulic acid, instead of unlabeled ferulic acid,
served as the substrate.
Analysis of coniferyl alcohol. The coniferyl alcohol was quantified by
ultraviolet spectroscopy, gas chromatography (GLC) and high performance
liquid chromatography (HPLC). GLC and HPLC were also used to determine
the purity of the coniferyl alcohol.

For quantification by UV spectroscopy, lO-20 ul of the coniferyl
alcohol sample was added to 3.0 ml of absolute ethanol and the absorbance

at 292 nm was measured with a Varian 634 S spectrophotometer (Varian

Instruments, Palo Alto, CA). The yield of coniferyl alcohol was

2 l I

calculated using an extinction coefficient of 5.85 x l0 l mole- cm-
(10).

GLC analyses were performed on a Varian 2440 gas.chromatograph
(Varian Instruments, Palo Alto, CA) which was interfaced with a Varian
CDS-lll computing integrator. A 2 m x 0.32 mm 1.0. stainless steel
column packed with 5% SE-30 on l00/120 mesh G-AW-DMCS was used. The
chromatograph oven temperature was held at 2l5°C or was programmed to
hold at l80 C for 2 min and then to increase to 280°C at 4°C min-1.
Samples of coniferyl alcohol solution in ethyl ether (IO-80 pl) were
added to a small vial, the solvent was evaporated and 100 pl of dioxane-

pyridine (l0:l) and 50 ul of BSA were added. The vial was then sealed

62

and allowed to incubate at room temperature for 30 min prior to
analysis. Coniferyl alcohol standards were prepared in an identical
manner. 1

HPLC analyses were performed using a Varian 5000 liquid chromato-
graph (Varian lAssociates, Palo Alto, CA) fitted with a Rheodyne model
7125 injector, 254 nm ultraviolet detector and interfaced with a
Spectra-Physics model 4100 computing integrator. A 30 cm x 4 mm MCH-lO
Micropac C18 reverse phase column (Varian Associates) was used with
50:50 methanol-water (flow rate 2.0 ml min -1) as the eluting solvent.
'For preparing samples for HPLC analysis an appropriate amount of coni-
feryl alcohol sample (dissolved in ethyl ether) was added to a vial, the
solvent was evaporated and 500 pl of methanol was added. Coniferyl al-
cohol standards were prepared in an identical manner.

Samples for gas chromatography-mass spectrometry analysis were
prepared as described above for GLC analysis. The analysis was
performed using a Hewlett-Packard 5840A gas chromatograph with a
Hewlett-Packard 5985 GC-MS (Hewlett-Packard Co., Palo Alto, CA). The
column packing was the same as that described above. A 6 ft x 0.25 in
glass column was used, and He at 29 ml min-1 was the carrier gas. The
chromatograph oven was temperature programmed to hold at 185°C for 2 min

1

and to increase at 6°C min- to 230°C. The injector temperature was

240°C.

Synthesis of dehydrogenative polymers of coniferyl alcohol. The volume
of the coniferyl alcohol solution in ethyl ether was reduced from 500 ml

' to 10-20 ml under a stream of air. A known amount of previously degassed
(boiled and bubbled with N2 while cooling) 0.01 M sodium phosphate

buffer (pH 6.5) was added. The remaining ethyl ether was evaporated

63

under a gentle stream of air. The procedure of Kirk et al. (10) was
used for the polymerization of the coniferyl alcohol except that the
quantities of the reagents used were proportionally altered depending on
the amount of coniferyl alcohol used. The synthetic lignins were stored
at -lO°C as suspensions in distilled water and were lyophilized as
needed before use.

Side chain (2'-I4C) labeled lignin was prepared from 14c-conifeny1
alcohol in a manner identical to that described above. The specific
radioactivity of the 14C-synthetic lignin was determined by adding a
known amount (approximately 50 pg) of this lignin to dioxane-based
liquid scintillation cocktail (DB-LSC, 6) to which a small amount of
water had been added (0.3 ml water to 1.5 ml cocktail). 50 ul of this
solution was then transferred to aqueous DB-LSC (0.5 ml water per 10 ml
DB-LSC) and the radioactivity was determined to within i 1% by counting
three replicate vials using a Searle Model 300 liquid scintillation
counter (Searle, Arlington Heights, IL).

Characterization of synthetic lignin. Elemental analysis (C,H,N) of the
synthetic lignin was determined using a Carlo-Erba model 1104 elemental
analyzer or by Galbraith Laboratories (Knoxville, TN). The oxygen
content was determined by difference [100-(%C + %H + %N)]. The values
for C, H, and 0 were then normalized to 100%.

Infra-red (IR) spectra of the synthetic lignin and milled wood
lignin (spruce-MWL, provided by Dr. T. K. Kirk) in KBr pellets
(approximately 2 mg of dry lignin per 200 mg KBr) were determined using
a Perkin-Elmer model 700 IR spectrophotometer (Perkin-Elmer, Norwalk,

CT).

54

Degradation of (2'-]4C) synthetic lignin pnghanerochaete chrysosporium.
A culture of Phanerochaete chrysosporium Burds. (ME-446) was provided
by Dr. T. K. Kirk, Forest Products Laboratory, U.S. Department of
Agriculture, Madison, WI and was maintained by periodic transfer on
malt GXtFACt agar. The inoculum consisted of a conidia suspension which
was prepared as described by Kirk et all (10) except 7-10 day old cul-
tures of E. chrysgsporium cultures were used and the final optical den-
sity at 600 nm of the condial suspension was 0.5 (1.8 cm light path).
the conidial suspensions were stored at -10°C until used and thawed

at room temperature prior to adding 0.5 ml per 10 ml of culture medium.

14

Degradation of C-synthetic lignin by P, chrysosporium was con-

ducted in a medium which contained the following per liter of distilled
water: KH2P04, 0.2 g; MgSO4 2
succinate, 1.46 g; D-glucose, 10.0 g; NH4NO3, 0.048 g; L-asparagine,

-7H 0. 0.05 g; tac12. 0.01 g; 2,2-dimethyl--

0.09 g; mineral solution (11), 1.0 m1; and vitamin solution (11),

0.5 ml. The final concentration of nitrogen was 2.4 mM_and the final
pH was adjusted to 4.5 with NaOH. The medium was dispensed (10 ml per
flask) in 125 ml Erlenmeyer flasks and sterilized by autoclaving at

121°C for 15 min.
The (2'-14C) synthetic lignin (specific activity of 2.8 x 105
dpm mg‘I) and identically prepared unlabeled synthetic lignin were

mixed to obtain a lignin preparation of desired specific activity.

14 5

This synthetic lignin and (U-ring- C) synthetic lignin (0.78 x 10 dpm

I, supplied to us by Dr. T. K. Kirk) were added separately to N,N-

mg'
dimethylformamide (DMF) to give 5% solutions. These DMF solutions were

slowly added to sterile distilled water (7 ‘ul DMF/0.5 ml water) and

65

one-half ml (0.35 mg) of these aqueous suspensions were added to each
flask. Using this procedure, no contamination of the cultures from the
addition of lignin was observed.

Culture flasks were closed with rubber stoppers which permitted
periodic flushing of the culture headspace as previously described (11)
except that the effluent tubing was connected to a 1 cc syringe barrel
with a 1 in 25 gauge needle. Every third day of the experiment the
flasks were flushed with COZ-free air at a rate of 60 ml min—1 for
45 min. The respired CO2 was trapped in 10 ml of a liquid scintil-
lation coctail which contained ACS (Amersham-Searle, Arlington Heights,
IL)-methanol-ethanol amine (Aldrich Chemical Co., Milwaukee, WI)

14

(50:40:10). The efficiency of C02 trapping using this procedure was

97% (L. J. Forney and C. A. Reddy, 1980, unpublished data). The

degradation data are expressed as % of added 140 recovered as 14CO2

after correcting for the efficiency of 14

CO2 trapping, and counting
efficiency (typically 85-89%) as determined by sample channels ratio
method. Values from four replicate flasks were used to calculate the

mean for each data point.

RESULTS

Synthesis of coniferyl alcohol. Ferulic acid reacts with N,0-bis-
(trimethylsisyl) acetamide (BSA) to form the trimethylsilyl (TMS) ester
of ferulic acid (Fig. l). The phenolic hydroxyl group of ferulic acid
also reacts with BSA to form a TMS ether. The ester was then reduced
by adding LiAlH4 with the concomittant formation of an alcohol group.
After allowing the reduction reaction to proceed for 12 h, l M_NaOH was
added to destroy the excess reducing agent and to regenerate the
phenolic hydroxyl group by hydrolyzing the phenolic ether bond. The
coniferyl alcohol was recovered as described in Materials and Methods.

The purity of the coniferyl alcohol, obtained was determined by
GLC and HPLC. Gas chromatography revealed that the coniferyl alcohol
sample was pure as evidenced by a single peak which had a retention
time identical to that of an authentic coniferyl alcohol. Temperature
programming of the chromatograph oven from 180°C (hold for 2 min) to
280°C at 4°C min'1 failed to Show the presence of any other compound
(data not shown). Using HPLC elution of the sample with 50% aqueous
methanol showed the presence of one major peak which had the same
elution volume as the coniferyl alcohol standard (Fig. 2). The
identity of the minor compound which eluted at 3.6 min in chromatograms
of both sample and standards is unknown although it is not ferulic acid
(which elutes at 1.4 min). Elution of the sample with 100% methanol
failed to reveal additional compounds in the ether extract (data not
shown). These results indicate the absence of any extraneous compounds

capable of absorbing at 254 nm (i.e., aromatic compounds).

66

67

The mass spectra of the coniferyl alcohol sample and coniferyl
alcohol standard showed nearly identical fragmentation patterns with
parent ions of mass 324 which corresponds to the mass of the TMS
derivative of coniferyl alcohol (Fig. 3). Presumably, one TMS group is
bonded to the alcoholic oxygen and a second to the phenolic oxygen of
coniferyl alcohol. The base peak, m/e = 73 is due to TMS ions. These
results indicate that the synthesis product is coniferyl alcohol.

The yield of coniferyl alcohol was routinely determined using UV
Spectroscopy and gas chromatography. The values obtained using these
two independent methods were in close agreement and averaged 47.1 t
3.0% (five trials).

Synthesis and characterization of synthetic lignin. The elemental
composition of the synthetic lignin was 62.2% C, 6.2% H and 31.4% 0
which agreed well with published values for milled spruce wood lignin
(9) and for synthetic lignin of Kirk et al. (10, Table l).

The infra-red spectra of the synthetic lignin and milled spruce
wood lignin were very similar (Fig. 4). However, as previously
reported for synthetic lignin the Shoulder at 1725 cm"1 in the spectrum .
of milled spruce wood lignin was absent in the spectrum of synthetic
lignin described here.

14C-synthetic lignin by Phanerochaete chrysosporium.

Degradation of
The usefulness of the (2'-14C) synthetic lignin obtained in this study as
a model substrate for studies on biological degradation of lignin was
evaluated by determining the rate at which it was degraded by the

typical white-rot fungus Phanerochaete chrysosporium. The rate of
14

 

degradation of (u c-ring) synthetic lignin, prepared by the

procedure of Kirk et a1. (10), was also determined to serve as a

68

comparison. The (2'-I4C) synthetic lignin was degraded at a rate

comparable to that of the (U-14

C-ring) synthetic lignin (Fig. 5). With
both the lignin preparations, ligninolytic activity appeared after 6 d
of incubation and increased over the next several days. The rate of

lignin degradation was very similar for the two lignin preparations.

DISCUSSION

In studies on the biochemistry of lignin degradation, substantial
quantities of coniferyl alcohol are needed to prepare synthetic lignin.
To simplify the chemical synthesis of coniferyl alcohol, we examined
the feasibility of reducing the terminal carboxyl group of ferulic acid
to form coniferyl alcohol. Previous attempts to directly reduce the
terminal carboxyl group of cinnamic acid derivatives has resulted in a
low yield of product (3) due to the susceptibility of the carbon-carbon
double bond in the propyl side chain to reduction when an acidic -OH is
adjacent to it. However, if the acid group is first converted to an
acid chloride or an ester, moderate yields of a,B-unsaturated aromatic
alcohols are possible (1,3,17). Since formation of trimethylsilyl
esters is simple and familiar to many scientists, it appeared that
reduction of the trimethylsilyl ester of ferulic acid to form coniferyl
alcohol might be the basis of a relatively Simple procedure. The
results show this to be the case.

The yield of coniferyl alcohol (47%) is typical for the reduction
of acid chlorides and esters of cinnamic acid derivatives (1,3,12,17).
It may be possible to increase the yield of coniferyl alcohol through
the use of other reducing agents and by employing more stringent
measures to exclude water from the silylation reactions and silylated
intermediates.

The data showed that the synthetic lignin obtained by polymeri-
zation of the coniferyl alcohol produced in this study, was similar in
elemental composition and gross structural characteristics to spruce
milled wood lignin and other synthetic lignins described in the

literature (9,10). Previous investigators have shown (U-I4C-ring) and

69

7O

(2'-]4C-sidechain) labeled lignins to be degraded at comparable rates
by E, chrysosporium. In agreement with these findings (11) we found
that P, chrysosporium degraded the (2'-]4C) synthetic lignin (prepared
using methods described here) at a rate comparable to that observed for

(u-I4c-ring labeled) synthetic lignin of Kirk (10).

LITERATURE CITED
Bazant, V., M. Capka, M. Cerny, V. Chvalovsky, K. Kochloefl, M.
Krauss and M. Malik. 1968.- Properties of sodium-bis-(Z-methoxy-
ethoxy)aluminum hydride. 1. Reduction of some organic functional
groups. Tetrahed. Lett. 2233303-3306.

Crawford, R. L., L. E. Robinson, and A. Cheh. 1979. ‘4

C-labeled
lignins as substrates for the study of lignin biodegradation and
transformation, p. 61-76. Ig_T. K. Kirk, T. Higuchi, and H.-M.
Chang (ed.). Lignin biodegradation: microbiology, chemistry and
applications. CRC Press, West Palm Beach, FL.

