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This is to certify that the

dissertation entitled

The Peroxidases of Phanerochaete Chrysosporium:
Culture Modelling and Application

presented by

Frederick Carl Michel Jr.

has been accepted towards fulﬁllment
of the requirements for

Ph.D. degreein Chem. Engr.

 

 

Major professor

Date 2/24/92

 

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cmmaﬁt

THE PEROXIDASES OF PHANEROCHAETE CHR YSOSPORI UM :
CULTURE MODELLING AND APPLICATION

by

Frederick Carl Michel Jr.

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Chemical Engineering

1991

63% /73 7

ABSTRACT

THE PEROXIDASES OF PMNEROCHAETE CHRYSOSPORIUM:
CULTURE MODELLING and APPLICATION
By:
Frederick Carl Michel Jr.

Many industrially signiﬁcant microbial products are produced during secondary
metabolism by fungal pellets. The white-rot basidiomycete Phanerochaete
chrysospon'lan produces extra-cellular lignin peroxidases (LIP) and manganese
peroxidascs (MN?) which have been implicated in the degradation of lignin and many
aromatic xenobiotics during secondary metabolism. A novel unstructured kinetic model
for mycelial pellet cultures of P. chrysosporiwn is presented which describes the culture
dry weight, respiration, glucose consumption, nutrient nitrogen uptake, pellet size and
oxygen effectiveness factor during the lag, primary growth, secondary metabolic, and
death phases. Intrinsic Michaelis-Menten kinetic parameters for respiration were
determined (Vmax= 03610.1 g/m3 pellet/s, Km= 0.51:0.3'g/m3) by measuring oxygen
concentration proﬁles using oxygen microelectrodes and were conﬁrmed by measuring
the rates of oxygen depletion and carbon dioxide evolution. The model shows good
agreement with experimental data from cultures with initial glucose concentrations of
5,000, 10,000, 11,000 and 15,000 g/rn3 and initial nutrient nitrogen concentrations of 2,
39 and 117 g/m3. Model simulations for air-ﬂushed, high nitrogen and large pellet
cultures, in which the production of LIPs and MNPs is greatly reduced, show that in
these cases the oxygen effectiveness factor is signiﬁcantly less than one.

The role of LIPs and W3 in decolorizing industrial and synthetic paper mill
bleach plant efﬂuents (BPE) containing soluble chlorolignin is studied by controlling the
levels of production of LIP and MNP using manganese, nutrient nitrogen and mutant

strains. Negligible BPE decolorization is exhibited by cultures which produce no LIPs or
MNPs. lip mutant cultures of P. chrysosporr’um, and wild-type cultures grown with 100
ppm manganese, produce MNPs but not LIPs but show signiﬁcant decolorization
activity. Also, high rates of BPB decolorization are seen in 3 day old cultures which
exhibit a relatively high level of MNP activity but little or no LIP activity. In cultures
with 0 ppm Mn(lI), high levels of LIPs but low levels of MNPs are produced and the rate
and extent of BPE decolorization is relatively low. These results indicate that MNPs play
a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.

To Emily

ACKNOWLEDGEMENTS

My sincerest thanks go to my advisor Professor Eric A. Grulke for his excellent
guidance, friendly manner and professional example and to Professor C.A. Reddy who
also played an integral part in my supervision and in my professional development.
Without their cooperation, patience and support this work would not have been possible.
I would also like to thank Professor Daina Breidis and Professor Hans E. Grethlein for
critically reading the manuscript and serving on my advisory commitee. Special thanks
go to Professor Grethlein who graciously provided desk and laboratory space. I learned
many of the experimental techniques used in this dissertation from Dr. Carlos Dosorctz
and Dr. S.B. Dass. I would like to thank them for their assistance and example.

I appreciate the ﬁnancial support provided by the Department of Chemical
Engineering, the Michigan Research Excellence Fund, Michigan Biotechnology Institute
and the Graduate Ofﬁce at Michigan State University.

My deepest thanks go to my parents whose love, support, encouragement and
guidance have supported me throughout my life.

Finally, I would like to acknowledge the Colonel Parker's regulars.

Table of Contents

List of Tables

 

List of Figures

 

Notation

 

 

Chapter I: Introduction
Chapter 11: Literature Review

 

2.1 Mycelial pellet culture modelling
a. Oxygen mass transfer limitations in mycelial pellets
2.2 Kraft bleach plant effluents (BPE)
a. Formation of BPEs
b. Treatment technologies
2.3 Degradation of BPE by white-rot fungi
2.4 Enzymes involved in lignin biodegradation and BPE decolorization
2.5 Research objectives

Chapter III: Theoretical .....

 

3.1 Kinetic model development

a. Lag phase

b. Primary growth phase

c. Secondary metabolism

d. Death phase
3.2 Determination of the rate limiting process in pellet culttnes

a. Estimation of external and intraparticle mass transfer resistances
3.3 Model for the intraparticle diffusion and reaction of oxygen

in mycelial pellets

a. Prediction of oxygen concentration profile in mycelial pellets

b. Calculation of the effectiveness factor for Michaelis-Menten kinetics

c. Unsteady state oxygen balance in sealed cultures
(1. Determination of the effective diffusivity in mycelial pellets
3.4 Model for the decolorization of plant efﬂuent

Chapter IV: Materials and Methods -- -

 

4.1 Microorganism
4.2 Culture conditions
a. Shake ﬂask culture conditions
b. Bioreactor culture conditions
4.3 Substrate assays
a. Glucose, carbohydrate, ammonium and other measurements
b. Oxygen proﬁle measurement
c. Manganese determination

iv

vii

12
12
14
15
18
21

22

22

28
27
28
28
31

31
32
34
35
39

41
41
41
41
42
43

4.4 Product Assays
a. LIP and MNP activity
b.pHoptimaofLIPandMNP
c. Pellet characteristics
d. SDS -PAGE electrophoresis
e. Fast protein liquid chromatography (FPLC)
4.5 Preparation and characteristics of Kraft bleach plant effluents
a. BPE color measurement
b. Solubility of BPE
c. Molecular weight of BPE
4.6 Computational methods

Chapter V:

 

Model for submerged, peroxidase producing, mycelial pellet cultures
of P. chrysosparium

Part I: Results
5.1 Characteristics of mycelial pellet cultures of P. chrysosporium
5.2 Estimation of mass transfer resistances in mycelial pellet cultures
a. Determination of the effective diffusivity in mycelial pellets
5.3 Determination of kinetic model parameter values
a. Kinetic coefﬁcients for the lag phase and primary growth phase
b. Kinetic coefficients for secondary metabolism
c. Kinetic coefﬁcients for the death phase
d. Mycelial pellet characteristics
5.4 Determination of kinetic coeﬁicients for the oxygen utilization model
5.5 Model test cases
5.6 Bioreactor studies

Part II: Discussion
5.7 Characteristics of cultures of P. chrysosporr‘um
5.8 Kinetic model for mycelial pellet cultures
5.9 Oxygen utilization in mycelial pellet cultures of P. cluysospon'wn
5.10 Kinetic model simulations
a. Air-ﬂushed cultures
b. High nutrient nitrogen cultrn'es
c. Large mycelial pellet cultures
d. Production of peroxidases by pellets with low effectiveness factors

51
51
55
58

62
62

65
74
78

79
79
79
82
85
85
88
88
91

Chapter VI: Application

 

Application of the peroxidases of P. chnsosporium to the
decolorization of Kraft bleach plant effluents (BPE)

Part I: Results
6.1 Decolorization of BPE by cultures of P. chrysosporium
6.2 Effect of varying MNP and LIP levels on BPE decolorization
6.3 BPE decolorization by lip and per mutant culttnes
6.4 In vitro decolorization of BPE

Part 11: Discussion
6.5 Role of MNPs and LlPs in the decolorization of BPE

Chapter VII: Conclusions and Recommendations

 

7.1 Conclusions
7.2 Recommendations

Bibliography

 

 

Appendices

92

92
92
100
106
111

113
113

118

118
119

120
129

Table 2.1

Table 2.2

Table 3.1.
Table 5.1

Table 5.2

Table 5.3
Table 5.4

Table 6.1

Table 6.2

Table 6.3

Appendix 1

Appendix 2.
Appendix 3

List of Tables

Effect of the rate of agitation on mycelial pellet size and the
production of lignin peroxidase (LIP) by nitrogen-limited
cultures of P. chrysosporium (from Michel er al., 1990).

Summary of studies of mycelial pellet growth and
respiration.

Literature values for the diffusivity of oxygen in water.

Observable moduli (O) for substrates of P. chrysospoﬁum
using worst case assumptions.

Physical characteristics of mycelial pellet cultures of
P. chrysosporiran.

Kinetic model parameter values.
Initial conditions for kinetic model test cases.

Apparent ﬁrst order rate constant for decolorization of
synthetic Kraft bleach plant effluent (BPE) by nitrogen-
limited cultures of P. chrysosporium as a function of the
day of BPE addition.

Apparent ﬁrst order rate constants for decolorization of
industrial Kraft bleach plant effluent (BPE) by P.
chrysosporium as affected by nitrogen and manganese levels
in the medium.

Decolorization of Kraft BPE by FPLC puriﬁed peroxidases
of P. chrysosporium in a cell ﬁee system.

Extracellular peroxidase activity in air-ﬂushed and oxygen-
ﬂushed agitated cultures of P. chtysosporiwn (as reported
by Dosorctz et 01., 1990).

Solubility of synthetic and Industrial BPE.

Effect of pH on the activity of lignin peroxidases and
manganese peroxidases.

58

61

74

102

111

131
132

Appendix 4

Appendix 5

Appendix 6

Appendix 7

Appendix 8

Appendix 9

Appendix 10

Appendix 11

Appendix 12

Appendix 13

Appendix 14

Appendix 15

Time course of nutrient nitrogen (ammonium), biomass
(culture dry weight). glucose. soluble carbohydrate.
extracellular protein. acetate and extracellular LIP and MNP
activity in agitated control cultures of P. chrysospon'wn
BKM- -l767.

Manganese concentration in soluble and insoluble fractions
of cultures of P. chtysosporiwn with three initial soluble
manganese concentrations as measured using atomic

absorption spectrophotometry.

Decolorization of BPE as a function of the day of BPE
addition.

Decolorization. culture dry weight and carbon dioxide
evolution by cultures of P. chrysosporiwn amended with
synthetic and industrial Kraft bleach plant efﬂuents (BPE).

Effect of nutrient nitrogen (39 and 390 g/m3) and
manganese (0. 12 and 40 ppm) on the decolorization of
BPE. the depletion of glucose and the production of
extracellular peroxidases.

Residual color (percent) in wild-type and mutant strain
cultures of P. chrysospon’wn (3,000 color units added).

Time course of the natural log of culture dry weight divided
by average initial culture dry weight (Ln [X/Xo.avg])
during lag and primary growth phases.

Pellet characteristics in nitrogen-limited agitated culture of
P. chrysosporl'um.

Oxygen concentration proﬁles in 1.2.4.5 and 12 day-old.
mycelial pellets of P. Chrysosporium measured using an
oxygen microelectrode.

Carbon dioxide evolution in mycelial pellet cultures of P.
chrysosporiwn.

Respirometer data for agitated cultures of P.
chrysosporium.Calculated value of the average Vmax.

Data for model test cases.

133

135

137

140

141

142

143

145

146

152

. Appendix 16
Appendix 17

Appendix 18

Appendix 19

Appendix 20.

Appendix 21

Appendix 22

Appendix 23

Sample output from shake ﬂask simulation computer
program.

Oxygen effectiveness factor as a function of mycelial pellet
radius and liquid phase oxygen concentration.

Glucose consumption and 002 evolution in air-ﬂushed
cultures as reported by Dosoretz at 01.. 1990.

Data for agitated cultures of P. chrysosporium grown with
methanol as the sole carbon and energy source.

Data for agitated cultures of P. chrysosporiwn grown with
ethanol‘ as the sole carbon and energy source.

Shake ﬂask simulation program listing.

Computer listing of oxygen proﬁle gradient simulation
program.

Data from bioreator studies.

155

158

161

162

163

164

166

167

Figure 2.1

Figure 2.2

Figure 2.3

Figure 3.1

Figure 3.2

Figure 3.3

Figure 4.1

Figure 4.2

Figure 5.1

Figure 5.2

List of Figures

Influences on mycelial pellet cultures from van Suijdam er
al.. (1982).

lignin peroxidase (LIP) and manganese peroxidase (MNP)
activities in air-ﬂushed and oxygen ﬂushed cultures from
Dosoretz er al.. (1990). Cultures were grown in nitrogen
limiwd conditions.

The process of brown pulp bleaching and the formation of
kraft bleach plant effluents (BPE) at a Michigan paper mill.

Summary of kinetic model equations for mycelial pellet
cultures of P. chrysosporiwn.

The process of mass transfer and reaction in mycelial pellet
cultures.

A schematic representation of an apparatus for measuring the
effective diffusivity in mycelial pellets.

Lignin peroxidase and manganese peroxidase enzyme
activities as a function of pH. Samples were buffered with
acetate (0.1 M)forpH4to7ortartarate(0.l M)forpH2to
3.5.

The solubilities of synthetic BPE and industrial BPE in
culture medium as a function of medium pH.

Biomass production. nitrogen and glucose utilization. and
production of exuacellular peroxidases in agitated. nitrogen-
limited (2.2 mM) cultures of P. chrysospon’um BKM-F-1767.
A. Time course of ammonium. glucose. and biomass (cultme
dry weight). Values represent averages for triplicate cultures.
B. Time course of lignin peroxidase (LIP) and manganese
peroxidase (MNP) activities and total extracellular protein
concentration.

C. Mycelial pellet size.

D. Carbon dioxide evolved and oxygen utilized.

Soluble and inrloluble manganese concentration as a function
of time in nitrogen-limited cultures of P. chrysosporl'um
initially containing 12 ppm and 40 ppm Mn(II).

11

23

29

37

45

49

52-53

56

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Plot of the logarithm of the biomass (X) divided by the
average initial biomass (Xo.avg) as a function of culture age
in nitrogen-limited mycelial pellet cultures of P.
chrysosporiwn. This plot was used for the determination of
pmax and ﬂag. Open diamonds are experimental data from
cultures on four different occasions. The line is a linear
regression ﬁt of the data points (um=3.9 x 10-5 s-l,
tlag=7.9 h).

Mycelial pellet radius in submrged cultures of P.
chrysosporiwn grown nitrogen-limited medium (Condition
2). solid line: kinetic model prediction. squares: experimental
data points.

Oxygen concentration proﬁle in a mycelial pellet of P.
chrysosporium (diam.=.00185 m) as determined using an
oxygen micro-electrode.

The solid line is as predicted by the kinetic model (Km=0.5
g/m3. Vmax=0o75 g/m3ls. De=2.9 x 10'9 m2/s).

Carbon dioxide evolution in nitrogen-limited mycelial pellet
cultures of P. chtysosporiwn (Condition 2). The solid bars
are as predicted by the kinetic model (Km=0.5 g/m3,
vm=o.76 g/m3/s. De=2.9 rt 109 m2/s.). Cross-hatched
bars are experimental data.

Typical plot of the rate of oxygen depletion (v g/m3/s) from
the culture ﬂuid versus the liquid phase oxygen concentration
(C1 g/m3) from nitrogen-limited. mycelial pellet cultures of
P. Chrysosporiwn as measured using a respirometer. Solid
line is a curve ﬁt using the method of Wilkinson (1961).
Open squares are experimental data from three consecutive
measurements.

The rate of oxygen uptake by cultures of P. chrysosporium as
a function of culture age as measured using a respirometer:

A. Experimental data.

B. Model prediction using kinetic parameter values from
Table 5.3.

C. Model prediction assuming an effectiveness factor of one
(Emm = l) at all times.

69

70

71

72-73

Figure 5.9

Figure 5.10

Figure 5.11

Figure 5.12

Figure 5.13

Figure 5.14

Figure 5.15

Figure 5.16

Culture dry weight (X). glucose concentration (G). and
nutrient nitrogen (ammonium) concentration (N) as a
function of time in cultures of P. chrysosporiwn gown under
various initial glucose and nutrient nitrogen conditions. Solid
lines are as predicted by the kinetic model. Open squares are
experimental data for glucose. closed diamonds are
experimental data for culture dry weight and open triangles
are experimental data for ammonium.

A. Condition 1; Go:- 5.000 g/m3, No= 39 g/m3.

13. Condition 2; oo= 10.000 g/m3, No= 39 g/m3.

C. Condition 3; oo= 15,000 g/m3, No: 39 g/m3.

D. Condition 4; 00:: 11.000 g/m3, No: 2 g/m3.

E. Condition 5; Go= 11,000 g/m3, No=117 g/m3.

CharacteristicsofastirredtankbioreactorcultureofP.
chrysosporr’wn gown in nitrogen limited medium.

The effect of the mycelial pellet density (p) on the biomass.
glucose uptake rate. oxygen effectiveness factor and mycelial
pellet radius as predicted by the kinetic model.

Three dimensional representation of the oxygen eﬂ‘ectiveness
factor (Emm) for mycelial pellets of P. Chrysospon’wn as a
function of the liquid phase oxygen concentration (C1) and

the characteristic pellet radius (R).

Total oxygen and carbon dioxide and oxygen eﬂ‘ectiveness
factor in nitrogen-limited cultures of P. Chrysosporium as
predicted by the kinetic model.

Glucose consumption and carbon dioxide production in air-
ﬂushed shake ﬂask cultures of P. chrysosporium as predicted
bytheldneticmodelaineandsolidbars)andasreportedby
Dosoretz er al. (1990) (open squares and cross-hatched bars).

Model simulation for cultures gown in high nutrient
nitrogen condition; 60:10000 g/m3, No=390 g/m3.

Kinetic model simulation for nitrogen-limited cultures of P.
chrysospoﬁwn with large pellets (R=3mm). (This case is
analogous to cultures of large pellets gown at low agitation
speeds as described by Michel er al.. 1990).

75-76

78

81

89

Figure 6.1

Figure 6.2

Figure6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Fir-Ire“

Figure6.9

Decolorization of Kraft bleach plant effluent (BPE) when
added to cultures on diﬁ‘erent days of incubation. Data for an
uninoculated control are also presented. Values represent
means for duplicate cultures.

The decolorization of synthetic BPE and industrial BPE by
nitrogen-limited. agitated cultures of P. chrysospan'wn BKM-
F- 1767. BPE was added to a ﬁnal concentration of 3.000
C.U.

SDS-PAGE of extracellular culture ﬂuid from P.
Chrysosporiwn cultures aged 2 to 9 days. Electrophoretic
mobility of FPLC-puriﬁed LIP isozymes (HZ. H6. H8. and
H10) and MNP isozyme H4 is presented.

SDS-PAGE proﬁle of extracellular ﬂuid from control
cultures on days 4 through 7 and from cultures which were
amended with (A) industrial BPE and (B) synthetic BPE.

The initial rate of synthetic Kraft bleach plant efﬂuent (BPE)
decolorization as a function of initial BPE concentration.
Synthetic BPE was added to nitrogen-limited cultures of P.
chnsaspan'wn 4 days after culture inoculation.

The effect of Mn(II) and nitrogen levels on LIP and MNP
activities of wild type P. chrysosporiwn. Values presented
are averages for triplicate cultures. A. MNP activity. B.
LIP activity.

SDS-PAGE of peroxidases in extracellular culture ﬂuid of P.
Chrysosporium. at the time of BPE addition. 4 days after
incubation at 39° C. Mn(II) concentrations were 0 ppm. 12
ppm and 100 ppm. respectively. in low. basal and high
Mn(II) nitrogen limited (2.4 mM N) cultmes. Nitrogen
sufﬁcient (24 mM N) cultures contained 12 ppm Mn(II).

The effect of Mn(II) and nitrogen levels on BPE
decolorization by cultures of P. chrysosporr’wn. BPE was
added on day 4 of incubation.

SDS-PAGE of peroxidases in extracellular culture ﬂuid of P.
chrysospon'um wild type strain BKM-F-1767. a lip mutant
strain and a per mutant strain at the time of BPE addition.

93

95

103

104

105

108

Figure 6.10

Figure 6.11

Figure 6.12

Lignin peroxidase (LIP) and manganese peroxidase (MNP)
activities in wild-type (BKM-F-l767). lip' mutant and per'
mutant cultures of P. chrysosporium. Cultures were gown in
low nitrogen medium which contained 12 ppm Mn(II).

Decolorization of BPE by P. Chrysosporl'um wild-type strains
ME446 and BKM-F 1767 and two mutants. per and lip of
ME446. BPE (3000 C.U.) was added to each of the cultures
on the peak day of MNP activity and the extent of BPE
decolorization was monitored each day for the next 5 days.
The values shown are averages of duplicate cultures on two
separate occasions.

Proposed reaction scheme for the degadation of BPE and the

deposition of manganese by manganese peroxidases (MNP)
of P. chrysospon’um.

xiv

109

110

116

Notation

 

Non-zero roots of equation in section 3.11

XV

Symbol Deﬁnition Unit
Bi Biot number (-)
C Oxygen concentration g[1113
Cb Uniform bulk solute concentration g/mB
Ci Oxygen concentration in pellet g1,1,3
Cg Gas phase oxygen concentration g/mB
C1 Liquid phase oxygen concentration g/m3
Cs Oxygen concentration at pellet surface g/m3
Csat Oxygen equilibrium concentration in media g/m3
C.U. Pt-Co Color Units (-)
D Diffusivity of oxygen in water m2/s
De Effective diffusivity of oxygen in pellets m2/s
Dslab Overall diffusivity in cell and gel matrix m2/s
E0 Effectiveness factor for zero order kinetics (-)
E1 Effectiveness factor for ﬁrst order kinetics (-)
E Effectiveness factor for oxygen (-)
Em Effectiveness factor for M.M. order kinetics (-)
fpg Glucose metabolism factor g/g
fr Glucose metabolism factor g/g
fx Glucose metabolism factor g/g
G Glucose concentration ”3
H Henry's law constant (-)
1 Index value for numerical method (-)
k apparent rate constant for decolorization dy-l
- kl Mass transfer coefﬁcient for oxygen m/s
Km Kinetic coeﬂ’rcient for oxygen 3,3,3
Ks Kinetic coefﬁcient for glucose g/m3
1 Gel matrix plate (or slab) thickness m
Mint Total solute diffused into slab at inﬁnite time 3,3,3
Mt Total solute diffused into slab at time t 8/1113
MW Molecular weight g/mol
a Number of steps in numerical method (-)
N Ammonium concentration g/m3
P Pressure atrn
pG Polysaccharide concentration m3
Q Ratio of total pellet volume to plate volume m3/m3
‘ln

Q02 Speciﬁc oxygen uptake rate

Qoznrax Maximum speciﬁc oxygen uptake rate
R Ideal gas law constant

R Characteristic pellet radius

r Distance ﬁ'om pellet center

R002 Bulk rate of carbon dioxide production
r02 Rate of respiration by pellets

R02 Bulk rate of oxygen consumption

R3 Bulk rate of glucose uptake

Rn Bulk rate of ammonium uptake

Rpg Bulk rate of polysaccharide synthesis
Rx Bulk rate of biomass production

T Temperature

tlag Length of the lag phase

v Reaction rate equation for oxygen

Vb, Head space volume

V1 Liquid phase volume

Vmax Rate constant for oxygen

Vpel Characteristic pellet volume

x Distance from the plate interface

X Biomass concentration (cell dry weight)
Xf Final biomass concentration

szpz C02 produced per oxygen consumed
Yg/Oz Glucose used per oxygen consumed
Yglx Growth yield coefﬁcient for glucose
Ynlx Growth yield coefﬁcient for nitrogen
Greek Symbols

a, Liquid volume divided by plate (or slab) volume
a; First order modulus

O Observed mass transfer modulus

n Pellet number

He Effective growth rate constant

um“ Growth rate constant

p Pellet density (dry weight/pellet volume)

xvi

s‘ 1
s" 1
m3 atmlmol/K

g/m3/s
g/m3/s
g/m3/s

CHAPTER]

INTRODUCTION

This study had two primary objectives reﬂecting the dual interests of the author.
Theﬁrstwastodevelopasimpleunsu'ucnuedldnedcmodeltodescribegowth.
respiration. substrate utilization and mass transfer in submerged batch cultures of the
white-rot fungus Phauerochaete cluysospan’um which describes the life-cycle phases of
primary gowth. secondary metabolism and death. One aim of the culture modelling was
to examine the effect of oxygen limitations on the production of the extracellular lignin
peroxidases (LIP) and manganese peroxidases (MNP) which are produced during
secondary metabolism and are primary components ofthe lignin degading system of this
organism. The second objective was to apply the lignin degading enzymes to the
treannent of paper mill bleach plant efﬂuents (BPE) which contain toxic chloroligrin
components and study the roles of LIP and MNP in BPE degadation.

The ligninolytic white-rot fungi such as Phanerochaere chrysosporiwn are the
primarydegadersofligninintheenvironmentThisorganismisoneofveryfew
organisms which can mineralize lignin completely to carbon dioxide (Kirk er al.. 1978).
In addition. P. chrysosparium can degade a wide range of xenobiotic chemicals
(Bumpus er al.. 1985) which are structurally similar to lignin such as DDT (Bumpus and
Aust. 1987). dioxins (Baron. 1984; Hammel er al.. 1986). chlorinated phenols (Lamar er
al.. 1990; Lin and Wang. 1990. Lackner er al.. 1991) and poly-aromatic hydrocarbons
(Hamrnel er al.. 1986). This ability is due to the non-speciﬁc nature of the lignin
degading system. P. chrysosporiwn produces two families of extracellular glycosylawd
heme Proteins. desisnawd lignin peroxidases (UPS) and MW P6101646“ (MNPS).
along with an Hzoz-generating system as the major components of its ligninolytic
system (Dass and Reddy. 1990; Gold er al.. 1989; Kirk and Farrel. 1987). This system is

2

expressed only during secondary metabolism and is triggered by carbon. nitrogen or
sulfur limitation (Jefferies et al.. 1981). Both LIPs and MNPs are families of isozymes.
They are heme glycoproteins which contain a single high spin ferric protoheme IX. The
LIPs catalyze the non-speciﬁc one-electron oxidation of non phenolic aromatic
substrates. including the Ca-Cp bond of lignin model compounds. yielding a multiplicity
ofﬁnalproducts (KirkandFarrel,1987). MNPs. on theotherhand. oxidize Mn(II) to
Mn(IIl). a highly reactive compound which oxidizes phenolic substrates (Gold er al..
1989. Paszczynski er al.. 1985. Kuwahara er al.. 1984). These enzymes are thought to be
directly involved in the rrrineralization of lignin by the white-rot fungi (Kirk and Farrel.
1987. Kuwahara er al.. 1984. ).

In submerged agitated cultures. P. chrysospon’um gows in the form of spherical
aggregates known as mycelial pellets (Burkholder and Sinnott. 1945). Studies have
shown that oxygen mass transfer limitations can inﬂuence the gowth kinetics of
mycelial pellets (Pirt. 1966; Metz and Kossen. 1977) and lead to oxygen starvation at the
pellet center (Schugerl er al.. 1983). An important engineering concept developed in
these studies was the oxygen effectiveness factor. deﬁned as the observed rate of oxygen
utilization divided by the rate expected without mass transfer limitations. Interestingly.
theproductionofLIPsandWPsbmehnuosporiwnhasbeen showntobegeatly
affected by the level of oxygen in the culture (Barlev and Kirk. 1981; Dosoretz er
al..1990a; Dosoretz and Grethlein. 1991) and by mycelial pellet size (Michel er al..
1990). However an analysis of mass transfer limitations in these cultrnes has not been
reported. One objective of this study was to determine the kinetics of oxygen mass
transfer and respiration in pellet cultures of P. chrysosporium and to examine the effect
ofoxygen limitations on the production of the LIPs and MNPs.

Although genes encoding LIP and MNP isozymes have been cloned and sequenced
(221mg er al.. 1991. de Boer et. al.. 1987; Tien and Tu. 1987; Naidu and Reddy. 1990).
introduction of these genes into simple. fast gowing microorganisms like Escherichia

3

coli and Saccharomyces cerevisiae has not resulted in the production of active LIPs or
MNPs. These organisms cannot glycosylate the LIPs or WPs and may not have been
able to fold the peroxidases properly or incorporate heme into the active site.

In order to produce active LIPs and MNPs for biotechnological applications.
bioreactor systems employing the white-rot fungi are necessary. For any bioreactor
design and scale-up. culture respiration. substrate utilization. biomass production and
product expression are inﬁrelated and are key design considerations. Previous models of
mycelial pellet culture growth and respiration largely consider only the primary (or
exponential) gowth phase of Aspergillus or Penicillium species. assume zero-order
kinetics for respiration and are tested against only one or two initial substrate conditions.
In practice. many industrially signiﬁcant products are secondary metabolites produced
after a limiting nutrient is exhausted. In addition Michaelis-Menten kinetics more
accruately describes the kinetics of microbial respiration. A central aim of this study was
to develop a simple unstructrned kinetic model. which describes the gowth. respiration.
substrate utilization and mass transfer in mycelial pellet cultures of P. chrysosporl’um
during the lag, primary growth, secondary metabolic and death phases.

In the paper industry. processes which chemically liberate residual lignin from
wood pulp require chlorine bleaching and result in approximately 3 billion gallons of
toxic and intensely colored waste efﬂuents annually (EPA report #600/8-80-042a). The
primary contributor to the color and toxicity of these streams is the bleach plant efﬂuent
(BPE) which contains high molecular weight, modiﬁed and chlorinawd lignin and its
degradation products (Boominathan and Reddy. 1991; Campbell. 1983; Huynh er al..
1985; Paice and Jurasek. 1984). This material is not readily degaded by conventional
bacterial water treatment processes (Boominathan and Reddy. 1991; Ericksson and
Kolar. 1985; Forney, 197 8) and typically enters receiving waters untreated. Cultures of
the white-rot fungi P. chrysosporiwn and Coriolus (Trametes) versicolor are able to

decolorize. dechlorinate and reduce the molecular weight of the chlorolignin component

4

of BPE. Although it has been assumed that the peroxidases of P. chrysosporium play a
role in the biodegadation of paper mill bleach plant efﬂuents. the actual contribution of
these enzymes has never been documented (Eaton et al.. 1983; Messner er al.. 1990;
Paice and Jurasek. 1984). A primary objective of this study was to determine the roles of
LIPs and MNPs in the degradation of BPE by P. chrysosporiwn.

