Phanerochaete chrysosporium in the Decolorization of
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p. 2368-2375 Vol. 57, No. 8
0099-2240/91/082368-08$02.00/0
Copyright C) 1991, American Society for Microbiology
Role of Manganese Peroxidases and Lignin Peroxidases of
Phanerochaete chrysosporium in the Decolorization of
Kraft Bleach Plant Effluent
FREDERICK C. MICHEL, JR.,' S. BALACHANDRA DASS,2'3 ERIC A. GRULKE,1
AND C. ADINARAYANA REDDY2,3*
Department of Chemical Engineering,' Department of Microbiology and Public Health,2
and Center for Microbial Ecology,3 Michigan State University,
East Lansing, Michigan 48824-1101
Received 6 March 1991/Accepted 28 May 1991
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The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in
decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by
a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was
seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is
blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the
activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization.
When P. chrysosportium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were
produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea
that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no
Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE
decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE
decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when
the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs
play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.
Billions of gallons of toxic and intensely colored waste detected in about 4 days and reaches a peak in about 6 to 7
effluents are released into the environment annually by the days while the MNP activity is detectable in about 2 days
pulp and paper industry (26). The primary contributor to the and reaches a peak in about 4 to 5 days of culture incubation
color and toxicity of these streams is the pulp bleach plant (10, 15). Bench scale reactor systems for BPE decolorization
effluent (BPE), which contains largely high-molecular- (6, 20) have been developed, but LIP and MNP activities
weight, modified and chlorinated lignin and its degradation have not been measured in these decolorization studies.
products (4, 6, 11, 24). Conventional bacterial water treat- BPE decolorization has been observed by Campbell (6) as
ment processes are relatively ineffective at removing these early as 2 days after culture inoculation, implying that
pollutants (4). However, ligninolytic white-rot fungi such as enzymes other than LIPs may be involved in this process.
Phanerochaete chrysosporium and Trametes versicolor can However, a basic understanding of the role of extracellular
efficiently decolorize and dechlorinate BPE (1, 6, 9, 11, 18, peroxidases in BPE decolorization by P. chrysosporium is
19, 27, 30). lacking. In this study, we determined the roles of LIPs
P. chrysosporium, when cultured under nitrogen-limited versus MNPs in the decolorization of BPE by P. chryso-
conditions, is known to produce two families of extracellular sporium. Our results indicate that MNPs play a more impor-
glycosylated heme proteins, designated lignin peroxidases tant role relative to the LIPs in BPE decolorization by this
(LIPs) and manganese peroxidases (MNPs), along with an organism.
H202-generating system as the major components of its
lignin-degrading system (7, 10, 15). The LIPs have Mrs of
38,000 to 43,000 and catalyze one-electron oxidation of a MATERIALS AND METHODS
variety of aromatic substrates to generate aryl cation radi-
cals which undergo subsequent nonenzymatic reactions to Microorganisms. The fungi used were P. chrysosporium
yield a multiplicity of final products (15). MNPs, on the other wild type (WT) strains BKM-F1767 (ATCC 24725) and
hand, have an average Mr of 46,000 and oxidize Mn(II) to ME-446 (ATCC 34541), a lip mutant derived from strain
Mn(III), which in turn oxidizes phenolic substrates to phe- ME-446 (3), and a peroxidase-negative mutant also derived
noxyl radicals (10). It has been assumed that LIPs and MNPs from ME-446 (14). All strains were maintained on 2% malt
play a role in the decolorization of BPE, but the actual extract agar slants, pH 4.5 (13).
contribution of these enzymes to BPE decolorization has Culture conditions. P. chrysosporium was cultured in basal
never been documented (9, 20, 24). low-nitrogen medium (12), which contained the following
Previous studies have shown that, in agitated nitrogen- (per liter): 10 g of glucose, 2.0 g of KH2PO4, 1.45 g of
limited cultures of P. chrysosporium, the LIP activity is MgSO4 7H2O, 0.132 g of CaCl2 2H20, 1 mg of thiamine
hydrochloride, 0.5 g of Tween 80 (not added in stationary
starter cultures), 1.2 mM D-diammonium tartrate, 20 mM
acetate (pH 4.5), and 0.4 mM veratryl alcohol. The following
*
Corresponding author. trace elements were also added (per liter): 0.14 g of nitrilo-
2368VOL. 57, 1991 DECOLORIZATION OF BPE BY P. CHRYSOSPORIUM PEROXIDASES 2369
triacetate, 0.070 g of NaCl, 0.007 g of FeSO4 7H20, 0.013 g water, and dried to a constant weight, and the cell mass was
of CoCl2 6H20, 0.07 g of ZnSO4 7H20, 0.0011 g of
-
calculated by difference. Ammonium concentration was
CuS04 5H20, 0.0007 g of AlK(SO4)2 12H20, 0.0007 g of measured by using an ammonia electrode (Orion model
H3BO3, and 0.0007 g of Na2MoO4- 2H20. Unless men- 95-12).