Cerny, M., J. Malek, M. Capra, and V. Chvalovsky. 1969.
Properties of sodium-bis-(2-methoxyethoxy)aluminum hydride. II.
Reduction of carboxylic acid and their derivatives. Coll. Czech.
Chem. Commun. 3451025-1032.

Freudenberg, K., and A. C. Neish. 1968. Constitution and bio-
synthesis of lignin. Springer-Verlag, New York, p. 47-122.
Freudenberg, K., and F. Niedercorn. 1956. Anwendung radioaktivar
Isotope bei der Erforschung des Lignins, VI: Herkunft der Iso-
hemipinsavre. Chem. Ber. 8952168-2173.

Hackett, v. F., w. J. Connors, T. K. Kirk, and J. G. Zeikus.
1977. Microbial degradation of synthetic lignins in nature:
lignin biodegradation in a variety of natural materials. Appl.
Environ. Microbiol. §§;43-51.

Haider, K. 1966. Synthese von 14C-ringmarkiersten phenolischen
lignin-spaltstucken und ligninalkoholen aus BaI4CO3. J. Labeled

Cmpds. II:l74-183.

71

10.

ll.

12.

l3.

14.

15.

72

Keyser, P., T. K. Kirk, and J. G. Zeikus. 1978. Ligninolytic
enzyme system of Phanerochaete chgysosporium: synthesized in
response to nitrogen starvation. J. Bacteriol. 135:790-797.
Kirk, T. K., and H.-M. Chang. 1974. Decomposition of lignin by
white-rot fungi. 1. Isolation of heavily degraded lignins from
decayed spruce. Holzforsch. 285217-222.

Kirk, T. K., W. J. Connors, R. D. Bleam, W. F. Hackett, and J. G.
Zeikus. 1975. Preparation and microbial degradation of synthetic
(MO-lignins. Proc. Nat. Acad. Sci. U.S.A. 32515-2519.
Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, and J. G.
Zeikus. 1978. Influence of culture parameters on lignin
metabolism by Phanerochaete chrysosporium. Arch. Microbiol.
1113277-285.

Lindeberg, 0. 1980. Synthesis of coniferyl alcohol and dihydro-
coniferyl derivatives using radical bromination with N-bromo-
succinimide as the key step. Acta Chem. Scand. B34:15-20.

Martin, J. P., and K. Haider. 1979. Biodegradation of 14

C-
labeled model and cornstalk lignins, phenols, model phenolase
humic polymers and fungal melanine as influenced by a readily
available carbon source in soil. Appl. Environ. Microbiol.
385283-289.

Neuhauser, E. F., R. Hartenstein, and W. J. Connors. 1978. Soil
invertebrates and the degradation of vanillin and cinnamic acid
and lignins. Soil Biol. Biochem. 195431-435.

Pearl, 1., and D. L. Beyer. 1951. Reactions of vanillin and its

derived compounds. XI. Cinnamic acids derived from vanillin and

its related compounds. J. Org. Chem. 16:216-220.

16.

17.

73

Trojanowski, J., K. Haider, and V. Sundman. 1977. Decomposition
of 14c-1abe1ed lignin and phenol by a Nocardia sp_. Arch. Micro-
biol. 1145149-153.

Yoon, N. M., C. S. Pak, H. C. Brown, S. Krishnamurthy, and T. P.
Stocky. 1973. Selective reductions. XIX. The rapid reduction of
carboxylic acids with borane-tetrahydrofuran. A remarkably
convenient procedure for the selective conversion of carboxylic

acids to the corresponding alcohols in the presence of other

functional groups. J. Org. Chem. 3852786-2792.

74

Table 1. Comparison of the elemental composition of the

synthetic lignin with that of other lignins

 

 

Type of lignin %C %H %0

Synthetic 1ignina 62.2 6.3 31.4

Synthetic 1igninb 53.2 5.8 31.0

Spruce milled wood 62.9 6.1 31.1
ligninc

 

aSynthetic lignin prepared by the procedures described in
this study.

bKirk et a1. (10).

CKirk and Chang (9).

75

Figure 1. Reaction sequence for the synthesis of coniferyl alcohol

from ferulic acid.

2922.4
9

«:00 Q

0...
z
:0

_
gauze

.i

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a

05.5 Q

o:
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.
o I
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.282 33.50
:0

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AT

$22.2. 13:33.0.20

 

77

Figure 2. Analysis by high performance liquid chromatography of coni-
feryl alcohol (a) standard and (b) that synthesized by the
methods described here. The detector was set at 0.04 AUFS
which allowed for the detection of trace levels of impuri-
ties although the signal.due to the presence of coniferyl

alcohol is off scale in both chromatograms.

78

0.04 l-

0.03 l-

0.02 F

0.01 "

1.1.1544,

LB.

1
2345

D.L.

2345

1

1

 

HINU'I’EB

79

Figure 3. Mass spectra of (a) coniferyl alcohol synthesized by the
methods described here and (b) that of an authentic

coniferyl alcohol standard.

 

 

 

 

 

 

 

 

 

 

 

3
E
I
g
v
§
8
'
a 3 a
fi'
° 3

SONVONHIV 3M1“!!! 96

150

100

(In/o) omu

81

Figure 4. Infra-red spectra of (a) synthetic lignin prepared by the
methods described here (——) and (b) milled spruce wood

lignin ( ---).

82

 

 

new

to; 3. H”

 

 

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8
8
d d I d d d d 8P
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BONVLLIISNVUL 96

83

14C) synthetic lignin (0), prepared

Figure 5. Degradation of (2'-
in this study, and (U-ring-14C) synthetic lignin of

Kirk (I) by Phanerochaete chrysosporium.

 

 

84

  

I4

 

   

 

2 0 O 6

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Ow0<¢0mn 2.20... U_._.mI.—.2>m 103’

 

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10

DAYS

CHAPTER II

The Involvement of Hydroxyl Radicals
Derived from Hydrogen Peroxide
in Lignin Degradation by the

White-Rot Fungus Phanerochaete Chrysosporium

 

SUMMARY

The possibility was investigated that hydroxyl radical I-OH)
derived from hydrogen peroxide (H202) is involved in lignin degra-
dation (I4C-lignin-—olAC02) by Phanerochaete chrysosporium. When 3.
chrysosporium was grown in low nitrogen medium (2.4 mM N), an in-
crease in H202 production in cell extracts was observed to coincide
with the appearance of ligninolytic activity and both activities
appeared after the culture entered stationary phase. The production

of -OH in ligninolytic cultures of P: chrysosporium was demonstrated

 

by a-keto-v-methiolbutyric acid dependent formation of ethylene.
Hydrogen peroxide-dependent ~0H formation was also shown in cell
extracts of ligninolytic cultures. The radical species was demon-
strated to be -0H by the -0H-dependent hydroxylation of p-hydroxy-
benzoic acid to form protocatechuic acid and by using 5,5-demethyl-l
-pyrroline-N-oxide (DMPO) and EPR spectometry to detect the produc-
tion of the nitroxide radical of DMPO. These reactions were inhi-
bited by -OH scavenging agents and were stimulated when azide was
added to inhibit endogenous catalase. Lignin degradation by P,
chrysosporium was markedly supressed in the presence of the -0H sca-
venging agents mannitol, benzoate and the nonspecific radical sca-
venging agent butylated-hydroxytoluene. The above results indicate
that ~0H derived from H202 is involved in lignin biodegradation by

P: chrysosporium.

 

85

86
Lignin is an integral part of all vascular plants and is among

the most abundant and widely distributed organic polymers in nature
(1, 47). Due to the importance of lignin in the biospheric carbon
cycle and the interest in the use of lignocellulosic materials for
the production of various fuels and chemicals (6), research on the
biodegradation of lignin has greatly accelerated in recent years. A
large number of basidiomycetous fungi, collectively known as white-
rot fungi, have been shown to degrade lignin extensively (3, 36).
Considerable progress has been made toward understanding the physio-
logical and nutritional parameters which affect lignin degradation
by white-rot fungi (S, 17, 31, 33, 35); however, little is known
about the basic biochemical mechanisms involved in the process.

In view of the molecular size and structure of lignin it is
unlikely that lignin is directly taken up by microbial cells. Hence,
it is reasonable to postulate that extracellular agents are involved
in the initial breakdown of the lignin polymer. Yet, extracellular
ligninolytic enzymes capable of extensively transforming lignin
have not been isolated from fungal cultures of wood decomposing
fungi (2, 3, 28, 36). Analyses of lignin residues following fungal
attack and solubilized products of lignin degradation have shown
that the extracellular degradation of lignin is oxidative in nature
(30, 34). These results further showed that the extracellular
agents attacking lignin agents are nonspecific and nonstereosolec-
tive as evidenced by the following observations: a) lignin is
extensively degraded despite the variety of intermonomer linkages

(l) and racemic asymmetric carbons in the polymer (480; and b) a

87
variety of structurally different lignins and lignin model compounds

are metabolized efficiently (15, 42). In consideration of the above
features, it appears unlikely that the reactions involved in the
initial degradation of the lignin polymer are mediated by specific
enzymes. Hence, it has been postulated that the actual extracellu-
lar agents attacking the lignin polymer might be activated oxygen
species (2, 28, 46) such as superoxide (02‘) or singlet oxygen
(102), however, conclusive evidence is lacking. Koenigs (37, 38)
observed hydrogen peroxide (H202) production by cultures of a number
of wood-decomposing fungi and suggested the possible involvement of
H202 in lignin degradation by white-rot fungi. In an effort to elu-
cidate the role of extracellularly produced activated oxygen species
in lignin degradation, we tested the hypothesis that hydroxyl radi-
cal (°OH) derived from H202 is involved in lignin degradation by
white-rot fungi. The above hypothesis can be rationalized by the
fact that the -OH is highly reactive and is known to react rapidly
with a wide variety of organic compounds in oxidative reactions (29,
52). This would be in keeping with the well established oxidative
degradation of lignin (30, 34). Furthermore, the -0H radical is
nonspecific, nonstereoselective (29, 52), and is known to partici-
pate in a number of biological reactions (8, ll, 44, 51). I

The -0H can be produced by one electron reduction of H202 (52)
and in biological systems is thought to be generated in two types of
reactions (8, 16, 25, 52). In the Fenton reaction (reaction 1),
H202 is reduced by Fe (II), or other reduced transition metals, to

produce -0H (52). In the iron-catalyzed Haber-Weiss reaction (25;

88
reaction 2), superoxide (05?) reduces Fe(III) to Fe(II) which in

turn reduces H202 to form -OH in a manner similar to that in reac-
tion 1. This reaCtion is greatly stimulated by chelators such as

EDTA (25).

l. Fenton Reaction

 

 

H202 + Fe(II) : -OH + 0H‘+ Fe(III)

2. Iron-catalyzed Haber-Weiss reaction

 

a. Fe(III)-chelate + 027 ——'-> Fe(II)-chelate + 02

b. Fe(II)—chelate + H202 ——> -OH + 0H— + Fe(III)-chelate

 

H202 + 02-,- : -OH + OH_ + 02

 

Phanerochaete chrysosporium causes a typical white-rot decay of

 

wood and has been used in numerous studies on the biochemistry of
lignin degradation (5, 17, 31, 33, 35). We used this organism in
this study to answer the following questions: (1) Is there a corre-
lation between the appearance of H202 production and ligninolytic
activity cells of f, chrysosporium? (2) Is ~0H produced by lignino-

 

lytic cultures of P: chrysosporium? (3) Does the addition of -OH

 

scavenging agents to ligninolytic cultures affect the rate of lignin
degradation?
Our results indicate that -0H, derived from H202, is produced in

cultures of P, chrysosporium and that -OH is involved in lignin

 

degradation by the fungus.

MATERIALS AND METHODS

nganism and culture conditions. Phanerochaete chrysosporium
Burds. ME446 (ATCC 34541) was provided by Dr. T. K. Kirk and was
maintained through periodic transfer on malt extract agar as pre-
viously described (35). Unless indicated otherwise, the fungus was
grown in shallow liquid cultures (25 m1 por 250 m1 Erlenmeyer flask)
in low N medium (35) which contained 0.6 up) NH4NO3 and 0.6 “[4.
asparagine 2.4 m!_N final concn.) The flasks were foam stoppered,
autoclaved for 15 min at 121°C and inoculated with a conidial sus-
pension in water (5% inoculum) which had been prepared from 7-10 d
old malt extract agar cultures as previously described (21). The
cultures were incubated in air (21% 02) at 39°C, without agitation
for various periods of time as indicated in the text.

Ligninolytic Activity. Lignin degradation was determined by
measuring the 14C02 produced during the metabolism of [2'-I4C]-
synthetic lignin. The [2'-I4C]-synthetic lignin was prepared as pre-
viously described (21) and 0.35 mg (39,300 dpm) of this lignin,
suspended in 0.5 ml of sterile water, was added to 10 m1 of sterile
low N medium. The medium was inoculated with 0.5 m1 of the conidial
suspension and the flasks were steppered prior to incubation with
manifolds which permitted periodic flushing of the culture head-
space. At 3 d intervals, the headspace of the cultures was flushed
with COZ-free air and the respired 14002 was trapped in a scintil-
lation cocktail containing 2-aminoethanol as previously described

(21).

89

90
H292 production. The specific activity for H202 production in

cell extracts of P, chrysosporium was determined using a modifica-

 

tion of the catalase-aminotriazole assay of Cohen and Somerson (10).

Cells of f, chrysospgrium were harvested after various periods of

 

incubation by centrifugation at 12,000 X g for 10 min, washed twice
with sodium phosphate buffer (50mg, pH 5.5), resuspended in 10 m1 of
the same buffer, and stored at -10°C until assayed. Freezing the
cells had little effect on H202 production by the cell extracts.