5
CHAPTERII

LITERATURE REVIEW

2.1 Mycelial pellet culture modelling

Filamentous fungi. like the lignin degading basidiomycete P. chrysosporium.
commonly gow in the form of spherical mycelial pellets in agitated submerged cultures
(Metz and Kossen.1977; Whitaker and Long. 1973). This form of gowth offers key
advantages in the large-scale manufacture of microbial products. Compared to
ﬁlamentous or single cell cultures. mycelial pellet cultrnes have lower media viscosities.
geater cell densities and require simple product separation techniques. In effect, the
organism is immobilized but free of the problems associated with artiﬁcial
immobilization matrices. Industrial scale processes utilizing mycelial pellets include the
production of citric acid using Aspergillus species (Sodeck er al..1981). the production of
antibiotics using Penicillin»: species (Whitaker and Long. 1973) the continuous
hydrolysis of raﬁinose in beet molasses using Marrierella vinacea (Kobayashi and
Suzuki. 1972) and the production of mushroom ﬂavorings (Litchﬁeld. 1977). Van
Suijdam er a1. (1982). have described the main inﬂuences on the gowth of mycelial
pellets (Figure 2.1). These include the pellet size and density. the biomass concentration,
the limiting substrate concentration. the dissolved oxygen concentration and the oxygen
effectiveness factor.

The gowth of P. chrysosporium in the form of mycelial pellets has long been
known (Burkholder and Sinnot. 1945). but Jager et al.. (1985) were the ﬁrst to produce
lignin and manganese peroxidases using mycelial pellet cultures of this fungus. Michel er
al. (1990) studied the characteristics of pellet cultures of P. chrysospon'wn and found
that the formation of mycelial pellets from a homogenized mycelial inocula was
complete within 8 hours after inoculation. and that the number of pellets formed and the

characteristic pellet size were functions of the rate of agitation. They showed that LIP

 

:lr Pellet density I

 

 

 

 

 

 

 

I................ ‘ 1....22'2331
l l . t

I Substrate balance I Oxygen balance I

 
 

 

 

Figure 2.1 Inﬂuences on mycelial pellet cultures ﬁom van Suijdam et
al.. (1982).

7

production was inhibited in cultures with both large pellets (diam.=6.6 mm) formed at
low agitation rates. and small pellets (diam.=l.3 mm) formed at high agitation rates
(Table 2.1).

Table 2.1 Effect of the rate of agitation on mycelial pellet size and the production of
lignin peroxidase (LIP) by nitrogen-limited cultures of P. chrysospon’um
(from Michel er al.. 1990).

 

 

 

RPM Characteristic Pellet LIP

(min-1) Diameter (mm) (U/l)
100 6.63 :1; 1.06 O
150 1.83 1; 0.32 341 :l: 73
200 I 1.291021 376;:33
250 12710.23 62:1: 100

 

 

 

Liebeskind er al. (1990) showed that for mycelial pellets of P. chrysosporium up to 5 mm
in diameter. the pellet volume fraction (and hence pellet density) was constant.
Oxygen mass transfer limitations in mycelial pellets

Oxygen mass transfer limitations within mycelial pellets can inﬂuence mycelial
pellet gowth kinetics and could explain the low levels of LIP produced by large pellets
of P. chrysosporiwn. Pirt (1966) was one of the ﬁrst scientists to deScribe "cube root"
gowth kinetics for mycelial pellets of Penicilliwn chrysogenwn. The cube-root gowth
model assumes that growth occurs only in an outer zone of the pellet due to intro-pellet
oxygen limitations. Phillips (1966) calculated the critical diameter for gowing mycelial
pellets of Penicillium. where oxygen depletion ﬁrst occurs at the pellet center by
assuming zero order kinetics for respiration. Yoshida er a1. (1968). developed a model

8

for oxygen consumption in pellets of Lentinus edodes using Michaelis-Menten kinetics.
They used an eddy diffusion term that was larger than the value of oxygen diffusivity in
water and reported that the speciﬁc oxygen consumption rate was a function of the depth
within the pellet. Kobayashi er al. (197 3) solved the differential equation describing
oxygen transfer by molecular diffusion in pellets of Aspergillus niger using Michaelis-
Menten kinetics and two other cases of respiratory activity. They used an approximate
solution for effectiveness factors in immobilized enzyme systems developed by Moo-
Young and Kobayashi (1972). In one case they assumed that mycelia can adapt to a
particular oxygen concentration by changing their maximum rate of oxygen consumption
(Vmax). Van-Suijdam er al. (1982) developed an unstructured gowth model for
mycelial pellets of P. chrysogenum in a bubble column. This model considered biomass
gowth. substrate depletion. respiration and pellet density. It incorporated functions for
mass transfer processes in both the pellet and in the bulk liquid and assumed zero order
kinetics for respiration. An empirical relationship for the gas-liquid mass transfer
coefficient (kla) was obtained which showed that the resistance to oxygen mass transfer
from the gas to liquid phase was negligible for pellet volume fractions less than 40%.

Wittler er al. (1986) investigated mechanisms of oxygen transfer for individual
mycelial pellets of P. chrysogenum. They used an oxygen microprobe to determine the
oxygen concentration proﬁle within and immediately outside of mycelial pellets under a
variety of different hydrodynamic and aeration conditions. Their results indicated that the
mass transfer of oxygen into mycelial pellets may occur by means of eddy diffusion.
molecular diffusion and convection. While the pellets used by Wittler er al. (1986) were
probably in secondary metabolism, the media used for oxygen proﬁle measurements
were different than the growth media due to calcium and ammonium ion interference
with the microprobe. Also. no attempt was made to correlate the respiration in individual
pellets to bulk culture behavior. A summary of previous models of mycelial pellet
gowth and respiration is presented in Table 2.2.

Table 2.2. Summary of studies of mycelial pellet growth and respiration.

 

 

 

Reference Organism Oxygen Kinetics Growth Phase
Yano er al.. 1961 A. niger zero-order primary
Philips. 1966 P. chrysogenum zero-order primary
Pirt. 1966 P. chtysogenwn cube-root primary
Yoshida er al.. 1967 Letinus edodes zero-order primary
T‘rinci. 1970 A. nidulans logarithmic
Moo-Young er al.. 1972 (enzymes) Michaelis-Menten
Kobayashi er al.. 1973 A. niger MM; adaptive primary
van Suijdam er al.. 1982 P. chrysogenwn zero-order primary and death
Wittler er a1n 1986 P. chryso&num zero-order data

 

 

Evident from this summary of previous studies is that only the primary or
exponential gowth phase is considered and secondary metabolism is largely ignored. In
practice. many important products are secondary metabolites expressed after a limiting
nutrient has been exhausted and primary growth has ended. Therefore an understanding
of the physiological state of the organism during this period is important. Also apparent
is that models for the gowth and respiration of cultures of the white-rot fungus P.
Chrysosporiwn have not been developed. Furthermore. in general. the kinetics of
respiration is assumed to be zero-order when in fact Michaelis-Menten kinetics more
accurately describes respiration by microorganisms in submerged culture conditions. This
is done for reasons of mathematic simplicity. The oxygen effectiveness factor for zero-
order kinetics is easily calculated (see for example Phillips. 1966) while calculation of
the effectiveness factor for Michaelis-Menten kinetics requires a trial and error iterative
technique (see Moo-Young and Kobayashi. 1972). Metz and Kossen (1977) summarized

10

some other problems associated with early studies of oxygen mass transfer and
respiration in mycelial pellets. In brief. they are that external mass transfer resistances
have not been considered, that oxygen measurements have been done under improper
conditions (e.g. not using the gowth mdia) and that unusual kinetic parameter values
have been used (e. g. unrealistic Km and diffusivity values).

One way of studying oxygen transfer and respiration by mycelial pellets is through
the use of oxygen microelectrodes. Revsbech et al. (1983) have reviewed the use of
micro-electrodes in microbial ecology. These improved Clark micro-electrodes feature a
small tip diameter of 2 um- 5 pm and allow the measurement of oxygen proﬁles in
whole tissues. cell cultmes and microbial ﬁlms without interference by calcium or
ammonium ions (as described by Wittler er al. 1986). In this study these devices were
used to directly measure oxygen concentration gradients within and immdiately outside
of mycelial pellets of P.’ chrysosporium. This allowed an accurate determination of
kinetic coefﬁcients for respiration and permitmd the study of respiration and oxygen
mass transfer under conditions essentially the same as culture conditions.

Although many studies have addressed oxygen limitations in mycelial pellets. few
have demonstrated the effect of oxygen starvation on product expression. Kobayashi and
Suzuki (1972) showed that in pellet cultures of Morrierella vinacea which produce a-
galactosidase. the speciﬁc enzyme activity decreased in pellets whose diameter was
geater than 0.25 mm. They concluded that in the larger pellets the transfer of oxygen
limited enzyme production. Mania and Ward (1989). showed that altering the fungal
mmphology from ﬁlamentous to pelleted gowth in the production of fumaric acid by
Rhizopus arrhr'zus. leads to a three-fold decrease in fumaric acid production. Dosorctz er
al. (1990a) showed that submerged mycelial pellet cultures of P. Chrysosporiwn. ﬂushed
daily with air. yield no lignin peroxidase and two thirds less manganese peroxidase as
compared to cultures ﬂushed daily with oxygen (Figure 2.2). Thus evidence indicates
that oxygen transfer limitations in mycelial pellets can limit product expression.-

11

180.00 3— “I 6,000

 

 

 

 

160.00 {- —°—w-

: —9—LlPoa --

140.00 S- ——o—u~m .

.. —rlt-—|mpoz :

120.00 3- -- 4,000
LIP ”0'00 T __ MNP
(Um 80.00 f- (”In

60.00 9- -- 2,000

40.00 3-

20.00 3- Z

.l
l l

 

 

 

r 0
0 50' 100 150 200 250 300 350

' 0.00

Hours

Figure 2.2 Ligrin peroxidase (LIP) and manganese peroxidase (MNP)
activities in air-ﬂushed and oxygen ﬂushed cultures from
Dosorctz et al.. (1990). Cultures were gown in nitrogen
limited conditions.

12

A primary aim of this study was to gain an understanding of the physiological
conditions which favor the production of the LIPs and MNPs by developing an
unstructured mathematical model for mycelial pellet cultures of P. cluysosporiwn. Key
requirements for the model were that it was valid during secondary metabolism,
incorporated Michaelis—Menten kinetics for respiration and considered mass transfer
limitations within the mycelial pellets.

2.2 Kraft bleach plant effluents (BPE).
Formation of BPEs

The process of paper manufacturing begins with a forest. After cutting. logs are
trucked to the mill, debarked and then chipped to uniform 1 inch squares. The chips are
steam heated, mixed with sulﬁde and caustic and cooked for 3 hours at elevated
temperature and pressure (340° F. and 165 psi at one Michigan mill). This process
dissolves 90% of the lignin present in the wood. When the chips are ﬂashed to
atmospheric pressure. they explode. and form brown wood pulp which has the color and
consistency of a wet gocery bag. The wood pulp is bleached to make white pulp for the
production of ﬁne printing papers.

Kraft bleach plant efﬂuents (BPE) are formed when residual lignin is removed
from the brown pulp during the bleaching process. This comes in a series of crosscurrent
stages using chlorine. caustic soda. sodium hypochlorite and chlorine dioxide (Figure
2.3). After each stage. dissolved impurities are washed from the pulp and blended
together. Collectively. this waste stream is known as the bleach plant efﬂuent (BPE)
which is part of the over 3 billion gallons of wastewater discharged by the paper industry
annually in the U.S (EPA report #600/8-80-042a). At one typical paper mill in
southwestern Michigan. 17.5 gallons of wastewater is generated per pound of paper
produced.

13

Wood
Chips Paper
1 BLEACH PLANT
Brown Pulp White Pulp

    

 

 

 

Basic EIluent Addie Efﬂuent
Kraft Bleach Plant Efﬂuent [BPE]

* high chlorine content
" high molecular weight
" dark brown color

Figure 2.3 The process of brown pulp bleaching and the formation of
kraft bleach plant efﬂuents (BPE) at a Michigan paper mill.

14

Chemically. BPE consists of chlorinated degadation fragments of lignin. The
molecular weight of these fragments ranges from 200 to over 100.000 daltons with high
molecular weight species predominating. The toxicity of this material has been debated
(Eriksson and Kolar. 1985). The predominantly high molecular weight of BPE makes it
difficult to enter cells and it is not directly toxic to aquatic species. However some of the
monomeric units of the BPE polymer. like chlorinamd phenols and guiacols. are known
to be toxic and carcinogenic. The rate of degadation of the high molecular weight
chloro-lignin polymer in the environment. however. is not known. BPE contains some
small molecular weight chlorinated aromatic compounds (Huynh er al.. 1985. Roy-
Arcand and Archibald. 1991) and dioxins. Additionally. the dark brown color of BPE is
aesthetically undesirable and limits the penetration of sunlight in streams and lakes.
Furthermore. BPE increases the chemical and biological oxygen demand of receiving
waters. Modiﬁcation of the paper bleaching process using oxygen rather than chlorine as
a bleaching agent may reduce the amount of BPE produced in the future. however
chlorine bleaching will still be necessary to produce ﬁne quality white paper as
exempliﬁed by the paper you now have before you (Messner er al.. 1990).

Treatment technologies for BPE

Many techniques for BPE treatment have been studied. however most pose
econorrric or technical diﬁ'lculties that have not been overcome. Dewatering and burning
BPE is dinicnlt since the high chlorine content requires special incinerators and dioxin
formation is a problem (Michigan paper mill personnel). Ultraﬁltration. carbon
adsorption. high molecular weight amine precipitation (Gupta and Bhattacharya. 1985)
and lime precipitation have proven too costly to implement (Royers er a1. 1985; Gupta
and Bhattacharya. 1985). Adsorption to ﬂy ash. an electrical power generation waste. has
beenshownto heeconorrricallyfeasiblebasedonlaboratoryscaletests (Guptaand
Bhattacharya. 1985) however this material is considered hazardous and in some cases

15

toxic. A treatment process called rapid inﬁltration. wherein color is adsorbed and
precipitawd by flow through soil has been successftu implemented at an industrial scale
in Canada (Swaney. 1983). None of these approaches destroys the BPE polymer in the
process. The chloro-lignin material is simply concentrated and land-ﬁlled or dispersed
into the environment and still poses a pollution problem

Recently. a laboratory scale chemical oxidation process has been tested which
shows promise (Sun et al.. 1989). In this process BPE is concentrated using ultraﬁltration
and is treated at 150° C. with sodium hydroxide and oxygen at 150 psi for 40 to 60
minutes. The process reportedly removes 70 to 80% of total organic chlorine and 60 to
70% of color and signiﬁcantly reduces the molecular weight of the BPE (Sun et al..
1989). However this process requires stoichiomenic quantities of chemical reagents.

Studies have shown that few. if any bacterial organisms signiﬁcantly degrade lignin
(Forney. 1978). Ericksson and Kolar et a1. (1985) measured the evolution of 14002
from a high molecular weight fraction of 14c-labelled synthetic BPE by bacteria isolated
from an aerated lagoon which received paper mill effluents. They reporwd that less than
4% of the labelled carbon was converted to CO; in 90 days. Nevertheless. as pointed out
by Roy-Arcand and Archibald (1991). the use of enzyme-based treatment would offer
distinct advantages over physical and chemical processes in that only catalytic and not
stoichiometric amounts of reagents would be needed.

2.3 Degradation of BPE by white-rot fungi.

P. Chrysosporiwn and other white-rot fungi play key roles in environmental lignin
degradation (Reddy. 1984). These organisms are the fastest known lignin degraders and
among them Phanerochaete Chrysospoﬁwn has been chosen as a model organism for the
study of lignin degradation (Kirk and Farrell. 1987; Boominathan and Reddy. 1991).
Kirk er al. (197 8) examined the culture parameters inﬂuencing the ligninolytic activity of
P. chrysosporium. ligninolytic activity increased 2 to 3 fold in oxygen ﬂushed cultures

16

as compared to air-ﬂushed cultures and was negligible in cultures sparged with 5%
oxygen. P. chrysosporiwn did not grow on lignin alone and ligninolytic activity was
dependent on the presence of a metabolizable carbon and energy source like glucose or
cellulose. Nitrogen sufﬁcient cultures (24 mM) exhibited only 25-35% of the ligninolytic
activity as compared to nitrogen-limited (2.4 mM) cultures.

Lundquist et al. (1977). ﬁrst demonstrated the potential of white-rot fungi to
degrade industrial lignin compounds. They showed that cultures of P. chrysorporr’um

could degrade synthetic 14c labelled bleached kraft lignins and other industrially
modiﬁed lignins to 14C02. Approximately 40% of the ring-labelled and 31% of the side

chain labelled carbon from synthetic BPE was recovered as labelled 14002 after 42 days

of treatment. Eaton er al. (1980) used stationary cultures of P. chrysosporiwn to degrade
the El stage effluent (ﬁrst alkaline stage after chlorination) of BPE which represents the
majority of the bleach plant color. This effluent was decolorized 60% in four days and
the biochemical and chemical oxygen demand of the efﬂuent was reduced by
apprOximately 40%. Huynh et al.. (1985) and Roy-Arcand and Archibald (1991) have
demonstrated that low molecular weight chlorinated components of BPE such as
chloroguiacols. chloroaldehydes and chlorophenols are removed by P. chrysosporiwn
and Coriolus versicolor during the decolorization process. In addition studies have
demonstrated that during the decolorization process. the molecular weight of the BPE
polymer is reduced (Lackner er al.. 1991).

Many examples of bioreactor systems for BPE treatment exist. Eaton er al. (1983).
Sundmann er a1. (1981) and Chang et al. (1983) presented a series of articles describing
the development of a rotating biological contactor system for BPE decolorization. The
patented process uses P. chrysosporium immobilized on rotating discs. The discs are
partially submerged in BPE which is diluted by the addition of growth nutrients.
Campbell (1983) evaluawd this process at an industrial scale and found that the cost for
treatment would be $18] metric ton dry pulp. The EPA is currently testing this mycelial

17

color removal (or MyCoR) process at the pilot plant scale (Susan Strohofer. personnel
communication). Another bioreactor system called MY COPOR. was developed by
Messner er al. (1990) and uses sintered glass bead immobilized P. chrysospon’um. This
system decolorized. dechlorinated and reduced the BOD of BPEs by 60%. 70% and 85%
respectively. An aerated lagoon process for BPE treatment by pellets of P.
clvysospon'wn has also recently been evaluawd (Prouty. 1989) in which a treatment cost
of $4/ metric ton dry pulp was reported. In addition small-scale bioreactor systems have
been developed for the production of LIP and MNP using stirred tank (Michel et al..
1990) air-lift (Bonnarme and Jefferies. 1990) and by immobilized fungus reactor
conﬁgurations (Linko. 1988).

Livernoche er al. (1983) studied the ability of a different white rot fungus to
decolorize BPE. They reported that Coriolus versicolor could decolorize BPE by 80%
after three days of incubation. Immobilization of C. versicolorin calcium alginate gel
beads increased the rate of decolorization. and a lab scale ﬂuidized bed reactor system
was successquy tested. Royer er al.. (1985) described a continuous process utilizing
mycelial pellets of Coriolus versr'color which decolorized E1 efﬂuents by 50% in 15 h to
30 h and was operated for 24 days. Archibald er a1. (1990) demonstrawd that a wide
variety of carbon somees could be used by C. versicolor during the decolorization
process including glycerol. ethanol and crude carbohydrate sources like molasses and
starch.

In summary. the white-rot fungi P. chrysospon’um and Coriolus versr’color can
rapidly decolorize. dechlorinate. and reduce the COD and BOD of bleach plant efﬂuents.
These organisms may prove valuable in industrial scale BPE waste treatment processes.

18

2.4 Enzymes involved in lignin biodegradation and BPE decolorization.

Phanerochaete chrysosporium produces a number of enzymes which are thought to
play a role in lignin degradation. These enzymes include the lignin peroxidases (LIP). the
manganese dependent peroxidases (MNP). glucose and glyoxal oxidases which are
involved in hydrogen peroxide production and intracellular enzymes which complete the
lignin mineralization process. Coriolus versicolor but not Phanerochaete chrysosporr’um
produces the copper containing phenol oxidases known as laccases.

Two independent groups ﬁrst isolated the enzymes which have come to be known
collectively as lignin peroxidase (Tien and Kirk. 1984; Gold and Glenn. 1984). The
extracellular lignin degrading enzymes (LIP) are secondary metabolites. ﬁrst isolated
from nitrogen-limited six day old cultures of P. chrysosporr'wn. LIP non-speciﬁcally
catalyzes several oxidations in the alkyl side chains of lignin related compounds
including the Ca-CB bond of the propyl side chain of lignin model compounds. It is a
heme containing glyco-protein requiring hydrogen peroxide for activity. Collectively
these isozymes were called ”ligninases". They share the ability to catalyze the oxidation
of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide. a MW of
between 39,000 and 43.000. cross reactions with polyclonal antibodies and pH optima of
2.5 to 3.0. The key reaction is the one electron oxidation of aromatic nuclei to produce
unstable cation radicals.

Manganese peroxidases are a much less studied group of isozymes found in the
culture ﬂuid of P. chrysospon’rmr. Kuwahara er al. (1984) and Tien and Kirk (1984) ﬁrst
isolated MNP. These enzymes are also glycosylated heme proteins but they catalyze the
oxidation of Mn(II) to Mn(IIl). a highly reactive intermediate. in the presence of
hydrogen peroxide. Mn(IIl) in turn can be stabilized by chelators. like lactate or
malonate. and diffuse from the enzyme active site to attack phenolic substrates (Gold.
1991; Lackner er al.. 1991).

19

The production of both manganese peroxidases and lignin peroxidases is regulated
by manganese and nutrient nitrogen. Bonnarme and Jeﬁeries. (1990). found that the
production of LIP varies inversely. and the production of MNP varies directly. with the
manganese (Mn(II)) concentration in the culture media. Other fermentation parameters.
such as glucose consumption and biomass production. are unaffected by manganese.
Brown et a1. (1990) reported manganese involvement in the transcriptional regulation of
MNP expression by P. chrysospoﬁwn. Gold (1991) has reported the identiﬁcation of
manganese recognition sequences in the promoter regions of manganese peroxidase
genes (but not lignin peroxidase genes).

The effect of nutrient nitrogen concentrations on ligninolytic activity is less clear.
Penn er a1. (1981) and Kirk at at. (1978) showed that in nitrogen depleted cultures of P.
Chrysosporiwn addition of ammonium or glutamate repressed ligninolytic activity. T‘ien
and Th. (1987) established that nitrogen regulation acts at the level of lignin peroxidase
gene transcription. LIPs were ﬁrst isolated from nitrogen-limited cultures of P.
chrysospoﬁwn (Tien and Kirk. 1983) but studies have reported ligninolytic activity in
carbon-limited cultures where nutrient nitrogen is present in excess (Jefferies er al..
1981) and have demonstrated LIP and MNP production in nitrogen sufficient cultures
with glucose (Linko. 1988; Tonon er al.. 1990) and glycerol as the carbon somoes
(Tonon er al.. 1990). In general. the addition of glutamate has been shown to repress
ligninolytic activity.

Beyond transcriptional level effects. the level of nutrient nitrogen has pronounced
affects on the physiology of P. chrysosporr’um. The level of biomass. the size of pellets
formed and the bulk rates of substrate utilization and respiration are much greater in
nitrogen sufﬁcient cultures as compared to nitrogen limited cultures (see Appendix 8).
Thus besides affecting peroxidase gene transcription. the level of nutrient nitrogen may
affect peroxidase production at a physiological level by altering culture conditions so that
the production of the peroxidases is unfavorable.

20

Laccases are produced by many white rot fungi and in particular C. versicolor.
These copper containing enzymes do not require hydrogen peroxide for activity and have
been implicated in the dechlorination of chlorophenols and chloroguaicols found in BPE
(Roy-Arcand and Archibald. 1991). In addition. laccases polymerize many phenol
containing substrates. Laccases have been implicated in the decolorization of BPE by
cultures of C. versicolor (Archibald er al.. 1990). '

Surprisingly. despite the numerous studies of the ligninolytic system and the
decolorization of BPE by the white-rot fungus P. chtysosporium the roles of the
extracellular LIPs and MNPs in BPE decolorization. dechlorination and degradation have
not been determined. Neither LIP nor MNP have been shown to decolorize BPE in vitro.
although both enzymes have been shown to degrade lignin model dimer compounds in
cell free systems (Kirk and Farrell. 1987). Paice and Jurasek (1984) demonstrated that
horseradish peroxidase (I-IRP) could catalyze the decolorization of BPE. The same
authors have claimed that peroxidases are not involved in the decolorization of BPE by
Coriolus versicolor (Archibald er al.. 1990) by demonstrating that oxygen scavengers.
hydrogen peroxide scavengers. catalase and compounds which affect peroxidase
expression such as veratryl alcohol. nitrogen and manganese have no affect on
decolorization of BPE by Coriolus versicolor. Recently. laccases of C. versicolor have
been shown to dechlorinate and polymerize chlorophenols and chloroguiacols found in
BPE (Roy-Arcand and Archibald. 1991).

Peroxidase activities in decolorization studies using P. chrysosporiwn are generally
not reported and demonstration of the involvement of LIP and MNP in the decolorization
of BPE has not been shown to date. One possible explanation for this is that the veratryl
alcohol oxidation assay. the simplest and most widely used assay for lignin peroxidase
activity. gives poor results when minute quantities of BPE are added to the assay mixture
(Michel. unpublished results). To a lesser extent. the presence of BPE also interferes with

assays for manganese peroxidase activity. A major focus of this study was to determine

21

the roles of the lignin and manganese peroxidases in BPE decolorization by P.
chrysospon'um. To accomplish this. the production of the LIPs and MNPs was controlled
using manganese. nutrient nitrogen and mutant strains which are defective in their ability
toproduceLIPandMNP(Boominathan eraL. l990;KimandReddy. l990)andthe
affect on BPE decolorization was examined. The activities of the extracellular
peroxidases were assayed in control cultures without added BPE and the presence of the
peroxidases in BPE amended cultures was conﬁrmed using gel electrophoresis.

2.5 Research Objectives

L To study mass transfer limitations in mycelial pellet cultures of P. chrysosporr'wn
and understand their effect on the production of LIPs and MNPs.

2. To develop an unstructured kinetic model describing growth. substrate utilization
and respiration by mycelial pellets of P. Chrysosporr'um timing the lag. primary growth.
secondary metabolism and death phases.

3. To apply the extracellular peroxidases of P. chrysosporr’um to the decolorization
of bleach plant efﬂuents and investigate the roles of MNP and LIP in BPE

decolorization.

CHAPTERIII

THEORETICAL

3.1 Kinetic Model Development

In this section the development of a kinetic model for batch cultures of mycelial
pellets of P. cluysosporr’um is presented. The model describes the unsteady-state glucose
(G). nutrient nitrogen (N). oxygen (C1). polysaccharide (pG) and carbon dioxide
concentrations as well as the culture dry weight (X). the characteristic mycelial pellet
radius (R) and the oxygen effectiveness factor (B). given the initial glucose. nutrient
nitrogen and oxygen concentrations and the initial inoculum dry weight. The model is
applicable to any mycelial pellet culture where intraparticle oxygen mass transfer is rate-
limiting.

Typically. nitrogen-limited cultures of P. chrysosporr’um proceed through four
phases. Following a short lag phase. a primary growth phase. during which the cultures
grow rapidly. When the limiting substrate (nutrient nitrogen) is exhausted from the
medium. and glucose is present in excess. the cultures enter a secondary metabolic phase
dining which the MNPs and then the LIPs are produced. Cell autolysis occurs during the
death phase. A set of rate equations is presented for each of these life-cycle phases.
Culture respiration. which is shown to be the rate limiting process under most conditions.
is described using a separate set of equations which are valid during both the primary
growth phase and secondary metabolism.

The critical model assumptions are that pellets are spherical. monodisperse and
of constant density during the primary growth and secondary metabolic phases. that the
transfer of oxygen into pellets occurs solely by means of molecular diffusion. and that
cell respiration follows Michaelis-Menten kinetics. A summary of the kinetic model
equations is presented in-Figure 3.1.

22

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WM

Relatively little change in the biomass. glucose or oxygen concentration is seen
immediately after inoculation in agitated submerged cultures of P. chrysosporium.
Dining this lag phase. tiny mycelial pellets form. The process of pellet formation.
involving the aggregation of hyphal fragments. is complete within eight hours after
culture inoculation and the total number of these spherical pellets does not increase after
this period (Michel er al..1990). Furthermore. these pellets exhibit a fairly uniform size
which is dependent on the rate of agitation. The total speciﬁc pellet volume and hence
pellet density. is constant for pellet diameters up to 0.005 m (Liebeskind et al..1990.
Michel et'al..l990). This is in contrast to previous studies of P. chrysogenwn where
pellet density varies with pellet size (Kobayashi et al.. 1973; van Suijdam er al.. 1982) or
position (Wittler er al.. 1986) as a result of autolysis at the pellet center. Thus. at a
speciﬁc biomass concentration the characteristic pellet radius and pellet volume can be

calculated using Equations 1 and 2.
(1)

 

 

x .1.
R= "'—4 3
37“)“
4 X (2)
Vpcl = §nR3 =3;

Primary Growth Phase

Growth proceeds exponentially after the lag phase if nutrients are non-limiting. The
growth rate and the uptake rate of nutrient nitrogen are given by the exponential growth
law and the yield coefﬁcient Y“,x which represents the grams of biomass formed per

gram of ammonium consumed (Equations 3 and 4).

25

R.-%’f-=u.x <3)

dN 4
Rn="a'{=Yn/x“ex ()

As reported by other investigators (Kobayashi et al.. 1973; Pin, 1966). the oxygen
concentration in the pellets may become growth limiting. The effective speciﬁc growth
rate (Fe) is related to the maximum growth rate (”max) by Equation 5 (vanSuijdam er

01.,1982).
“e ‘ E ”max (5)

where the oxygen effectiveness factor (B) is deﬁned as the observed rate of
respiration divided by the rate that would be observed without mass transfer limitations.
The calculation of the effectiveness factor is described in section 3.3. If the eﬂ‘ectiveness
factor is near unity. the biomass concentration can be calculated as a function of time by
integrating Equation (3).

X 6
1n[376}=llmllxﬂ-tlag)=ltlmaxt'l~*maxtlag ()

This is a linear equation such that a plot of Ln(X/X0) versus time during the
primary growth phase will reveal "max and tlag as its slope and x-intercept respectively.
Glucose serves as both a carbon and energy source for P. Chrysospon'ran during
primary growth. It is used for energy via respiration and for biomass production
(Equation 7).