The cells were thawed as needed, homogenized with a glass tissue
homogenizer and ruptured by a French pressure cell at 20,000 psi.
Unbroken cells and cell debris were removed by centrifugation at
12,000 X g for 10 min and the supernatant cell extract was used for
the H202 assays. The reaction mixture contained: 125 ug catalase
(bovine heart, 5500 u ng-l; Sigma Chemical Co., St. Louis, MO), 11
umol glucose, 50 umol 3-amino-l,2, 4-triazole (Sigma Chemical Co.)
and 0.9 ml sodium phosphate buffer. The reactions were initiated by
adding 0.1 ml of cell extract and were incubated at 39°C for 5 to
120 min. At various times, 200 ul samples were removed and placed
in 1.0 m1 of 0.45 M_ethanol to inhibit the further formation of non-
catalatic Hzoz-catalase-aminotriazole complexes. After 10 min at
room temperature, 0.5 ml of this solution was added to 4.5 m1 of

6 mM_H202 in sodium phosphate buffer and incubated for 180-295 s at
0°C (on ice) followed by termination of the reaction by the addition
of 1.0 ml 6 N H2304. The residual H202 was determined by titration
with 7.5 “5". KMnO4. The assay was standardized by substituting glu-
cose oxidase (B-D-glucose: 02 l-oxidoreductase, E.C.l.1.3.4) from

Aspergillusniger (Sigma Chemical Co.) for the cell extract, in the

91

reaction mixture. Glucose oxidase solution was desalted prior to
use by passing through a Sephadex G-25 column. Boiled cell extracts
served as negative controls.

Enumeration of conidia and fungal cell mass determinations.
The number of conidia (aleuriospores) formed in cultures of 3:.EEEXT
sosporium was determined in triplicate cultures on ll consecutive
days beginning on d 3. The cultures were diluted with an equal
volume of water and homogenized in a Waring blender for 30 sec. A
portion of the homogenized culture was placed in a haemocytometer
and the conidia were counted using phase contrast microscopy.

The growth of E, chrysosporium in low N medium was determined

 

by gravimetric determination of fungal cell mass. On 14 consecutive
days, starting on day l, three replicate l0 ml cultures in low M
medium were filtered through tared 0.22;:m micropore filters (Milli-
pore Corp., Bedford, MA). The cells on the filter were washed with
10 ml of l0 mfl_2,2-dimethylsuccinate buffer (pH 4.5) and were dried
to a constant weight under vacuum at 60°C. The cell mass was calcu-
lated by difference.

Determination of -0H formation in cultures. The -OH is known
to react with <rketo-1bmethiolbutyric acid (KTBA) to produce ethy-
lene gas (l2). To detect -0H formation in cultures of E, chrysos-
jggium_the organism was grown in 5 ml of low N medium contained in
50 ml foam stoppered serum vials. After l4 d of incubation the foam
stoppers were replaced with rubber septa and the vials were sealed
with aluminum caps. To different flasks, 0.5 ml of water or the -OH
scavenging agents benzoate (5 mg_final concn.) or mamnitol (50 mfl_

final concn.) were added and the reactions were initiated by

92
injecting 0.5 ml KTBA (3.3 mM_final concn.). Identical but auto-

claved fungal cultures also served as negative controls. Reactions
were terminated at various times after reincubation by injecting 1.0
ml 6 N_H2504 per vial. The amount of ethylene gas present in 0.2 cc
samples of the headspace of each vial was determined using a Varian
2440 gas chromatograph (Varian Associates, Palo Alto, CA) equipped
with a 2 m X 3.2 mm 0.0. stainless steel column packed with Porapak
N (Waters Associates, Milford, MA) and a flame ionization detector.
The carrier gas was N2 at 30 ml min". The detector gas flow rates
were 300 ml air min'1 and 30 ml H2 min'z. The chromatograph oven
was maintained at 200°C, with the injector and detector both set at
l40°C.

CX-distilled water. Distilled water, used to prepare reagents
for hydroxylation (Table 3 and 4) and EPR experiments (Fig. 3) was
freed of trace minerals and other contaminants by passage through a
Milli-Q water purification system (Millipore Corp., Bedford, MA) and
then through a column packed with Chelex-lOO resin (Sigma Chemical
Co., St. Louis, MO). This purified distilled water is hereafter
referred to as CX-distilled water. Solutions of Fe(II) were pre-
pared in CXedistilled water and sparged with either N2 or Ar to
minimize the dissolved 02 concentration. All organic reagents solu-
tions were adjusted to pH 4.5 prior to use.

Preparation of cell extracts. Cell extracts (used in experi-

 

ments described in Table 2 and Fig. 3) were prepared from l4 d old
cultures of E, chrysosporium. The cells were harvested by centrifu—
gation (l2,000 x g for 10 min), washed once with 20 ml of 10 mM_
2,2-dimethylfiuccinate» (pH 4.5), once with 20 ml of CX-distilled

water, and finally resuspended in 20 ml of the latter. Glass beads

93

(0.5 mm; Scientific Products, Romulus, MI) were then added to the
cell suspension (ten times the weight of the cell pellet) and the
cells were ruptured by blending in a precooled Haring blender for
3-30 sec periods separated by 30 sec cooling periods. The unbroken
cells and glass beads were removed by centrifugation for l0 min at
48,200 xg. The cell extracts were held on ice and used within 3 h.
Protein determinations were made using the procedure of Lowry et al.
(4l).

Assay of ~0H4production by measuring hydroxyltion of p-

 

hydroxybenzoicacid. The -OH is known to react with various aromatic
compounds resulting in the hydroxylation of the aromatic ring (52).
Thus, the production of -0H was determined by measuring hydroxyl-
ation of p-hydroxybenzoic acid (pHB) to form 3,4-dihydroxybenozic
acid (protocatechuic acid; PCA). The basic reaction mixture con-
tained l0 umol p-hydroxybenzoic acid (Aldrich Chemical Co., Mil-
waukee, N1), 10 umol glucose, 0.1 umol FeClz or FeCl3, l umol EDTA,
200 ul glucose oxidase (0.06 u ml") or 200 ul cell extract and
CX-distilled water to 1.0 ml. Where indicated mannitol (20 or 200
umol), benzoate (2 or 20 umol), a-keto-v-methiolbutyric acid (2 or
l0 umol), or 0.l umol NaN3 were also added to the reactions. The
reactions were preincubated at 39°C for at least 10 min, initiated
by adding cell extract or glucose oxidase, incubated for 120 min and

were terminated by adding 100 ul 6 N H2504, pcA was identified as

the reaction product by comparison with authentic PCA (Aldrich
Chemical Co., Milwaukee, NI) using high pressure liquid chromato-
graphy (HPLC) as described below and gas chromatography using pre-
viously described methods (2l).

94

The amount of PCA in the reactions was determined by HPLC
using a Hibar II RP-18 (250 mm X 4.6 mm) column (Anspec, Inc., Ann
Arbor, MI) with water/methanol/acetic acid (200:20:10) at 2.0 ml
min‘1 as the eluting solvent. The chromatograph was comprised of a
Rheodyne Model 7125 injector (Rheodyne, Inc., Berkeley, CA), a
Milton-Roy pump (Milton-Roy Corp., Riveria Beach, FL) and a Labor-
atory Data Control UV detector with a 254 hm filter (Model LDC III,
Laboratory Data Control, Riveria Beach FL).

Detection of -0H by EPR spectometry. The -DH rapidly reacts
with the nitrone spin trap 5,5-dimethyl-l-pyrroline-N-oxide (DMPO)
(Aldrich Chemical Co., Milwaukee, WI) and the resultant DMPO-0H
adduct can be detected by EPR spectometry (18). The DMPD was
purified prior to use by vacuum distillation and stored at -10°C.
The reaction mixture in a final volume of 1 ml contained: 0.1 ml
cell extract, 10 umol glucose, 0.1 umol FeClz, 1.0 umol EDTA, 0.1
umol NaN3, 60 umol DMPO and 30 mM NaCl. Where indicated, l00 umol
ethanol, 50 umol mannitol, 100 umol benzoate or 10 umol p-hydroxy-
bonzoic acid were also added. The reactions were initiated by the
addition of Fe(II) and the spectra were recorded immediately using a
Varian Century E122 EPR spectrometer (Varian Associates, Palo Alto,
CA) set at the following conditions: 3370 G magnetic field, 9.41208
GHz, 30 mN microwave power, 100 kHz modulation frequency, 2.5 modu-
lation amplitude, 0.25 s time constant and 8 min scan time.

In a second experiment, the -0H was generated chemically by
the use of Fenton's reagent and the DMPO-0H adduct formed was
detected as described above. The reaction mixture contained in a

final volume of 1 ml; 60 umol DMPD, 0.l umol FeClz, 0.l umol H202

95
and 30 mM_NaC1. The spectrum was recorded using 15 mN microwave

power and a 2 min scan time.

Effect of -0H scavenging agents on ligninolytic activity. The
effect of -0H scavenging agents on the metabolism of lignin by E,
chrysosporium was determined by measuring the amount of 14C02 pro-
duced following the additions of [2'-l4c] synthetic lignin to
ligninolytic cultures of the fungus, in the presence or absence of
-0H scavenging agents. After 13 d of incubation, 0.5 ml of a
synthetic lignin suspension (described above) and either 0.5 m1 of
10 m!_dimethylsuccinate (pH 4.5), 50 mM mannitol, 500 mM mannitol,
10 mM benzoate, 10 mM butylated hydroxytoluene (BHT), or 1 mM BHT,
were added to the cultures. The flasks were then stappered with
sterile gassing manifolds and reincubated. After 24 and 48 h of
incubation, the respired 14C02 was flushed from each flask and quan-
tified as described above. Four replicate flasks were used for each

treatment and the results are expressed as the mean :_S.E.

To rule out the possibility that the inhibition of [14CJ-syn-
thetic lignin degradation was due to nonspecific inhibition of
fungal metabolism by mannitol, benzoate or BHT, the effect of these
compounds on the metabolism of D-[U-14CJ-glucose was determined in a
manner identical to that described above except that D-[U-14C] glu-
cose (ZZLfl, 1.02 X 105 dpm, Schwarz/Mann, Orangeburg, NY) was sub-

' stituted for[14C]-synthetic lignin.

RESULTS

Relationship between culture growth phase, ligninolytic acti-
vity and H292 production. In low N media, ligninolytic activity of

f: chrysosporium has been shown to be derepressed after primary

 

growth ceases due to nitrogen starvation and apparently is a secon-
dary metabolic event (17,33). If 00H derived from H202 was involved
in the degradation of lignin, one would anticipate little H202 pro-
duction in the primary growth phase and an increase in specific
activity of H202 production to occur in concert with the appearance
of ligninolytic activity, after the culture entered the stationary
phase. The results showed that this was in fact the case. After 2
d of incubation, the nitrogen in the culture was exhausted (data not
shown), fungal growth slowed beginning on d 3 and the culture
appeared to enter stationary phase after 6 d (Fig. l). The for-
mation of conidia in the cultures followed the end of primary growth
and reached a maximum of l X 106 ml'1 on d 8, further indicating
that the culture was in stationary phase. Lignin degradation began
on d 5 or 6 and increased rapidly thereafter. There was little
H202 production activity (3-8 nmol min"1 mg protein“) in extracts
of cells from 6-7 d old cultures, i.e., before the appearance of
ligninolytic activity. Once the culture entered the stationary
phase, the specific activity for H202 production increased and
reached a maximum level of approximately 70 nmol in 20 d old
cultures (Fig. l). Boiled cell extracts did not produce H202.

These results indicate that a temporal relationship existed

between the appearance of H202 production activity and ligninolytic

96

97
activity. Since 13-14 d old cultures were actively ligninolytic and
contained high levels of H202 production activity (or approximately
50 nmol min"1 mg protein“) they were used in the experiments
described below.

Demonstration of -OH in cultures of P. chrysosporium. If -0H
derived from H202 was involved in lignin degradation, the radical

should be demonstrable in ligninolytic cultures of P, chrysosporium.

 

Appreciable levels of ethylene were produced in 14 d old cultures
(Fig. 2) after adding KTBA, thus demonstrating that -DH is produced
in the extracellular environment of actively ligninolytic cultures
of P, chrysosporium. The fact that the observed ethylene production
from KTBA was a -0H dependent process was confirmed by showing that
it was inhibited in the presence of -0H scavenging agents benzoate
or mannitol. Nhen 10 mM_benzoate or 50 m! mannitol was present in
the culture, the production of ethylene from KTBA was inhibited by
94 and 15%, respectively (Table 1). When cultures were autoclaved
prior to adding the KTBA or when KTBA was not added to the culture,
no ethylene was produced.

When 3, chrysosporium was grown in a high N medium (identical
to low N medium except that the N content was increased to 24 mM).
less than 1% of the added [‘4CJ-synthetic lignin was recovered as
14C02 in the first 14 d of incubation whereas identical cultures
grown in low N medium degraded or approximately 14% of the added
lignin. If there is a correlation between -0H production and lignie
nolytic activity by P, chrysosporium, less ethylene would be
expected to be produced when KTBA was added to cultures grown in

high N medium. The results showed that cultures grown in high N

98
medium produced approximately loo-fold less ethylene from KTBA than

cultures grown in low N medium (data not shown).

Hydroxylation of p-hydroxybenzoic acid to protocatechuic acid
by_;QH, Since the above results suggested that the -0H radical may
play a role in ligninolytic activity we sought to obtain several
independent lines of evidence to demonstrate conclusively that the
radical species in question was -0H. In one experiment the
-0H-mediated hydroxylation of p-hydroxybenzoic acid (pHB) to form
protocatechuic acid (PCA) was investigated using cell extracts of P,
chrysosporium (9, Table 2). The -DH may be produced through the
reduction of H202 by Fe(II). Alternatively, the cell extracts may
produce 053 or other reductants capable of reducing Fe(III) to
Fe(II) which in turn reduce H202 and yield the -DH as in the iron-
catalyzed Haber-Weiss reaction (25). Therefore, the ability of
Fe(II) and Fe(III) to participate in -OH formation by cell extracts
was examined.