7
Rg = - Sid—(3 = RguuvimkIII + ngwt ( )

The amount of glucose used for biomass formation during primary growth can be
calculated using the yield coefﬁcient Y”. which represents the grams of glucose
consumed per gram of biomass produced (Equation 8).

a; (8)
REM = Yslx at

26
The rate of glucose uptake for respiration under aerobic conditions is proportional
to the rate of oxygen utilization. The yield coefﬁcient Ym represents the grams of
glucose consumed divided by the grams of glucose respired (Equation 9).

Rgruplradon = Yum R02 (9)

Secondary metabolism

In nitrogen-limited cultures. the onset of secondary metabolism coincides with the
depletion of nutrient nitrogen from the medium (Jefferies et al..l98 l). The production of
extracellular MNPs and then LIPs occurs only during this phase. The production of these
enzymes as well as the total ligninolytic activity (MC-lignin to 14C0?) is signiﬁcantly
enhanced by high oxygen partial presslnes (Barlev and Kirk.198l; Dosorctz er al..1990a;
Dosorctz and Grethlein.l991; Reid and Seifert.1982; Kirk and Farrell.l987). In the
classic case. no cell growth occurs during this phase. However. in cultures of P.
chrysosporiwn. the culture dry weight slowly increases after nitrogen is depleted.
Polymerization of glucose in the media to cell-bound and soluble B,1-4 glucan has been
shown to occur (Dosorctz er al..l990a;. Dosoretz and Grethlein. 1991; Leisola er
al..l982) which is also affected by the oxygen partial pressure (Dosorctz er al..l990a).
The primary metabolic activity of the cell during this period. however. is respiration. The
total rate of glucose uptake during secondary metabolism is given by Equation 10.

d0 (10)

The glucose metabolism factors fx. fpg and fr represent the fractions of glucose
consumd used for biomass. polysaccharide synthesis and respiration. respectively.

during secondary metabolism (Equations 11 a.b.c).
(1 1a)

Rgum" = -f.'d—t

27

99 (11b)
Rgpolyo = 'fvs dt

Rwa- = 'fr9£'- Yuoz Rm (11c)

The total rate of glucose consumption is given by Equation 12.

d6 12
Rs--- d.=-f.d.- rm». YuozRos "

Simplifying Equation 12 gives the total rate of glucose consumption as a function

of the rate of culture respiration (Equation 13).
Ram-1% = [ﬂkm “3’
Death Phase
In aerobic batch cultures of many fungi. a decline in cell mass occurs due to cell

autolysis when the carbon energy source is exhauswd. In nitrogen limited cultures of P.
chrysospon‘um, proteases (shown to degrade LIPs and MNPs) are released during the
beginning of the death phase (Dosoretz and Grethlein.l991). Although glucose has been
deplewd. carbon dioxide evolution continues during the death phase. To account for this
the assumption was made that polysaccharide produced during secondary metabolism is
used as an energy source by non-autolyzed cells (Equation 14).

(14)
R =" ddt -k,,X

Therateofcelldryweightdecreaseisexpressedusingaﬁrstorderrateconstant
(knot) (Equation 15). The amount of biomass remaining after all biological activity has

ceased is Xf. this is assumed to be a fraction of the maximum biomass attained.

we k...<x a) 05>

28

During the death phase oxygen is no longer a limiting nutrient and the effectiveness
factor is not needed. To provide maintenance energy. polysaccharides formed during
secondary metabolism are respired (Equation 16).

__1_ 9E (16)
R02:'[Yuoz ' dt

3.2 Determination of the rate limiting process in mycelial pellet cultures of P.
chrysospoﬂum

Previous studies of mycelial pellet growth and respiration have shown that the
mass transfer of oxygen into mycelial pellets may become diffusion limited. A schematic
representation of the process of oxygen mass transfer in mycelial pellet cultures is
presented in Figure 3.2. Oxygen must continuously move from the gas phase to the liquid
phase. from the bulk liquid to the pellet surface. and from the pellet surface to the
interior portion of the pellet where respiration occurs. Due to its low solubility in water
(32.9 g/m3 in water at 39° C). oxygen can quickly become depleted within the mycelial
pellet. The mass transfer of substrates is analyzed for mycelial pellet cultures of P.
chrysosporiumin the following section.

Estimation of external and intraparticle mass transfer resistances.

The contributions of the gas-liquid. liquid-pellet, and intro-particle mass transfer
limitations can be estimated by direct measurement of gas and liquid phase substrate
concentrations. and by calculating the dimensionless Biot number (Bi) and observable
modulus (O) (Bailey and Ollis. 1986). If the rate of oxygen transport from the headspace
to the liquid phase is much greater than the bulk rate of oxygen consumption. the
concentration of oxygen in the liquid phase and head-space can be related using Henry's
law (Equation 17).

29

liquid pellet

 

Cs(air) = am am”
' Cg(02) = 32.9 glm3

Figure 3.2 The process of mass transfer and reaction in mycelial pellet
cultures.

30

c1 = H Ch, (17)

The value of the Henry‘s law constant can be estimated by dividing the solubility of
oxygen in cell-free media by the ideal gas concentration in the headspace (Equation 18).

H Csatm ' (18)
‘ MWP

The external and intraparticle mass transfer resistances can be compared based on

the Biot number (Bailey and Ollis. 1986) (Equation 19).
k, R (19)
Bi 3 3—D—e'

For Biot numbers greater than 100. the effects of external mass transfer resistance
are not signiﬁcant (Bailey and Ollis. 1986).

The dimensionless observable modulus (Equation 20) can be used to estimate the
relative importance of the intraparticle mass transfer resistances of subsu'ates without
prior knowledge of intrinsic kinetic parameters (Bailey and Ollis. 1986).

 

. . [a

._._ De C1
For values of O less than 0.3 the effectiveness factor is approximately one and the
diffusion and reaction of substrate in the pellet is reaction limited. For 0 greater than 0.3.

intraparticle diffusion resistance limits the reaction rate (Bailey and Ollis. 1986).

31

3.3 Model for the intraparticle diffusion and reaction of oxygen in mycelial pellets.
If diffusion occurs only by means of molecular diffusion. the diffusion and reaction
of oxygen in a spherical mycelial pellet can be modelled using the following second-
order differential equation and boundary conditions.
£3. Q19. 2 9.9]

r dr (21)

@r=R. C=C8

ﬂ-
@r=0. dr' -0

De is the effective diffusivity of oxygen in the mycelial pellet and v is the rate of
oxygen utilization for respiration. Various methods have been used to solve the pseudo-
steady state form (dC/dt=0) of this differential equation (Kobayashi et al..197 3. M00-
Young and Kobayashi.l972. van Suijdam er al..1982. Yano et al..1961). The solutions
are commonly represented in terms of an effectiveness factor. deﬁned as the observed
reaction rate divided by the rate without mass transfer limitations. In sealed shake ﬂask
cultures the pseudo-steady state assumption is not valid over long time periods (> 1/2 hr)

since the oxygen concentration and mycelial pellet size change with time.

Prediction of oxygen concentration profile in mycelial pellets.

During the time period when a microelectrode is being used to measure the
oxygen proﬁle in a mycelial pellet. the pseudo-steady state form of Equation 21 can be
used. A ﬁnite difference approach was used to approximate this form of Equation 21
(Carnahan ct al..1969).

 

Ci-l'zci+ci+l + Ci-l-l'Ci-l _ _y_ (22)
Al’2 iAl’z - De

The boundary conditions are C0=C1 and Cn=Cs. Solving for Ci.” gives an

expression for the oxygen concentration proﬁle in the pellet (Equation 23).

32

c... = [.13.] [2e . g- th... . sag] <2»

By choosing an appropriate model for respiration. the concentration proﬁle within
the pellet can be calculated using a shooting algorithm. The oxygen concentration at the
pellet center (where dC/dr = 0) in the range zero to Cs is used to start the algorithm and
the surface concentration is calculated. The center concentration is changed until the
calculated surface concentration converges to the known value (Cn=C3). If a small step
size (n=100) is used this approximationt yields an accurate solution (<0.01% error) to the
pseudo-steady state form of Equation 21. The oxygen concentration proﬁle can be
measured using an oxygen microelectrode (Revsbech and Ward. 1983). Intrinsic kinetic

parameters are determined by ﬁtting experimental data to Equation 23.

Calculation of the effectivens- factor for Michaelis-Menten kinetics.
The generalized effectiveness factor (B). valid for any rate equation and spherical
geometry. can be calculated at time t using Equation 24.

z (V(Ct) (Isa ' 1113) ) (24)
E = 2°

 

v(c.) R3

For most microbial cultures. the Michaelis-Menten equation adequately describes
the respiration kinetics (Equation 25).

Vmax C1 (25)
v . [Km + C1]

Although Equation 23 provides a simple means of estimating kinetic parameter
values from oxygen proﬁle data. calculation of the oxygen effectiveness factor at each
succesive time point in the integrated model using Equation 24 proved very time

33

consuming (over 2 hours for one batch culture simulation using an IBM Model 50 2
computer). Therefore. for the integrated kinetic model the approximation derived by
Moo-Young and Kobayashi (1972) was used to calculate effectiveness factors for
Michaelis-Menten kinetics (Em) at successive time increments. This approximation is
based on the more easily calculated zero-order (E0) and ﬁrst order (E1) eﬁ'ectiveness

factors (Equation 26).
MICHEALIS-MENT'EN ORDER:

Km

E0 —
Estu= + [$1131 (26)

1+ [or]

 

If the intrinsic Michaelis-Menten kinetics are known. the zero order (E0) and ﬁrst
order (E1) effectiveness factors can be easily calculated using Equations 27 and 28

(Bailey and Ollis.l986).

ZERO ORDER:

 

Vmax 1

(27)

6DeCs 1 Vmax 1
BO 1' 0m: f°‘"' R[wees]2 >1

 

 

1 R Vmax
El = ﬂail; where: 4) = '3’ [De 2 (28)

34

Using the effectiveness factor. the rate of oxygen uptake by pellets of P.
chrysosparium (r07) can be calculated based on the liquid phase oxygen concentration

(C1) (Equation 29). A Monod expression is added to account for the decrease in the

respiration rate at low glucose concentrations.

rm= BuuLLM fa] [as—G— 4. o] <29>

The bulk rate of respiration (R0,) can be calculated using the characteristic pellet
volume (vpel)v and the pellet number (11) (Equation 30).

R02 = 1'02 vpal Ti (30)

During respiration. glucose is metabolized to carbon dioxide. The rate of C02

produced can be calculated using a yield coefficient ( Rom = Ycozm R02; where Ym
= 44/32 a 1.38). The carbon dioxide balance assumes that all 002 accumulates in the gas
phase.ThetotalamountofCOzingramswhichhasaccumulatedinthe gasphaseis

given by equation 31.

t

C02 = 1.38 VII
0

 

Vmax C1]
dt (31)

x
p E“ [Km+Cl

Unsteady state oxygen balance in sealed cultures.

In sealed shake ﬂask cultures the gas phase is ﬂushed daily with oxygen. As the
oxygen is consumed for respiration. the total amount in the ﬂask decreases. The amount
of oxygen in the gas phase plus the amount in the liquid phase equals the total oxygen in
the ﬂask at time t (Equation 32).

35
total 02 = c. vl + ch, v... (32)

Since the gas and liquid phase concentrations are near equilibrium. the Henry's law
relation can be substituwd into Equation 32 to give the following expression (Equation
33).

totalo. . C.(vl + HVI.) (33)

A differential expression for the change in the liquid phase oxygen concentration as
a function of time is obtained by combining Equation 33 and the bulk rate of respiration

(R0,).
El ML.

dt = v. + H v... (34)
Thus an approximate solution to Equation 21 can be obtained by numerically
integrating Equation 34 and recalculating the respiration rate. the effectiveness factor and
the liquid phase oxygen concentration at each successive time point. The mycelial pellet

radius can be determined based on the substrate balances. the biomass balance and the
pellet density.

Determination of Effective Diffusivity of Oxygen (De) in Mycelial Pellets.
The value of the diffusivity of oxygen within the mycelial pellet is critical to the
accurate determination of the kinetic parameters for respiration. However. a number of

different values for the pure solute diffusivity of oxygen in water D (Doz-Hp) have been
reported in the literature (Table 3.1).

36

Table 3.1 Reporwd values of the diffusivity of oxygen in water.
15vo r

 

 

(mzls) (C) Reference Comments
2.9 x10’9 39° Perry eraL. 1984 reportederrori20%
1.8 x 10-9 20° Yano etal..1961 cites Perry etal. 1950
1.8 x 10-9 20° Kobayashi etal. 1973 cites Yano etal..1961
2.1 x 10.9 25° Jordan etaL. 1956 direct measurement.
1.7 x 10'9 25° Jordan et al.. 1956 10% sucrose solution.
3.2 x 10'9 M. Phillips. 1966 assumes 0.85 void fraction

3.4 x 10-9 39° Wilke-Chang equation value (see Perry et. al. 1984)
2.2 x10'9 n.r. Bailey and Ollis. 1986
2.0x10'9 25° Wittler etal., 1986

 

 

 

n.r.- value not reported
Because of the uncertainty in this value and the possibility of a reduced value of the

diffusivity within the pellets due to a void fraction less than unity. the diffusivity of
oxygen within mycelial pellets of P. chrysospoﬁum was measured directly.

A stirred bath technique was used to determine the effective diffusivity in mycelial
pellets based on a method described by Clesand er al. (1988) developed for the
measurement of glucose diffusivity in cell matrices. Crank (1975) has detailed various
stirred tank experiments for the determination of effective diffusivities. In one such
experiment a slab initially free of solute is contacted with a well stirred solution (Figure
3.3). By measuring the decrease of the solute concentration in the liquid phase. the
uptake rate by the slab and the diffusivity of the solute can be calculated. Assuming
Fickian diffusion and a well mixed liquid phase. the concentration in the slab can be

represented using the following equation and boundary conditions.

37

 

 

 

 

 

V
or = I 0 VI
Valah
Delonlzed water
0 = total pellet volume “WM“: 3'" 5‘"

 
  
 
 

 

slab volume

De 2 X Agarwitfl suspended.

killed mycelial pellets

       

k‘.1 {I r'z'u A 0

Magnetic Stir plate

Figure 3.3 A schematic representation of an apparatus for measuring the
effective diffusivity in mycelial pellets.

38

Q 92S
d. = Dslab dxz

(35)

B.C.s C=Oatt=0. C=Cbat x=0. 95§=0 at x=l

where l is the slab thickness. Cb is the uniform bulk solute concentration in the
liquid and x is the distance from the interface. A mass balance on the liquid phase gives
Equation 36.

dCb dCb
“17.1? = ”slab [alts-=0 ‘36)

B.C. Cb=Co att=0

where a is equal to V1/V slab: the liquid volume divided by the slab volume. These

Equations can be solved using a Laplace transformation (Crank. 1975). The solution is
writtenintermsofMt. theamount ofsolutein the slab altimetandMinfthe amount of

solute in the slab when the slab and liquid are in equilibrium where qn are non-zero

 

 

positive roots of Equation 38.
inﬁnity
.35:— 3 1_ 2a(1+a) [-Dslttbt 2] (37)
Minﬁnity 1+rx-l-r12qn2 exp 12 q“
n=l '
tan (in = ‘a Go (38)

Thus. if Mt/Minf is known at time t. then Dslabt the effective diffusivity in the slab
canbecalcuhtedsincemtandlareknown.

39

To determine De (the effective diffusivity in the pellets) a theoretical relationship
derived by Maxwell (1973) can be used. This heat transfer equation was derived for the
effective conductivity in a medium composed of periodically spaced spheres. The
diﬁusivity analog. where no reaction is taking place. is given by Equation 39.

+6 ’ 2Q[&'%] (39)

eats-s1

D

2

De
D a
De

In this equation. D is the diffusivity of oxygen in water. Dslab is the overall slab
effective diffusivity. De is the effective diffusivity in the spheres (or pellets) and Q is the
ratio of the total pellet volume to the slab volume. By plotting Dslab/D for a number of
different values of Q. a curve is generated which is a function of De. The curve can be ﬁt
by selecting the appropriate value of De. According to Cresand et.al. (1988) the
uncertainty in the value determined using the above approach is approximately 5% if the

error in the concentration measurements is minimal.

3.4 Model for the decolorization of Kraft bleach plant efﬂuent.

The decolorization of BPE was modelled using an apparent ﬁrst order rate constant.
Aﬁrstordermodelwas usedbecause the initialrateofdecolorization was foundtobe
proportional to the initial BPE concentration. for synthetic BPE concentrations below
4000 C.U. (see results). This model was also used for reasons of mathematical simplicity.
The rate of decolorization was modelled using the Equation 40.

(40)

9% = k(C.U. - C.U.f)

The value of the apparent rate constant (k) was determined ﬁtting experimental data to
the integrated form of Equation 40.

CHAPTERIV

MATERIALS AND METHODS
4.1 Microorganisms.

The microorganisms used in this work were strains of the white-rot basidiomycete
Phanerochaete Chrysosporium. The wild-type strains used were BKM- - 1767 (ATCC
24725) and ME446 (ATCC 34541). In addition mutant strains derived from NIB—446
were used. These had the phenotypes lip“ (Boominathan et al.. 1990) and lip' and mnp'
(Kim and Reddy. 1990). All strains were obtained from the laboratory of Dr. C.A.
Reddy. of the Department of Microbiology and Public Health. Michigan State University
and were maintained on 2% malt agar extract slants. pH 4.5 (Kelley and Reddy. 1986).

4.2 Culture conditions.

Experiments were conduced in shake ﬂask cultmes using asceptic techniques. The
culture conditions used were as described by Michel er al. (1991). The deﬁned culture
medium contained diammonium tartrate (2 to 117 g/m3 ammonium) as the nitrogen
source and glucose (5000 to 15000 g/m3) as the carbon source. The media also contained
2000 g/m3 of KHzPO4. 1450 g/m3 of MgSO4*7H20. 132 g/m3 of CaClz*2H20. 1.0
glm3 thiamine hydrochloride. 500 g/m3 Tween 80 (not added in stationary starter
cultures). 20 mM acetate (pH 4.5) and 0.4 mM veratryl alcohol. The following trace
elements were also added: 142 g/m3 of nitrolotriacetate trisodium salt. 70 g/m3 of NaCl.
36.9 g/m3 (12 ppm) of MnSO4*HzO. 12.6 g/m3 of CoC12*6HzO. 7.0 g/m3 of
FeSO4‘7H20. 7.0 g/m3 of ZnSO4*7H20. 1.1 g/m3 of CuSO4*5H20. 0.7 g/m3 of
A1K(SO4)2*12H20.0.7 g/m3 of 1131303 and 0.7 g/m3 of NazMoO4'l'21-I20. Unless
mentioned otherwise. all the media contained 12 ppm Mn(II) as MnSO4. In some

experiments Mn(II). added as MnSO4. was varied to give 0. 12. 40 or 100 ppm.

41

Shake flask culture conditions.

Inoculated media (85 ml) were dispensed into sterile 250 ml rubber stoppered
Erlenmeyer ﬂasks. The inoculum consisted of a 10% (v/v) of homogenized mycelia
which was grown in static cultures using the media described above with 10,000 g/m3
glucose and 39 g/m3 ammonium (as diammonium tartrate) but without Tween 80. All
culturesweregrownat39°C.Thecultureswereagitatedat 173 RPMonashakertable
(New Brunswick Gyrotory G-10) with 2.5 cm of eccentricity. The headspace (165 ml) of
the ﬂasks was ﬂushed daily with oxygen for approximately 3 minutes at a ﬂow rate of 1
l/min (Michel er al. 1990).

Bioreactor conditions.

The bioreactor system used consised of a 5 liter Bioﬂo II (New Brunswick Sci. Co.)
with a total volume of 6.3 liters. The culture conditions were as described by Michel er
al. (1990). Agitation was provided by a marine impeller at a rate of 100 to 350 rev min'
1. no bafﬂes were used. The reactor contents were sampled through the top-plate. For dry
weight measurements a sample volume of greater than 50 ml was used. 002 evolution
was measured by sealing the reactor without sparging for 24 hours. and sampling through
the top plate using a pressure-10k syringe (Precision Sampling Corporation.) The
respiration rate was also measured by transiently stopping the oxygen ﬂow and
measuring the decrease in the oxygen concentration in the culture ﬂuid with time using a
galvanic oxygen electrode.

4.3 Substrate Assays.

Glucose was measured as reducing sugar by the dinitrosalicylic acid (DNS) method
using D-glucose as the standard (Miller. 1959). Samples were centrifuged and diluted
and a 3.0 ml of a prepared DNS reagent solution was added. The assay mixture was

42

boiled for 5 minutes. cooled and the 595 nm absorbance was measured against an assay
mixture blank.

Ammonium concentration was determined using an ammonium electrode (Orion
model#95-12). Total carbohydrates were determined using the phenol-sulfuric acid assay
as described by Leisola er al. (1982). For determining mycelial dry weight cultures were
vacuum ﬁltered through tared GF/C grade ﬁlter paper. rinsed with 100 ml deionized
wateranddriedat105°Ctoaconstantweight

The oxygen concentrations in the culture ﬂuid and head space were measured using
a polaragraphic oxygen electrode (Ingold. model #531) which was calibrated with
oxygen and nitrogen sparged deionized water at 39° C. Respiration rate measurements
were made by pouring one entire culture (85 ml) into a hydraulically full stirred vessel
(rcspirometer) and measuring the decrease in oxygen concentration with time using a
stripchartrecorderwithachartspeedonOcm/horsocm/h. Stirring was providedbya
magnetic stir bar rotating at approximately 120 min'l. The rate of oxygen depletion was
determined by measuring the slope of the oxygen depletion curve. .

Acetate. ethanol and methanol concentrations (see Appendix 19 and 20) were
determined using gas-liquid chromatography (V arian. model 3700. ﬂame ionization
detector). Samples were centrifuged. mixed with 4 parts 3N phosphoric acid and a
volume of 5111 was injected. The injector. column and detector temperatures were 140°
C. 125° C and 150° C. respectively. Peak areas were measured using an integrator
(Hewlett-Packard. model HP-3396A).

Oxygen profile measurement

Oxygen proﬁles within mycelial pellets were measured using oxygen
microelectrodes with tip diameters of approximately 3 pm generously donated by Dr.
Peter Revsbech of the Center for Microbial Ecology (Revsbech and Ward. (1983). The
electrode was connected to a chemical microsensor (picoammeter) and was calibrated
with oxygen and nitrogen. sparged into distilled water. To determine the oxygen

43

concentration proﬁle. a pellet was removed and placed onto a plate of agar-solidiﬁed
extracellular culture ﬂuid (15 ml). Liquid extracellular culture ﬂuid (15 ml) was added to
the slmface of the agar and the system was equilibrated to air. Oxygen measurements
were made by inserting the microelectrode into the pellet using a micromanipulator (0.01
mm gradations). In some cases a well was made in the agar gel using a Pasteur pipet. The
microelectrode penetration procedure was monitored using a stereo dissecting
microscope. The micromanipulator was also used to measure the diameter of the pellet
being probed. The procedure was conducted in a 39° C. incubator room

The gas in the headspace was sampled with a pressure-10k syringe (Precision
Sampling Corporation) and carbon dioxide was measured using a gas chromatograph
(GOW-MAC series 350. thermal conductivity detector) ﬁtted with a Porapak Q 80/ 100
column (helium carrier gas. column temperature 90° C). A sample volume of 1 to 5 [.11
was injected.

Manganese determination.

Manganese was measured using atomic absortion spectroscopy (V arian SpectrAA
model AA-20) at a wavelength of 279.5 nm. One entire culture (85 ml) was ﬁltered
through GF/C grade ﬁlter paper to remove pellets and precipitated manganese solids.
The ﬁltrate. or soluble manganese fraction. was used undilued. dilued 1:3 or diluted
1:10 with DI. water (depending on initial manganese concentration). The precipitaed
and mycelially bound manganese was solubilimd by adding an equal volume (85 ml) of
0.1 M HCl to the ﬁlter paper and stirring. Once the insoluble manganese had dissolved.
this fraction was collected by vacuum ﬁltration through the same ﬁlter paper.

4.4 Product Assays.
LIP and MNP activity.

LIP activity was determined spectrophotometrically by measuring veratryl alcohol
oxidation to veran'yl aldehyde at pH 2.5. room temperature (23° C.) and a wavelength of
310 nm as described by Tien and Kirk (1984). MNP activity was measured as described
previously at a wavelength of 610 nm (Kuwahara er al.. 1984; Michel er al.. 1991) at 30°
C. Thesamplevolumewas 10to40tll.thepras4.5 andthereactiontimewas4min.
A unit of MNP activity was deﬁned as one pmol of phenol red oxidized per liter per
minute. using an extinction coefﬁcient of 4460 M'1 cm'l (Michel er al.. 1991). MNP
activity was also assayed using remazol blue. 1040 111 of sample was added to 400 [.11 of
buffer (125 mM). 400 ill of5 g/l chicken albumin. 100 pl of 0.17 g/l MnSO4 and 200 ill
of 0.25g/l remazol blue. The reaction was started by adding 100 pl of 1 uM H202. The

absorbance difference at 595 nm was measured over a 4 minute period.

The total protein concentration of the culture ﬁltrate was measured using the
Bradford Method employing bovine serum albumin as a standard. A commercially
available reagent was used (Protein Assay. Bio-Rad. Richmond CA).
pH optima of LIP and MNP.

The pH optima of LIP and MNP were determined by buffering day 6 culture ﬂuid.
containing high levels of both LIP and MNP activity. with tartarate (10 mM) or acetate
(20 mM) to 10 pH values between 2 and 7.2. The two enzymes exhibited different pH
optima. LIP activity peaked at pH 2.5 and MNP peaked at pH 4.5 to 5.0. Interestingly,
MNP activity at pH 2.5 was reduced by more than 80% compared to its activity level at
pH 4.5. Also. LIP activity at pH 4.5 was reduced by more than 90% compared to its
activity level at pH 2.5 (Figure 4.1).

45

 

 

—°— veratryl alcohol
oxidation assay
—0— phenol red oxidation
_ my
-—¢— remazol blue oxidation
assay
1 _r MNP
up 7

0.9 --

0.8 ~-

0.7 ~-

0.6 ~-

Normalized
Enzyme 0.5 ~-
Activity

0.4 ~-

0.3 '1‘-

O.2 1' '

0.1 ~-

0 Irrirrrjrrr%riT=rWI|rr -‘

 

1 2 3 4 5 6 7
pH

Figure 4.1 Lignin peroxidase and manganese peroxidase enzyme
activities as a function of pH. Samples were buffered with
acetate (0.1 M) for pH 4 to 7 or'tartarate (0.1 M) for pH 2 to
3.5.

Pellet characteristics.

To measure pellet size. pellet density and pellet number (concentration). a known
volume of culture ﬂuid with pellets was porned into a Petri dish. photographed and
enlarged. The total number of pellets was counted and the diameters of a group of twenty
pellets were measured. scaled and averaged using the Sauter equation (Michel et al..
1990). There was little difference in the values of the Sauter characteristic pellet radius
and the radius determined by simply averaging the measured radii. Pellet sizes were also
measured using a micromanipulator and a stereo dissecting microscope. ‘
SDS-PAGE electrophoresis.

Extracellular proteins were identiﬁed using sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) as previously described (Kelley and Reddy. 1986).
Samples (2.0 ml) were removed from cultures and concentrated to 200 til using a
Centricon ultraﬁltration unit (Amicon Div.. W. R. Grace 8: Co.. Danvers. MA) with a 10
kD molecular weight cut-oﬂ'. The concentrated samples were applied to a 4%
stacking/10% running gel and the proteins were stained with Coomasie brilliant blue.
Fast protein liquid chromatography.

To separate and purify MNP and LIP proteins. the extracellular ﬂuid from active. 4
or 6 day-old cultures was frozen overnight to precipitate polysaccharides. centrifuged.
concentrated to approximately 5% of the original volume by ultraﬁltration. and dialyzed
against 2 liters of 10 mM acetate buffer (pH 6). The concentrated enzyme solution was
applied a Mono Q column (Pharmacia. Uppsala, Sweden) at a ﬂow rate of 0.2 to 1.0
mllminandelutedusingagradientofacetatebufferfrom 10tho 1.0M. Heme
proteins were deteced by continuous monitoring at 409 nm. Fractions (0.5 ml) were
colleced using a programmable fraction collector.

47

4.5 Preparation and characteristics of Kraft bleach plant effluents

Two types of Kraft bleach plant efﬂuent (BPE) were used. an industrial BPE
obtained from a U.S. pulp mill and a synthetic BPE. The synthetic BPE was prepared
from a high grade (less than 1% ash) commercially available Kraft lignin (Indulin AT,
Westvaco Co.) as described by Lundquist et al.. (1977). Brieﬂy the procedure employed
was as follows: Indulin AT (3.83 g). was dissolved in 150 ml of 0.14 M NaOH. 25.5 ml
of 0.6 M H2SO4 was added with stirring. and the lignin material was precipitated.
Chlorine water [668 ml of 8.1% (v/v) Chlorox] was added. and the solution was agitated
for 1 h in a shaker bath at 25° C. The solution was removed from the shaker bath and
stripped with nitrogen gas via a gas dispersion tube. to remove excess chlorine. After 2
h.213mlof1.0MNaOHwasadded.andthemixturewasincubatedfor2hinashaker
bath at 30° C. Next. the solution was acidiﬁed to pH 2.5 using 0.6 M H2804
(approximately 128 ml) and deionized water was added to bring the mixture to a volume
of 1.5 l. The efﬂuent generated using this protocol was considered typical of the efﬂuent
from an industrial kr'aft bleach plant (Lundquist er al.. 1977).

Both industrial and synthetic BPE were concentrated under vacuum using a
Rotavapor-R (Brinkmann) to approximately 12% of their original volume. The pH was
adjusted to 4.5 using 1.0 M NaOH and the solutions were stored at 4° C until used. After
concentration the efﬂuents contained 56.000 to 69.000 standard platinum cobalt units of
color (C.U.). The concentrated BPE solutions were generally added to cultures of P.
chlysosporiwn to give a ﬁnal concentration of 3.000 C.U (624 C.U. = 1 g BPE/liter).
Typically. industrial bleach plant efﬂuents contain 3.000 to 10.000 C.U.

48

BPE Color measurement.
The measurement of decolorization of both fungal-treated and untreated BPE was

based on the NCASI standard method (NCASI. 1971). The standard Platinum-Cobalt
color solution (500 color units) was prepared by dissolving 0.2492g of K2PtC12 and 0.2

g of CoClz-GHZO in a mixture of 20 m1 concentrated HCl and 50 ml of DJ. H20. After

dissolving. the mixture was diluted to total volume 200 ml.