As shown in table 2, the amount of PCA formed in complete
reaction mixtures containing Fe(II) (44.7 pmol PCA ml") was much
greater than that in complete reaction mixtures containing Fe(III)
(14.0 pmol PCA ml“). The lower amount of PCA formed in reactions
containing Fe(III) was probably due to the need to reduce Fe(III) to
Fe(II) required for the reductive cleavage of H202 to form the ~0H.
When the cell extract was omitted from reaction mixtures containing
Fe(II) 14.5 pmol PCA m1-l was formed. No PCA was formed on the
deletion of cell extract from reaction mixtures containing Fe(III).
The formation of PCA in the former reaction mixtures was probably

due to the autooxidation of Fe(II).

99

The -0H scavenging agents benzoate, mannitol and KTBA were all
shown to inhibit the formation of PCA in reaction mixtures contain-
ing either Fe(II) or Fe(III) (Table 2) indicating that the formation
of PCA from pHB in these reaction mixtures was a -0H dependent pro-
cess.

Endogenous catalase activity in cell extracts was found to be
completely inhibited by 0.1 all NaN3. Inhibition of catalase by the
inclusion of NaN3 in the reaction mixture would allow for the pro-
duction of a higher concn. of H202 and in turn, higher levels of -0H
production. In the presence of 0.1 nil NaN3, the formation of PCA in
reactions containing either Fe(II) or Fe(III) increased two-fold and
three-fold, respectively.

The formation of PCA in reactions containing Fe(II) or Fe(III)
decreased on deletion of glucose or EDTA. Nhen pHB, cell extract or
iron was deleted, or when boiled cell extract was used in these
reaction mixtures, little or no PCA, other than that resulting from
Fe(II) autooxidation, was formed.

The above results suggest that the hydroxylation of pHB to
form PCA was a -0H dependent process and that the -OH was derived
from H202. To independently confirm these conclusions, an experi-
ment identical to that described above was performed except that
glucose oxidase and glucose were substituted for cell extract as the
source of H202. The results showed that comparable amounts of PCA
were formed when the specific activities for H202 production were
similar in reaction mixtures containing cell extract or glucose oxi-

dase (Tables 2 and 3). The formation of PCA in reaction mixtures

100
containing glucose and glucose oxidase was also inhibited by ben-

zoate, mannitol or KTBA and was greatly stimulated by the addition
of glucose and EDTA. PCA formation was negligible when Fe(II) was
deleted. In contrast to results obtained using cell extract, the
inclusion of 0.1 mM_NaN3 resulted in no increase in the amount of
PCA formed in reactions containing glucose oxidase, since there was
no catalase present. The results of the previous experiment sug-
gested that PCA formation in reaction mixtures containing Fe(III)
may be dependent on the reduction of Fe(III) to Fe(II) by cell
extract. If so, no PCA formation would be expected when Fe(III) was
substituted for Fe(II) in the present experiment. Results showed
that this was the case (data not shown) indicating that there was no
mechanism for reducing Fe(III) to Fe(II) in these reactions.

Demonstration of -0H production using DMPO. The -DH reacts
with DMPD to produce a relatively stable nitroxide radical (18).
The DMPO-0H adduct can be detected by EPR spectrometry and identi-
lfied by its characteristic g-value and hyperfine splitting constants
of the resultant spectrum. The EPR spectrum of reactions containing
H202, Fe(II) (Fenton's reagent; reaction 1) and DMPO showed a quar-
tet with a symmetrical 1:2:2:l signal intensity, a g-value of 2.006
and hyperfine splitting constants offiAN=Ag=14.9 G indicating the
production of the DMPO-0H adduct (Fig. 3a). The EPR spectrum of
complete reaction mixtures containing cell extract (Fig. 3b) was
very similar to that observed in Fig. 3a indicating that ~0H was
produced in these reactions.

The -DH reacts with ethanol to form the o-hydroxyethyl radical
which in turn can react with DMPO to produce a stable DMPO-(a-hydro-

xyethyl) adduct, the spectrum of which is distinct from that of the

101
DMPD-DH adduct (18). When 100 nmol ml'1 of ethanol was added to the

complete reaction mixture, the DMPO-(a-hydroxyethyl) adduct which
had hyperfine splitting constants of AN . 15.8 G and AH = 22.8 G was
observed (Fig. 3c). These results confirm the identity of the radi-
cal species being produced as ~0H.

Production of -OH in the complete reaction mixture was depen-
dent on the presence of Fe(II) (Fig. 3d) and cell extract (data not
shown) and did not occur when boiled cell extract was added to the
reaction mixture (Fig. 3e). As shown above, the inhibition of cata-
lase in cell extract by NaN3 allows for higher levels of °0H to be
produced. Thus, the intensity of the signal was markedly diminished
when NaN3 was omitted from the reaction mixture (Fig. 3f). The pro-
duction of -DH in the absence of EDTA was approximately one-third
that seen in complete reaction mixtures (Fig. 39) and was essen-
tially eliminated in the absence of glucose (Fig. 3h). The for-
mation of the DMPO-0H adduct was inhibited when mannitol, benzoate
or pHB were added to the reactions (Figs. 3i, 3j, and 3k, respec-
tively). The spectrum observed with mannitol was presumably that of
the DMPD-mannitol radical adduct (J. Bucher, M. Tien, T. Moorehouse,
and S. Aust, manuscript in preparation). The results indicate the
H202-dependent formation of -OH in cell extracts from ligninolytic

cultures of E, chrysosporium.

 

Inhibition of lignin degradation by :DH scavengingaggnts,
The results presented above showed a relationship between ligninoly-
tic activity, H202 production activity, H202-dependent -0H produc-
tion by cell extracts and -DH production in cultures of P, 5232:
sosporium. If -DH is involved in lignin degradation, then one would

102
expect the degradation of lignin to be inhibited by -OH scavenging

agents. The results (Table 4) showed that the ~0H scavenging
agents, mannitol and benzoate and the nonspecific radical scavenging
agent BHT, markedly inhibited lignin degradation during the 24 h
following their addition to ligninolytic cultures. There was
little difference in the inhibition observed after 24 h vs. 48 h of
incubation. BHT and benzoate were the most effective scavenging
agents examined. Ligni n degradation was inhibited 59—62% by 0.1 “[4.
BHT and 91% by 1.0 mM_BHT. Benzoate was almost as effective as BHT.
Lignin degradation was inhibited by 34-40% and 48-53%, respectively,
when mannitol was added at final concn. of 5 11M and 50 ni_4_. Thus,
much higher concentrations of mannitol were necessary for signifi-
cant inhibition of lignin degradation as compared to those of ben-
zoate and BHT.

An alternate, albeit less likely, explanation for the above
results is that the inhibition of lignin degradation by mannitol,
benzoate and BHT was due to a nonspecific inhibition of cellular
metabolism and that these agents have little or no specific effec on
lignin degradation. To investigate this possibility, the effect of
these agents on the metabolism of D-[U-14CJ-glucose by identical
cultures of P, chrysosporium was determined. As shown in Table 5,
they had little or no effect on the metabolism of D-[U-14CJ-glucose.
Benzoate (lmM final concn.) slightly inhibited glucose metabolism;
however, the inhibitory effect on 14C02 release from [14C1-synthetic
lignin was significantly greater. In addition, these compounds were
shown to have no effect on the production of H202 by cell extracts

(data not shown).

DISCUSSION

In this study, extracts of cells from stationary phase cul-
tures of P, chrysosporium, grown in low N medium, were shown to pro-
duce significant amounts of H202. This finding extends the list of
wood-decomposing fungi which are known to produce H202 (37, 38).
Hydrogen peroxide is known to be a product of numerous enzymatic
reactions (37). It has also been shown to be produced nonenzymati-

cally by dismutation of hydroperoxy radicals (the conjugate acid of

02.) as shown in reaction 3 (7).
(3) 1100- + H00- —~ 02 + H202 k = 8.6 x 105 M-ls-l

Certain metals and metal complexes (25, 39) as well as superoxide
dismutase (EC 1.15.1.1; 8, 22) have been shown to catalyze reaction
3. For example, Cugg catalyzes this reaction at a rate of 2 X

109 M‘ls". Significant levels of metal ions are known to be pre-
sent in habitats of white-rot fungi, thus the formation of H202 from
053 in nature may occur through reaction 3. However, whether or b

not 053 is a precursor of H202 in cultures of E, chrysosporium is

 

not known.

A temporal relationship between the appearance of H202 produc-
tion activity and ligninolytic activity was observed in cultures
grown in low N medium. In agreement with previously published
results (33), ligninolytic activity appeared in response to nitrogen
starvation and after the culture had entered the stationary phase.

Similarly, H202 production increased rapidly following the cessation

of primary growth and after the formation of conidia in the culture

103

104
was initiated. R. L. Kelley and C. A. Reddy (manuscript in prepar-

ation) obtained similar results using an independent assay for H202
production (Hzoz-dependent peroxidatic oxidation of o-dianisidine).
Furthermore, both ligninolytic activity and H202 production were
seen in cultures grown in low N medium regardless of whether or not
lignin was present in the medium. Collectively, these data strongly
suggest a close correlation between H202 production and lignin
degradation by the fungus.

In the presence of reduced transition metals H202 is readily
reduced to form -OH (26, 51, 52). The production of -OH in ligni-

nolytic cultures of P, chrysosporium was indicated by the production

 

of ethylene from KTBA. Inhibition of the reaction by mannitol and
benzoate, known ~0H scavenging agents (25, 26, 50, 51), confirmed
that the production of ethylene observed in the ligninolytic
cultures is a -0H-dependent reaction. Furthermore, -0H was barely
detectable in cultures grown under conditions in which lignin was
not degraded, indicating a link between ~0H production and lignino-
lytic activity.

The production of -OH in cell extracts from ligninolytic cul-
tures was demonstrated by the -OH dependent hydroxylation of pHB.
The hydroxylation of pHB to PCA in reactions containing cell extract
required the presence of either Fe(II) or Fe(III). The production
of -0H in reactions containing Fe(II) and cell extract probably pro-
ceeds through a Fenton-type reaction. In reactions containing
Fe(III), the cell extract apparently reduces Fe(III) to Fe(II)
followed by the production of -0H through a Fenton-type reaction.
The reduction of Fe(III) by cell extract may be mediated by 053 (13,

50) or by other reactions of endogenous metabolism.

105
The validity of the production of -0H from H202 was supported by the

fact that a 2-3 fold stimulation in -0H production was observed when
the endogenous catalase activity in the cell extract was inhibited
by 0.1 mM_NaN3 (Table 3). Furthermore, PCA was formed in identical
reactions when glucose oxidase and glucose were employed as a source
of H202 instead of cell extract. In reactions in which glucose oxi-
dase and glucose were used to generate H202 (Table 4), PCA was
formed only in the presence of Fe(II), and not when Fe(III) was
substituted for Fe(II). This observation is consistent with the
conclusion that the cell extract can reduce Fe(III) to Fe(II). Pre-
vious investigators have found that the reductive cleavage of H202
to form ~0H can be enhanced in the presence of metal chelates (25).
Similarly, in these experiments, the formation of PCA was stimulated
in the presence of EDTA.

Verification of -OH production in cell extracts was obtained
by using EPR spectrometry. The production of the DMPO-OH adduct via
the addition of -0H to DMPO was dependent on the addition of cell
extract, DMPO, glucose, Fe(II) and NaN3 to the reaction mixtures.
The observed requirements for NaN3, which would allow for higher
concentrations of H202 in the reactions, and Fe(II) is additional
evidence that the production of -0H proceeds through the reductive
cleavage of hydrogen peroxide with Fe(II) serving as the electron
donor. Based on the above results, it appears that in ligninolytic

cultures of P, chrysosporium the production of -0H occurs through

 

the reduction of H202 produced by the fungus.
The -DH scavenging agents mannitol and benzoate and the non-

specific radical scavenging agent BHT significantly inhibited the

106
degradation of [2'-]4C] synthetic lignin but had little or no effect

on the metabolism of D-[U-14CJ-glucose or the production of H202 by
the fungus. The results strongly suggest that the inhibition of
lignin degradation by these compounds is due to their ability to
rapidly react with -0H and indicate that -DH is involved in the

degradation of lignin by E, chrysosporium.

 

The involvement of -OH in lignin degradation seems reasonable
in that a relatively nonreactive species, H202 (24, 29) is produced
and excreted by the fungus. Recent studies (R. L. Kelley and C. A.
Reddy, 1981, unpublished data) have shown that various culture para-

meters which affect lignin degradation by P, chrysosporium similarly

 

affect the production of H202 and 00H in.a manner which is consist-
ent with the involvement of -DH in lignin degradation. Furthermore,
the involvement of -DH in lignin degradation would explain the lack
of success by previous investigators in isolating enzymes capable of
extensively transforming lignin and the inability of cell-free
culture filtrates to degrade lignin (27, 28, 36). For example,
hydroxylation of the aromatic rings within lignin has been shown to
occur during the extracellular transformations of the polymer by
white-rot and brown-rot fungi (34, 36), yet monooxygenase or dioxy-
genase activity has not been demonstrated in the cell-free culture
filtrates of these fungi (3, 36). As demonstrated previously (52)
and in this study, ~0H readily adds to aromatic rings. Indeed, -0H
reacts nonspecifically and rapidly with many organic compounds in
highly exothermic reactions (29, 52). This would account for the
apparent low specificity and oxidative nature of the ligninolytic
system of white-rot fungi previously reported (15, 30, 34, 36).