To measure color. samples were diluted in phosphate buffer (pH 7.6). centrifuged
for 15 min at 10.000 x g. and the absorbance at 465 nm was measured as previously
described (NCASI. 1971). The conversion factor used was 4015 std Pt-Co color units
(cu) per 1.0 absorbance unit at 465 nm (cm-1). Color on the mycelium was measured
by homogenizing 10 parts buffer to 1 part centrifuged mycelial pellets. and then ﬁltering
through GF/C grade ﬁlter paper (Whatman Paper Ltd.. Maidstone. England). In some
instances adjustment of the pH of fungal treated BPE led to the precipitation of
extracellular polysaccharides. When this occured the data were disregarded.

Solubility of BPE

The solubility of both synthetic and industrial BPE was determined as follows; 2.0 ml of
concentraed BPE was added to 9.0 m1 of complete media. The pH was adjusted to
values between pH 2.5 and pH 7 using 1 M HCl or 1 M NaOH. These solutions were
mixed vigorously. allowed to sit for 30 minutes. mixed again and then cetrifuged for 10
minutes at 14.000 x g. After centrifugation. 0.5 ml aliquot each supernatant was
immediately removed and mixed with 1.0 ml of phosphate buffer (pH 7.6). The
absorbance of each sample was then measured at 465 nm. Industrial BPE was more
soluble at pH 2.0 to 7.0 than synthetic BPE prepared as described above (Figure 4.2).

49

14000

11111

12000
1mmsenama

1111:

10000

11111

8000
Oak»

Unﬂs

[till

6000

Synthetic BP

4000

2000

llrllrrlllrrrl

 

 

Figure 4.2 The solubilities of synthetic BPE and industrial BPE in
culture medium as a function of medium pH.

50

Molecular weight of BPE.

The molecular weight of the colored material in the plant and synthetic BPE was
estimated using ultraﬁltration. Samples of BPE (1 mi. 1200 C.U.) were mixed with 3 ml
0.1M KOH to solubilize all precipitates. After centrifugation (no precipitates observed) 2
ml of the solutions were applied to disposable ultracentrifuge tubes with a molecular
weight cut-off of 10.000 daltons. After centrifugation for 2 hours both the upper (>10000
MW) and lower ((10000 MW) fractions were diluted back to the original 2 ml using
buffer (pH 7.6) and 465 nm absorbance was measured. For borh industrial and synthetic
BPEs. more than 90% of the color remained in the upper part of the tube indicating that
most (>90%) of the colored material in the BPE had a molecular weight greater than
10.000 daltons.

4.6 Computational methods.

The complete kinetic model equations were analytically or numerically integrated
using Euler's method with a time increment of 1 h. A BASIC computer program
calculated X. G. N. C. pG. EMM.r total culture 002. total culture Oz and characteristic
pellet radius (R) as a function of time (Appendix 21). The data were transferred to a
spreadsheet (EXCEL) and plotted. This was compared to experimental data for X. G. N.
total culture CO; and pellet size (Appendix 15). The Michaelis-Menten effectiveness
factor was determined using the method of Moo-Young and Kobayashi (1972).

Another computer program. (Appendix 22) calculated the oxygen concentration
gradient within a mycelial pellet. given the pellet radius and liquid phase oxygen
concentration. This program solved the Equations for diffusion and reaction of oxygen
within a mycelial pellet using Michaelis-Menten kinetics. A shooting algorithm was
used with Newton-Raphson convergence. The program was used to determine values of
Vmax and Km as described by Michel er al. (1992a).

CHAPTER V

A model for submerged, peroxidase producingnnycelial pellet cultures
of Phanemchaete chlysosporium

Part I: Results

5.1 Culture characteristics of mycelial pellet cultures of P. chtysosporium.

Patterns of growth (culture dry weight and pellet radius). glucose and nitrogen
(ammonium) depletion. carbon dioxide evolution and oxygen depletion. and the
production of extracellular peroxidases were determined for nitrogen-limited. mycelial
pellet cultures of P. chrysosporium BKM-F-1767 (Figure 5.la-d). The organism grew
rapidly dining the ﬁrst 24 hours after inoculation (doubling time=5 .2 h) and the nutrient
. nitrogen (ammonium) concentration decreased steadily to undetectable levels within 24
Chorus. The glucose concentration in the mdium decreased in a linear fashion between
days 2 and 8 of incubation and was completely depleted by day 10 (Figure 5.1a). The
total culture carbohydrate concentration (Appendix 4) closely followed the glucose
concentration between days 0 and 5. From day 5 to day 9. total carbohydrates exceeded
total culture glucose due to the formation of polysaccharides. Between days 1 and 9 there
was a steady increase in culture dry weight from approximately 1,000 g/m3 on day 1 to
approximately 2.000 g/m3 on day 9. Concurrently. the size of the mycelial pellets
increased from an average diameter of 1.5 mm to 2.1 mm (Figure 5.1c).

Figure 5.1

52

A Dm’mwlm

l¢~SOII>

onene-O

\.

-g°°d'~tnl

 

 

 

01.19 ems

1”

5%

1U

 

 

 

Biomass production. nitrogen and glucose utilization. and
production of extracellular peroxidases in agitated, nitrogen-
limited (2.2 mM) cultures of P. chrysosporiwn BKM-F-1767.
A. Time course of ammonium, glucose. and biomass (culture
dry weight). Values represent averages for triplicate cultures.
B. Time course of lignin peroxidase (LIP) and manganese
peroxidase (MNP) activities and total extracellular protein
concentration.

C. Mycelial pellet size.

D. Carbon dioxide evolved and oxygen utilized.

 

 

53

 

 

 

 

 

 

C
T6000
25‘" pelletotamerettmm) . ~~5000
I. I
2" ........ I. - --4ooo
- I I - .' Culture
Pellet .. I I u ...... " ,."9 dry
Diameter 1'5 ~. .......... .. 3000 weight
mm
( ) 1- ch 0° 0 «2000mm
8 ° ° 0 o
0.5 ‘- ctrltrrteotywaghtwms) -- 1000
0L I I I I I I I ' ' ' o
0 23456789101112
Days
D
2000"
1800‘
1600-
1400 --
1200-
g/m3ldy1000
800-
600-
400--
200--
0 t t
0 3456789101112

54

After glucose depletion on day 10. the culture dry weight decreased rapidly. probably
due to cell autolysis and/or cell wall polysaccharide utilization (Boominathan and Reddy.
1991; Kirk and Farrell, 1987). Acetate. which is used as a buffer and has been shown to
increase the production of LIP and MNP compared to other buffers (Michel et al.. 1988).
was not detectable after 3 days (Appendix 4).

The concentration of 002 in the headspace (measured daily before culture
oxygenation) also gradually increased during secondary metabolism from day 1 to day 9.
The depletion of oxygen from the headspace paralleled the evolution of 002 with a
nearly 1:1 OglCOz molar ratio. characteristic of aerobic metabolism (Figure 5.1d).

The extracellular peroxidases of P. chrysospan'wn are widely implicated in lignin
degradation and in the detoxiﬁcation of a variety of toxic aromatic compounds (Kirk and
Farrell. 1987; Lamar er al.. 1990; Valli et al.. 1991. Eaton. 1984. Bumpus er al.. 1985;
Lin and Wang. 1990). Therefore. the time courses of MNP and LIP activities as well as
total extracellular protein were determined. The depletion of nutrient nitrogen (Figure
5.1a) coincided with the onset of secondary metabolism and the production of
extracellular peroxidases (Figlne 5.1b). In agreement with other studies (Dosorctz er al..
1990a. Michel et al.. 1991). MNP activity was ﬁrst detected in the extracellular culture
ﬂuid between day 2 or 3 of incubation, increased to a maximum on day 4 or 5. and
declined to low levels by day 11 (Figure 5.1b). LIP activity. on the other hand. ﬁrst
appeared between days 4 and 5. reached a maximum between days 6 and 7 and then
rapidly declined. This decline in LIP activity during secondary metabolism has been
reportedtobeduetodegradationofLIPproteins byaprotease induced under starvation
conditions (Dosorctz er al.. 1990b). The observed loss of peroxidase activity between
days 8 to 11 also corresponded to the depletion of glucose in the culture ﬂuid. Total
extracellular proteins. which are initially high due to mycelial disruption during
homogenizing. declined throughout the primary growth phase but gradually increased

55

during secondary metabolism on days 5 through 8 as the MNP and LIPs were produced
(Appendix 4).

The concentration of soluble manganese in cultures declined to low levels one day
after MNP's were ﬁrst produced (Figme 5.2). The appearance of a brown mycelial
coloration coincided with this decrease in soluble manganese. Most of the manganese.
which was soluble before MNPs were produced. was found in an insoluble fraction
associated with the mycelia by day 5. Cultures without manganese in the medium did
not form the brown coloration. nor did high nitrogen cultures containing 12 ppm Mn.
where no peroxidase activity was detected. Thus manganese deposition in these cultlues
seems to be related to the production of MNP and not due to simple physical
precipitation. After the addition of BPE (see chapter 6). the brown coloration was
observed to diminish substantially and then reappear after the BPE had been decolorized.

5.2 Estinration of mass transfer resistances in mycelial pellet cultures of P.
chnsosporium.

The overall resistance to the mass transfer of substrates in a submerged mycelial
pellet culture (Figure 3.1) can be examined in terms of a series of resistances involving a
gas ﬁlm and a liquid ﬁlm for the gas liquid interface. a liquid ﬁlm around the mycelial
pellets and intraparticle diffusion (Kobayashi et al.. 197 3). In most studies of respiration
by mycelial pellets the oxygen concentration at the pellet surface (Cs) is assumed to be
equal to the bulk liquid concentration (C1) (Metz and Kossen.1977). This assumption has

rarely been validated. In theory. the relative importance of the ﬁlm mass transfer
resistance can be estimated by calculating the Biot number (Equation 19).
Experimentally. the bulk and surface concentrations of oxygen can be directly measured
using oxygen microelectrodes. The Biot number for oxygen. assuming a hypothetical

worst case of very small pellets (R=0.5 mm) and a low value for the mass transfer
coefﬁcient (k1 = 0.0005 mls). is 250. For a Biot number greater than 100. the external

56

 

 

 

 

45 [ 12 ppm Mn(II)
4o ..
35 " —°—aolublo
30 -- +lnsoiuble
Mn 25 "
(pm) 20 ..

 

 

 

45

40 PPM ”"00

 

”n 25" —°—aoluble
(ppm)20” +mmb

 

 

 

 

 

15.)-

10..

5... '

o ..................................................
012345678910

Figure 5.2 Soluble and insoluble manganese concentration as a function
of time in nitrogen-limited cultures of P. chrysosporiwn
initially containing 12 ppm and 40 ppm Mn(II).

57

mass transfer resistance is negligible compared to the intraparticle mass transfer
resistance (Bailey and Ollis. 1986) and the pellet surface concentration is approximately
equal to the bulk liquid concentration. Measurements using an oxygen microelectrode
conﬁrmed this ﬁnding. There was a negligible decrease in the oxygen concenn'ation as
the microelectrode was moved from the bulk liquid phase to the pellet surface. Likewise.
for both ammonium and glucose substrates. the Biot number indicated that the substrate
concentration at the pellet surface was not signiﬁcantly different from the bulk liquid
substrate concentration.

To conﬁrm Henry's law equilibrium for oxygen (Equation 18). the gas phase and
liquid phase oxygen concentrations were measured in active cultures using a
polarographic oxygen electrode. The liquid phase oxygen concentration was never
signiﬁcantly different than the corresponding gas phase equilibrium concentration. This
implies that the rate of oxygen transfer into the liquid phase by means of diffusion is
much faster than the bulk rate of culture respiration and that Equation 18 is valid. Van
Suijdam er al. (1982) reported that in mycelial pellet cultures of P. Chrysogenlan. the
gas-liquid mass transfer coefﬁcient (k1,) dropped rapidly above pellet volume fractions
of 40%. In this study the pellet volume fraction never exceeded 10%.

The dominant mass transfer resistance in shake ﬂask cultures of P. chrysospoﬁwn
was the intraparticle diffusion of oxygen. This was shown by calculating the observed
moduli (O) which compares the rate of reaction to the rate of substrate diﬂ‘usion
(Equation 20). For O values less than 0.3 the resistance to intraparticle mass transfer is
negligible and the process is reaction limited. For O values greater then 0.3. the process
is diffusion limited. Typical observed oxygen uptake rates with worst case assumptions
of large pellets (R=.0015 m) and liquid phase oxygen concentration of 1.5 g/m3. gives a
O value of 10.5 (Table 5.1). By comparison. the observable modulus for glucose is 0.16
using typical observed glucose uptake rates with worst case assumptions of large pellets
(R) 1.5 mm) and a low bulk glucose concentration (G<500 g/m3). The observed

58

modulus for ammonium during primary phase growth using worst case assumptions is
0.13 (Table 5.1).

Table 5.1. Observable moduli for substrates of P. chrysosporium using worst case

 

 

 

 

 

assumptions.
Oxygen Glucose Ammonium
v (g/mB pellet/s) 0.18 0.29 0.01
131! (m2/s) 2.9 x 10-9 9 x relo 2.1 x 10-9
C1(g/m3) 1.5 500 3
R (m) '7 0.0015 0.0015 0.0008
O 10.5 0.16 0.13

 

 

aPuresolntedlﬂuivityinwateru”CfmmPerryud.(l984).

Determination of the effective diffusivity of oxygen in mycelial pellets of P.
chrysosporium A

The effective diffusivity (De) of oxygen in mycelial pellets was determined using a
stirred cell apparatus as described in section 3.2. A group of pellets which had been
killed by suspension in 1% glutaraldehyde was immobilized in 2% agar. The suspension
waspouredintothe bottomofa50ml graduatedcylindertoZOml (l= 0.06m) andafter
the gel had solidiﬁed. nitrogen satmated liquid was added above the gel. the apparatus
was sealed. and the system was allowed to equilibrate for 3 days. The liquid was then
replaced by an equal volume of liquid ((1:1) which was air satmated. A polaragraphic
oxygen electrode was placed into the liquid and the apparatus was sealed. A small
magnetic stir bar resting on the gel matrix mixed the liquid phase. The decrease in the
oxygen concentration was monitored using a strip chart recorder for six hours. Five
different pellet volume fractions were used corresponding to Q values of from 0 to 0.4.
Since the effective diffusivity of oxygen in a 2 wt% agar gel has been shown to be 99%

59

of the value in water (An Lac er al.. 1986). the diffusivity of oxygen in the 2% agar slab
without pellets was assumed to be 2.9 x 10'9 mzls.

The results using this procedure were disappointing when compared to theoretical
predictions. During some runs the calculaed diffusivity in the slab (Dslab) was too large
(greaerthanD).Forotherrunsthediffusivitywas lessthanDby twoordersof
magnitude. These inconsistent results were attributed to problems eliminating all oxygen
bubbles from the diffusion cell apparatus. the effect of pressure on the oxygen electrode
output. the diffusion of air into the apparatus and the sloughing away of the agar gel from
the cylinder. After many attempts this technique was abandoned.

To try and overcome these problems. the diffusivity of glucose into the slab was
measured. This technique eliminated the problems associated with the oxygen electrode.
the air bubbles and the diffusion of air into the apparatus. Glucose was assayed using the
DNS assay. Initially the slab was free of glucose and contained 20 vol% pellets (Q=0.2).
To start the experiment 30 ml of a 5 g/l glucose solution was added above the slab (asl)
andtheapparatuswasplacedina39°Croomwith stirring. After48 hours. asamplewas
removed and compared to the initial sample. The value of Mt/Minf calculated based on
experimental data was 0.432 and the calculated value for the diffusivity of glucose in the
pellet-agar matrix slab was 9.5 x 10-10 m2/s. This compares wen to the literature value
for the diffusion of glucose in water of 9 x 10-10 m2/s (Cresand er al.. 1988). Subsequent
attempts to measure the effective diffusivity were unsuccessful due to deterioration of the
gel matrix over the 48 hour period. Since the diffusivity of glucose into this 20 vol%
pellet agar slab was not signiﬁcantly different than the diﬁ'usivity of glucose in water. the
effective diffusivity of glucose in mycelial pellets of P. chrysosporiwn (De) was assumed
to be equal to the pine liquid diffusivity (D). At this point the effective diffusivity of
oxygen in the pellets was assumed to be equal to the pine liquid diffusivity of oxygen.
thus for all culture modelling the value of the effective diffusivity used was 2.9 x 10-9

60

m2/s which is the value given by Perry er al. (1984) and Jordan et al. (1956) for the
diffusivity of oxygen in water at 39° C.

5.3 Determination of kinetic model parameter values

The kinetic parameter values for the kinetic model were determined in nitrogen-
limited cultures as described in section 5.1. The physical characteristics of these cultures
were as presented in Table 5 .2.

Table 5.2. Physical characteristics of agitaed. submerged mycelial pellet culture
of P. chqsosporiwn.

 

 

 

 

Characteristic Value
Temperature 39° (3
liquid Phase Volume (V1) 8.5 x 10-5 m3
Head Space Volume (Vhs) 1.65 x 10-4 m3
Pellet numbera (‘1) 7.3 3; 1.1 x 105 m'3
Pellet densityb (p) 6.513.0x 104 g/m3
Diffusivityc (De) 2.9 x 10-9 m2/s
Henry's law constant (H) 37.8
Solubilityof oxygen at 39° C 32.9 $11113

 

' Valnaswareuparimrntallydaerm'nadndrqortedara mangonenanduddeviation.

b Valneuaedformmodallim 3; themgedexperimenrallyobaervedvalnss.
cLhumrlcvahrefortiediffusivityofoxygalinwatarat39°C(l’erryare]. 1984)

A summary of the determined growth model parameter values is presented in Table 5.3.
The methods used to determine the model parameters are described in the following
sections (5.3 and 5.4).

61

Table 5.3. Kinetic model parameter values.

 

 

 

Parameter Definition Value Equations
Respiration
Vmax Michaelis-Menten coeﬁ'icient 0,75 5: o. 10 m3 pellet/s 25. 27. 28. 29
Km Michaelis-Menten coefﬁcient 0,5 :1: 0,3 g/m3 25 . 26. 28. 29
Yum Theoretical yield coefﬁcient 0.92 g/g 9. 13. 16
YCO2JOZ Theoretical yield coefﬁcient 1.38 g/g 31
Prinrary Growth
tug length of the lasphm 2810.2 x104 s 6
“max Growth rate constant 39 :l: 0.3 x 10-5 8-1 5. 6
Y” Growth yield for glucose 0.92 1 0.1 g/g 8
Yn/x Growth yield for ammonium 0.04 i 0.1 g/g 4
Secondary Metabolism
fpg Polysaccharide from glucose 0.04 i 0.03 g/g 11a. 12. 13
f" Biomass from glucose 0.12 :l_- 0.01 g/g 11b. 12 13
fr Glucose for respiration 0.84 3; 0.03 g/g 11c
Death Phase
kaut Autolysis rate constant 25 i 05 x 10—6 8-1 15
Polysaccharide utilization constant 04 i 0.1 x 10-6 s-l 14

"ps

 

 

62

Kinetic coefﬁcients for the lag phase and primary growth phase.

To determine pm and the length of the lag-time (tlag). the natural log of the
biomass divided by the average initial biomass concentration was plotted for the ﬁrst 24
hours after inoculation (Figure 5.3). The slope and the x-intercept were determined using
a linear regression technique (Appendix 10). As given by Equation 6. the length of the
lag time (x-intercept) was 7.9 h and the maximum growth rate constant pm (slope).
was equal to 3.9 e'5 s‘l.

Yield coefﬁcients during primary metabolism were determined as follows. The
yield coefﬁcient for nutrient nitrogen (Y n/x. Equation 4) was calculated by dividing the
total nutrient nitrogen consumed. by the cell dry weight. at the end of the primary growth
phase. The yield coeﬁ'icient for glucose (Equation 8) was estimated based on the
following assumptions. In fungi. inorganic nutrients account for 1% to 10% of the dry
biomass (Aiba er al..l973). In nitrogen limited cultures. nutrient nitrogen accounts for
only 1% to 3% of the biomass. Therefore. the yield coefﬁcient for glucose (Y g/x) dining
balancedgrowthwassetat0.92 g/g.

Kinetic coefficients for secondary metabolism.

The glucose metabolism factors (Equations 11 a.b.c) are used to determine the fate
of glucose as it is used for polysaccharide production. biomass and respiration during
secondary metabolism.

f pg polysaccharides

 

__£L___. biomass

— 1

Glucose

fr

 

g respiration

63

3.5 1'

3--

2.5 '-

2 -

I

In (X/Xo) 1 .5

 

 

‘4 8 12162024 28 32

Hours

Figure 5.3 Plot of the logarithm of the biomass (X) divided by the
average initial biomass (Xo.avg) as a function of culture age
in nitrogen-limited mycelial pellet cultures of P.
chrysosporl'um. This plot was used for the determination of
pmax and ﬂag. Open diamonds are experimental data from
cultures on four different occasions. The line is a linear

regression ﬁt of the data points (Pmax=3-9 x 10'5 s‘l.
tlag=7.9 h).

64

Dosorctz er al.(l990a) showed that in submerged oxygen ﬂushed cultures initially
containing 10.000 g/m3 glucose. approximately 400 g/m3 of soluble polysaccharide is
formed by the time of glucose depletion. Hence. a ﬁgure of 4% was used for the glucose
consumed for soluble polysaccharide synthesis (fpg=0.04). A small amount of biomass is
produced during secondary metabolism corresponding to approximately 12% of the
glucose consumed (fr-=0. 12). The balance of the glucose consumed is used for respiration
(fr=0.84). The values of these factors are assumed to be ﬁxed for the purposes of the

model, however in reath these factors may change due to oxygen starvation or other
metabolic or environmental conditions (see discussion).

The yield coefﬁcients for oxygen and carbon dioxide from glucose used for
respiration (Equations 9 and 31). were calculated based on the overall stoichiometry of
respiration (Ycopjozz 1.38, Yg/Ozs .92).

C6H1206+502 > 6002+ 6H20
Kinetic coefficients for the death phase.
The rate constant for autolysis (kaut) was estimaed by curve ﬁtting biomass data

 

aferglucosewasdepletedfromthemediumtoaﬁrstorderdeathraeEquation
(Equation 15). The rate constant for polysaccharide utilization (kpg) was calculated by

measuring the rate of 002 evolution after glucose was depleted from the medium using
the yield coefﬁcients for respiration (Equations 14 ande and assuming that all 002
evolved is due to polysaccharide respiration. The ﬁnal culture dry weight (Xf) was
determined by measuring the biomass concentration 20 days after no respiration was
detectable (Equation 15).
Mycelial pellet characteristics

The pellet density (p) was calculated from Equation 1 using experimental values
for the characteristic pellet radius. pellet number and culture dry weight. Using the
constant constant pellet density assumption. the model predicted characterisitic pellet
radius (R) was calculated. The time course of the experimentally determined pellet radii

65

and the model predicted pellet radii using parameter values from Table 5 .3 showed good
agreement (Figure 5.4). Dming the death phase. pellet density decreased due to autolysis.

5.4 Determination of kinetic coefficients for the oxygen utilization model.

Three different approaches were used to determine the intrinsic Michaelis-Menten
kinetic parameters for respiration; Vmax and Km (Equation 25). The values of both
Vmax and Km were determined by ﬁtting data for the oxygen concentration proﬁle in
mycelial pellets as determined using a microelectrode with the model Equation for the
oxygen concentration proﬁle (Equation 23). The value of Vmax was also determined by
measuring the rate of oxygen depletion from the bulk liquid and calculating Vmax using

W'rlkinson's method. The values of Vmax and Km determined by oxygen proﬁle
modelling were checked by comparing the evolution of C02 predicted by the integrated

kinetic model with experimental data for C02 evolution.

The oxygen concentration proﬁles within individual mycelial pellets were
nteasttred using an oxygen microelectrode as described in Methods. the data (see
Appendix 12) were plotted using EXCEL software and compared to oxygen proﬁle data
generated using a computer simulation (see Equation 23 and Appendix 22). Values of
Vmax and Km in the computer simulation were changed until the computer simulation
proﬁle most closely resembled the experimental data. Using this approach. the range of
values of Vmax were 07610.10 g/m3/s and the range of values of Km were 05:03
g/m3. A typical oxygen concentration proﬁle and the computer simulation curve which
most resembled the experimental data is presented in Figure 5.5. In pellets with a
diameter greater than 1 mm. the oxygen concentration decreased to undetectable levels
well before the pellet center. However. the data were taken in an air atmosphere. In
shake-ﬂask cultures. the oxygen partial pressure was generally greater than 0.5 atrn and
the rate of autolysis due to oxygen starvation was probably negligible. since both culture
dry weight and the rate of respiration increased during secondary metabolism

1

0.0016 -

0.0012 t

 

Pellet
Radius 0.0008 -
R (M)

 

0.0004 -

 

 

Hours

Figure 5.4 Mycelial pellet radius in submerged cultures of P.
chlysosporr'um grown nitrogen-limited medium (Condition
2). solid line: kinetic model prediction. squares: experimental
data points.

67

The evolution of carbon dioxide was measured by determining the concentration of
CO; in the culture headspace every 24 hours before oxygenation. The data (see
Appendix 13) were compared to 002 evolution as prediced by the integrated kinetic
model. The model curve using the parameter values presented in Table 5.3 (Vmﬁx value
of 0.76 g/m3/s, Km of 0.5 g/m3 and pellet density value of 65000 g/m3 pellet). closely
followed the 002 production data (Figure 5.6) except on the ﬁrst day. This may be due
to a fasterrate of metabolism during the growth phase.

Respiraﬁonratemeasmementswerealsomadebypomingoneenﬁrecultmeintoa
full stirred vessel (respirometer) and measlning the decrease in the liquid phase oxygen
concentration (C1) with time using a polarographic oxygen electrode (Ingold. model
#531) (see Appendix 14). To determine the rate of oxygen uptake (v). the slope of the
depletion curve was measured at various oxygen concentrations (Figure 5.7). The
apparent Vmax and apparent Km values were ﬁt using the method of Wilkinson (1961).
During primary growth. the apparent value of Vmax was 0.04 g/m3 media/s. Dtuing
secondary metabolism the apparent Vmax values ranged from 0.014 to 0.031 g/m3
mdia/s while the apparent Km ranged from 1.5 to 8 g/m3. The apparent Vmax dropped
to 0.0073002 g/m3 media/s one day after the culture glucose level dropped below 1,000
g/m3 and to 0.0001¢.0001 g/m3 media/s ten days later. By using appropriate pellet
density and cultlue dry weight values. the oxygen depletion Vmax were averaged to give
the speciﬁc apparent Vmax (0.771023 g/m3/s) (see Appendix 14). Note that mass
transfer effects are not considered in the apparent Vmax and apparent Km values;
however. if the effectiveness factor is near unity (the case at high C1) the apparent Vmax
and the intrinsic Vmax values should be comparable.

Some evidence indicated that oxygen transfer may occur by mechanisms other than
diffusion in the respirometer apparatus. The rate of depletion of oxygen measured daily
in cultures of P. chrysosporr’wn was plotted against the liquid phase oxygen
concentration (C1) (Figlne 5.8a). The overall oxygen depletion rate (Rog) increased daily

68
with culture age until culture glucose was exhaused (Day 10). This agrees with C02
evolution data which also increased daily during secondary metabolism (see Figure 5 .6).
However. the depletion rate remained near the apparent Vmax value calculated using
Wilkinson's method until the oxygen concentration was below 5 g/m3. A model
simulation (Fig 5 .8b). indicates that respiration should have decreased from the apparent
Vmax at around 20-30 g/m3 oxygen due to intraparticle mass transfer limitations as
represened by the effectiveness factor. One possible explanation of this ﬁnding is that
thestirringrateintherespirometeris sorapidthatpelletsaredisruptedtoaﬁlamentous
form causing the effectiveness factor to become unity. In this case. the oxygen uptake
rate would appear as it does in Figure 5.8c. This behavior more closely resembles the
experimental results. Filaments of mycelia were observed after a period of stirring on
most occasions. Another possibility is that the stirring rate in the respirometer induced

convective diffusion into the pellets by causing the pellets to contract and expand.

69

 

 

 

 

10 T

8 -—

6 ..

02
(911113)

4 --

2 _.
'i

0 ﬂ
0 0.00025 0.0005 0.00075 0.001

r (m)

Figure 5.5 Oxygen concentration proﬁle in a mycelial pellet of P.
chtysospon'um (diam.=.00185 m) as determined using an
oxygen micro-electrode.

The solid line is as predicted by the kinetic model (Km=0.5
g/m3, vm=o.76 g/m3/s, De=2.9 x 109 m2/s).

0.2 70

Model
0.16 predicted C02 evolution
0.12 ‘
C02 ‘
evolved \ i
(g) ‘ i ‘
0.08
1 ‘ [
0.04 1 ’
l l ‘ i
o i

1234567891011-12
Culture Age (days)

Mean and one 5.0.
(2 to 4 mplbatu on three separate nee-aloha)

COZ
evolved

(9)

 

 

1234567891011‘12
Culture Age (days)

Figure 5.6 Carbon dioxide evolution in nitrogen-limited mycelial pellet
cultures of P. chrysosporium (Condition 2). The solid bars

are as predicted by the kinetic model (Km=0.5 g/m3,
vm=076 g/m3/s. De=2.9 x 10-9 mZ/s.) Cross-hatched

bars are experimental data.

0.024 -

71

I

Vmax j a

(g/m3/s)

Figure 5.7

 

 

-.

30

Cl (g/m3)

Typical plot of the rate of oxygen depletion (v g/m3/s) from
the culture ﬂuid versus the liquid phase oxygen concenu'ation
(C1 g/m3) from nitrogen-limited, mycelial pellet cultures of
P. chrysosporium as measured using a respirometer. Solid
line is a curve fit using the method of Wilkinson (1961).

Open squares are experimental data from three consecutive
measurements.