107

Recently, Amer and Drew (2) reported that 0E3 produced by the
white-rot fungus Coriolus versicolor, was “exported“ to the extra-
cellular environment and speculated that 053 may be involved in
lignin degradation by this fungus. The mechanism by which 053 was
produced in cultures of C, versicolor was not established. However,
it is known that 053 is produced in several enzymatic reactions (8,
22) and can also be formed through the oxidation of H202 by ~0H (52)

as shown in reaction 4.
(4) 011. + H202 — H20 + 1102- k = (1.2-4.5) x 107 h-ls-l

Superoxide radicals have been implicated in many "oxygen
radical“-mediated processes including lipid peroxidation (32, 50),
depolymerization of polysaccharides (43) and phagocytosis by poly-
morphonuclear leucocytes (4). However, it has long been known that
053 is relatively nonreactive in aqueous systems (13, 16) and the
ability of 023 pg: §g_to initiate the above processes has recently
been questioned (16, 22). It has been proposed that essentially all
of the in yltrg_manifestations of 053 can be explained by the
involvement of 053 in the iron-catalyzed Haber-Weiss reaction
(Reaction 2; 16, 22) to produce -0H. Thus, if 053 is involved in
lignin degradation, it is possible that it serves as a reductant for
the production of -0H, via the iron-catalyzed Haber-Neiss reaction
(Reaction 2). f

The involvement of singlet oxygen (‘02) in the photochemical
oxidation of lignin and humic acids is well established (23, 49).

Involvement of 102 in lignin degradation by E, chrysosporium was

 

108
first suggested by Nakatsubo et al (46). However, no evidence for

the production of 102 by E, chrysosporium was provided and it is not

 

clear at the present time how and if this organism produces 102.

The enzymatic.production of 1O2 is unlikely since 22.5 kcal mol'1 is
necessary to excite 02 from its triplet ground state (3 2) to the
lowest energy singlet state (1 Ag) (29). It has been proposed that
102 is produced abiologically through the spontaneous dismutation of
053 (40; reaction 5) and the uncatalyzed Haber-Neiss reaction (40;

reaction 6).

(5) 05° + 0§°-—*H202 + 02

(6) 05° + H202——*-0H + H0 + 02

Recent studies by Foote et a1 (20) have shown that less than 0.07%
of the 02 formed in reaction 5 would be in the 1a state and the
possibility that any 102 is formed has been questioned by Millson
and Kearns (45). Furthermore, the uncatalyzed Haber-Weiss reaction
(reaction 6) has been shown to proceed very slowly with a rate .
constant of < 1 M“S‘1 and is probably of little importance in a
biological systems (7, 14). Therefore, it would appear unlikely
that significant levels of 102 are produced in ligninolytic cultures

of E, chrysosporium through reactions 5 or 6.

 

Singlet oxygen has been shown to be produced in some systems
during the propagation and termination of radical chain reactions
(19). For example, it is generally accepted that 102 is produced
during the breakdown of’ a-hydroxy hydroperoxy acid or peroxy radi-

cals which can be produced after an initial reaction of a compound

109

with some activated oxygen species (19, 50; reaction 7). These
reactions have been shown to occur in the peroxidation of lipids,

for example.

 

(7) R00- + Roo- :» noon + 102

Thus, the inhibition of lignin degradation by P, chrysosporium by

 

the 102 trap anthracene-9,lO-bis-enthanesulfonic acid (AES) observed
by Nakatsubo et a1 (46) may be due to the quenching of 102 produced
during radical pr0pagation or termination reactions which probably
occur in lignin degradation. It should also be pointed out that AES
is likely to scavenge -0H due to the extended n electron system of
the molecule and this may explain, at least in part, the observed
inhibition of lignin degradation.

The results presented here indicate that the -0H derived from
H202 plays an integral role in the decomposition of lignin by P,
chrysosporium and perhaps other white-rot fungi. The specific reac-
tions in which ~0H participates are not known at this time. How-
ever, we suggest that the primary role of -OH is in the initial oxi-
dation and depolymerization of the lignin polymer which occur in
the extracellular environment to produce low molecular weight
moieties. Some of the low molecular weight moieties which result

could then be taken up by the fungal cells and metabolized further.

110

Table 1. Effect of -0H scavenging agents on the production of ethylene

gas from KTBA by cultures of P, chrysosporium.a

 

 

 

Treatment nmoles ethylene ml"1c % of complete
Completeb 18.36 1 0.71 100

+ 50 mM Mannitol 15.69 :_0.24 65.5

+ 5 mM Benzoate 1.16 :_0.04 6.3

- KTBA 0.00 0.0
Boiled cultures 0.00_ 0.0

 

aCultures were grown for 14 d in 5 ml volumes in low N medium contained
in 50 m1 bottles (see Materials and Methods).

bThe complete reaction mixture consisted of a 14 d old culture to which
KTBA (3.3 “34. final concn.) had been added.

cThe amount of ethylene produced per ml of headspace during 10 h of

incubation; numbers were expressed as mean :_S.E.

111

Table 2. The effect of -0H scavenging agents and azide on the hydroxy-
lation of p-hydroxybenzoic acid to form protocatechuic acid by cell

extracts of P, chrysosporium.

 

 

Protocatechuic Acid Formedb

 

 

Final (10'12 moles ml"1 reaction mixture)
concn.
Addition (mM) 0.1 mM Fe(II) 0.1 mM Fe(III)
Completea - 44.7 14.0
Benzoate 2 41.2 10.5
Benzoate 20 21.0 4.1
Mannitol 20 34.5 10.5
Mannitol 200 7.9 4.5
KTBA ' 2 41.9 12.1
KTBA 10 17.8 7.1
NaN3 - 85.9 44.2
- Fe - 6.1 3.9
- Glucose - 38.3 3.4
- p-hydroxybenzoate - 0.0 0.0
- EDTA - 18.5 4.5
+ Boiled Cell Extract - 19.2 2.2
- Cell Extract - 16.3 1.1

 

aThe complete reaction mixture contained: 10 nmol p-hydroxybenzoic
acid, 10 nmol glucose, 0.1 nmol FeClz 0R FeCl3, 1 nmol EDTA, 200 1
cell extract (74 nmol H202 min-1 ml cell extract-l) and CX-distilled
water to 1.0 m1.

bAfter 120 min incubation at 39°C.

112

Table 3. Effect of -0H scavenging agents and azide on the hydroxyla—
tion of p-hydroxybenzoic acid to form protocatechuic acid in reactions

containing glucose oxidase.

 

 

Addition Einal _Protocatechu1c Acid Formedb
oncn. - (101 moles ml reaction mixture)
(mM)

Completea - 33.0

Benzoate 2 24.2

Benzoate 20 19.5

Mannitol 20 26.3

Mannitol 200 17.8

KTBA 2 18.8

NaN3 . 0.1 28.8

- Fe(II) - 1.1

- Glucose - 18.5

- p-hydroxybenzoate - 0.0

- EDTA - 12.1

- Boiled enzyme - 14.9

- Enzyme - 16.3

 

aThe composition of the complete reaction mixture was the same as in
Table 2 except 200 pl glucose oxidase (60 nmol H202 min‘1m‘ glucose
oxidase solution‘) was substituted for cell extract.

bAfter 120 min incubation at 39°C.

113

Table 4. Effect of -0H scavenging agents on ligninolytic activity

and Phanerochaete chrysosporium.a

 

% of controlb

 

 

 

 

14C-lignin 14C-glucose
Addition Final Concn. 24 hC 48 h 24 h 48 h
(mM)

Complete - 100 100 100 100
+ Mannitol 5 60 66 114 121

+ Mannitol 50 47 52 88 99

+ Benzoate l 13 16 72 86

+ BHT 0.1 38 41 91 98

+ BHT 1.0 9 9 92 94

 

aComplete reaCtion mixtures consisted’of7cultures of’E, chrysosporium

 

were grown for 13 d in low N medium to which the -0H scavenging agents

and either [14C] lignin or [14C] glucose had been added. The metabo-

lism of these substrates was assayed after 24 and 48 h of incubation

by quantification of the respired 14C02,

b% of control a dpm (no additions)-dpm (with addition) x 100

 

dpm lno additionSl

cTime interval after following addition of -0H scavenging agent.

Fig.

114

Relationship between the specific activity for H202 production
in cell extracts (‘), the metabolism of [25140] synthetic
lignin to [14602] (.1 and mycelial dry weight (:11. g.

chrysosporium was grown in low N medium as described in

 

Materials and Methods.

 

 

 

115

slate-0910.0... 3...!

 

 

.10.... :83! to 238.5

116

Fig. 2 The production of -OH in ligninolytic cultures of P, 5551:
sosporium. The -DH was detected by the -0H-dependent for-
mation of ethylene following the addition of «1-k8t0-‘Y
-methiolbutyric acid (KTBA) (3.3 mM final concentration) to
cultures grown for 14 d in low N medium. The ethylene pro-
duced was detected using gas chromatography and is expressed

as nmoles ethylene per m1 of headspace.~

 

117

 

 

I

 

 

    

P

m .6. M 2 0 am 3 4 2 00

7.5 mzmfizem moose

HOURS

Fig 3.

118

Electron paramagnetic resonance spectra of DMPD-DH adduct
formed in reactions containing H202 and Fe(II) (Fenton's
reagent or cell extract (B-K) as described in Materials and
Methods. Fenton's reagent, A; complete, B; plus 100 mM.etha—
nol, C; minus Fe(II), 0; plus boiled cell extract, E; minus
azide, F; minus EDTA, G; minus glucose, H; plus 50 mM_man-
nitol, 1; plus 100 mM_benzoate, J; and plus 10 m! p-
hydroxybenzoate, K. the following gain settings were used

(a) = 6.3 x 102, (b) = 1.6 x 104, (C)-(K) = 4.0 x 104.

 

 

10.
ll.
12.

13.
14.

15.

16.

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Finkelstein, E., Rosen, G. M., and Rauckman (1980) Arch. Biochem.

Biophys. 29931-16
Foote, C. S. (1976) in Free Radicals in Biology, Vol. II ed. N. A.

 

 

Pryor (Academic Press, New York, NY) pp. 85-133
Foote, C. S., Shook, F. C., and Abakerli (1980) J. Amer. Chem.
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(in press)

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Gellerstedt, G., Kringstad, K., and Lindfors, E. L. (1978) in
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eds. B. Ramby and J. F. Rabele (John Wiley and sons, New York, NY)
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Halliwell, B. (1974) New Phytol. 12:1075-1086

 

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Halliwell, B. (1978) FEBS Letters 255238-242

 

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CHAPTER III

Ultrastructural Localization of Hydrogen Peroxide
Production in Ligninolytic Cells of

Phanerochaete chrysosporium

 

ABSTRACT

Previous studies have shown that the hydroxyl radical derived
from hydrogen peroxide (H202) is involved in lignin degradation by
Phanerochaete chrysosporium. In the present study, the Ultrastructural
sites of H202 production in cells from ligninolytic cultures was demon-
strated by cytochemically staining cells with 3,3'-diaminobenzidine
(DAB). Hydrogen peroxide production, as evidenced by the presence of
deposits of oxidized DAB, appeared to be localized in the periplasmic
space of cells from ligninolytic cultures grown for 14 d in nitrogen-
limited medium. When identical cells were treated with DAB in the pre-
sence of aminotriazole, periplasmic deposits of oxidized DAB were not ob-
served, suggesting that the deposits of oxidized DAB resulted from the
HZOZ-dependent peroxidatic oxidation of DAB by catalase. Cells from cul-
tures grown for 3 or 6 d in nitrogen-limited medium or for 14 d in ni-
trogen-sufficient medium had little ligninolytic activity, low specific
activity for H202 production and did not contain periplasmic oxidized
DAB. The results suggest a positive correlation between the occurence
of oxidized DAB deposits in the periplasmic space, the intensity of the

staining reaction, the specific activity for H2 2 production in cell ex-

tracts and ligninolytic activity.

124

INTRODUCTION
Previous studies have shown that the ligninolytic system of
Phanerochaete chrysosporium is synthesized after the cessation of
primary growth and in response to nitrogen starvation, apparently as a
part of secondary metabolism (an idiophasic event; 10). Furthermore, a
temporal relationship between the appearance of ligninolytic
activity and hydrogen peroxide (H202) production activity in nitrogen-

1imited cultures of E, chrysosporium has been demonstrated (7).

 

Hydrogen peroxide-derived hydroxyl radical (dMi) was shown to play an
integral role in lignin degradation as evidenced by the fact that
benzoate and mannitol, which scavenge -OH, inhibited 11'9'”n
degradation (7).

Hydroxyl radical has been shown to be highly reactive and to
cause damage to cellular constituents such as proteins, lipids and DNA
(1,5,13). In many cells these effects are minimized by compartmental-
izing the H202 production activity and catalase within subcellular
organelles such as peroxisomes and glyoxysomes (17). In light of the
observation that high levels of H202 are produced by cell extracts of

ligninolytic cultures of P3 chrysosporium (contrasted with minimal

 

levels of H202 produced by non-ligninolytic cultures) it was of
interest to determine the subcellular location of the H202 production

activity in E, chrysosporium. Such information would be valuable in

 

understanding how H202 production is localized in E, chrysosporium in a
way to minimize its cytotoxic effects, yet allows for the involvement
of H202 in the production of -OH in the extracellular environment.

In this study, the ultrastructural sites of H202 production

activity in cells of P, chrysosporium were demonstrated by cyto-

125

126

chemically staining the cells with 3,3'-diaminobenzidine (DAB). The
results of this study suggest that HZOZ-production activity, as evi-
denced by the presence of deposits of oxidized diaminobenzidine (DAB)
is localized in the periplasmic space of cells from ligninolytic cul-

tures but not in cells from nonligninolytic cultures.

MATERIALS AND METHODS

Organism and culture conditions. Phanerochaete chrysosporium Burds.
ME 446 (ATCC 34541) was obtained from T. K. Kirk (Forest Products
Laboratory, U. S. Department of Agriculture, Madison, WI) and was
maintained through periodic transfers on malt extract agar slopes as
previously described (11).

The composition of the nitrogen-limited basal medium used for
these experiments has been described previously (11) and contained

0.6 mM asparagine plus 0.6 mM NH4NO 2.4 mM N). High N medium was

3(
identical to the basal medium except that it contained 6 mM asparagine
plus 6 mM NH4NO3 (24 mM N). The medium (50 ml) was dispensed into

500 ml Erlenmeyer flasks, which were then foam-stoppered and autoclaved
for 15 min at 121°C. The medium was inoculated with a conidial
suspension of E, chrysosporium in water as previously described (6).
The cultures were incubated in air at 39°C, without agitation, for
various periods of time as indicated in the text.