0.03

0.025

0.005

Figure 5.8

 

72

Day

:IVJ_‘_J_L;IJA

 

1 r v r r l r I r v I I v I r l r 012

0 10 20 30 + 14

 

 

 

Oxygen Concentration (g/m3)

The rate of oxygen uptake by cultures of P. chrysosporium as
a function of culture age as measured using a respirometer:

A. Experimental data.

B. Model prediction using kinetic parameter values from
Table 5.3.

C. Model prediction assuming an effectiveness factor of one
(Emm = l) at all times.

73

 

 

 

 

 

 

 

0.03
0.025
0.02
v
o. 15
(g/m3ls) 0
0.01
0.005
0
0 10 20 30
. Oxygen Concentration (glm3)
C
0 03 Day
__.__ 1
0.025 ——D-— 2
3:32::2:2::i:1:2“:1::15::11:2::::::::::::::::::::::: -—a
0'02 /.-...eeeooeaeeeeeeeeeoeeeeue ‘
v ‘ . . ........................... 5
(glm3/s) 0.015 E: .. ............................ 6
’35: I..IIIIIIIIII-IIIIIlIIIIIIII
0.01 3:5,: -' __._ 7
X"
0.005 7:)
O l . . % . . . . A—. .
0 10 20 30

Oxygen Concentration (glm3)

74

5.5 Model test cases

The complete kinetic model Equations describing growth, substrate utilization and
respiration in shake ﬂask cultures of P. Chrysosparium were integrated and solved using a
computer program (Appendix 21). Euler's method was used to approximate the
differential Equations with a time increment of one hour. The kinetic parameters
determined in the previous two sections were used (see Table 5 .3). Batch cultures were

grown with ﬁve diﬂ'erent initial conditions to test the model ﬁt (Table 5 .4).

Table 5.4 Initial conditions for model test cases.

 

 

Ammonium Glucose
Condition Description (g/m3) “(£32
1 Low glucose 39 5 .000
2 Nitrogen-limited 39 10.000
3 High glucose 39 15 .000
4 Very low nitrogen 2 11,000
5 3X basal nitrogen 117 11,000

 

 

 

In one set of experiments, three different initial glucose concentrations were used
(conditions 1.2 and 3). The model closely predicted the glucose and biomass data for the
three conditions (Figure 5 .9). Nutrient niuogen was completely depleted under each
condition within 24 hours. The initial level of glucose had little effect on the specific rate
of glucose or nitrogen uptake. the culture dry weight or the pellet size. The initial glucose
concentration primarily affected the length of secondary metabolism. In low glucose
cultures (condition 1). the onset of the death phase occured just 120 hours after
inoculation. In high glucose cultures (condition 3) the length of secondary metabolism
was extended by approximately 80 hours compared to basal glucose cultm'es and the '
onset of the death phase did not occur until 300 hours after culture inoculation.

75

(glm3) Condition 1 (glm3)

 

 

 

 

Figure 5.9 Culture dry weight (X). glucose concentration (G), and
nutrient nitrogen (ammonium) concenu'ation (N) as a
function of time in cultures of P. clvysosporium grown under
various initial glucose and nutrient nitrogen conditions. Solid
lines are as predicted by the kinetic model. Open squares are
experimental data for glucose, closed diamonds are
experimental data for culture dry weight and open u'iangles
are experimental data for ammonium.

A. Condition 1; Go: 5,000 g/m3, No: 39 g/m3.
B. Condition 2; Go: 10,000 g/tn3, No: 39 g/m3.
C. Condition 3; Go= 15,000 g/m3, No= 39 g/tn3.
D. Condition 4; Go=11,000 g/m3, No: 2 g/tn3.

E. Condition 5; Go: 11,000 g/m3, No=ll7 g/tn3.

76

X6

 

 

 

 

 

Condition4 0
(9&3, (am)
-~1E+04
~9ooo
L
-6000
'15;-
10-L “3000
5‘r
A‘ O ——'
o . i i i : o
0 so too 150 200 250 300 350
Hours

 

 

 

x - G Condition 5

 

 

 

 

0 50100150200250300350400450
Hour:

77

For conditions 1.2 and 3. the MNP activity reached a maximum of 2.500 W! on day
3. TheLlPactivityincondition 1 cultures reachedamaximumof l40U/l on day 5. In
conditions2and3,LIPactivityreachedamaximumof260U/land310U/lbetween
days 5 and 6 respectively. A rapid decrease in biomass concentration and the
disappearanceofMNPandLIPactivitycoincidedwiththetimeofglucose depletion.
This occurred at hour 140, 220 and 300 for the low glucose (condition 1), basal glucose
(condition 2) and high glucose (condition 3) cultures respectively. Under identical initial
conditions. Jones (1990) has shown that this kinetic model accurately predicts culture dry
weight and glucose consumption by cultures of P. chrysospon’wn in a rotating biological
contactor. when corrections are made for slab geometry.

A second set of initial conditions was teswd in a subsequent experiment (conditions
4 and 5) to determine the model's ability to predict changes in the nutrient nitrogen
concentration. When cultrnes were grown with very low levels of nuu'ient niu'ogen, (2
g/m3, condition 4), little total biomass was produced and glucose was consumed slowly
(Figure 5.9d). In cultures grown in 3 times the basal level of nutrient nitrogen (117 g/m3
ammonium. condition 5), nutrient nitrogen was depleted within 30 hours, glucose was
depleted within 190 beans and the biomass concentration reached 3,000 g/m3 (Figure
5.9e). Thus the level of nuu'ient niu'ogen primarily affected the biomass level at the end
of the primary growth phase. The biomass level. in turn, affected the rate of glucose
metabolism and respiration. The integrated kinetic model accurately predicted the
experimental data under these conditions.

Cultures grown in 39 g/m3 ammonium (condition 2) produced the highest LIP
activity (220 ml) even though less biomass was produced than in the higher niu'ogen
cultures (condition 5). In condition 4 and 5 the LIP activity decreased to undetectable
levels one day after the glucose concentration dropped below 400 g/m3.

78

5.6 Bioreactor studies

Cultures were grown in a 6.8 l stirred tank reactor (BioFlo II, New Brunswick
Scientiﬁc Co. Inc.) with a 2.3 1 operating volume. This system had the same liquid to
headspace volume ratio as the shake ﬂask cultures described above. Oxygen was
provided by daily ﬂushing of the headspace of the reactor with oxygen, and the daily
evolution of C02 was measured. Compared to control cultures grown in shake ﬂasks, the

bioreactor culture produced the same amount of biomass and had a similar pH profile but
consumed glucow and evolved C02 much slower (Figure 5.10). The bioreactor culture
produced signiﬁcantly less MNP and no LIP. The large scale production of LIPs and
MNPsissﬁHhampaedbythemabiﬁtywproducemepaoxidasesmascalablemacmr
configuration. This is primarily due to the organisms sensitivity to agitation (Michel er
al., 1990). The production of MNPs appeared to be less sensitive than the production of

LIPs to culture and agitation conditions.
12000 -- -- 2500

 

 

 

 

10000
8000
1500
6000
e .5 “P 1000
4000 -L .
. -- 500
. 4— Daily carbon dioxide evolution
2°00 " .' . tel-“31m
o 1'11=II1T%rIII:rfWIJlrrrﬁll‘srii o
0 2 4 6 8 10 12

Days
5.10 Characteristics of a stirred tank bioreactor culture of E. W

79
PartII:Discussion

5.7 The life-cycle of cultures of Phaucmchaete ehrysosporium

Nitrogen-limited, peroxidase producing cultures of P. Chrysosporiwn (Figure 5.1)
displayed the classic characteristics of the microbial life-cycle. For approximately 8
hours immediately after inoculation. no signiﬁcant growth occurred (Figure 5.1a). After
8 hours. the biomass increased exponentially (Figures 5.1a and 5.3) until the nutrient
niuogen was depleted. During secondary metabolism (or idiophase), the biomass, the rate
of 002 evolution and oxygen depletion. and the mycelial pellet radius (Figure 5.4)
increased slowly until the glucose was depleted from the medium. Both the lignin
. peroxidases (LIP) and manganese peroxidases (MNP) were produced during this period.
Depletionofthecarbon sourcefromthe mediumonday lOcoincidedwithadecreasein
the biomass, a substantial decrease in the rate of culture respiration and the disappearance
of peroxidase enzyme activity. Clearly, the cultures proceeded through four distinct
phases (Figure 5.1); a lag phase where little growth or metabolism occurred, a primary
growth phase where growth was exponential and nutrient nitrogen and glucose were
depleted, a secondary metabolic phase where glucose was depleted and culture
respiration reached its highest rate, and a death phase where the biomass and metabolic
activity diminished rapidly.
5.8 Kinetic model for mycelial pellet alltures

An integrand model based on the life-cycle phases accurately predicted a wide
spectrum of culture characteristics using a minimal number of model parameters. In total,
ten independent model parameters were used to describe the life-cycle of mycelial pellet
cultures of P. chrysospoﬁum (Table 5.3). These included growth rate constants, substrate
utilization factors and respiration kinetic parameters. The reported parameter values are
those which best ﬁt the control culture data. Error estimates for the determined parameter

values represent the range of observed values and are not standard deviations.

80

The ”delayed" exponential growth law (Figure 5.3) predicted the level of biomass
at the beginning of secondary metabolism within 10% of the experimentally determined
value. however experimental data were not accurate enough to distinguish between
different growth models such as the Monod model, the cube-root model or other models.
This is due to error in the biomass determination of about ;|; 10% (see van-Suijdam er al..
1982 for more discussion of this). However, unlike other commonly used growth models.
the integrated kinetic model accounted for the depletion of limiting subsu'ates which
uiggered the transition between life-cycle phases.

The model accruately predicted the pellet radius as a function of time when the
pellet number (n) and pellet density (p) was known (Figure 5.4). A function relating
pellet density and pellet size has been used by other investigators to account for the
decrease in the pellet density which occurs as a result of autolysis within the mycelial
pellets (Y ano et al., 1961, van-Suijdam et al., 1982). A relationship of this type was not
used here because previous studies have shown that for pellets of P. chrysosporiwn pellet
density is constant up to pellet radii of 0.0025 mm. However, the error in the pellet
number and size determinations were fairly large ( SD. = j; 15%) but the pellet size did
not change substantially as it did in other studies (for instance in Yano er al., (1961)
pelletsfromlessthanlmmtogleaterthan10mmwerestudied).Thiswasduetothe
short period of growth in the nitrogen limited cultures. In addition, a wide range of pellet
densities were observed for different experimental runs. Densities as low as 30,000 g/m3
to as high as 90,000 g/m3 were observed. The reason for this variation is not known. The
effect of pellet density variation on the biomass, effectiveness factor, glucose uptake rate
and pellet radius as predicted by the kinetic model is presented in Figure 5.11 . As pellet
density decreases, the characteristic pellet size increases. Increases in pellet size results in
lower oxygen effectiveness factors for respiration (Figure 5.11 ).

81

 

 

 

 

 

 

 

 

 

 

    

 

 

 

‘ 0.001
. Mycelial Pellet accummml . 0.0005
-r

......... o
0 50 100 150 200 250 300

Figure 5-11 The effect of the mycelial pellet density (p) on the biomass,
glucose uptake rate, oxygen effectiveness factor and mycelial
pellet radius as predicwd by the kinetic model.

82
5.9 Oxygen utilization in mycelial pellet cultures of P. chrysosporium.

The intrinsic kinetics parameters for respiration, Vmax and Km, determined by
three separate methods were in agreement. The values of the Michaelis-Menten kinetic
parameters for respiration determined by modelling the oxygen concentration proﬁle
within pellets of P. chrysospoﬁwn (Figure 5.5) closely agreed with CO; evolution data
(Figure 5.6) when the same parameters were used. To a lesser extent, oxygen depletion
data (Figure 5.7 and 5.8) also agreed with kinetic parameter values determined by proﬁle
modelling. A wide range of kinetic parameter values (SD.=30% of mean) were obtained
using Wilkinson's method and data ﬁom the respirometer (Vmax = 0.77 :l: 0.23 g/m3/s)
(see Appendix 14). This may be due to disruption of the pellets within the respirometer
apparatus (Figure 5.8). This agreement. and the fact that closure of the oxygen mass
balance was obtained, gives added credibility to the kinetic parameter values determined
for culture respiration. ‘

In previous studies the oxygen utilization by fungal pellets has been studied only
during the primary growth phase (Table 2.2). The rate constants for respiration in these
previous studiesareofthe sameorderofmagnitudeasthose determinedhere forP.
chnsosporiwn. In the zero order model for pellets of P. chrysogenwn developed by
Phillips (1966) a rate constant of 0.56 g/m3/s was reported. Van Suijdam er al. (1982)
reported a maximum oxygen uptake rate of approximately 0.8 g/m3/s for pellets of P.
' chrysagenum. These values compare to a Vmax value for pellets of P. chrysosporr’wn of

0.76 g/m3/s. Both Yano er al. (1961) and Kobayashi et al.(l973). reported that the
maximum speciﬁc oxygen uptake rate (Qozmax) for ﬁlamentous A. niger is 6.1x10'5

gig/s. By comparison, for pellets of P. Chrysosporiwn QOzmax was 1.2x10‘5 g/g/s. It

should be noted that the respiration kinetics determined in this work were for secondary
metabolism and not for primary growth as in the previous studies.

The low Km value for respiration of 05:03 g/m3 found for pellets of P.
chrysospon'wn is also in line with other studies. The value of Km reported for pellets of

83

A. niger by Kobayashi ct aI.(l973) was 0.1 g/m3 and yeast cells typically have a Km of
approximately 0.05 g/m3 (Bailey and Ollis, 1986). Because of the very low value of Km
for oxygen, a zero-order model (Km=0) for respiration has been used by some
investigators (Phillips, 1966; van Suijdam er al., 1982; Yano er al., 1961; Wittler er al.,
1986) to model respiration in mycelial pellets. If a zero-order model is assumed for
pellets of P. chrysosporium, then a critical pellet radius can be calculated where the
center of the pellet ﬁrst becomes oxygen starved. For mycelial pellets of P.
chrysosporiwn, this radius is 0.86 mm in oxygen saturated medium and 0.40 mm in air
saturated medium.

Wittler er al. (1986) showed that in a ﬂow reactor, oxygen transfer in pellets of P.
chrysogenum may occur by means of convection and eddy diffusion as well as by
molecular diffusion. These methods of oxygen transfer were not considered in the model
presented here, where in mildly agitated shake ﬂask cultures were studied. However, in
larger scale fermentations, with sparging and more turbulent mixing conditions, reactor
hydrodynamics may signiﬁcantly inﬂuence the oxygen mass transfer within the mycelial
pellets. Respirometer results indicated that convection may play a role in oxygen mass
transfer is rapidly agitated cultures of P. chrysosporiwn (see Figure 5.8)

For Michaelis-Menten kinetics, a three dimensional surface (Figure 5.12 ) provides
a tool for quickly estimating the oxygen effectiveness factor in mycelial pellets of P.
chrysosporium as a function of the characterisitic pellet radius (R) and the liquid phase
oxygen concenu'ation (C1). The most favorable conditions for aerobic metabolism are
represented by the plateau of the surface where the oxygen effectiveness factor (B) is
near one. This region corresponds to small pellet radii and high dissolved oxygen

concentrations.

84

     

       
    

    

               

‘15. :11“*‘\ \\ ‘®\\“\\\\\\{\\\\\\\\\\{\\\I\\\\\
W§WW§r
\ w \

\
\\

            
  

 

\ \ \
\ \t\\\\\\\\\\\\\\\ \ \

        
  

   

 

    
          

  

Cs
(g/m3 )

Figure 5.12 Three dimensional representation of the oxygen effectiveness
factor (Emm) for mycelial pellets of P. chrysosporiwn as a
function of the liquid phase oxygen concentration (C1) and

the characteristic pellet radius (R).

85

5.10 Kinetic model simulations

Models for LIP and MNP production are difﬁcult to develop without detailed
knowledge of the metabolic pathways involved and their genetic regulation. To study the
effect of oxygen limitations on peroxidase production, computer simulations based on the
kinetic model were conducted for various conditions in which the production of LIP and
MNP is adversely affected. The time course of carbon dioxide evolution, oxygen
depletion and the effectiveness factor in basal nitrogen-limited cultures is presented in
Figure 5.13. In these cultures, the eﬂ’ectiveness factor remains near unity during primary
growth (thus validating the assumption made for Equation 6) and drops to approximately
0.6 during secondary metabolism on day 9. Nearly 50% of the oxygen in the headspace is
depleted between each oxygenation cycle. This simulation clearly reveals the unsteady
state nature of fermentations in sealed shake-ﬂasks.
Air-ﬂushed cultures

In air-ﬂushed cultures no LIP and 66% less MNP is produced. Furthermore,
Dosorctz er al. (l990a) found that in air-ﬂushed submerged cultures of P. chrysosporium,
the rates of glucose depletion and carbon dioxide production decreased by 33% and 70%
respectively compared to oxygen ﬂushed cultures, while the biomass concentration
reached 2,900 g/m3 by day s and soluble polysaccharides doubled. A model simulation
for air-ﬂushed cultures using the kinetic parameters from Table 5.3 closely predicted the
data for carbon dioxide production reported by Dosorctz et al. (l990a) but under-
predicted the glucose utilization rate and the biomass concentration. The oxygen
effectiveness factor for air-ﬂushed cultures ranged from 0.1 to 0.6. Thus in air ﬂushed
cultures, more cells are oxygen limited and unable to respire glucose so the yield
coefﬁcients for glucose metabolism may be different. By adjusting the glucose
metabolism factors accordingly (fpg=0.25, fx=0.3) the model closely predicted the
results for C02 evolution, glucose consumption and biomass production as reported for

86

0'5 :Hﬁ’h'i "t " ' i -------- T 1
‘ “ll “5‘ 'il‘ :1 a 'l .
: i \i ‘t:‘\ :‘l 1“ :1 ( I.I Emm :
~~ ("\1"t1"‘1 “08
0'4. \‘thr‘d‘H _ .
TOtal t l: u ‘: l‘: “: .-
Culture 3 ' l 1‘; 1‘. r: : '
Oxygen 0.3 -- i i __ 05
(9) - :
and ; 02 i Emm
Carbon 0.2 :- -: 0,4
Dioxide - . .
(9) I ‘ ‘ _ ‘ ' :
0-1 j r, .i is! i‘ {g :n! ii , a: 0.2

 

 

 

Figure 5.13 Total oxygen and carbon dioxide and oxygen effectiveness
factor in nitrogen-limited cultures of P. chrysosporium as
predicted by the kinetic model.

87

0.15 ‘- - 10000

0.12 .2. _
j - 7500

Carbon 0.09 :- - a
Dioxide ; _ Glucose
Evolution ‘ . . 5000 (g/m3)

(g) 0.06 ‘- -

I I I

l J

 

C
e .. ee ..
a. at -e e
a -o no u ..
ee . .
1 .e': .t c'o 'e o'e'
.. cc or a. co er
I. 0» Cl .5 his I.
be -I II I. l. .0 II
00 It Or .0 ' C. 0" O. I.
e. a... De .e_a a... '0’ '0‘: a... e v
0 av ro- . ..
.' n. be are .e en te I. or be ..
e; or no .e~ e. ec‘ set .0 e. to
. at e. co co I. e. on .. .. l ,.
0‘ .0 I. .......... . .. "
-. e- ee be or ee ’0 II -I be to
on >o- er ee oe- es ee Ie- .- e! I
Q " O0 O0 00 e. or be cc . be o.
II III I. .. I.l .. .. ... . .. .
nc co so as or no ee on ea 1 ee
00 -O- .0 do ten .6 at 00- co in
- o. 0.0 .e.e 0.0. 'e.e. '0‘. a... .0... e .e e
e r .
as ea or to 00 00 In ' ee on
. e- co oe~

 

01234567891011
Days '

Figure 5.14 Glucose consumption and carbon dioxide production in air-
ﬂushed shake ﬂask cultures of P. chrysosporiwn as predicwd
by the kinetic model (line and solid bars) and as reported by
Dosorctz et al. (1990) (open squares and cross-hatched bars).

88

air-ﬂushed cultlues by Dosorctz er al. (1990a) (Figure 5.14 ). This ﬁnding suggests a
relationship between the level of polysaccharides produced and the dissolved oxygen
concentration within the mycelial pellets.

High nutrient nitrogen cultures.

A simulation for batch cultures grown in high nitrogen (390 g/m3 ammonium)
illustrates the effect of nutrient nitrogen sufﬁciency on pellet cultures of P.
chrysosporium. According to the model simulation, culture glucose is exhausted much
faster in these cultures and autolysis begins between days 2 and 3 (Figure 5.15). Thus,
under this condition the cells are no longer in a secondary metabolic state when LIPs and
MNPs are normally produwd in control cultures. The fed-batch administration of glucose
to such cultures to prolong secondary metabolism may not ameliorate this situation since
the pellets have already grown to a large size (>2 mm diameter) and have an oxygen
effectiveness factor of approximately 0.2 so that much of the center of the pellet is
oxygen starved.

Large mycelial pellet cultures

A model simulation for cultures in which large mycelial pellets form (pellet
diameter = 6 mm) is presented in Figure 5.16. This case is analogous to cultures grown
under low rates of agitation (see Michel er al., 1990). According to the model simulation,
under this condition, nutrient nitrogen is depleted within 50 hours after inoculation. Also,
during secondary metabolism, the rate of glucose depletion is markedly slower than the
rate typically found in cultures with small pellets. The oxygen effectiveness factor drops
precipitously to a value of 0.1 during the primary growth phase as the pellet size
increases (Figure 6.6).

20000

18000 --
16000 '-

1 4000

1 2000 -

10000
8000
N, G, X 6000

4000 .
2000 '

Figure 5.15

 

 

89

  

 

 

 

uwmswo)
- - - - Gram-3)

"'— Xlslm3)
—Emrn

 

 

 

 

 

* 0.8
“ 0.6

0.4
0.2

-0.2

- -0.4

Model simulation for cultures gown in high nutrient
nitrogen condition; 60:10000 g/m3, No=390 g/m3.

 

 

 

 

 

 

 

 

1 VT " 0.005
0.9 -- -- 0.0045
0.8 -- .. 0.004
0.7 + Hump) '- 0.0035
0.6 r- 0.003

Emm 0.5 . - 0.0025(2)
0.4 r- - -r 0.002
0.3 -- -- 0.0015
0.2 r- 0.001
0.1 *- 0.0005
0 .ﬁ 1 vi - 0

figure 5.16

 

 

 

Kinetic model simulation for nitrogen-limited cultures of P.
chrysosporium with large pellets (R=3mm). (This case is
analogous to cultures of large pellets gown at low agitation
speeds as described by Michel et al., 1990).

91

Production of peroxidases by mycelial pellets with low effectiveness factors.

Cultures of P. Chrysasporl’um, for which the kinetic model predicted a low oxygen
effectiveness factor (E<0.4), produce much lower levels of LIP and MNP activity. For
example, Dosorctz er al. (1990a) showed that in air-ﬂushed cultures, (where the model
predicts an oxygen effectiveness factor ranging ﬁom0.l to 0.3), no LIPs and one third
less MNPs were produced. In high nitrogen cultures where the oxygen effectiveness
factor was less than 0.2 (Flglue 5.16) no LIPs or MNPs were produced (see Figure 6.8).
Furthermore, Michel er al., (1990) found that large pellets (6 mm diam), formed using
low agitation rates, and for which the kinetic model predicts effectiveness factors of
around 0.1, fail to produce LIPs. By comparison, cultures of P. chrysospoﬁltm where a
high effectiveness factor is predicted by the kinetic model (small pellets and liquid phase
oxygen concentrations near saturation), produce high levels of both LIP (200 to 300 WI)
and MNP (1000 to 2500 Ull). Therefore, oxygen limitations in mycelial pellets of P.
chrysosporium appear to have a signiﬁcant affect on the level of LIPs and MNPs
produced. Unlike genetic regulation of peroxidase expression by manganese, veratryl
alcohol and nitrogen, the effect of hyperbaric oxygen on cultures of P. Chrysosporium
appears to have a physiological basis.

CHAPTERVI

Application of the peroxidases of Phancrochactc chrysospoﬁum
to the decolorization of Kraft bleach plant effluents (BPE)

Part I: Results

6.1 Decolorization of BPE by cultures of P. chrysosporium.

Kraft pulp is typically bleached with chlorine and its oxides (Eriksson and Kirk.
1985). The efﬂuent from this bleaching operation is called bleach plant efﬂuent (BPE).
Combined chlorination stage and extraction stage efﬂuents from industrial and artiﬁcial
sources were used in this study. The chromophoric (or color causing) material in these
efﬂuents has a high molecular weight and a low solubility in water. For both BPEs
studied, over 90% of the color containing material had a molecular weight geater than
10,000 daltons. The solubility of synthetic and industrial BPE decreased with pH from
7.0 to 2.0. Synthetic BPE had a lower solubility than indusuial BPE (see Figure 4.1). At
pH 4.5, the solubility of synthetic BPE was approximately 4,000 C.U. corresponding to
approximately ‘7 g BPE/l. Industrial BPE had a relatively high solubility. The solubility
of indusuial BPE was geater than 14,000 C.U. at pH 4.5 (Figure 4.1).

Industrial and synthetic BPE were added to niuogen-limited cultures of P.
chrysospon’wn. Addition of synthetic BPE to nitrogen-limiwd cultures (as described in
section 5.1), on days 2 through 7 caused rapid decolorization of the BPE material (Figure
6.1). It was of interest that decolorization occurred on days 3 and 4 when no LIP activity

was detectable in control cultures. The rate of decolorization reached a maximum

93

 

 

 

 

 

 

 

"‘ Day2
G Day3
'2‘ Day4
4000 -- 0 Days
: 1‘ Day6
‘ ﬂ- 0 7 .
‘ x/X—X/X\X—-—X\X 3’
3000 “ ' . ‘X' Nolnoculum
. I
Color :_
Units 2000 .
I
1000 -- - '
0 s ...... i . i . .. pr:
0 2 4 6 8 10

Figure 6.1 Decolorization of Kraft bleach plant efﬂuent (BPE) when
added to cultures on different days of incubation. Data for an
uninoculated control are also presented. Values represent
means for duplicate cultures.

94

between day 4 and 5 when MNP activity also reached its highest level and showed little
or no increase in rate when the LIP activity reached its maximum between days 6 and 7.
Thus, the rate of decolorization temporally paralleled MNP, rather than LIP activity.
Industrial BPE showed a similar pattern of decolorization compared to synthetic BPE
when it was added on day 4 (Figure 6.2). Incubation of synthetic or indusuial BPE with
autoclaved or ﬁlter sterilized culture ﬂuid from day 6 cultures failed to show signiﬁcant
decolorization activity.

Apparent ﬁrst order rate constants (see discussion of the ﬁrst order model below)
for daily decolorization data are presented in Table 6.1.
Table 6.1 Apparent ﬁrst order rate constant for decolorization of Kraft BPE by
cultures of P. chtysospoﬁum as a function of the day of BPE addition.

 

 

Day k
of BPE Addition Apparent Rate Constant (dy-l)
2 0.35
3 0.45
4 0.70
5 0.75
6 0.75
7 0.75
s 0.80

 

 

 

The rates are highest (0.75 to .80 d'l) on days 5 to 8 when both MNP and LIP are
present. However, relatively high rate constants of 0.35, 0.45 and 0.70, respectively,
were observed with 4 and 5 day-old cultures, which had little or no LIP activity. It was

of interest that the apparent rate constant for BPE decolorization on day 4 of incubation

95

 

 

 

 

 

 

 

 

100 .
- + Industrial BPE
30 1 . . 0 Synthetic BPE
60
Residual
Color (%) .
4o ..
.205-
o i . i . i i 4.
0 1 2 3 4 5

Days

Figure 6.2 The decolorization of synthetic BPE and industrial BPE by
nitrogen-limited. agitated cultures of P. chrysosporium BKM-
F-l767. BPE was added to a ﬁnal concentration of 3,000
C.U.

96

(when MNP activity is at or close to its maximum and the LIP activity is negligible) is
>90% of that observed on days 6 and 7, when both LIPs and MNPs are present at
relatively high levels.

In cultures to which BPE was added, LIP activity was never detected and MNP
activity was difﬁcult to detect by enzymatic assay, even after dialysis. Therefore, the
pattern of extracellular LIP and MNP production was studied using SDS-PAGE
electrophoresis. In ageement with activity assays, in control cultures without added
BPE, MNP protein band (Mr=43,000 to 46,000) appeared ﬁrst (between days 2 and 3)
whereas the LIP protein bands were not seen clearly until day 5 (Figure 6.3). On days 5
to 9 multiple MNP and LIP protein bands were evident in the culture ﬂuid (Figure 6.3).
Dass and Reddy (1990), have shown that in acetate buffered cultures the predominant
MNPproteinproducedwasH4 (43-46kD) andthemajorIJPproteins producedwereHZ
and H6 (38-40 kD). Thus the primary MNP and LIP proteins will form discreet bands on
an SDS-PAGE gel.

Although no LIP activity could be detected in industrial and synthetic BPE
amendedculttnes,theLIPandMNPproteinbands weresimilartotheproteinbands
observed in control cultures (Figure 6.4). The protein band representing MNP proteins
was evident on days 4 through 7 while the protein bands representing LIP proteins were
evident on days 5 through 7. Thus although LIP and MNPs are not always detectable by
enzymatic assay, the proteins are present in the exuacellular culture ﬂuid of BPE
amended cultures. I

Concentrations of between 1,000 and 8,000 C.U. were added to nitrogen-limited
cultures of P. chrysosporiwn on day 4. The initial rate of synthetic BPE decolorization
was ﬁrst-order below BPE concentrations of 4000 C.U. and zero-order above 4,000 C.U.
This is in ageement with the ﬁndings of other investigators (Campbell et al., 1983; Yin
er al., 1989). Attempts to ﬁt the data using a Michaelis-Menten kinetic model did not
give good results so a ”ﬁrst order below 4000 C.U.] zero-order above 4000 C.U." model

 

97

Mmmmmlﬁo 28450100
91,400

  

42,7”

31 a”.

21 .ree

SDS—PAGE of extracellular culture ﬂuid from P.
chrysosporium cultures aged 2 to 9 days. Electrophoretic
mobility of FPLC-purified LIP isozymes (H2, H6, H8, and
H10) and MNP isozyme H4 is presented.

Figure 6.3

98

Control Industrial BPE
Day Day
4 5 6 7 M 5 6 7

1
i

 

\

“aneoézr

it
E!
s
.5

 

.-
_I

    

Control, Synthetic BPE

 

Figure 6.4 SDS-PAGE proﬁle of extracellular ﬂuid from control
cultures on days 4 through 7 and from cultures which were
amended with (A) industrial BPE and (B) synthetic BPE.

 

 

160

140

rLlrrrl
l

120 'r

100 :-

Initial
Rate 80
(C.U./hr)

60

40

lllllﬁllllljl

20

 

0

Figure 6.5

\

 

-h—
l—

1 J l
rﬁlrrf] [till

2000 4000 6000 8000 10000
Initial Concentration (C.U.)