Qytochemical stainingytechniques. Hydrogen peroxide production activity

 

in cells of E, chrysosporium was demonstrated by cytochemical staining

 

with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Aldrich Chemical
Co., Milwaukee, WI) using a procedure modified from Van Dijken and
Veenhuis (19). The cells were harvested by centrifugation at 12,000 X
g for 10 min, and washed once with 50 mM sodium phosphate (pH 5.5).

The pellet was resuspended in the same buffer, homogenized with a glass
tissue homogenizer and centrifuged as above. The cells were fixed in
3% glutaraldehyde in 0.1 mM cacodylate buffer (pH 7.0) and were treated
with aerated DAB solutions [2 mg DAB and 100 nmoles glucose per ml of

sodium phosphate buffer (70 mM; pH 6.6) at 39°C for l h]. In control

127

128

experiments, glutaraldehyde-fixed cells were preincubated for 30 min in
phosphate buffer (described above) containing 50 nmoles 3-amino-1,2,4-
triazole (aminotriazole, Sigma Chemical Co., St. Louis, MO) per m1.
These cells were treated with DAB solution identical to that described
above except that it contained an additional 50 nmoles aminotriazole

-1. After 30 min, the cells from both treatments were transferred to

m1
fresh DAB solutions. These DAB-treated cells were post-fixed with 2%
0504 in 0.1 M sodium cacodylate buffer (pH 7.0) at room temperature for
45 min, washed with the same buffer and held in 1% aqueous uranyl
acetate overnight. The uranyl acetate was decanted and the cells were
embedded in 1% Noble agar. A thin layer of solidified agar was cut
into 2 mm squares, dehydrated in a graded alcohol series and propylene
oxide followed by embedding in Poly-bed (Polysciences, Warrington, PA).
After sectioning, the specimens were post-stained for 10 min with 2%
uranyl acetate and 3 min with lead citrate and examined using a Philips

EM 300 electron microscope.

5292 production. The specific activ1ty for H202 production 1n cell

 

extracts of E, chrysosporium was determined using a modified catalase-

aminotriazole assay as previously described (3,7).

14 . 14

C-synthetic 1ignin. The degradation of (2 - C)-
5 14

Degradation of

synthetic lignin (1.0 X 10 dpm mg-]) to CO2 was determined as

described previously (6).

RESULTS
Cytochemical staining of Hage_production activity. DAB is peroxidati-
cally oxidized by catalase or peroxidase in a HZOZ-dependent reaction
to form an osmiophilic polymer of oxidized DAB (14,16) which becomes
electron dense after treating with osmium. Hence, any subcellular
locations which are the sites of catalase or peroxidase and H202
production activity will appear as dark regions in electron micrographs
(14). This is the basis of the cytochemical staining employed in this
study to visualize sites of H202 production activity in cells of E.

chrysosporium.

 

Experiments were conducted to determine the relationship between
levels of ligninolytic activity, H202 production and the presence of
deposits of oxidized DAB within the cells. Less than 1% of the 14C-
synthetic lignin was degraded during the first 6 d of incubation

whereas 5.1% and 11.8% of the added ‘4

C-lignin was degraded after 10
and 14 d incubation, respectively, by cells grown in nitrogen-limited
medium (Table 1). Furthermore, extracts prepared from cells grown for
3, 6, 10, and 14 d in nitrogen-limited medium had specific activities
(nmol min'1 mg protein-1) for H202 production of 2.9, 3.1, 22.1, and
52.3, respectively (Table 1). Thus only cells from 10 and 14 d-old
cultures of P, chrysosporium, which were actively ligninolytic, had
significant levels of H202 production activity whereas 6 d cultures did

not have appreciable levels of ligninolytic activity or H202 production

activity. When cells from 10-14 d-old nitrogen-limited cultures were

stained with DAB, electron-dense deposits of oxidized DAB were

observed between the cell wall and cytoplasmic membrane of the
fungal cells (Fig. 1A and C). In contrast, in cells from 3 and 6 d-old

129

130

cultures these periplasmic deposits were not seen (Fig. 1D and E).

The deposits of oxidized DAB were found to be distributed rather evenly
along the length and circumference of the cells (Fig. 1A and C). The
H202 production activity of cells grown in nitrogen-limited cultures
for 10 d was less than half that found in cells after 14 d incubation
(Table 1). As one might expect, the staining intensity and number of
deposits of oxidized DAB in the periplasmic space of 10 d-old cells was
qualitatively less than that observed with 14 d-old cells (Fig. 1C).
These data suggest a positive correlation between the numbers of peri-
plasmic deposits of oxidized DAB which appear to be the sites of H202

production, and ligninolytic activity of E, chrysosporium.

 

Aminotriazole is a known inhibitor of catalase (15). Therefore,
when cells from 10 d and 14 d-old nitrogen-limited cultures were in-
cubated with DAB in the presence of aminotriazole, deposits of oxidized
DAB would not be expected to be formed if only catalase (and no peroxidase)
was present in the periplasmic space. The results (Fig. 1B) indicated
that the electron-dense regions observed in the electron micrographs
(Fig, 1A and 1C) were due to the peroxidatic oxidation of DAB by cata-
lase.

Little lignin degradation occurred in cultures of E, chrysosporium

 

when grown in high nitrogen medium (Table 1). Also the specific activity
for H202 production decreased in these cultures from 12.5 on d 3

to 6.1 on (114. Furthermore, in these 14 d-old cultures the specific
activity for H202 production was approximately 8-fold lower than

that observed in similar cells grown in nitrogen-limited medium.

If there was a positive correlation between the number of deposits of
oxidized DAB periplasmic and ligninolytic activity, as indicated by

the above results, then one would expect few or no periplasmic

131

deposits in cells from 14 d-old nonligninolytic cultures of E, chryso-
sporium, grown in high N medium. The results showed that this was the

case (Fig. 1E).

DISCUSSION

Lignin was not degraded during the first six days of incubation

in cultures of E, chrysosporium grown in nitrogen-limited medium (2.4

 

mM N) and the H202 production activity of these cells was also low.
Similarly, cells from 14 d-old cutlures of E, chrysosporium grown in
high nitrogen medium (24 mM N) degraded less than 1% of the added
.14C-synthetic lignin and exhibited low levels of H202 production activity.
Deposits of oxidized DAB were not observed in the periplasmic space of
cells from either of the above nonligninolytic cultures. The develop-
ment of ligninolytic activity in nitrogen-limited cultures was concomi-
tant with an increase in the specific activity for H202 production, which
was in turn correlated with the appearance of periplasmic deposits of
oxidized DAB. There was no apparent difference in the number of cyto-
plasmic oxidized DAB-positive microbodies between cells from lignino-
lytic cultures and nonligninolytic cultures. These observations suggest
that the H202 production activity associated with lignin degradation is
localized in the periplasmic space of cells from ligninolytic cultures
(L.J. Forney and C.A. Reddy, unpublished data). Most, but not all of

the hyphae examined from 14 d-old culture contained deposits of oxidized
DAB in the periplasmic space. In cells from 10 d-old cultures, the
deposits of oxidized DAB were observed in a lower proportion of the

cells examined and, when present, were less numerous and smaller (Fig.

1C) than in 14 d-old cells.

132

133

In the presence of H202, the oxidation of DAB can be accomplished
by several enzymes, including peroxidase and catalase (14,16). When
cells containing high specific activities for H202 production were
treated with DAB in the presence of aminotriazole, deposits of oxi-
dized DAB were not observed in the periplasmic space of the cells.
Since aminotriazole specifically inhibits catalase (15), these data
indicate that peroxidatic oxidation of DAB was most likely effected by
catalase and was not an artifact of the staining procedure. Further-
more, if significant leVels of peroxidase had been present in the peri-
plasmic space, deposits of oxidized DAB would have been observed in the
presence of aminotriazole. Since this was not the case, little or no
peroxidase was present in the periplasmic space.

Catalase and H202 prodUction activities are known to be located in
subcellular organelles such as peroxisomes and glyoxisomes (8,17). The
definition of deposits of exodized DAB in the periplasmic space
of cells used in this study suggests that the H202 production activity
is present in membrane-limited organelles. However, due to the
intensive oxidation of DAB it was not possible to determine with cer-
tainty whether or not these activities are contained within a unit-
membrane. Further studies are needed to clearly demonstrate this
point.

The mechanism by which E, chrysosporium produces H202 has not been
established. However, wood decomposing fungi have been shown to
produce numerous enzymes, including various carbohydrate oxidases (12)
cellobiose-quinone oxidoreductase (18) and aromatic alcohol oxidases
(4,9), which oxidize various substrates with the concomitant production

of H202. Hydrogen peroxide could also be produced through the

134

enzymatic or chemical dismutation of superoxide radicals (1.2).
Further investigations are needed to determine the specific
enzymatic reaction(s) which play a role in H202 production by E,
chrysosporium.

To our knowledge, these results represent the first time that H202
production activity and catalase have been demonstrated to be localized
in the periplasmic subcellular structures of a fungus. This unusual

arrangement would seem to be advantageous to E, chrysosporium. With

 

the H202 production activity located outside the cytoplasmic membrane
the fungus is able to avoid high levels of H202 in the cytoplasm where
it could participate in cytotoxic processes, yet the enzymes responsi-
ble for the production of H202 are retained by the cell. Furthermore,
the H202 could readily diffuse from the cells whereupon it could
undergo reductive cleavage to produce -OH which is known to be

involved in lignin degradation.

LITERATURE CITED
Brawn, K., and I. Fridovich. 1981. Superoxide radical and
superoxide dismutases: threat and defense. Acta. Physiol.
Scand. Suppl. 492:9-18.
Bielski, B.H.J., A. 0. Allen. 1977. Mechanism for the dispropor-
tionation of superoxide radicals. J. Phys. Chem. 81:1048-1050.
Cohen, G., and N. L. Somerson. 1969. Catalse-aminotriazole
method for measuring secretion of hydrogen peroxide by micro-
organisms. J. Bacteriol. 98:543-546.
Farmer, V. C., M.E.K. Henderson, and J. D. Russell. 1960.

Aromatic alcohol oxidase activity in the growth of Polystictus

 

versicolor. Biochem. J. 74:257-262.

 

Fong, K., P. B. McCay, J. L. Poyer, B. B. Keele, and H. Misra.
1973. Evidence that peroxidation of microsomal membranes is
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activity. J. Biol. Chem. 248:7792-7797.

Forney, L. J., and C. A. Reddy. 1982. Biological studies on
1ignin degradation: A simple procedure for the synthesis form
ferulic acid of coniferyl alcohol, a precursor of synthetic.
1ignin. Appl. Environ. Microbiol.

Forney, L. J., and C. A. Reddy. 1982. The hydrogen peroxide-
dependent formation of hydroxyl radicals and their apparent
involvement in lignin degradation by Phanerochaete chrysosporium.
J. Biol. Chem.

Halliwell, B. 1974. Superoxide dismutase, catalase and gluta-
thione peroxidase: solutions to the problems of living with

oxygen. New Phytol. 73:1075-1086.

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136
Higuchi, T. 1980. Microbial degradation of dilignols as lignin
models, p. 171-193. In_T. K. Kirk, T. Higuchi, H-m. Chang (eds.),
Lignin biodegradation: microbiology, chemistry and potential
applications, CRC Press, West Palm Beach, FL.
Keyser, P., T. K. Kirk, and J. G. Zeikus. 1978. Ligninolytic
enzyme system of Phanerochaete chrysosporium: synthesized in the
absence of lignin in response to nitrogen starvation. J. Bacteriol.
135:790-797.
Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, and J. G.
Zeikus. 1978. Influence of culture parameters on lignin
metabolism by Phanerochaete chrysosporium. Arch. Microbiol.
117:277-285.
Koenigs, J. W. 1972. Production of extracellular hydrogen
peroxide by wood-rotting fungi. Phytopathol. 62:100-110.
Kong, S., and A. J. Davison. 1980. The role of interactions
between 02, H202, '0H, e' and 027 in free radical damage to
biological systems. Arch. Biochem. Biophys. 204:18-29.
Lewis, P. R. 1977. Other cytochemical methods for enzymes, p.
225-287, Ig_A. M. Glauert (ed.), Practical methods in electron
microscopy, Vol. 5, North Holland/American Elsevier, New York.
Margoliash, E. A., A. Novogrodsky, and A. Schejter. 1960.
Irreversible reaction of 3-amino-l,2,4-triazole and related
inhibitors with the protein of catalase. Biochem. J. 74:339-348.
Seligman, A. M.,.M. J. Karnovsky, H. L. Wasserkrug, and J. S.
Hanker. 1968. Nondrop ultrastructural demonstration of cyto-
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17.

18.

19.

137

Tolbert, N. E. 1971. Microbodies - peroxisomes and glyoxisomes.
Ann. Rev. Plant Physiol. 22:45-74.

Westermark, U., and K. E. Eriksson. 1974. Cellobiose-quinone
oxidoreductase, a new wood-degrading enzyme from white-rot fungi.
Acta. Chem. Scand. 828:209-214.

Van Dijken, J. P., and M. Veenhuis. 1980. Cytochemical

localization of glucose oxidase in peroxisomes of Aspergillus

 

n1ger. Eur. J. Appl. Microbiol. Biotechnol. 9:275-283.

138

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139'

FIGURE LEGEND

Figure 1. Transmission electron micrographs of P3 chrysosporium showing

 

details of cell wall (CW), periplasmic area and cytoplasmic
membrane (CM). Deposits of oxidized DAB (see text for
details) are indicated by arrows. Bar = 0.1 um. Cells grown
in nitrogen-limited medium for 14 d (A; 90,100X), 10 d (C;
113,4OOX), and 6 d (D; 56,7OOX); (B) is identical to (A)
except for the addition of aminotriazole to the staining
solution (60,000X); cells grown for 14 d in medium not

limited in nitrogen (E; 80,000X).