The initial rate of synthetic Kraft bleach plant efﬂuent (BPE)
decolorization as a function of initial BPE concentration.
Synthetic BPE was added to nitrogen-limited cultures of P.
Chrysosporium 4 days after culture inoculation.

100

was employed (Figure 6.5). The ﬁrst order rate constant (k) ranged from 0.8 to 0.5 dy‘l
in this experiment. Interestingly, the concentration where the model switched ﬁom zero-
order to ﬁrst-order (4000 C.U.) kinetics coincided with the solubility of the synthetic
BPE at pH 4.5 (see Figure 4.2). This ﬁnding suggests that the decolorization system acts
on soluble BPE at a much faster rate than on insoluble BPE and that the reaction is
pseudo-ﬁrst order with respect to soluble BPE. Industrial BPE had a much higher
solubility than synthetic BPE at pH 4.5 (>>l4,000 C.U., see Figure 4.2). Previous kinetic
models developed for BPE decolorization also use transition from zero-order to ﬁrst-
order kinetics (Campbell et al., 1983; Yin at al., 1989) but do not consider BPE
solubility. Both Campbell et a1. (1983) and Yin ct al.(l989) used industrial BPEs which
presumably have relatively high solubilities. However Yin er al. used an initial BPE
concentration of 20,000 C.U. which may be near the solubility limit of indusuial BPE at
pH 4.5. Campbell reporwd that saturation of the fungal decolorization system occurred at
between 4,000 and 6,000 C.U. in the MyCoR system. A ﬁrst-order model was used to ﬁt
rate data in subsequent experiments where a ﬁnal concenu'ation of 3,000 C.U. of
synthetic or industrial BPE was added to cultures.

6.2 Effect of varying manganese concentration on MNP and LIP production and
BPE Decolorization.

It has been well-documenwd that high levels of niuogen in the medium repress LIP
and MNP production in culnues of P. chnsosporium (Gold er al., 1989; Kirk and Farrell,
1987). More recently, manganese levels in the medium were shown to have a dramatic
effect on the levels of production of MNPs and LIPs (Bonnarme and Jcﬁ‘eries, 1990;
Brown et al., 1990; Perez and Jefferies, 1990). High levels (40 ppm) of Mn(II) were
shown to completely suppress LIP and enhance MNP production, whereas in the
complete absence of Mn(II), no MNPs were produced but LIP production was essentially
normal (Bonnarme and Iefferies, 1990; Brown et al., 1990; Perez and Jefferies, 1990).

101

Varying concentrations of Mn(II) were added to niuogen limited (2.4 mM) cultures
of P. chrysosporium to determine the relative contribution of LIPs versus MNPs to BPE
decolorization, The aim was to manipulate LIP and MNP levels using manganese and
study the effect of these variations on BPE decolorization. Three levels of Mn(II) were
added to cultures: 0, 12 and 100 ppm corresponding to low, basal and high Mn(II)
levels. Basal Mn(II) cultures exhibiwd high MNP and LIP activity reaching maxima on
days 4—5 and 6-7, respectively (Figme 6.6a,b). Low Mn(II) culttnes exhibited relatively
low MNP activity compared to high Mn(II) cultures and reached a maximum on day 4.
In high Mn(II) cultures LIP activity was not detected but MNP activity was greater than
that in basal Mn(II) cultures.

In high niuogen (24 mM nitrogen) cultures containing 12 ppm Mn(II) neither MNP
nor LIP activity was detected (Figure 6.6a,b) and no LIP and MNP bands were seen on
the SDS-PAGE gel (Figme 6.7). SDS-PAGE analysis of the culture ﬂuid ﬁom high
manganese cultures [100 ppm Mn(II)] and basal manganese cultures [12 ppm Mn(II)]
showed a single intense protein band (Figure 6.7) which appears to be an MNP protein
based on its molecular size and the fact that neither the high Mn(II) nor the basal Mn(II)
cultures displayed LIP activity on the day of BPE addition, i.e., on day 4 of incubation.
In low manganese cultures on the other hand, faint bands corresponding to LIP and MNP
proteins were seen.

When BPE was added to 4 day-old basal Mn(II) and high Mn(II) cultures,
decolorization proceeded at a high initial rate (Figure 6.8). Signiﬁcantly slower rates of
decolorization were observed in low Mn(II) and high niuogen cultures. A mass balance
analysis 4 days after the addition of BPE showed approximately 8% decolorization in
high nitrogen (nitrogen sufﬁcient) cultures containing 12 ppm Mn(II), 27%
decolorization in low Mn(II) cultures, 75% decolorization in high Mn(II) cultlues, and
85% decolorization in basal Mn(II) cultures. The apparent ﬁrst order rate constants

(Table 6.2) for decolorization of BPE in cultures containing different levels of nitrogen

102

and Mn(II), showed that when LIP and MNP are not present little decolorization was
observed. The absence of LIPs cultures had minimal effect on the decolorization rate
whereas decreasing the activity of MNPs had a pronounced effect on the decolorization
rate even when the LIPs were present (Table 6.2).

Table 6.2 Apparent ﬁrst order rate constant for decolorization of BPE by P.
chrysosporiwn as affecwd by niuoggl and manganese levels in the medium.

 

 

 

Nitrogen level Mn(II) Predominant Decolorization rate
(mM) (ppm) peroxidases constant (dy-1)b
24.0 12 none 0.01
2.4 0 LIP 0.10
2.4 12 LIP, MNP 0.70
2.4 100 MNP 0.65

 

 

bDiﬁerencesinmtecoustanmofgeatathan0.070y—lmeconsidaedsigniﬁcant

103

  
  
 

<> 2.4 mM NH4 ﬁr 2.4 mM NH4 G 24 mM Ni-l4 + 24 mM NH4

Oppm Mn(II) 12ppm Mn(II) 100ppm Mn(II) 12ppm Mn(II)
2500 I
2000 }_
1500 }

MNP
U/l ‘
1000 :*

 

 

300 j"

200 ;

LIP
WI

100 }

 

 

 

.. .. .. , .. ., ::.ﬁh
0 2 4 6 8 10
Days

Figure 6.6 The effect of Mn(II) and niuogen levels on LIP and MNP
activities of wild type P. Chrysosporium. Values presented
are averages for triplicate cultures. A. MNP actrvrty. B.
LIP activity.

 

 

Figure 6.7

104

 

SDS-PAGE of peroxidases in extracellular culture ﬂuid of P.
Chrysosporiwn, at the time of BPE addition, 4 days after
incubation at 39° C. Mn(II) concenu'ations were 0 ppm, 12
ppm and 100 ppm, respectively, in low, basal and high
Mn(II) nitrogen limited (2.4 mM N) cultures. Niuogen
sufﬁcient (24 mM N) cultures contained 12 ppm Mn(II).

+ 24 mM NH4
12ppm Mn(ll)

100

Residual
Color (%)

25'

75 --

50 --

 

Figure 6.8

105

 

 

0 2.4 mM NH4 ‘0' 2.4 mM NH4 0 2.4 mM NH4
Oppm Mn(II) ' 12ppm Mn(II) 100ppm Mn(II)
Lo
. O
O
‘0
A

 

The effect of Mn(lI) and nitrogen levels on BPE
decolorization by cultures of P. chrysosporium. BPE was
added on day 4 of incubation.

106

6.3 BPE decolorization by lip and per mutant cultures.

To ﬁu'ther investigate the importance of extracellular peroxidases in BPE
decolorization, two mutant strains (per and lip) and two wild-type strains (ME-446 and
BKM-F 1767) of P. chrysosporium were compared based on their ability to decolorize
BPE. The per mutant lacks the ability to produce exu'acellular peroxidases based on
several lines ofevidcnce. First, no MNP or LIP activity was detected in culture ﬂuid of
per mutant gown in low nitrogen basal mdium whereas under identical conditions both
MNPs and LIPs are produced in large amounts by the wild type. When concentramd
culture ﬂuid from the per mutant cultures was fractionated by SDS-PAGE, no LIP or
MNP bands were evident (Figure 6.9). In the same gel, however, concentrated culture
ﬂuid from 4~day-old cultures of the lip mutant and the wild type showed a single band
(corresponding to MNP). Moreover, FPLC analysis of the concentrated exu'acellular
ﬂuid from the per mutant cultures showed no LIP or MNP peaks. N o physiological
difference was found between the per mutant and the wild type (aside ﬁom a lack of
MNPandLIPproduction). Thepermutantconsumedglucoseatthesamerateasthe
wild type, produced the same amount of cell biomass and formed mycelial pellets similar
in size to those of the wild type. The tip mutant has been characterized previously
(Boominathan et al., 1990). It produces MNPs and H202 but lacks the ability to produce
the LIPs. The wild type strains MB-446 and BKM-F 1767 produce both LIPs and
MNPs.

BPE was added to the per mutant, lip mutant, and wild-type cultures two days after
MNPs ﬁrst appeared (generally day 4 of incubation) and the extent of decolorization was
monitored daily for the next 5 days. The presence (or absence) of extracellular
peroxidases was determined by SDS gel electrophoresis of concenu'ated culture ﬂuid
(Figure 6.9) and by LIP and MNP assays of control cultures without added BPE (Figme
6.10). SDS gel results showed that in lip and wild-type cultures a single protein band (at
46,000 MW) corresponding to MNP was present at the time of BPE addition. In the per

107

mutant culture, neither LIP nor MNP protein bands were evident (Frogure 6.9). The
MNP activities in the ME446, BKM- -1767, lip mutant and per mutant cultures were
3,550, 2,200, 2,050 U]! and 0 U/l, respectively at the time of BPE addition (Figure 6.10).
Only the wild-type cultures produced LIP activity (Figme 6.10).

The per mutant, exhibited negligible decolorization of BPE (Figure 6.11) while the
wild-type strains exhibited rapid decolorization of BPE. The lip mutant (which produces
MNPs only) showed about 80% of the decolorization activity exhibited by ME446.
These results, consistent with the other data presented above, indicate that the
extracellular peroxidases are important for BPE decolorization and that the MNPs play a
predominant role in BPE decolorization by P. clvysosporium.

Further evidence of the involvement of MNP as opposed to LIP in BPE
decolorization is given by the culture pH dining decolorization. As determined in
Methods (see also Appendix 3), the MNPs have their maximal activity at pH 4.5 while
LIPs havetheirpeakacﬁvityatpH2.5.LIPactivityatpH4.5isreducedbymorethan
90% compared to it's maximal activity. In the decolorization cultmes the pH ranges from
4.5 t05, therange wheretheMNPs aremostactive andLIPactivityisreduced.

108

Culture Condition
am am
Basel Low

“II (II) Mn(II) Marker LIP- Por-

-——— _—.-

 

20,000 - a—r.

Figure 6.9 SDS-PAGE of peroxidases in exuacellular culture ﬂuid of P.
Chrysosporium wild type su'ain BKM-F-l767, a lip mutant
suain and a per mutant strain at the time of BPE addition.

109

MNP
‘1" 1000 ~-

 

 

0123456789101112_

 

0 1 2 3 4 5 8 7 8 9 10 11 12

Figure 6.10 Lignin peroxidase (LIP) and manganese peroxidase (MNP)
activities in wild-type (BKM-F- 1767), lip" mutant and per'
mutant cultures of P. chrysospan’um. Cultures were gown in
low nitrogen medium which contained 12 ppm Mn(II).

 

110

 

 

 

 

 

 

 

‘0' PER- mutant
0 LIP- mutant
-*-M50m
I BKM-F-1767
100 . ‘
1 sea
80
j_ .
Residual 3 .
Color (%) -
40 j" .
20
o d : .4 . : a. : ..:
0 1 2 3 4 5

Days

Figure 6.11 Decolorization of BPE by P. chrysosporium wild-type strains
ME446 and BKM-F 1767 and two mutants, per and lip of
ME446. BPE (3000 C.U.) was added to each of the cultures
on the peak day of MNP activity and the extent of BPE
decolorization was monitored each day for the next 5 days.
The values shown are averages of duplicate cultures on two
separate occasions.

111

6.4 In vitro decolorization of BPE.

A brief study was conducted of the ability of isolated MNPs and LIPs to decolorize
BPE. Culture ﬂuid from 5-day old nitrogen limimd cultures with both LIP and MNP
activity was collected and concentrated using ultraﬁlu'ation. This concentrated (10:1)
enzyme solution was applied to an FPLC column and the MNPs and LIPs were eluted
using an acetate gradient. The eluate was distribuwd using a fraction collector and
individual fractions were assayed for LIP and MNP activity. A 300 ill aliquot of the
MNP ﬁ'action (no LIP activity) and a 300 Ill aliquot of a LIP fraction (1110) (no MNP

activity) were added to test tubes containing 900 pl of BPE (722 initial color units), 40 pl
of 8mM H202 and 60 pl of 5.2 g/l MnSO4 and incubated overnight at 37 C. Control

cultures with H202 only, with MnSO4 and H202, and with H202, MnSO4 and
concentrated 5-day old culture ﬂuid were also prepared. The results are shown in Table
6.3. Compared to the LIP and MNP activity in day 6 nitrogen limited cultures, the
peroxidase activities in the in vitro experiments were a factor of three higher.

Table 6.3 Decolorization of Kraft BPE by FPLC puriﬁed peroxidases of P.

chUsosporium in a cell ﬁee system.

 

 

 

 

Condition Residual Color (%)a
H20 10011
W304: H202 99 i 2
11411804112029me 73 :4
MnSO4. H202. puriﬁed LIP (1110) 110 j; 1
MnSO4, H202, day 5 culture ﬂuid 66 i 5

 

a Vduuueuansforuiplicemrampluionemdcviniou.

112

The puriﬁed MNP preparation decolorized the BPE by approximately 27% in 20 hours.
Similarly, 5-day old concenu'awd culture ﬂuid containing both LIP and MNP activities
decolorized the BPE by 34% in 20 hours. When puriﬁed LIP H10 was added to the BPE the
color increased by 10% in 20 hours. Neither water, nor H202 and MnSO4 showed any
decolorization activity. In further experiments to demonstrate in vitro decolorization no
decolorization activity was observed and this avenue of study was abandoned. (Note:
Recently, Lackner et al. (1991) have demonstrated in vitro decolorization of BPE by MNP
andMn(III).Intheirreaction systemthe hydrogenperoxide wasaddedataslowrateto
obviate enzyme deactivation. Furthermore, Michael Gold (personal communication) has
indicated that acetate, which was used as a buffer, may interfere with the reaction cycle).

113

Part 11: Discussion

6.5 Role of MNPs and LIPs in the decolorization of BPE.

Earlier studies have shown that P. chtysospon'um as well as some other white-rot
fungi rapidly decolorize BPE. Sandman er al. (1981) showed that BPE from the ﬁrst
alkali extraction stage after chlorination (E1 efﬂuent) was decomposed to low molecular
weight colorless products. P. chrysospon'wn was shown to degade signiﬁcant amounts
of l4C-labeled bleached kraft lignin to 14002 (4,19). Campbell (1983) and Eaton et al.
(1983) developed the MyCoR process which utilizes P. chrysosporium immobilized on
partially submerged rotating discs to decolorize indusuial BPE. Yin er al. (1989)
examined the kinetics of BPE decolorization in the MyCoR process and indicated that
BPE decolorization consisted of three distinct stages. The LIP or MNP activities were
not reported in any of these previous studies. Furthermore, although Paice and Jurasek
(1984) have shown that horseradish peroxidase can catalyze BPE decolorization, and
Archibald et al.(1990) have implicated laccases of C. versicolor in BPE decolorization,
the role of LIPs and MNPs of P. chrysospariwn in the decolorization process has never
clearly been established. '

Results indicate that the exu'accllular peroxidases of P. chrysosporium play a key
role in BPE decolorization by this organism. Little or no BPE decolorization was seen
when P. chrysospon’um was gown in high niuogen mdium which blocks production of
both LIPs and MNPs (Figure 6.8) suggesting that these peroxidases are required for
decolorization activity. This is independently supported by the experiment with the per
mutant which lacks the ability to produce LIPs and MNPs but produces H202
comparable to the wild-type (Boominathan and Reddy, 1991). The fact that the per
mutant shows negligible BPE decolorization (Figure 6.10) is consistent with the idea that
extracellular peroxidases play a key role in BPE decolorization.

114

A determination of the relative conuibution of LIPs versus MNPs to BPE
decolorization was an important focus of this investigation. Several lines of evidence
indicated that the MNPs play a predominant role in BPE decolorization by P.
chrysospoﬁum. First, a high degree of BPE decolorization was observed in the high
Mn(II) wild-type culttnes and in the lip mutant cultures, in which high levels of MNP
activity, but no LIP activity, was seen (Figures 6.8 and 6.10) and LIP proteins were not
evident on SDS-PAGE gels (Figures 6.7 and 6.9). Second, very low levels of BPE
decolorization were observed in wild-type cultures gown without Mn(II) in which high
levels of LIP and very low levels of MNP activity were seen (Figure 6.8). Third, BPE
decolorization activity temporally paralleled the appearance of MNP rather than LIP
activity in wild-type cultures containing 12 ppm Mn(II) (see Figure 6.1). For example,
relatively high rates of BPE degadation are observed on days 3 and 4 of incubation in
basal nitrogen-limited medium at which time little or no LIP activity is seen and LIP
proteins were not present in the culture ﬂuid (Figure 6.4). These results, in combination
with results showing that peroxidases are necessary for decolorization to occur,
conclusively show that MNPs play a more important role than LIPs in decolorizing BPE.

The results of this study indicating a minor role for LIPs and a major role for MNPs
is an interesting mum to earlier studies which showed that the LIPs play a major role
in the degradation of synthetic 14C-1ignin to 14002 whereas the MNPs play a minor role
in this process (Boominathan er al., 1990; Perez and Jefferies, 1990). Boominathan at al.
(1990) showed that the lip mutant of P. chrysosporirmr, which lacks the ability to
produce LIPs but produces a full complement of MNPs, exhibits only about 16% of the
ligninolytic activity of the wild-type indicating that MNPs play a minor role in lignin
degradation by P. chrysospon'tmr. Perez and Jefflies (1990) showed that high levels of
MNP activity but no LIP activity was seen when P. chrysosporilan was gown in
nitrogen-limited medium in the presence of 39.8 ppm Mn(II). These cultures exhibited
only about 10 to 11% ofthe lignin degradation ability as compared to cultures grown in

115

low N medium containing 0.35 ppm Mn(II), which supports production of high levels of
LIP but negligible levels of MNP activity. These apparent differences in contributions of
LIPs versus MNPs to lignin degadation versus BPE degadation are quite interesting and
unexpecwd. The observed diﬁ‘erences in activities may reﬂect the diﬁ‘erences in the two
substrates (i.e. lignin and BPE). BPE is predominantly soluble, chlorinated, and partially
degaded whereas the structure of DHP more closely resembles the insoluble native
lignin polymer.

In conclusion, the results indicate that extracellular peroxidases are important for
BPE decolorization by P. chrysosporilan and that MNPs play a predominant role and
LIPs play a relatively minor role in this decolorization process.

The appearance of a brown mycelial coloration has been used as a visual indicator
of the onset of ligninolytic activity in cultures of P. Chrysospon'um. This brown
coloration coincides temporally with the rapid disappearance from the culture ﬂuid of
soluble manganese (Figme 5.2) and with peak MNP activity (Figure 5.1b). When BPE is
added to cultures with this brown coloration, the color disappears for a period of two or
three days while the decolorization process occurs and then gadually reappears.
Therefore based on these ﬁndings, and the results of Lackner er al. (1991), a simple
reaction scheme was been postulated for the enzymatic oxidation of BPE and the
formation of insoluble manganese by MNPs (Figure 6.12). Divalent manganese (Mn(II)),
which is a component of the culture media, is enzymatically converted to reactive
bivalent manganese (Mn(III)) by MNP and hydrogen peroxide. The trivalent manganese
non-speciﬁcally oxidizes the BPE polymer leading to a variety of products and in the
process is reduced back to divalent manganese. In the absence of oxidizable substrates
like BPE or lignin, trivalent manganese, formed by the action of MNP, is converted via
hydrolyzation and disproportionation to divalent and tetravalent manganese (Lackner et

al., 1991). Tetravalent manganese can react to form insoluble manganese salts such as

manganese dioxide (MnOz). These insoluble manganese salts give the pellets their

116

characteristic brown coloration during the onset of ligninolytic activity. This study is the
ﬁrst to relate a decrease in the concentration of soluble manganese with the appearance

of the brown coloration on the mycelial pellets and adds credence to the idea that the
brown coloration is due to deposits of solid MnOz.

117

Degraded

BPE ‘—V__' “1"" —1

MNP + H202

BPE —JL— Mn:[lll] «J

polymer

 

 

MnOz «e Mn(N]
[solid ] 02

Figure 6.12 Proposed reaction scheme for the degradation of BPE and the
deposition of manganese by manganese peroxidases (MNP)
of P. chrysosporium.

CHAPTER 7
Conclusions and Recommendations
7.1 Conclusions

1. An integrated model based on the life-cycle of P. chrysosporiwn accurately
predicts a wide range of culture characteristics including culture dry weight, substrate
utilization rates and potential oxygen limitations in mycelial pellets, during primary
growth and during secondary metabolism, when LIPs and MNPs as well as many
industrially signiﬁcant products are produced. The model suggests a relationship between
the level of polysaccharides produced and the dissolved oxygen concentration within the
mycelial pellets.

2. Analysis of the mass transfer limitations in nitrogen limited, peroxidase
producing mycelial pellet cultrues of P. Chrysosporiwn indicates that intra-particle
oxygen mass transfer can be rate limiting timing secondary metabolism. Cultrnes which
have oxygen effectiveness factors signiﬁcantly less than one (E<0.4) produce little or no
MNP or LIP. Thus, the effect of oxygen appears to have a physiological as opposed to a

genetic basis.
3. Extracellular peroxidases play a key role in the decolorization of Kraft bleach

plant effluents (BPE) by P. chrysosporium. MNPs play a predominant role, and LIPs a

relatively minor role, in the process of Kraft bleach plant effluent decolorization.

118

119

7.2 Recommendations

1. Since oxygen limitations within mycelial pellets appear to limit the production of
the extracellular peroxidases, methods for producing small pellets or thin mycelial ﬁlms
which are not oxygen lirrrited should be studied.

2. The decolorization of BPE using whole cultmes of P. chrysosporium requires a
long period of incubation before the decolorization activity begins. The large volume of
efﬂuents generated by the paper industry would require enormous fermentation treatment
volumes. Also, the production of the peroxidases is very sensitive to agitation, oxygen
tension, nutrient composition and the presence of extracellular proteases. One alternative
approach for BPE treatment would be to develop an immobilized enzyme decolorization
system using MNPs so that enzyme production and BPE treatment could be decoupled.
Immobilized enzymes would have a potentially longer life-span requiring less total
enzyme production. A recommendation is that future work focus on the Met

development of an in vitro decolorization system. This system would involve enzymes
and cofactors such as H202 and manganese. '

3. Industrial BPE streams are relatively dilute. To reduce the fermentation system
volume it may be beneﬁcial to concentrate the BPE stream using ultraﬁltration,
precipitation or adsorption. The relative toxicity of the low and high molecular weight
ﬁ'action must be considered.

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128

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APPENDICES

(see list of tables for index)

130

Appendix 1

Extracellular peroxidase activity in air ﬂushed and oxygen flushed agitated cultures
of P. chrysospoﬁum as reported by Dosorctz et al., l990a.

 

 

 

Data for Figme 2.2
LIP activity MNP activity
(U/l) (U/l)
Time

(11) air 02 air 02

0 0.00 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00
48 0.00 0.00 0.00 0.00
72 0.00 0.00 0.00 2.37
96 0.00 70.00 0.21 3.67
120 0.00 145.00 1.47 3.27
168 0.00 165.00 1.30 1.64
192 0.00 149.00 1.07 1.47
216 0.00 135.00 0.90 1.35
240 0.00 101.00 0.00 0.62
264 0.00 72.00 0.00 0.00
288 0.00
312 0.00

 

 

 

 

 

131

Appendix 2

Solubility of Synthetic BPE as a function of pH.

 

 

 

 

 

 

 

Data for Figure 4.1
Synthetic BPE
pH 1 Color Units
3 1511
3.56 2022
4 2204
4.5 2749
5 3749
5.88 6669
Industrial BPE
pI-l Color Units
2.23 7079
2.95 11092
3.39 13887
3.85 13592b
4.53 14832"
5.10 148741)
5.85 148109

 

 

 

b maximum possible soluble color rsooo C.U. (data not used).

Effect of pH on the activity of lignin peroxidases and manganese peroxidases.

132

Appendix 3

 

 

 

 

 

 

Data for Figure 4.2
LIP activity MNP activity
veratryl Phenol Remazol

pH alcohol Red Blue

oxidation assay assay
2.05 0.544 0.050 0.014
2.57 1.000 0.150 0.037
3.05 0.913 0.202 0.060
3.57 0.522 0.360 0.1 14
4.05 0.217 0.948 0.928
4.53 0.109 0.915 0.622
5.08 0.043 0.487 0.538
5.56 0.021 0.402 0.083
7.13 0.001 0.272 0.011

 

 

133

Appendix 4
Time course of nutrient nitrogen (ammonium), biomass (culture dry weight),
glucose, soluble carbohydrate, extracellular protein, acetate and extracellular LIP
and MNP activity in agitated control cultures of P. cluysosparium BKM-F-l767.

Data for Figure 5.1

 

 

 

 

 

 

 

Time Ammonium Dry Weight Glucose
(d) (rag/l) (all) (34) pH
0.00 39.10 0.11 10.64 4.50
0.33 25.40 0.13 9.77 4.43
0.79 2.74 0.64 8.85 4.48
1.00 0.00 0.90 8.54 4.45
2.00 1.07 ' 8.38 4.52
3.00 1.13 7.16 4.98
3.92 1.39 5.91 4.68
5.00 1.36 4.23 5.18
6.00 1.49 3.52 4.84
6.92 1.57 2.38 4.53
8.00 1.87 0.34 4.52
8.96 1.98 0.11 4.61
10.04 1.91 0.08 4.84
10.96 1.51 0.07 5.06
11.92 1.33 5.17
12.96 1.14
Time MNP“ LIP“
(days) (U/l) S.D. (U/l) SD.
1.8 0 0 0 0
2.7 774 681 0 0
3.7 1215 421 0 0
4.6 1423 743 15 20
5.6 1182 716 35 49
6.7 982 199 230 15
7.5 700 175 243 3
8.6 699 72 240 1
10.0 273 64 120
10.7 10 ' 0 69
11.5 0

 

 

 

 

 

avaloesarernetrnst’artriplicatecultrrresononeoccasian

 

134

 

 

 

 

 

Appendix 4 (cont)
Glucose Carbo- pH Acetate Biomass
Time hydrates
(d) gyms) (5M3) (mM) (gm3)
0.00 12290 12130 4.45 21.55 60
0.92 11800 11410 5.14 7.86 890
1.08 11850 10660 5.80 2.50 1100
2.04 10200 10160 3.59 0.71 1450
2.92 9970 9570 5.18 0.34 1580
3.88 8770 8280 5.37 1.10 1780
4.92 6840 6750 5.38 0.15 1690
6.00 4990 5250 5.30 1800
7.17 3400 4170 5.31 1920
7.33 2380 3070 5.43 1790
8.29 1290 1860 5.36 2060
8.92 560 760 5.27 0 2300
9.92 350 180 5.51 1950
11.08 200 40 5.55 1760
Time Protein LIP MNP
(d) (ﬁlms) (Um (U11)

0.00 88.7

0.92 25.9

1.08 22.5 0

2.04 8.3 0

2.92 12.0 0 450

3.88 21.9 12 866

4.92 26.5 145 811

6.00 24.3 163 582

7.17 24.8 185 433

7.33 23.8 189 415

8.29 24.3 177 388

8.92 17.5 99 234

9.92 16.7 62 44

11.08 13.0 28 0

 

 

 

 

 

 

 

135

Appendix 5

Manganese concentration in soluble and insoluble fractions of cultures of P.
chnsorparium with three initial soluble manganese concentrations as measured

using atomic absorption spectrophotmetry. (SD. = 1.4 ppm)

 

 

 

 

 

 

 

 

Mn Mn total Mn
soluble insoluble (sum)
Time ﬁ'action ﬁ'action
M (mm) (mm) (m)—
0 ppm MnSO4 added at inoculation.
0 0.05 0.01 0.06
2 0.05 0.02 0.07
4 0.02 0.00 0.02
6 0.02 0.02 0.04
8 0.04 0.01 0.04
12 ppm MnSO4 added at inoculation.
0 13.16 0.09 13.25
1 13.30 0.25 13.54
2 11.03 0.52 11.55
3 11.95 0.75 12.70
4 11.19 1.05 12.24
5 7.63 4.57 12.20
6 3.53 8.79 12.32
7 4.36 6.31 10.67
8 3.52 7.04 10.56
9 2.06 8.91 10.97
40 ppm MnSO4 added at inoculation
0 43.21 0.86 44.07
1 36.23 2.26 38.48
2 40.68 1.46 42.14
3 36.70 2.27 38.97
4 42.37 1.94 44.30
5 5.82 30.64 36.45
6 5.03 34.98 40.01
7 3.45 36.66 40.11
8 3.31 37.74 41.05
9 4.68 29.73 34.41

 

 

 

 

 

136

Appendix 6

Decolorization of BPE as a function of the day of BPE addition. '
BPE was added to approximately 3,000 C.U. on days 2 through 8 and the 465 nm
absorbance (pH 7.6) was measured.

 

 

 

 

Data for Figure 6.3
Color Units (C.U.)

Hour Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 uninoc.