 

 

 

140

 

APPENDICES

APPENDIX 1

APPENDIX 1

Speculation on the involvement of oxygen radicals in
lignin degradation by wood decomposing fungi

How microorganisms degrade a polymer as structurally complex as
lignin is a very intriguing area of research. It has become clear, based
on the results of earlier studies,that 1ignin is metabolized in a manner
different from that of other biopolymers (14, 23). In this paper, some
of the relevant structural features of lignin and the apparent require-
ments they may impose on the ligninolytic systems of microorganisms are
discussed.

Within plant tissue, lignin exists in a matrix with hemicelluloses
(1, 35). Fungal hyphae are probably unable to penetrate the intact
lignin-polysaccharide.matrix. and so much of the polymer probably re-
mains physically inaccessible to the fungus. Furthermore, lignin is in-
soluble in water (25), has a high molecular weight (1, 3, 25) and is
three dimensional (1), thus making transport into the cell unlikely.
These features of lignins in plant tissues would appear to dictate that
the agents which attack lignin be extracellular and freely diffusible.

In consideration of the structural complexity of the lignin poly-
mer, the agents responsible for lignin degradation probably have rather
broad substrate specificity. More than 24 different intermonomer link-
age types have been shown to occur in 1ignin (1), many of which are not

readily hydrolyzed (1, 25). Due to the nonenzymatic free radical

141

142

mechanism by which the polymer is formed, the linkages are nonrepeating
and are randomly distributed throughout the polymer (1). Furthermore,
the polymer is racemic despite the presence of asymmetric carbons in the
aliphatic three carbon side chains (33). Indeed, each 1ignin molecule
can be considered to be structurally unique. Yet despite the structural
complexity of lignin, white-rot fungi are able to extensively oxidize
and degrade (greater than 90%) the polymer (1, 8, 20, 21). These
findings suggest that most, if not all, of the intermonomer linkages in
lignin can be attacked by white-rot fungi.

Certain general observations suggest that at least certain com-
ponents of the ligninolytic system have relatively low specificity. Lig-
nin decomposing fungi are capable of degrading structurally different
lignins including lignins in various genera of plants (e.g. gymnosperm,
angiosperm and grass lignins), within different tissues of the same
plant (e.g. early, late and compression wood) (20) and industrially
modified lignins (e.g. kraft lignins and lignosulfonates) (26). Thus,
the ligninolytic system is able to recognize structurally different
polymers as substrates. Furthermore, Keyser §t_al_(19) suggested that
the relatively low rates of lignin degradation by fungi, and the appar-
ent inability of lignin to serve as a growth substrate for fungi, may
be indicative of the low activity of the ligninolytic system and, in-
directly, that the system possesses low specificity.

.As discussed above (see Literature Review), cell-free culture
filtrates of wood decomposing fungi are low in protein (3, 15) and are
unable to extensively transform 1ignin (14, 15). In fact, conclusive
evidence for the involvement of any enzyme in lignin degradation is

lacking.

143

Thus it would appear that the agent(s) which effect the initial
transformations of lignin should be extracellular, freely diffusible,
and be relatively nonspecific and nonstereoselective in their mode of
attack. These apparent requirements have led to the suggestion that the
extracellular transformations of lignin which occur during its degrada-
tion by white-rot fungi may be mediated by "activated oxygen species"

such as oxygen radicals (2, 11, 14, 28).

Oxygen chemistry

The ground state of dioxygen is a triplet state with one electron
pair shared and with two unpaired electrons occupying different anti-
bonding orbitals but having the same spin quantum number (16). Con-
certed reactions between dioxygen in its ground state and organic com-
pounds, which normally are in a singlet state, are spin-forbidden and
therefore kinetically unfavorable unless dioxygen is first activated
(16). This can be accomplished in at least three ways, by: (1) Trans-
ferring energy to ground state oxygen to excite it to a singlet state

1 129); (2) The reaction of oxygen with the unpaired

(either 13 or
d electrons of a transition metal or stable organic free radical (as
in monooxygenases and dioxygenases; 16) or (3) Partial reduction of
oxygen to form superoxide radical (02'-), hydrogen peroxide or hydroxyl

radical (eOH) (Reaction 1; 12).

 

 

Oxygen radicals have been implicated in numerous important biological

processes (12) including phagocytosis by polymorphonuclear leucocytes

144

(4, 32), lipid peroxidation (18, 34), inflammatory responses (e.g.
certain types of arthritis) (27) and xenobiotic and drug metabolism

(5, 7). It has also been suggested that oxygen radicals may have a role
in aging (30), spontaneous mutations in DNA (6, 36) and carcinogenesis

(30).

The involvement of hydroxyl radicalin.liqnin.degradation .

The mechanism by which oxygen radicals might be produced in cul-
tures of white-rot fungi were discussed earlier (see Chapter II). Evi-
dence presented here (see Chapter II) indicated that i0H plays an inte-
gral role in lignin degradation by P. chrysosporium and perhaps other
wood-decomposing fungi. The -OH is a strong oxidant and reacts nonselec-
tively with many organic compounds at rates limited by diffusion (37).
The following reactions which have been shown to occur during lignin
degradation by white-rot fungi could be effected by -OH (9, 14, 29, 37):
(a) Hydroxylation of aromatic rings; (b) Aromatic ring cleavage; (c) Oxi-
dation of carbons a to aromatic rings to carbonyls; (d) Cleavage of C-C
single bonds; (e) Oxidation of carbons to carboxylic acids; and (f) De-
methylation of methoxyl groups. Furthermore, the involvement of -OH in
lignin degradation may explain why the process is apparently obligately
aerobic (13)'(see Literature Review) and why investigators have been
unable to isolate enzymes capable of extensively degrading lignin (14).

In consideration of the apparent involvement of .OH in lignin de-
gradation, it now appears possible to offer reasonable interpretations

of some of the earlier results on lignin degradation.

145

Inhibition of lignin degradation by o-phthalate
Fenn and Kirk (10) have shown that twice as much lignin is de-

graded by E. chrysosporium when 10 mM_2,2-dimethylsuccinate was used to

 

buffer the medium as compared to that observed with 10 mM_o-phthalate
as the buffer. The degradation of methoxyl, side-chain and ring labelled

lignins to 14

CO2 were inhibited to a similar degree in the presence of
o-phthalate but the oxidation of glucose, acetovanillone and apocynal
were unaffected (10). In other experiments, 3, chrysosporium was grown
in medium buffered with 2,2-dimethylsuccinate for 6 d at which time

d 14C-synthetic lignin were

o-phthalate (9 mM_final concentration) an
added. The degree of inhibition of lignin degradation observed was ap-
proximately the same as when the cultures were grown from dtjin medium
containing o-phthalate. Similar results were obtained when cyclohexi-

mide (an inhibitor of eucaryotic protein synthesis) was added to the

14C-synthetic lignin.

cultures 1 h prior to the addition of o-phthalate and
These results indicate that o-phthalate inhibits the existent ligninoly-
tic system.

Hydroxyl radical rapidly react with aromatic compounds

(k-109-101014'ls'1; 37). o-Phthalate is an aromatic compound and as

such would be expected to efficiently scavenge .OH. Since .OH have been
shown to play a role in lignin degradation, the inhibition of lignin

degradation by o-phthalate may be due to its ability to scavenge .OH.

Cosubstrate requirement for lignin degradation
Data available to date indicate that lignin does not serve as
a carbon source for growth of white-rot fungi (22). A cosubstrate ap-

pears to be required not only for growth but also to support 1ignin

146

14C-syn-

degradation (17, 31). _However, studies using specifically labelled
thetic lignin have shown that lignin carbon does enter into central meta-
,bolic pathways in white-rot fungi since it was incorporated, with re-
arrangement of the labelled carbons, into the secondary metabolite vera-

tryl alcohol (17) and was respired as 14

CO2 (17, 19, 31). It is possible
that the function of the cosubstrate, in addition to providing carbon

and energy for cell growth, is to serve as a source of reducing equiva-
lents for the partial reduction of O2 to 02'-, or H202 which then lead

to the production of ~OH. The latter in turn mediate the degradation

of lignin.

Effect of oxygen partialypressure

Dioxygen has been shown to aggravate the damage caused in biologi-
cal systems by oxygen radicals. For example, Kong and Davison (24)
examined the effect of various partial pressures of oxygen on the re-
sultant increase in permeability in the plasma membrane of erythrocytes
to glyceraldehyde-3-phosphate following the production of radicals by
y-irradiation. When erythrocyte suspensions were incubated in an atmos-
phere of 0.2 and 1.0 atm of oxygen, the membrane permeability increased
2.8 and 60-fold respectively, as compared to anaerobic conditions.
The observed increase in damage caused by oxygen was thought to be due
to increased branching of radical chain reactions. Similar effects may
in part explain the higher rate of lignin degradation by E, chrysosporium
when incubated under higher partial pressures of oxygen (0.4-1.0 atm)
as compared to air or 0.05 atm of oxygen. Following initiation by -OH,
the increased branching of radical chain reactions in lignin which would

be likely to occur in the presence of high partial pressures of oxygen,

147

may allow for more extensive oxidation and depolymerization of the
polymer. The depolymerization of lignin is presumab1y the rate-limiting
step in lignin degradation. As a result, the more extensive depoly-
merization of lignin under high partial pressures of oxygen would in turn
results in more rapid metabolism of lignin carbon to C02.

Thus, the data obtained in previous investigations appear consis-
tent with the involvement of .OH in the degradation of lignin by wood-
decomposing fungi. Further studies are needed to determine the specific
reactions effected in 1ignin by -0H, the mechanism of lignin depolymeri-
zation, identification of the depolymerization products which are sub-
sequently metabolized by the fungi, and what, if any, other microbially
produced agents are involved in the extracellular degradation of the

polymer.

IO.

11.

LITERATURE CITED

Adler, E. 1977. Lignin chemistry-past, present and future.
Wood Sci. Technol. 11:169-218.

Amer, G.I. and S.W. Drew. 1980. The concentration of extra-
cellular superoxide radicals as a function of time during
lignin degradation by the fungus Coriolus versicolor.
Dev. Ind. Microbiol. 22:479-484.

 

Ander, P. and K.E. Eriksson. 1978. Lignin degradation and utili-
zation by microorganisms. Prog. Ind. Microbiol. 1421-58.

Barbior, B., R. Kipnes, and J. Curnutte. 1973. Biological defense
mechanisms. Evidence for the participation of superoxide
in bacterial killing by xanthine oxidase. J. Lab. Clin.
Invest. 52:741-744.

Borg, D.C., K.M. Schaich, J.J. Elmore, Jr., and J.A. Bell. 1978.
Cytotoxic reactions of free radical species of oxygen.
Photochem. Photobiol. 28:887-907.

Brawn, K. and I. Fridovich. 1980. Superoxide radical and super-
oxide dismutases: threat and defense. Acta Physiol. Scand.
Suppl. 492:9-18.

Cohen, G. 1978. The generation of hydroxyl radicals in biological
systems: toxicological aspects. Photochem. Photobiol.
28:669-675.

Cowling, E.B. 1961. Comparative biochemistry of the decay of
sweetgum sapwood by white-rot and borwn-rot fungi. U.S.D.A.
For. Ser. Tech. Bull. 1258, 79 pp.

Fee, J.A. 198D. Superoxide, superoxide dismutase and oxygen
toxicity, p. 209-247. Ig_T.G. Spiro (ed.), Metal ion acti-,
vation of dioxygen. Wiley-Interscience, New York, NY.

Fenn, P. and T.K. Kirk. 1979. Ligninolytic system of Phanerochaete
chrysosporium: inhibition by o-phthalate. Arch. Microbiol.
123:307-309.

 

Forney, L.J., C.A. Reddy, M. Tien and 5.0. Aust. 1982. Hydrogen
peroxide-dependent formation of hydroxyl radicals and their
involvement in lignin degradation by Phanerochaete
chrysosporium. J. Biol. Chem. (submitted).

 

148

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

149

Fridovich, I. 1980. The biology of oxygen radicals. Science
201:875-880.

Hackett, W.F., W.J. Connors, T.K. Kirk and J.G. Zeikus. 1977.
Microbial degradatidn of synthetic 14C-labelled lignins
in nature: lignin biodegradation in a variety of natural
materials. Appl. Environ. Microbiol. 33:43-51.

Hall, P. 1980. Enzymatic transformations of lignin: 2. Enzyme
Microb. Technol. 2:170-176.

Hall, P., W.G. Glasser and S.W. Drew. 1980. Enzymatic transfor-
mations of lignin, p. 33-49.1 Ig_T.K. Kirk, T. Higuchi and
H.-m. Chang (eds.) Lignin biodegradation: microbiology,
chemistFy and potential applications, CRC Press, West Palm
Beach, L.

Hamilton, G.A. 1974. Chemical models and mechanisms for oxygen-
ases, p. 405-451. Ig_0. Hayaishi (ed.), Molecular mechanism
of oxygen activation, Academic Press, New York, NY.

Jeffries, T.W., S. Chqj and T.K. Kirk. 1981. Nutritional regu-
lation of ligninfidegradation in Phanerochaete chrysosporium.
Appl. Environ. Microbiol. 42:290-296.

Kellogg, E.W. and I. Fridovich. 1975. Superoxide, hydrogen
peroxide and singlet oxygen in lipid peroxidation by a
xanthine oxidase system. J. Biol. Chem. 250:8812-8817.

Keyser, P., T.K. Kirk and J.G. Zeikus. 1978. Ligninolytic enzyme
system of Phanerochaete chrysosporium: .synthesized in the
absence of lignin in response to nitrogen starvation. J.
Bacteriol. 135:739-745.

 

Kirk, T.K. 1971. Effects of microorganisms on lignin. Annu.
‘ Rev. Phytopath. 9:185-210.

Kirk, T.K. and H.-m. Chang. 1975. Decomposition of lignin by
white-rot fungi. II. Characterization of heavily degraded
lignins from decayed spruce. Holzforschung 28:218-222.

Kirk, T.K., W.J. Connors and J.G. Zeikus. 1976. Requirement for
a growth substrate during lignin decomposition by two wood—
rotting fungi. Appl. Environ. Microbiol. 32:192-194.

Kirk, T.K. 1981. Toward elucidating the mechanism of action of
the ligninolytic enzyme system in basidiomycetes, p. 131-
148. _I__11A.,Hollaender (ed.), Trends in the. biology of fer-
mentation for fuels and chemicals, New York, NY.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

150

Kong, S. and M.J. Davison. 1989. The role of interactions between
0 , H O , .OH, e , and O in free radical damage to biologi-
cgl s§s€ems. Arch. Bioc em. Biophys. 204:18-29.

Lai, Y.Z. and K.V. Sarkanen. 1971. Isolation and structural
studies. p. 165-240. In K.V. Sarkanen and C.H. Ludwid (eds.)
Lignins: occurrence, formation, structure and reactions,
Wiley-Interscience, New York, NY.

Lundquist, K., T.K. Kirk and W.J. Connors. 1977. Fungal degrada-
tions of kraft and lignosulfonates prepared from synthetic
14C-lignins. Arch. Microbiol. 112:291-296.

McCord, J.M 1974. Free radicals and inflammation: protection
of synovial fluid by superoxide dismutase. Science 185:
529-532.

Nakatsubo, F., 1.0. Reid and T.K. Kirk. 1981. Involvement of
singlet oxygen in the fungal degradation of lignin. Bio-
chem. Biophys. Res. Commun. 102:84-91.

Nofre, C., A. Cier and A. Lefier. 1961. Activation de l'oxygene
moleculaire en solution aqueuese par les metalliques et
leurs chelates. Bull. Soc. Chim. Fr. 530-535.

Pryor, N.A. 1978. The formation of free radicals and the con-
sequences of their reactions jg_vivo. Photochem. Photobiol.
28:787-801.

Reid, I.D. 1979. The invluence of nutrient balance on lignin
degradation by the white-rot fungus Phanerochaete chrysosporium.
Can. J. Bot. 57:2050-2058.

Root, R.K., J. Metcalf, N. Oshima and B. Chance. 1975. H 02
release from human granulocytes during phagocytosis 1.
Documentation, quantitation and some regulating factors.
J. Clin. Invest. 55:945-955.

Shimada, M. 1980. Stereobiochemical approach in lignin biode-
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T. Higuchi, H.-m. Chang (eds.) Lignin biodegradation:
microbiology, chemistry and potential applications, CRC Press,
West Palm Beach, FL.

Tien, M. and S.D. Aust. 1982. Hydroxyl radical involvement in su-
peroxide-dependent lipid peroxidation. Arch. Biochem.
Biophys. (in press).

Vance, C.P., T.K. Kirk and R.T. Sherwood. 1980. Lignification as
a mechanism of disease resistance. Ann. Rev. Phytopathol.
18:259-280.

36.

37.

151

Van Hemmen, J.J. and W.J.A. Meuling. 1975. Inactivation of bio-
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and their dismutation products: singlet molecular oxygen
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Walling, C. 1975. Fenton's reagent revisited. Acc. Chem. Res.
8:125-131.

APPENDIX 2

APPENDIX 2

Assay of Ligninolytic Activity (MC-lignin -14

by Phanerochaete chrysosporium:
Evaluation of methods for the.recovery and
quantification of 4C02

C02)

 

Introduction

The biodegradation of lignin is freqUently assayed by determining

f 14 14

C-synthetic lignin or C-(lignin labelled) lignocellu-

4

the amount 0

lose respired as 1 C02 (2). Due to the low rates of degradation ob-

served, it is sometimes necessary to measure very low levels (SO-100 dpm)
of radioactivity and thus it is critical that the methods used to re-
cover and quantitate the 14CO2 be carefully optimized and employed.

These studies were undertaken to evaluate the effect of several para-

meters on 14CO2 quantification.

14

Evaluation of methods to trap CO

2

Two different methods for trapping 14

CO2 were evaluated. In one
method, the 14CO2 was trapped directly in KOH and in the second method
14CO2 was flushed from the headspace of the flask and trapped in scin-
tillation cocktail which contained one of several organic amines. In
the reaction mixtures used in these experiments, the 14CO2 was produced

14

by acidification of alkaline solutions to which NaH CO3 had been

added.

152

153

14 14

1. Use of KOH to trap C02. To evaluate the trapping of CO2 directly
in KOH, a known amount of NaH14CO3 in 5 ml of slightly alkaline water

was added to a 50 m1 Erlenmeyer flask which was then closed with a

serum stopper from which a small polypropylene well (Kontes, Co.,
Vineland, NJ) containing 300 pl 0.5 M KOH was suspended. To acidify

the reaction mixture, one ml of 1 M HCl was injected through the serum

stopper into the NaH14

CO3 solution. The reactions were allowed to equili-
brate for at least fourhours at room temperature after which the KOH

from the center well was transferred to 10 ml of scintillation cocktail
which contained ACS (Amersham-Corp., Arlington Heights, IL)-water-O.2

M KOH (200:15:2) (3). The amount of 14CO2 recovered was determined

by liquid scintillation counting. The counting efficiency of the scin-
tillant was determined to be 89.3% using a 14C-toluene standard. Ex-

perimental results showed.that 97.1% of the added NaH14

as 1400

CO3 was recovered
2.

Upon addition of acid to the medium, the equilibrium between CO2
(g), HCO3' and CO3: shifts in favor of C02(q). This would favor

14C-

the recovery of 14C02, produced through microbial metabolism of
lignin, which may be dissolved in the medium.

In some instances, acidification of the medium or reaction mix-
ture is not desired. The following experiments were conducted to de-
termine how effectively 14002 could be recovered when NaH14CO3 was added
to a buffered medium (pH 6.8), which was not subsequently acidified.
Ten ml of 20 mum-Po4 buffer (pH 6.8) and aliquots of NaH14co3
were placed in 50 m1 Erlenmeyer flasks which were immediately sealed

with serum stoppers as described above. One ml amounts of 1.0 M HCl

154

were added to some of the flasks and no1additions were made to the re-
maining flasks. After allowing 48 h for equilibration of the gases,
the trapped 14CO2 was quantified. There was no difference in the ef-

fectiveness of 14CO2 recovery between the acidified medium and the buf-

14 14

fered medium with 96.0% of the added NaH CO3 recovered as

14

002 from
acidified reactions and 96.9% recovered as CO2 from reactions which were
not acidified.

14

2. Trapping of CO2 by flushing of headspace. Previous investigators

have shown that the p02 in the culture atmosphere markedly affects the

 

rate and extent of lignin degradation by E, chrysosporium (1). In the
procedure described above, the flask atmosphere .Was' not replenished with
air and remains closed to the atmosphere except for the short periods
of time required to change the CO2 traps (serum stoppers and 0.5 M

KOH in the center well). This would probably result in the partial
depletion of oxygen in the culture headspace which would have a dele-
terious effect on lignin degradation. Furthermore, the effectiveness
of 14CO2 recovery using this method was compromised in some instances
due to the poor fit between the serum stopper and the Erlenmeyer flask.

The following experiments were conducted to evaluate the trapping

14 14

of CO2 by flushing the flask headspace and trapping the CO2 in scin-

tillation fluid which contained an organic amine. The experiments were
conducted by placing 200 pl of NaH14CO3 in a dry 125 ml Erlenmeyer flask
and closing the flasks with rubber stoppers fitted with 2-5 mm glass
tubes. One of the tubes extended into the flask to within 1-2 cm of the

acid solution and the second tube extended to the bottom of the stopper.

On the outside of the flasks, both tubes were connected via latex

155

tubing to glass wool-plugged filters and the flasks were closed by
clamping the rubber tubes. Ten ml of 1 M HCl. was then injected into the
flasks through the rubber stopper using a syringe with an 18 guage needle.

To flush the 14

CO2 present in the flask headspace, the tube which ex-
tended into the flask was connected to a COZ-free air supply and the
second tube was connected to a 1 ml syringe barrel fitted with a 1 in
25 guage needle. The syringe barrel was then placed in a polypropylene
scintillation vial containing 10 ml of liquid scintillation cocktail.
The CO2 was scrubbed from the air used to flush the flasks by bubbling
it through 0.02 M NaOH which contained 0.002% phenol red and then
through distilled water to remove aerosols containing NaOH. The latex
tubing delivering the COz-free air was then connected to a gassing mani-
fold constructed of brass aquarium gang valves. The flasks were flushed
for 60 min at a flow rate of 60 ml min'l. Flushing at 75 or 100 ml
min'1 did not improve recoveries of 14CO2 (data not shown).

The overall efficiencies (counting efficiency and their ability
to trap 14C02) of four liquid scintillation counting (LSC) cocktails
were determined. The following LSC cocktails were examined: (a) ACS
(Amersham-Searle, Arlington Heights, IL)-methanol-ethanolamine (Aldrich
Chemical Co., Milwaukee, WI) (5:4:1); (b) 3a20 (Research Products In-
ternation, Elk Grove Village, IL)-methanol-ethanolamine (5:4:1); (c)
Count-sorb (Research Products International); and (d) ACS:methanol:
phenethylamine (Eastman Kodak Chemicals, Rochester, NY). The results
showed that the overall efficiency of LSC cocktails (a), (b), (c)
and (d) were 94.8%, 93.8%, 92.8% and 90.1%, respectively. Thus, all the
scintillation cocktails were efficient. Cocktails (c) and (d) were

excluded from further consideration since phenethylamine and Count-

156

sorb are rather expensive and phenethylamine is noxious relative to
ethanolamine. Cocktail (a) uses ACS, a xylene based, water compatable
LSC cocktail; whereas cocktail (b) uses 3820, a toluene based, liquid
scintillation cocktail which is not compatable with aqueous samples ex-
cept at very low concentrations of water. Therefore, cocktail (a) was
chosen for future use on the basis of its greater versatility in bio-

logical experiments.

Effect of flushing time on the recovery of 14CO2

To optimize the recovery of 14002 during the flushing of cultures,
the 14CO2 was produced through the metabolism of CH314COONa by
Nocardia s2, DSM 1069 (4). The basal medium used contained perl
of distilled water: yeast extract, 0.1 g; NaZHPO4, 1.5 g; NaH2P04,
1.5 9; NaCl, 0.25 g; (NH4)ZSO4, 1.25 g; MgSO4-7 H20, 0.01 g; FeSO4-7

4 dpm). The final

H20, 0.002 g; and sodium acetate, 1.36 g (8.6 X 10
pH of the medium was adjusted to 6.8, dispensed into 125 ml Erlenmeyer
flasks (10 ml/flask) and sterilized by autoclaving at 121°C for 15 min.
The flasks were inoculated with 0.5 m1 of a 24 h old culture

which had been grown in the same medium containing nonradioactive ace-
tate and closed with sterile rubber stoppers as described above. The
cultures were then incubated for 16 h at 30°C on a rotary shaker water
bath. To quantify the respired 14C02, the flasks were flushed with
COZ-free air for a total time of 60 min. After 15, 30 and 45 min of
flushing, the CO2 traps (scintillation vials with cocktail a) for some
of the flasks were replaced with fresh CO2 traps. Thus, the amount of
14CO2 trapped after 15, 30, 45 and 60 min was determined. After flush-

ing the flasks, 200 pl of the culture was removed and placed in a vial

157

containing 10 ml of LSC cocktail (a). The radioactivity present in the
vials was determined by liquid scintillation counting using a Searle
Model Delta 300 liquid scintillation counter (Searle, Arlington Heights,
IL) with preset discrimination cartridge (3H and 14C cartridge).

During the 16 h incubation period, approximately 61% of the

added CH314COONa was respired as 14

C02. After 15, 20, and 45 min of
flushing, 87, 93, and 96% respectively of the total 14002 produced

had been flushed from the flasks and trapped in the CO2 traps (Table 1).
Flushing the flasks for 60 min did not significantly increase the re-
covery of 14CO2 compared to that observed with 45 min flushing. Total
recovery of the added 14C was greater than 95%. Based on these data,

a 45 min flushing time (equivalent to 7 total flushings of the headspace)

14

was determined to be optimal for maximal recovery of CO2 and to ensure

adequate aeration of the culture.

Determination of quench correction curves

A quench correction curve for cocktail (a) (ACS-methanol-ethano-
lamine, 5:4:1) was constructed using the sample channels ratio method.
Ten ml of cocktail (a) was added to five replicate vials. 14C-Toluene
(lOO ul, 2.125 X 104dpm, ICN, Davis, CA) was added to each vial. The
radioactivity present (counts per minute, cpm) and the sample channels
ratio were determined as described above. In four successive trials,
30 ul of CH3C1 was added to each vial and the cpm determinations were
repeated. All determinations of radioactivity were made to i 1% and the
counting efficiencies were calculated. The regression equation des-
cribing the plot of counting efficiency vs sample channels ratio was

y = -51,74 x +1132.23(r2 = -O.98). The plot of the counting efficiency

158

vs sample channels ratio (SCR) was nonlinear at SCR values greater than
0.920 and therefore, the equation should not be used when the SCR is

larger than this value.

159

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160

LITERATURE CITED

Bar-Lev, 5.5. and T.K. Kirk. 1981. Effects of molecular oxygen on
lignin degradation by Phanerochaete chrysosporium. Biochem.
Biophys. Res. Commun. 99:373-378.

Crawford, R.L., L.E. Robinson and A.M. Cheh. 1980. 14c-Labened
lignins as substrates for the study of lignin biodegradation
and transformation, p. 61-76. Ig_T.K. Kirk, T. Higuchi and
H.-m. Chang (eds.) Lignin biodegradation: microbiology,
chemistry and potential applications, CRC Press, West Palm
Beach, FL.

Huskisson, N.S. and P.F.V. Ward. 1978. A reliable method for
scintillation counting of 14CO2 trapped in solutions of sodium
hydroxide, using a scintillant suitable for general use. Int.
J. Appl. Rad. and Isotopes 29:729-734.

Trojanowski, J., K. Haider and V. Sundman. I977. Decomposition of
14C-labelled lignin and phenols by a Nocardia_§g. Arch.
Microbiol. .114:149-153.

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