2.4 2878
42 2491 2972
69 1579 2552 , 2972
90 1170 1575 2915 3048
113 871 999 1325 2681 3010
137 621 674 867 1030 2650 2991
160 465 522 662 723 1151 2934
188 492 401 416 651 507 3029
207 280 291 314. 412 386

 

 

137

Appendix 7

Decolorization, culture dry weight and carbon dioxide evolution by cultures of P.
chrysasporium amended with synthetic and industrial Kraft bleach plant effluents

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(BPE).
Data for Figtne 6.2
Industrial BPE Synthetic BPE
Time Color 96 Color 96
(days) (C.U.) residual (C.U.) residual
4.00 2885 j; 398 100 3622 i 85 100
4.10 2548 :1: 187 42 2076 J: 114 38
4.88 1209 :l: 18 28 1373 :1: 36 26
5.71 819 1 51 21 927 1102 19
7.25 614 18 699 17
Biomass 002
evolved
(8’1) (mM/dy)
N 0 Industrial Synthetic No Industrial Synthetic
Day BPE BPE BPE BPE BPE BPE
3 34.5 34.5 34.5
4 1.26 1.26 1.26 43.2 43.2 43.2
5 1.35 1.55 1.66 54.3 43.9 47.9
6 1.31 1.48 1.53 56.1 50.2 40.1
7 1.40 1.55 1.6 41.1 36.1 23.9

 

 

 

 

138

Appendix 8

Effect of nutrient nitrogen (39 and 390 g/m3) and manganese (0, 12 and 40 ppm) on
the decolorization of BPE, the depletion of glucose and the production of

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

extracellular peroxidases.
(Data for Figtne 6.6 and 6.8)
lignin Peroxrdase' Activity (U/l)
Time (days) 0.00 3.08 4.63 5.58 6.67
12 ppm Mn 0 0 15 :20 35 :49 230 :14
OppmMn 0 0 118113 129 :18 63 :27
40 ppm Mn 0 0 0 0 9 19
Time (days) 6.67 7.50 8.58 9.96 10.67
12 ppm Mn 230 _-t_-14 243 i3 240 :1 120 :1 69
OppmMn 63127 4119 2216 20114 1113
40 ppm Mn 9 :9 101 3:21 215 3; 2 91 :23 67 1:19
Manganese Peroxrdase' Activity (0]!)
Time (days) 0.9 2.67 3.69 4.63 5.58
'12 ppmMn o 1780 1260 1370:40 2430 1990130
0 ppm Mn 0 0 170 110 70 120 180 :50
40 ppm Mn 0 730 i800 1170 1150 1370 :20 1950 :60
Time (days) 6.67 7.50 8.58 9.96 10.67
12 ppm Mn 1170 :10 760 :60 630 11 320 i1 10 :1
0 ppm Mn 430 180 280 180 500 190 230 180 220 110
40 ppm Mn 1380 120 1460 3:140 1400 :30 100 :40 70 _-I;110

 

 

 

139

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Appendix 8
(cont.)
Color (C.U.)
Time (day) 3.83 4.04 4.50 5.00 5.58 5.92
‘ Nitrogen Suﬁ. 3329 2335 2210 2062 1954
12 ppm Mn 3618 2704 1954 1642 1431 1295
0 ppm Mn 3488 2937 2573 2545 2409 2579
40 ppm Mn 3738 2602 1812 1528 1437 1392
Time (dy) 6.67 7.50 8.79 9.96 16.50
Nitrogen Suﬁ. 2039 1539 1812 1750 1982
12 ppm Mn 1233 943 880 795 250
0 ppm Mn 2374 1937 2187 2159 1522
40 ppm Mn 1159 1062 846 704 528
Glucose (g/l)

Time (days) 0 0.94 1.71 2.67 3.69 4.63
Nitrogen Suﬁ. 10 7.9 +0.3 5.8 +0.1 2.2 +1.1 0.78 +0.1 0.24 +0.1
Low Nitrogen 10 8.5 +0.1 7.9 +0.1 6.5 +0.5 5.8 +0.1 5.6 +0.1

Time (days) 5.58 6.67 7.50 8.58 9.96 10.67
Nitrogen Suﬁ. 1.1 +0.1 0.5 +0.1 0.6+0.1 0.7 +0.1 0.5 +0.1 0.5 +0.1
Low Nitrogen 4.7 +0.2 3.0 +0.2 2.3 +0.3 1.4 0.8 0.6

 

 

 

140

 

 

Appendix 9
Residual color (percent) in wild-type and mutant strain cultures of P. chrysaspoﬁum
(3,000 color units added).
Data for Figure 6.11
Time
after BPE

addition per lip BKM-F-

(Days) mutant mutant ME-446 1767
0 100 100 100 100
1 101 63 50 41
2 92 ' 52 38 31
5 90 43 26 21

 

 

 

 

141

Appendix 10

Time course of the natural log of culture dry weight divided by average initial
culture dry weight (Ln [X/Xo,avg]) during lag and primary growth phases.

 

 

 

 

Data for Figure 5.3
Ln [X/Xo,avg|
Time Line ﬁt

(3) Data W= 3.9 3'1)

0 -0.00467 -1. 13848

0 -0.24106 - 1.13848

0 -0.02237 -1.13848

0 0.118947 -1.13848

0 0.108557 -1.13848
28800 0.118947 -0.01056
28800 -0.01348. 001056
28800 -0.05 87 3 -0.01056
28800 -0.02237 -0.01056
34128 0.249567 0.198105
34992 0.242056 0.231942
48600 0.692856 0.764884
55800 0.784587 1.046864
68688 1.72232 1.551608
68340 1.399019 1.540329
68170 1.708023 1.531305
75600 1.401172 - 1.822308
86400 2.066737 2.245278
86400 2.165689 2.211441
86400 2.169885 2.279116

 

 

142

Appendix 11

Pellet characteristcis in nitrogen limited agitated culture of P. chrysospoﬁum

 

 

Data for Figure 5.4
' Pellet“ P611“
Time Radius Time Number

(11) (mm) (b) m‘3
22 0.795 :1; 0.13 22 7674
26 0.786 :1: 0.14 26 6953
49 0.813 :1: 0.13 49 7980
49 0.783 3; 0.13 49 5552
70 0.796 1 0.14 70 6830
118 0.783 10.15 70 7316
144 0.877 d; 0.14 118 9635
172 0.847 :1: 0.10 144 7716
176 0.935 1; 0.20 172 7472
199 0.913 :3; 0.14 176 6329
199 0.808 1; 0.11 199 7694
214 0.897 1: 0.17 199 4982
g 214 1.010 :1; 0.26 214 7597
238 0.955 :1: 0.20 214 6305
266 1.039 3; 0.15 238 8317
263 8780

Mean: 7320 1- 1129

 

 

 

 

avaluesareaveragesamnestandarddeviatianfaragroupoftweotypellets.

143

Appendix 12

Oxygen concentration profiles in mycelial pellets of P. chnsospodum.
Experimental data for pellets grown in nitrogen-limited medium and removed at the time
indicated. Values at the top of each column represent the diameters of the individual
mycelial pellets. Values were determined using an oxygen microelectrode.

 

 

 

 

 

 

DAY 1
2.15 mmj 1.85 m I 1.65 mm 1.85 mm

Depth 02 02 02 Depth 02
(mm) (3,232 W232 £3,232 (mm) (5'23)
0.00 6.61 6.84 6. 80 0.00 6. 84
0.05 5.17 5. 99 6.06 0.06 5. 83
0.10 3.42 4. 35 4.28 0. 08 5. 05
0.15 2.33 2.88 2.95 0.10 4.59
0.20 1.48 1.63 2.02 0.13 3. 65
0.25 0. 86 0.70 1.09 0.18 2. 57

0. 30 0. 31 0.16 0.54 0.23 1. 56
0. 35 0.08 0.00 0.16

 

 

 

 

 

 

 

 

 

 

 

 

 

DAY2
2.35 m I 2.30 ml 1.55 mm | 1.15 mm F210 m l 1.65 m I 2.00 mm

Dapth 02 02 02 02 02 02 02
0.00 6.34 6.92 6.73 6.54 6.73 6.73 6.73
0. 05 5.38 5.38 5.96 5.38 5.77 4.81 5. 38
0.10 4.23 4.23 5.19 3.27 3.84 3.08 4.04
0.15 3.27 3.27 4.23 1.54 1.92 1.73 2. 31
0.20 2.31 2.31 3.65 0.77 0.77 0.77 1.35
0.25 1.54 1.35 2.88 0.19 0.38 0.38 0.58
0.30 0.58 2.50 0.00 0.00 0.00 0.00
0.40 0.19 1.73

0.45 0.00 0.00

 

 

 

144

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Appendix 12~(cont.)
DAY 4
, 1.85mm] 1.85mm12.20mml 2.20mm
Depth 02 02 02 02
(mm) (#23) W23) (ms) 15/232
0.00 6.69 6.40 6.72 6.44
0. 05 5.84 5.46 5.59 5.02
0.10 4. 28 3. 86 4.17 3.69
0.15 2. 78 2. 45 2.93 2.56
0. 20 1. 62 1.41 1.98 1.69
0. 25 0. 75 0.59 1.12 0.95
0. 30 0.19 0.19 0.56 0.47
0. 35 0.02 0.00 0.20 0.19
0. 40 0.00 0.05 0.00
0. 45 0.00
DAY 5 DAY
12
1.65 mtg 2.35 m I 1.85 m I 2.35 mm 2.65 mm 2.65 mm
Depth 02 02 02 02 02 02
(mm) (#232 $3,332 Ss£m32 £36232 ‘ SEQ32 £3932
0.00 6.92 6. 92 6. 23 6.46 6. 00 6.40
0. 05 5. 67 4. 84 5. 44 5. 80 6. 20
' 0.10 5.44 4.06 3.51 4.43 5.60 5.80
0.15 4.80 2.86 2.31 3.51 5.40 5.60
0.20 3. 60 1 9.8 1.38 2.77 5.30 5 .45
0.25 2. 49 1.15 0.55 1.75 4.90 5.40
0. 30 1.61 0.60 -0.09 1.01 4.60 5.30
0. 35 0. 92 0.05 0.00 0.28 4.40 5.00
0.40 0.74 -0.09 4.20 4.90

 

 

 

 

145

 

 

 

 

 

 

 

Appendix 13
Carbon dioxide evolution and Oxygen depletion in mycelial pellet cultures of P.
chrysosporium.
Data for Figure 5.6
C02 evolved
Time Data Model
(days) (a) L
1 0.0914 0.0229
2 0.1076 0.1043
3 0.1238 0.1137
4 0.1361 0.1218
5 0.1456 0.1283
6 0.1264 0.1329
7 0.1365 0.1351
8 0.1336 0.1325
9 0.1092 0.1139
10 0.0585 0.0531
11 0.0266 0.0369
12 0.0245 0.0297
13 0.0239
Data for Figure 5.1c
002 02
Time evolved depleted
(Days) gm3medin/dy) (g/m3 media jdy)
0.92 1207
2.04 1420
2.92 1633
3.88 1796 1418
4.92 1922 1525
6.00 1668 1155
7.17 1802 1054
8.29 _ 1763 1093
8.92 1441 1267
9.92 772 553
10.96 352 382
12.00 323 103

 

 

 

 

146

Appendix 14

Respirameter Data for agitated cultures of P. chrysospoﬁum.

(C1 8 oxygen concentration in media, g/m3)
(v = oxygen uptake rate by whole culture, g 02/ m3 medial s)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Data for Figtne 5.8
time a- 19 hours 43 horas 69 horas 91 horns
C1 v Cl v Cl v 01 v
m3) (g/m3/s) (M3) was Ms) (glm3/s) (8111131 (g/m3/s)
14.50 0.0183 13.10 0.0142 6.26 0.0126 29.33 0.0146
13.18 0.0172 10.55 0.0137 5.36 0.0114 26.70 0.0133
12.03 0.0155 8.16 0.0126 4.61 0.0097 24.56 0.0119
10.96 0.0137 6.02 0.0105 3.96 0.0086 22.41 0.0128
10.05 0.0132 4.37 0.0086 3.38 0.0080 19.94 0.0142
9.06 0.0126 2.92 0.0069 2.80 0.0065 17.30 0.0151
8.24 0.0114 1.90 0.0047 2.44 0.0052 14.50 0.0142
7.42 0.0103 1.24 0.0037 2.06 0.0043 1220 0.0137
6.76 0.0103 1.81 0.0029 9.56 0.0146
5.93 0.0103 1.65 0.0023 6.92 0.0137
5.27 0.0086 25.91 0.0137 4.61 0.0119
4.70 0.0080 23.45 0.0141 2.64 0.0092
4.12 0.0080 20.83 0.0150 1.32 0.0073
3.54 0.0074 18.04 0.0150
3.05 0.0063 15.41 0.0146
2.64 0.0057 12.79 0.0146
2.22 0.0052 10.17 0.0146
1.90 0.0040 7.54 0.0128
1.65 0.0034 5.58 0.0109
1.40 0.0034 3.61 0.0091
2.30 0.0061
1.41 0.0049
Wilkinson's Method
| Vmﬂm Vmgm VW V
I 0.075 + 0.027 0.035 + 0.003 0.021 + 0.001 0.018 + 0.001
I KM Kmm Kmart KL.‘
I 23.3 + 10.0 15.2 + 2.3 6.3 + 0.5 3.9 + 0.6 ,

 

 

 

 

147

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Appendix 14 (cont)
time = 116 hours 143 hours 164 horn's 212 hours
C1 v v C1 v C1 v
_(s/m3) (g/m3/s) M3) (glm3/s) (Ema) (g/m3/s) (m3) (g/m3/s)
29.33 0.0146 19.12 0.0146 19.45 0.0128 8.90 0.0174
26.70 0.0133 16.48 0.0151 17.14 0.0133 5.77 0.0149
24.56 0.0119 13.68 0.0156 14.67 0.0137 3.54 0.0103
22.41 0.0128 10.88 0.0156 12.20 0.0137 2.06 0.0064
19.94 0.0142 8.08 0.0151 9.72 0.0151 1.24 0.0046
17.30 0.0151 5.44 0.0137 6.76 0.0156 12.85 0.0275
14.50 0.0142 3.13 0.0110 4.12 0.0119 7.91 0.0238
12.20 0.0137 1.48 0.0073 2.47 0.0087 4.28 0.0169
9.56 0.0146 0.49 0.0055 0.99 0.0082 1.81 0.0110
6.92 0.0137 0.33 0.0082
4.61 0.0119 ,
2.64 0.0092
1.32 0.0073
7 Wilkinson's Method 8 _
| v | Vmax,“ | v I anxm__l
I 0.015 + 0.001 I 0.017 + 0.001 I 0.014 + 0.001 I 0.044 + 0.011
| Kmem | Kmm | ngm I K‘mL_|
I 1.4 + 0.2 I 1.4 + 0.3 I 1.0 + 0.4 I 8.1 + 4.0

 

 

 

148

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

234 hours 260 hours 284 hours 332 hours
C1 v C1 v C1 v C1 v
.Ja/m3) (glm3/s) (a/m3) (g/m3/s) Ja/m3) (g/m3/s) (a/m3) (8/m3/2)
14.17 0.0064 20.44 0.0073 28.68 0.0087 21.75 0.0069
11.87 0.0066 17.80 0.0064 25.54 0.0085 19.28 0. 0069
9. 39 0.0066 15.82 0.0053 22.58 0.0080 16.81 0.0062
7. 09 0.0066 14.01 0.0053 19.78 0.0080 14.83 0. 0053
4. 61 0.0066 12.03 0.0057 16.81 0.0082 13.02 0.0050
2. 31 0. 0064 9.89 0.0062 13.84 0.0078 11.21 0.0050
18.46 0. 0101 7.58 0.0066 11.21 0.0073 9.39 0.0050
14.83 0. 0096 5.11 0.0069 8. 57 0.0069 7.58 0.0050
11.54 0.0092 2.64 0.0060 6.26 0.0066 5. 77 0.0050
8. 24 0.0087 0.82 0.0050 3. 79 0.0064 3. 96 0.0048
5. 27 0. 0082 28.35 0.0096 1 .65 0. 0060 2. 31 0.0046
2. 31 0.0066 24.88 0.0089 14 .01 0.0073 0.66 0.0046
0.49 0.0050 21.92 0.0080 11.37 0.0071 17.14 0.0050
19.12 0.0073 8. 90 0.0066 15.33 0.0048
16.64 0.0071 6.59 0.0066 13.68 0.0048
14.01 0.0071 4.12 0. 0066 11.87 0. 0050
11.54 0.0066 1.81 0. 0053 10. 05 0. 0050
9.23 0.0066 0.33 0.0041 8. 24 0.0050
6.76 0.0066 6.43 0.0048
4.45 0.0064 4.78 0.0048
2.14 0.0055 2.97 0.0048
0.49 0.0046 1.32 0.0046
g , Wilkinson's Method ,
I “when I “my; I “my I “WLI
| 0.009 + 0.001 | 0.007 + 0.002 | 0.008 + 0.001 I 0.007 + 0.000
mm mean I ““322 mm.__|
L1+07 03+01 | 10+02 ~0l+01

 

 

 

 

 

 

149

 

 

 

 

 

 

 

 

Appendix 14 (cont)
time=72 hours 96 hours 120 hours
Cl v C1 v C1 v
Jug g/m3/s s/m3 g/m3/s 1,1,3 g/m3/s
16.20 0.01918 7.34 0.01111 25.79 0.01463
14.82 0.01854 5.33 0.01032 23.15 0.01463
13.53 0.01598 3.62 0.00847 20.52 0.01488
12.52 0.01343 2.29 0.00635 17.80 0.01614
11.60 0.01279 1.33 0.00450 14.71 0.01665
10.68 0.01279 0.67 0.00291 11.80 0.01564
9.76 0.01215 0.29 0.00159 9.08 0.01463
8.93 0.01151 0.10 0.00106 6.54 0.01236
8.10 0.01151 6.57 0.01085 4.63 0.00984
7.27 0.01087 4.62 0.00995 3.00 0.00757
6.54 0.01023 2.99 0.00786 10.08 0.01816
5.80 0.00959 1.79 0.00566 6.81 0.01488
5.16 0.00927 0.95 0.00386 4.72 0.01059
4.47 0.00831 0.40 0.00238 3.00 0.00832
3.96 0.00799 0.10 0.00169 7.26 0.01261
3.31 0.00831 14.19 0.01641 4.99 0.01135
2.76 0.00703 11.24 0.01614 3.18 0.00878
2.30 0.00639 8.38 0.01588 1.83 0.00605
1.84 0.00575 5.53 0.01402 1.00 0.00391
1.47 0.00511 3.33 0.01045 0.43 0.00214
1.10 0.00448 1.76 0.00725 0.23 0.00073
0.83 0.00384 0.72 0.00437 0.16 0.00035
0.55 0.00320 0.19 0.00296 1.73 0.00580
0.37 0.00192 10.38 0.01588 0.91 0.00353
0.28 0.00160 7.53 0.01429 0.45 0.00189
11.14 0.00972 5.24 0.01191 0.23 0.00126
9.39 0.01023 3.24 0.00979 1.91 0.00530
7.46 0.01023 1.71 0.00701 1.09 0.00353
5.71 0.00895 0.71 0.00423 0.64 0.00189
4.24 0.00742 0.19 0.00291 0.41 0.00126
3.04 0.00639
1.93 0.00511
1.20 0.00358
0.64 0.00243
0.32 0.00128
, ,Wilkinson's Method
Vmax a“, 0.022 + 0.008 0.022 + 0.001 0.019 + 0.001
Km 8.0 + 1.2 4.3 + 0.6 3.7 + 0.5

 

 

150

 

 

 

 

 

 

 

 

Appendix 14 (cont)

144 hours 168 home 192 hours

C} v C1 v C1 v
1133 8,133/8 m3 HEB/s 4,1113 8033/8
25.80 0.01635 7.25 0.01555 12.18 0.02055
24.63 0.01700 4.45 0.01308 10.70 0.02149
23.35 0.01635 2.55 0.00919 9.08 0.02009
22.27 0.01700 1.15 0.00601 7.80 0.01822
20.91 0.01471 0.38 0.00424 6.46 0.01775
20.15 0.01700 24.82 0.00177 5.25 0.01588
18.46 0.02158 24.69 0.00265 4.17 0.01401
17.05 0.01929 24.43 0.00619 3.23 0.01215
15.68 0.01897 23.80 0.01149 2.42 0.00981
14.31 0.01929 22.78 0.01502 1.82 0.00794
12.90 0.01691 21.63 0.01591 1.28 0.00701
5.79 0.01588 20.49 0.01856 0.81 0.00514
4.90 0.01144 18.96 0.02298 0.54 0.00346
4.14 0.01046 17.18 0.02474 0.31 0.00318
3.39 0.00981 15.40 0.02519 27.58 0.01495
2.73 0.00948 13.55 0.02563 26.50 0.01635
2.02 0.00817 11.71 0.02563 25.22 0.01775
1.55 0.00621 9.86 0.02474 23.95 0.01728
1.13 0.00490 8.14 0.02298 22.74 0.01962
0.85 0.00392 6.55 0.02121 21.12 0.02149
0.57 0.00392 5.09 0.01944 19.64 0.02055
' 6.45 0.01635 3.75 0.01679 18.16 0.02149
5.27 0.01570 2.67 0.01370 16.55 0.02242
4.19 0.01419 1.78 0.01060 14.93 0.02242
3.23 0.01243 1.15 0.00751 13.32 0.02336
2.40 0.01053 0.70 0.00557 11.57 0.02336
1.71 0.00844 0.34 0.00353 9.96 0.02242
1.19 0.00661 0.19 0.00212 8.34 0.02055
0.76 0.00562 18.45 0.02563 7.00 0.01868
0.38 0.00536 16.61 0.02519 5.65 0.01775
9.51 0.01831 14.83 0.02519 4.44 0.01495
8.19 0.01962 12.98 0.02519 3.50 0.01308
6.69 0.01962 11.20 0.02519 2.56 0.01121
5.37 0.01766 9.35 0.02430 1.88 0.00888
4.14 0.01635 7.70 0.02165 1.28 0.00747

Wilkinson's Method
Vmax m, 0.021 + 0.001 0.032 + 0.001 0.029 + 0.001
Km 2.0 + 0.3 3.5 + 0.3 4.2 + 0.3

 

 

 

 

151

 

 

 

 

 

 

 

 

 

 

Appendix 14
. . s 02 . 8 02 I
CalculatedvaluesofVmaxrnunltsof Im3s fromunrtsof Im3media sI°
Vmax app Km app Vmax X
Day g/m3 media/s 4&3 g/m3/s (£3)
1 0.075 23.00 *
2 0.035 15.00 *
3 0.022 8.00 0.97 1250
3 0.021 6.31 0.84 1375
4 0.022 4.30 0.88 1375
4 0.018 3.87 0.66 1500
5 0.019 3.69 0.70 1500
5 0.015 1.35 0.51 1625
6 0.021 2.07 0.71 1625
6 0.017 1.36 0.52 1750
7 0.032 3.52 1.00 1750
7 0.014 0.91 0.41 1875
8 0.029 4.23 0. 85 1875
9 0.044 8.10 1.21 2000
10 0.009 1.08 * *
11 0.007 0.33 * 1'
12 0.008 0.97 "' *
14 0.007 0.07 * *
I mean Vmax I 0.77 3; 023 I

 

'dlanatnudmlydataforaacoodarymetabolirmwasmidered.

152

 

 

 

 

 

 

 

 

Appendix 15
Model test cases.
Experimental data for Figure 5 .9
Condition 1
Time Glucose Dry Weight
0*) (811113) (rial.
0 5689 90
7.92 4954 108
18.9984 4033 464
24 3268 998.8
48 2837 1052
72 2008 1236
93.84 918 1416
120 78.6 1370
144 13.1 1215
150 25 1239
165.84 15 1125
192 1080
214.8 818
240 666
285.84 602
Condition 2
Time Glucose Dry Weight
(h) m3) . (8,232
0 10600 114
8 9770 129
19 8840 641
24 8540 904
48 8380 1071
72 7160 1133
94 5910 1388
120 4230 1359
144 3520 1493
150 2960
165 2370 1571
192 339 1865
215 114 1984
240 80 1910
263 60 1512
311

 

 

 

1140

 

153

 

 

 

 

 

 

 

 

 

lAppendhr15(cont)
ICandﬂhmrz

'Thne Aunnunﬁunr (Bacon: lJnvaeupu
(b) (m3) (m3) (21:132. _.

0 33J3 10090 129
10 23:7 147
28 143 9790 705
38 0 874
53 8960 1223
67 1240
76 9276

98 7109 1333
120 6227 1373
149 1440
168 4729 1513
192 3293

222 1539 1736
245 547

262 238.9 2017
285 54

338 1521
432 1247

(kuuﬂﬂknr3

'Ihne (Hucose anylvehﬂu
(b) m3) (m3)

0 17520 112

8 14980 113

19 14470 632

24 13280 1003

48 14400 1107

72 12290 1220

94 10700 1475
120 10480 1632
144 7760 1477
150 7450

166 6730 1584
192 5290 1823
215 4170 1952
240 2900 2129
263 1700

286 660 2548
311 327

336 1980

 

 

 

154

 

 

 

 

 

 

 

 

 

Appendix 15 (cont)
Condition 4
Time Ammonium Glucose Dry Weight
(11) (g/m3) (g/m3) (yin?)
0 2.89 11090 118
9 2.05 114
28 2.26 10380 174
38 131.8
53 10100 275.3
67 90.5
76 10732
98 9997 261.2
120 10900 400
168 9755 476.5
192 9400
222 8970
245 9050
262 8950
285 9090 742.3
310. 8830
337 8102
432 7900 906
Condition 5
Time Ammonium Glucose Dry Weight
(h) (g/m3) (g/m3) (gm32
0 117 11000 127.7
10 102 145.9
28 10.6 8993 1180
38 2.49 2202
53 1 7260 2568
67 1.14 2660
76 0 6250 2805
98 5375 2952
120 3383 3430
149 3480
168 57 3 4006
192 74
222 80 2879
262 2496
338 2094
432 1739

 

 

 

Appendix 16

155

Sample output from shake flask simulation computer program.

Model data for Figln'e 5.9, Condition 2

 

 

 

 

Model parameter values
Ynx umax Vmax Km fr . fx fpg Kant t] p De
0.048 0010039 0.76 0.5 0.84 0.12 0.04 2.5E-06 7320000 65000 2.9E-09
T N G X C1 Oztatal 002 R pG
h “3 m3 m3 Emm. m3 8 g m m3
4 39 11000 100 1 32.75 0.20709 0.00194 0.000369 0
8 39 11000 100 1 32.53 0.20568 0.003878 0.000369 0
12 35.2 10911 174.55 0.989 32.22 0.20375 0.006537 0.000444 0
16 28.5 10755 304.1 0.98818 31.69 0.20039 0.011153 0.000534 0
20 16.9 10485 529.55 0.9873 30.77 0.19455 0.019179 0.000643 0
24 -3.2 10015 921.6 0.98619 32.98 0.2085 0.033109 0.000773 0
28 0 9859.1 940.29 0.98666 31.01 0.19608 0.017077 0.000778 6.2312
32 0 9700.5 959.32 0.98573 29.01 0.18344 0.034456 0.000784 12.573
36 0 9539.3 978.67 0.98402 26.98 0.17058 0.052138 0.000789 19.024
40 0 9376.8 998.17 0.95477 24.93 0.15763 0.06995 0.000794 25.523
44 0 9217.8 1017.3 0.9084 22.92 0.14495 0.087386 0.000799 31.884
48 0 9064.4 1035.6 0.98737 32.98 0.2085 0 0.000804 38.016
52 0 8890.1 1056.6 0.98646 30.78 0.1946 0.019108 0.000809 44.988
56 0 8712.9 1077.8 0.98353 28.54 0.18047 0.038543 0.000815 52.08
60 0 8534.8 1099.2 0.95094 26.3 0.16627 0.058065 0.00082 59.202
64 0 8361.1 1120 0.90195 24.11 0.15242 0.077107 0.000825 66.149
68 0 8194 .6 1140 0.8449 22.01 0.13914 0.095364 0.00083 72.809
72 0 8036.6 1159 0.98729 32.98 0.2085 . 0 0.000835 79.129
76 0 7842.7 1182.3 0.98532 30.53 0.19304 0.021261 0.00084 86.887
80 0 7647.7 1205.7 0.95336 28.07 0.17749 0.042641 0.000846 94.687
84 0 7457.5 1228.5 0.9035 25 .67 0.16233 0.06349 0. 000851 102.29
88 0 7275.5 1250.3 0.84507 23.38 0.14781 0.08345 0. 000856 109.58
92 0 7103.5 1271 0.78272 21.21 0.13409 0.10231 0.000861 116.45
96 0 6942.3 1290.3 0.98721 32.98 0.2085 0 0.000865 122.9
100 0 6729.4 1315.9 0.96161 30.29 0.19153 0.023338 0.000871 131.42
104 0 6521.1 1340.9 0.91287 27.67 0.17492 0.046179 0.000876 139.75
108 0 6321.5 1364.8 0.85413 25.15 0.159 0.068062 0.000881 147.73
112 0 6133 1387.4 0.79076 22.77 0.14397 0.088731 0.000886 155.27
116 0 5956.9 1408.5 0.72612 20.55 0.12993 0.10804 0.000891 162.32

 

 

 

156

 

 

 

 

 

Appendix 16 (cont)
'1' N o x a, 02mm ca; 11 p6
0') (xi-113) (sin-3) Emm. (1123) (s) (s) ("0 m3)
116 0 5956.9 1408.5 0.72612 20.55 0.12993 0.10804 0.000891 162.32
120 0 5793.6 1428.2 0.97372 32.98 0.2085 0 0.000895 168.85
124 0 5565.9 1455.5 0.92881 30.11 0.19035 0.024955 0.000901 177.96
128 0 5347.2 1481.7 0.87121 27.35 0.17291 0.04894 0.000906 186.71
132 0 5140.3 1506.5 0.80762 24.74 0.15641 0.071626 0.000911 194.98
136 0 4947 1529.7 0.74201 22.30 0.14099 0.09282 0.000916 202.71
140 0 4768.2 1551.2 0.67697 20.05 0.12673 0.11243 0.00092 209.87
144 0 4603.6 1571 0.94861 32.98 0.2085 0 0.000924 216.45
148 0 4365 1599.6 0.8942 29.97 0.18948 0.026152 0.000929 225.99
152 0 4138.8 1626.7 0.83166 27.12 0.17144 0.050959 0.000935 235.04
156 0 3927.1 1652.1 0.76589 24.45 0.15456 0.07417 0.000939 243.51
160 0 3731.2 1675.6 0.7 21.98 0.13893 0.095657 0.000944 251.35
164 0 3551.3 1697.2 0.63599 19.71 0.12459 0.11538 0.000948 258.54
168 0 3387 1716.9 0.9204 32.98 0.2085 0 0.000952 265.11
172 0 3142.3 1746.3 0.86087 29.894 0.18899 0.026826 0.000957 274.9
176 0 2913 1773.8 0.79647 27.002 0.1707 0.05197 0.000962 284.07
180 0 2700.6 1799.3 0.73098 24.323 0.15377 0.075257 0.000966 292.57
184 0 2505.8 1822.7 0.66683 21.86 0.13823 0.096623 0.000971 300.36
188 0 2328.4 1844 0.60554 19.62 0.12408 0.11608 0.000974 307.46
192 0 2167.3 1863.3 0.89199 32.98 0.2085 0 0.000978 313.9
196 0 1925.9 1892.3 0.83163 29.93 0.18925 0.026473 0.000983 323.56
200 0 1703 1919 0.76933 27.12 0.17148 0.050904 0.000987 332.47
204 0 1499.7 1943.4 0.70797 24.56 0.15527 0.073197 0.000992 340.6
208 0 1316 1965.5 0.64938 22.24 0.14061 0.093347 0.000995 347.96
212 0 1151.1 1985.2 0.59465 20.16 0.12747 0.11142 0.000999 354.55
216 0 1003.9 2002.9 0.8657 32.98 0.2085 0 0.001002 360.44
220 0 794.1 2028.1 0.81309 30.33 0.19177 0.022998 0.001006 368.83
. 2% 0 610.49 2050.1 0.76309 28.02 0.17713 0.043128 0.00101 376.17
228 0 454.13 2068.9 0.71802 26.05 0.16467 0.060269 0.001013 382.43
232 0 325.35 2084.3 0.67938 24.42 0.1544 0.074387 0.001015 387.58
236 0 223.58 2096.6 0.64795 23.14 0.14629 0.085544 0.001017 391.65

 

 

157

 

 

 

 

Appendix 16 (cont)

r N a x c, Oztotal 002 R [)0
(11) (31.1.3) (g/m3) Emm. (3&3) (a) (s) 0“) (s/m3)
240 0 146.97 2105.7 0.84707 32.98 0.2085 0 0.001019 394.72
244 0 76.158 2114.2 0.8298 32.08 0.20286 0.007761 0.00102 397.55
248 0 0 2069.2 1 31.18 0.19712 0.015648 0.00102 375.02
252 0 0 ”25.7 1 30.31 0.19159 0.023255 0.00102 353.29
256 0 0 1983.8 1 29.46 0.18625 0.030592 0.1!)102 332.34
260 0 0 1943.4 1 28.65 0.1811 0.037669 0.00102 312.13
264 0 0 1904.4 1 32.98 0.2085 0 0.00102 292.63
268 0 0 1866.8 1 32.22 0.20371 0.“)6583 0.00102 273.83
272 0 0 1830.5 1 31.49 0.1991 0.012931 0.(X)102 255.7
276 0 0 1795.6 1 30.79 0.19464 0.019055 0.00102 238.21
280 0 0 1761.8 1 30.11 0.19035 0.02496 0.(X)102 221.34
284 0 0 1729.3 1 29.45 0.1862 0.030657 0.1X)102 ”5.07
288 0 0 1697.9 1 32.98 0.2085 0 0.1!) 102 189.38
292 0 0 1667.6 1 32.37 0.20465 0005299 0.1!) 102 174.25
296 0 0 1638.4 1 31.78 0.20093 0.010409 0.00102 159.65
300 0 0 1610.3 1 31.21 0.19735 0.015338 0.(X)102 145.57
304 0 0 1583.1 1 30.67 0.19389 0.020092 0.00102 131.99
308 0 0 1556.9 1 30.14 0.19055 0.024677 0.1K) 102 118.9
312 0 0 1531.7 1 32.98 0.2085 0 0.00102 106.27
316 0 0 1507.3 1 32.48 0.2054 0.004265 0.“) 102 94.086
320 0 0 1483.8 1 32.02 0.20241 0.“)8379 01X) 102 82.336
324 0 0 1461.2 1 31.56 0.19952 0.012346 001102 71.104
328 0 0 1439.3 1 31.12 0.19674 0.016173 0.00102 60.075
332 0 0 1418.2 1 30.69 0.19405 0.019864 0.1!)102 49.533
336 0 0 1397.9 1 32.98 0.2085 0 0.00102 39.366
340 0 0 1378.3 1 32.58 0.206 0.003433 010102 29.56
344 0 0 1359.4 1 32.20 0.20359 0.(X)6744 0.00102 20.103
348 0 0 1341.1 1 31.84 0.20127 0.1119938 0.(X)102 10.981

 

 

 

158

Appendix 17

Oxygen effectiveness factor as a function of mycelial pellet radius and liquid phase
oxygen concentration

Data for Figlne 5.12

 

Oxygen Concentration (g/m3)
am 10 20 30 4g go 6.0 7.0 8.0 2.0 100 11.0
000006 0990 0997 0.999 0999 1.000 1.000 1.000 1.000 1.000 1.000 1000
00001 0964 0989 0995 0.997 0998 0999 0999 0999 0999 1000 1000
000015 0931 0978 0989 0994 0996 0997 0998 0998 0999 0999 0999
00002 0713 0965 0983 0990 0993 0995 0996 0.997 0998 0998 0999
000025 0536 0841 0976 0985 0990 0993 0995 0996 0997 0.997 0.998
00003 0420 0.664 0869 0.980 0986 0990 0993 0994 0.995 0996 0.997
000035 0341 0531 0711 0862 0968 0988 0991 0993 0.994 0.996 0.996
00004 0286 0.435 0584 0722 0841 0936 0.988 0.991 0993 0.994 0.996
000045 0244 0.364 0487 0606 0716 0814 0.897 0.961 0991 0993 0.994
00006 0212 0.310 0.412 0513 0.610 0700 0783 0857 0920 0968 0993
000065 0188 0268 0.354 0440 0624 0605 0681 0752 0.817 0876 0.926
00006 0168 0235 0308 0381 0454 0525 0594 0660 0722 0780 0833
000065 0162 0209 0271 0534 0.398 0460 0522 0581 0.638 0.692 0.744
00007 0138 0187 0240 0295 0351 0406 0461 0514 0566 0.616 0664
000075 0127 0169 0215 0263 0.312 0361 0410 0458 0506 0560 0596
00008 0117 0164 0194 0237 0280 0524 0367 0410 0452 0494 0534
000086 0109 0141 0176 0214 0263 0291 0330 0369 0407 0.445 0482
00009 0102 0130 0.161 0.195 0229 0264 0299 0534 0.369 0.403 0437
000096 0096 0120 0148 0178 0209 0241 0272 0504 0.336 0367 0598
0001 0089 0112 0137 0164 0192 0220 0249 0278 0.306 0.336 0364
000105 0084 0104 0127 0.151 0.176 0202 0228 0256 0.281 0307 0333
00011 0080 0098 0118 0140 0163 0187 0211 0236 0259 0283 0307
000115 0076 0092 0110 0l30 0151 0173 0.195 0217 0239 0261 0283
00012 0072 0086 0103 0122 0141 0.161 0.181 0201 0222 0242 0263
000125 0069 0082 0097 0114 0131 0.150 0168 0187 0206 0225 0244
00013 0066 0078 0091 0107 0123 0.140 0157 0176 0192 0210 0227
000135 0063 0074 0086 0101 0116 0131 0147- 0.163 0180 0.196 0212
00014 0.060 0070 0082 0096 0.109 0.123 0.138 0.153 0.168 0184 0.199
0.00145 0068 0067 0078 0090 0.103 0116 0130 0.144 0158 0172 0187
00015 0066 0064 0074 0086 0097 0.110 0122 0136 0149 0.162 0176
000156 0064 0.061 0070 0081 0092 0.104 0.116 0128 0.140 0163 0165
00016 0062 0059 0067 0077 0087 0098 0110 0121 0133 0.144 0.156
000165 0060 0066 0064 0073 0083 0093 0104 0116 0126 0137 0.148
00017 0048 0064 0062 0070 0079 0089 0099 0109 0119 0129 0140
000176 0047 0062 0069 0067 0075 0085 0094 0103 0113 0123 0133
00018 0046 0060 0067 0064 0072 0081 0.089 0098 0108 0.117 0.126
000185 0044 0049 0065 0061 0069 0077 0085 0094 0102 0111 0120
00019 0043 0047 0063 0069 0066 0074 0082 0090 0098 0.106 0114
000195 0041 0045 0061 0067 0063 0071 0078 0086 0093 0101 0109
0002 0040 0044 0049 0066 0061 0.068 0075 0082 0089 0097 0104
000206 0039 0043 0047 0063 0069 0066 0072 0079 0086 0093 0100
00021 0038 0041 0046 0061 0.066 0062 0069 0076 0082 0089 0096
000215 0067 0040 0044 0049 0064 0060 0066 0072 0079 0086 0092
00022 0036 0.039 0043 0047 0052 0068 0064 0070 0076 0.082 0088
000225 0036 0038 0041 0046 0051 0.066 0061 0067 0073 0.079 0.084
00023 0036 0037 0040 0044 0049 0.064 0069 0064 0070 0.076 0081
000236 0064 0036 0039 0043 0047 0062 0067 0062 0067 0073 0078
00024 0063 0036 0088 0042 0046 0.050 0.055 0060 0065 0070 0075
000246 0032 0034 0037 0040 0044 0049 0.053 0.058 0063 0068 0073
0.0025 0032 0033 0036 0039 0043 0047 0.051 0.056 0065 0070

159

Appendix 1? (cont)

Oxygen Concentration (g/m3)

3 E} 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 29.0 21.0 22.0
0m 1M 1M 1M 1M 1M 1M 1M 1M 1M I.” 1.000

0M1 1000 1M 1M 1M 1M 1M 1M 1.!!!) 1M 1M 1.1!!)

0.0002 0.999 0.999 0.999 0.999 0.999 0 999 0.999 1M 1M 1 (I10 1 000
0.11125 0.998 0.998 0.999 0.999 0.999 0 999 0.999 0.999 0 999 0.999 0 999
0M3 0.997 0.998 0.998 0.998 0.998 0 999 0.999 0.999 0.999 0 999 0 999
OM35 0.997 0.997 0.997 0.998 0.998 0 998 0 998 0.999 0 999 0.999 0.999
00004 0.996 0.996 0.997 0.997 0.997 0 998 0.998 0.998 0 998 0 998 0 999
0.011145 0.995 0.995 0.996 0.997 0 997 0.998 0 998 0 998 0 998 0 998
0m 0994 0.995 0.996 0.996 0 997 0.997 0.997 0.998 0 998 0.998
0.11355 0.966 0.992 0.995 0.996 0 996 0.996 0.997 0 997 0 997 0 998
0% 0.881 0923 0985 0995 0996 0.996 0.996 0997 0 997 0997
0W5 0.792 0.837 0.915 0 947 0 973 0.992 0.996 0 996 0 997 0 997
0.01!” 0.710 0.754 0.835 0.871 0 904 0 933 0.959 0 979 0 994 0 996

0N1 0.392 0.420

m105 0.359 0.385 0.436 0.460 0.485 0.509 0.533 0.556 0.580 0.602
00011 0.331 0.355 0.401 0.424 0.447 0.470 0.492 0.514 0.536 0.557
0.111115 0.3“ 0.327 0.371 0.392 0.414 0.435 0.456 0.476 0.496 0.517
OM12 0.283 0.303 0.344 0.364 0.384 0.403 0.423 0.442 0.461 0.480
W125 0.263 0.282 0.319 0.338 0.357 0.375 0.393 0.411 0.429 0.447
0.11113 0.245 0.263 0.298 0.315 0.332 0.349 0.366 0.383 0 400 0 417
0.111135 0.229 0.245 0.278 0.294 0.310 0.326 0.342 0.358 0.374 0.390
0.“)14 0.214 0.230 0.245 0.260 0.275 0.290 0.3“ 0.321 0.335 0.350 0.365
0.111145 0.201 0.215 0.230 0.244 0.258 0.272 0.287 0.301 0.315 0.329 0.343
0.0015 0.189 0202 0.216 0.229 0.243 0.256 0.269 0.283 0.296 0.309 0.322
0.111155 0.178 0.191 033 0.216 0.228 0.241 0.254 0.266 0.279 0.291 0.303
0.“)!6 0168 0.180 0.192 0.204 0.216 . 0.227 0.239 0.251 0.263 0.275 0.286
0.1!)165 0159 0.170 0.181 0.192 0.204 0.215 0.226 0.237 0.248 0.259 0.271
0.11117 0.150 0.161 0.172 0.182 0.193 0.203 0.214 0.225 0.235 0.246 0.256
001175 0.143 0.153 0.163 0.173 0.183 0.193 0.203 0.213 0.223 0.233 0.243
0CD" 0.136 0.145 0.154 0.164 0.173 0.183 0.192 0.202 0.211 0.221 0.230
0.111185 0.129 0.138 0.147 0.156 0.165 0.174 0.183 0.192 0.201 0.210 0.219
00119 0.123 0.131 0.140 0148 0.157 0.166 0.174 0.183 0.191 0200 0.208
0.111195 0.117 0.125 0.133 0.141 0.150 0.158 0.166 0.174 0.182 0.190 0.198
0” 0.112 0.120 0.127 0.135 0.143 0.151 0.158 0.166 0.174 0.182 0.189
000205 0.107 0.114 0.122 0.129 0.136 0.144 0.151 0.159 0.166 0.173 0.181
0M1 0102 0.109 0.116 0.123 0.130 0.138 0.145 0.152 0.159 0.166 0.173
OM15 0.098 0.105 0.111 0.118 0.125 0.132 0.138 0.145 0.152 0.159 0.165
00022 0.094 0.101 0.107 0.113 0.120 0.126 0.133 0.139 0.146 0.152 0.158
011325 0090 0.096 0.103 0.109 0.115 0.121 0.127 0.133 0.140 0.146 0.152
00023 0M 0.093 0M9 0.104 0.110 0.116 0.122 0.128 0.134 0.140 0.146
0.11235 0.084 0.089 0.095 0100 0.1“ 0.112 0.117 0.123 0.129 0.134 0.140
0.11124 0.081 0.086 0.091 0.097 0.102 0.107 0.113 0.118 0.124 0.129 0.135

OM45 0.078 0.133 0.088 0.093 0.098 0.103 0.109 0.114 0.119 0.124 0.129
0.4135 0.075 0.130 0.085 0.090 0.195 0.100 0.105 0.110 0.115 0.120 0.125

 

0.!!!“

0.11115

OM175

 

0.732

0.217

0.197
0.188

0.18
0.172
0.165
0.158
0.152
0.146

0.14
0.135

0.13

0.158
0.151
0.146

0.14
0.135

0.178

0.151
0.145
0.14

160

 

Appendix 1? (cont)
Oxygen Concentration (31103)
26 27 28 29 30 31 32
1000 1000 1000 1000 1.000 1.000 1.000
1000 1000 1000 1.000 1000 1000 1000
1000 1000 1.000 1000 1.000 1000 1.000
1000 1000 1000 1000 1000 1000 1000
1000 1000 1000 1000 1.000 1.000, 1000
0999 0999 1000 1.000 1000 1000 1000
0999 0999 0.999 0.999 0999 0.999 0999
0999 0999 0999 0.999 0.999 0.999 0999
0999 0.999 0.999 0.999 0999 0999 0999
0999 0999 0999 0.999 0999 0.999 0999
0998 0998 0998 0999 0999 0999 0.999
0998 0998 0.998 0998 0.998 0.999 0999
0998 0998 0998 0.998 0.998 0998 0.998
0997 0998 0998 0.998 0.998 0.998 0.998
0997 0997 0.997 0.998 0998 0.998 0.998
0979 0.991 0.997 0.997 0997 0998 0.998
0924 0943 096 0.974 0986 0995 0.998
0862 0883 0903 0.922 0.939 0954 0.968
0801 0822 0843 0.863 0.882 09 0917
0743 0764 0785 0005 0824 0343 0861
0.689 0.71 073 0.749 0769 0.787 0805
064 0659 0.679 0.698 0716 0736 0.752
0595 0613 0632 065 0668 0686 0.703
0554 0571 0589 0606 0.623 064 0.657
0516 0533 056 0566 0583 0599 0615
0482 0.498 0514 053 0545 0661 0576
0451 0466 0481 0.496 0511 0626 064
0423 0437 0.452 0466 048 0.493 0607
0397 0411 0424 0.438 0.451 0.464 0.477
0374 0387 0399 0.412 0.425 0437 0449
0352 0365 0377 0389 04 0.412 0424
0333 0344 0356 0367 0378 0389 0401
0314 0325 0336 0347 0358 0368 0379
0.298 0308 0318 0329 0339 0349 0359
0282 0292 0302 0312 0321 0331 034
0268 0277 0287 0296 0306 0314 0323
0266 0264 0272 0281 029 0299 0307
0242 0251 0259 0268 0276 0284 0293
0231 0239 0247 0256 0263 0271 0279
022 0228 0236 0243 0251 0259 0266
021 0218 0225 0232 024 0247 0254
0201 0208 0215 0222 0229 0236 0243
0192 0199 0206 0213 0219 0226 0233
0184 0191 0197 0204 021 0216 0223
0.177 0.183 0189 0.196 0201 0207 0214
0169 0.175 0181 0.187 0193 0.199 0205
0163 0168 0174 0.18 0.186 0191 0.197
0.156 0162 0167 0.173 0.178 0.184 0189
015 0.156 0161 0166 0171 0.177 0182
0145 0.15 0.155 016 0.165 017 0.175

161

 

 

Appendix 18
Glucose consumption and C02 evolution in air-ﬂushed cultures as reported by
Dosorctz et al., 19900.
Data for Figure 6.4
Time ' 002 Glucose

(h) (3; (m3)

0 0 10000
2.4 0.0267 9500
48 0.0313 8700
72 0.0340 8000
96 0.0354 7000
120 0.0408 6800
144 0.0394 6000
168 0.0399 5800
192 0.0405 4500
216 0.0381
240 0.0326
264 0.0326

 

 

 

 

162

 

 

 

 

 

 

 

 

 

 

Appendix 19
Data for agitated cultures of P. chasesporium grown with methanol"I as the sole
carbon and energy source.
Time Biomass Acetate Methanol
(dais) M M (all) DH
0 0.187 1.2 10 4.6
1 0.352 0.441 8.61 6.03
2 0.540 0.043 7.59 6.56
4 0.507 0.036 6.77 6.53
5 0.521 0.018 6.21 6.58
6 0.515 0 5.52 6.55
7 0.539 4.39 6.56
12 0.378 2.06 6.71
13 0.336 1.96 6.65
Time Protein MnP LiP
(days) (mg/l) (W1) (W1)

1 22.2 0 0

2 21.9 0 O

4 20.6 0 0

5 21.0 0 0

6 21.9 0 0

7 23.2 0 2

12 31.8 0 4

13 39.0 0 2

 

 

l"Culture conditions were as described for condition 2 cultures, except that methano1(10 M) was
mbstimtedforglucoseandniuogenmfﬁcientconditionswcreusedam m3 ammonium).

 

163

 

 

Appendix 20
Data for agitated cultures of P. chrysospoﬁum grown with ethanol. as the sole
carbon and energy source.
Time Protein MNP LIP
(days) (mgﬂ) DH (W1) (WI)
0.00 4.50

1.00 8.56 6.12
2.00 8.07 6.32
3.00 6.01 6.21

 

 

 

 

 

 

4.00 4.36 5.93 0 0
5.33 3.83 5.21 0 0
6.08 2.35 5.37 40 0
8.00 5.80 5.51 530 0
10.04 4.94 5.50 360 0
13.92 3.21 5.70 230 0
Time 002 Ethanol Biomass NH4
(dim) (3) (3’1) (6’1) (m)
0.00 0 7.67 0.13 39
1.00 0.063 8.09 0.38 0
2.00 0.039 7.11 0.55 0

3.00 0.028 3.53 0.78
41!) 0.036 2.68 0.81
5.33 0.040 2.47 1.01
6.08 0.049 2.03 1.08

 

7.04 0.042

811) 0.052 1.60 1.33

9.00 0.036

10.04 0.039 1.33 1.52
11 0.035

13.92 0.040 0.40 1.55

 

 

 

’cmmmmmwmumudtacmmmzcm.mmmamw was
WfamcmmmﬂmgrownﬁmWOOgMMmesolewbmmm
cmunufaﬂedmgrowinmﬁonuymnbxhﬂaschuuesmdmaefaeagimwdwmmnm
inoculated.

This program was used to generate all model data (Figures 5.4, 5.6, 5.9, 6.3, 6.4, 6.5).

164

Appendix 21

Shake ﬂask simulation pmgram listing

2 REM SHAKE FLASK SIMULATION
5 OPEN 'c:\windows\moddat\datan79.xls' FOR OUTPUT AS ll

 

DIM C(SOO).X(500),N(500),R(500),OZlSOO),PG(500).C02(SOO).OZTOT(500)
DIM S(100).SS(4),SO(4),V(100),GI(10),PN(S)
REM ** constant physical values '*

VLIQ-.000085:VHS-.000165:TLAG-0:PNUH-7320‘1000:PDBNS-GSOOO!
H-37.8:DELT-3600:DEOZ-2.9E-O9

REM ** model parameter values ‘*

YNX-.048:A-.84:B-.12:C-l-A-B:F-.4:YC0202-44/32:1026-176/6/32
VH-.76:KM-.5:UHAX-.OOOO39:KAUT'.0000025:KPG-.5:KS-400:DES-2.98-09

60508 4000

60508 5000

REM ** initial conditions (all units are seconds, neters or grass) '*
FOR II-l TO 1

6(01-11000: N(0)-39: 02(01-39. 2: X(O)-100: 96(01-0: 002401-0: OZTOT(O)-. 2085
“1(11-39: 81(21'117: "1(31'3: N02-l
11101-141 (II)

RE“ itiitititiittitiﬁttitittﬁi L‘g ph‘.. 0......titﬁtttitﬁttﬁtilﬁtt.ﬁt

608 2-1 20 rnac
0121-610) arm-14(0):oz1r1-oz1o1:xtr1-x(0) :Pctr1-2101:c02(r)-c02(0)
RiT)-(3/4/3.14159/PNUH/PDBNS'X1T))‘(1/3)
VPELS=X(T)/PDERS*VLIQ
DOZDT--(VH*02(T-l)/(KM+O2(T-1))1‘iVPBL3/VLIO)
ozror(r)-ozror(r-1)+VLro-natrtnoznr
02(r1-ozror(r7Itvnro+a~vns1
coz12)-c02(r-l)-44/32*ooznr-oatr*VLro
60508 3000
NEXT 2
REM ttttttttttttttttttﬁtttti pri-ary growth ph.se titttttttttttttttttttittt
X(T)-X(T-1)*BXP(UMAX*NOZ*DBLT1
8(2)-N<r-1)-rux*uunx*uoz*xtr)*oanr
VPELS-X(T)/PDBNS*VLIQ
DOZDT--N02*(VH*02(T-1)/(KH+02(T-1)))‘(VPBLSIVL101/(KS/6(T- 11+1)
nsncr-. 92*(X(T-1)-X(T))+DOZDT*YOZG*DBLT
PG(T)-PG(T-1)
G(T)-G(T-1)+DELGT
R(T)-(3/4/3.14159/PNUM/PDBNS'XiT)1 (1/3)
I? nor ((INTiT/24)- (212411-01 runs:
02202121-02202101:coztr)-c02107
GOSUB 4000
0020 260
OZTOT(T)-02TOT(T-1)+VLIO*DBLT*DOZDT
02(r)-ozror(r)/(ero+a*vaS)
C02(T)-C02(T-l)-44/32*DOZDT*DBLT'VLIO
60503 3000
00503 1000
r-r+1:rr (r>350) runs 760
IF (N(T-1)>O) nun (G(T-1)>O) runs 150
RE“ titttttttttttttttttttt secondary O'th ph‘se tttttttttttttttstttttttt
éssgormttrurtr/241- (212417-01 runs 413:
02202121-02201101:c02(r1-c02101:coro 420
OZTOT(T)-02TOT(T-1)‘(VPELS*VH*OZ(T-l)/(KH+OZ(T-1)))*DBLT*N02/(KS/G(T-1)+1)
02(r)-ozror(r)/(vnro+a*vust
c02(r1-44/32*(02202101-ozror1211
ooznr--u02*(VM302(r)Itxu+oztr)17*(VPBLSIVLIQ)ItKSIctr-11+1)
DELGT-DOZDT*DELT*.8958/A
c121=c(r-1)+oaLcr
PG(T)-PG(T-1)-C'DBLGT
X(T)-X(T-1)-B*DELGT
8127-(3/4/3. 14159/Puuu/Poaus*x(r)) (1/3)
VPELS-X(T)IPDBNS*VLIQ
60808 1000
00803 3000
r-r+1:rs (r>350) runs 760
It G(T-1)>80 7888 400

 

999

165

XF=F‘X(T-l)

REM aaaaaaaaaaaaeeaaaaaaaaaaaaaa death phase aaaaaaaaaaaaaaaaaacaaaaaaaaaa

DXDT=‘KAUT‘(X(T-II'XF)20PCDT3‘KPG'KAUT'(X‘T’1)‘XF)

X‘T)'X(T-l)+DXDT‘DELT

R(T)'R(T-1):N02'l

PG(T)‘PG(T‘1)+DPGDT.DELT

IF NOT ((INT(T/24)’(T/24))‘O) THEN 650

OZTOT(T"02TOT(O)3C02(7)'C02(0)

GOSUB 4000

6070 560

OZTOT‘T)‘OZTOT(T‘1)+DPGDT'(176/5’32)
02(T)-02TOT(T)/(VLIQ*H*VHS)
C02(T)-44/32.(02TOT(0)'OZTOT(T’)

60508 3000

T3T+IZIF ‘T>350) THEN 760

IF PG(T-1)>0 THEN 600

BRASS G,X,N,R'02, PG,C02 0210

DIN G(500),X(500) “(500) R(500)o 02(500) PG‘SOO) COZCSOO), 02TOT(500)

"02'1

PRINT .1,

NEXT 11

RE“ iﬁittttttﬁtttttﬁt.tittititﬂitiﬁﬁﬁttﬁittﬁttﬁﬁltittttiﬁtﬁtiﬁﬁii

00 REM * oxvqen effectiveness factor subroutine * (02(t). RCt):NOZ)*

3010
3015
3020
3030
3040
3070
3100

THIBLE-erl[3*(VN/KNIDBS)‘5

ALPHA-KN/OZiT)

IE (TNIELE>S) THEN 1530
:ggggg-{EXP(THIELB)-EXP(-TRIBLE)lI(EXPCTBIBLB)+EXP(-TNIBLE)). :GOTO 1550
El-1/THIELE‘((1/TANE3T-(1/3/TNIELB)))

PNI-G*DES*02(T)Iva/R(T)/R(T)

IF (PHI<1) THEN 16

80-1: GOTO 1700

EO-l-(l-PHI)‘(3I2)

NOZ-(EO+ALPER‘E1)/(1+ALPEA)

GOTO 1490

RE" ttttttttﬁﬁttItttlﬁttttttttitit.

REN ‘*** printing subroutine ******

PRINT USING “Ill“;T:

PRINT USING ' IOI.I';N(T);02(T):

PRINT USING ' lll...’l';02TOT(Tl;C02(T):NOZ;

PRINT USING ' lllll.l';PG(T):G(T);X(Tl3

PRINT USING ' I.lllllll';R(Tl

IF NOT ((INTiT/4l-(T/4ll-0) THEN 3200

PRINT .1,USING ' l...l.l““';T,N(T),G(T),X(T),N02,02(T),OZTOT(T),C02(T),R(

T),PG(T)
3200

4000
4002

4010
5000
5005
BUB

5010
5015
5020

RETURN

REM *"‘* LEGEND SUBROUTINE '*"‘**

PRINT ' t NH4 02 02tota1 C02 n02 PG 6 x
R

RETURN

REM **'*"‘ initial output subroutine *"'*'******'

PRINT 41,:PRINT Il,‘ TNX UNAX VN . KN A B C RAUT pelnum peld
DeO

PRINT :1: USING ' I..III““";YNX,UNAX,VH,KH,A,B,C,KAUT,PNUH,PDENS,DES
PRINT

PRINT .1,“ T(h) N(9/n3) G(g/n3) X(g/n3) n02 02(9/I3) OZTOT(9) C02(g)

R(l) Pqu/I3l'
5030 RETURN

166

Appendix 22
Mycelial pellet oxygen concentration gradient simulation program

This program was used to model oxygen proﬁle data obtained using the oxygen

microelectrode (Figure 5.5).
REM aeaaaaaaa 91:09:“ eaaeaeataeeaa

REM Solves the equations for diffusion and reaction of oxygen within a
REM spherical pellet. Michaelis-Menten kinetics for oxygen is assumed. A
REM shooting algorithm is used with Newton-Raphson convergence.
REM
DIM 8(200). DIM 88(4) :DIM 80(4) :DIM V(200) :DIM RI(200)
OPEN 'c: \pel rad. 818' FOR OUTPUT AS I1
10 DES-2. 9E- -0 :VM-. 76: KM-. S
12 SSURF-lo

oumaunw

13 CLS
14 PRINT:PRINT:PRINT:PRINT ' “‘**""‘*‘* N VS R **‘*“**‘*‘**":PRINT
16/ PRINT ' vnax-';VM;'g/n3ls, Kn-';KM;'g/n3, Des-';DE8:'n2/s ss-';SSURP;
U “3.

R-3/1000/2

26 orvrsrons-rur(100)
27 DR-R/DIVISIONS
30 50(1)-ssuar:50(3)-13—30
3s 50(2)-(30(1)+50(3))/2
40 ron x-i TO 3
SO 8(0l-SO(K)
60 S(1)-SO(K)
100 son I-1 TO DIVISIONS
105 V(I)-VM*S(I)/(KM+S(I))
120 S(I+1)-(I/(I+l))*((2'S(I)+S(I-l)*(1/I-l))+V(I)*DR‘Z/DES)
200 nsxr I
220 SS(K)-S(DIVISIONS)
230 NEXT x
240 CONVERGENCE-asst(SS(Z)-ssuar)/ssunr)
250 It couvaacsncs<. 001 runs 600
300 IF ssunr>55(2) THEN 340
310 80(1)-80(2)
coro 35

340 50(3)-SO(2)

350 GOTO 35

600 VSUM-0:RI(0)-0

605 FOR J-1 TO DIVISIONS

607 RI(J)-J*DR

610 IF S(J)<0 THEN V(J)-0

640 PRINT ' 02 (g In3 ) - ';

641 PRINT USING 'III. IIIII';S(J);
642 PRINT ' r m)-

643 PRINT USING “I. IIIII';RI(J)

645 PRINT I1, USING“ II. IIII““ :;S(J). RI(J)
660 VSUM-VSUM+V(J)‘(RI(J)‘3-RI(J-1 ) 3)

720 NEXT

800 EEFACTOR‘VSUM/(R‘3'VM*SSUR£I(KM+SSURP))

:05 PRINT:PRINT ' r (n) effectiveness factor as calc 30 ca
c-

810 PRINT USING ' II.IIIIIII';B*.13EEPACTOR:

815 PRINT USING ' III.IIIIIIIII':SS(2)380(2)
1000 END

HICHI N

1| @ﬁﬂﬂlﬂiﬂﬂlﬂfm@ﬂjﬂlﬁﬂf

 

 

3