tioned otherwise, all media contained 12 ppm of Mn(II) as SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel
MnSO4. In some experiments the Mn(II) concentration was electrophoresis (SDS-PAGE) was carried out as described
varied to give 0, 12, or 100 ppm. High-nitrogen medium was previously (13). Samples (2.0 ml) were removed from cul-
identical to the basal low-nitrogen medium described above tures and concentrated to 20 ,lp, using a Centricon ultrafil-
except that it contained 12 mM diammonium tartrate. tration unit (Amicon Div., W. R. Grace & Co., Danvers,
Media (85 ml) were dispensed into sterile, rubber-stop- Mass.) with a 10,000-molecular-weight cutoff. The concen-
pered, 250-ml Erlenmeyer flasks, inoculated with a 10% trated samples were applied to a 4% stacking-10% running
(vol/vol) mycelial inoculum [grown in low-nitrogen medium gel, and the proteins were stained with Coomassie brilliant
described above without added Mn(II)], and agitated at 173 blue.
rpm with daily oxygenation (21).
Color measurement. The measurement of decolorization of
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both fungus-treated and untreated BPE was based on the RESULTS
standard method of the National Council of the Paper Growth, substrate utilization, and decolorization of BPE.
Industry for Air and Stream Improvement (23). Samples Patterns of growth (cell dry weight), glucose and nitrogen
were diluted in phosphate buffer (pH 7.6) and centrifuged for (ammonium) depletion in the medium, and production of
15 min at 10,000 x g, and the A465 was measured as extracellular peroxidases in nitrogen-limited cultures of P.
described previously (23). The conversion factor used was chrysosporium BKM-F1767 are presented in Fig. 1A. The
4,015 standard platinum-cobalt color units (CU) per A465 of organism grew rapidly during the first 24 h after inoculation
1.0. Color on the mycelium was measured by homogenizing (doubling time, 5.2 h), and the culture nitrogen decreased
1 part of centrifuged mycelial pellets in 10 parts of buffer and steadily to undetectable levels within 24 h. The glucose
then filtering the mixture through GF/C-grade filter paper concentration in the medium decreased in a linear fashion
(Whatman Paper Ltd., Maidstone, England). between days 2 and 8 of incubation and was completely
Kraft BPE. Synthesis of BPE from Indulin AT (Westvaco) depleted by day 10 (Fig. 1A). Between days 1 and 9 there
was performed as described by Lundquist et al. (19). Briefly, was a slow but steady increase in dry weight which de-
the procedure used was as follows: Indulin AT (3.83 g) was creased substantially after glucose depletion on day 10,
dissolved in 150 ml of 0.14 M NaOH, 25.5 ml of 0.6 M H2SO4 probably due to cell autolysis or cell wall polysaccharide
was added with stirring, and the lignin material was precip- utilization or both (4, 15).
itated. Chlorine water (668 ml of 8.1% [vol/vol] Chlorox) was The extracellular peroxidases of P. chrysosporium are
added, and the solution was agitated for 1 h in a shaker bath widely implicated in lignin degradation and in detoxification
at 25°C. The solution was removed from the shaker bath and of a wide variety of toxic aromatic compounds (4, 15, 17, 29).
stripped with nitrogen gas via a gas dispersion tube, to Therefore, the time courses of MNP and LIP activities were
remove excess chlorine. After 2 h, 213 ml of 1.0 M NaOH determined in parallel with a study of the decolorization
was added, and the mixture was incubated for 2 h in a shaker activity. The depletion of nitrogen (Fig. 1A) coincided with
bath at 30°C. Next, the solution was acidified to pH 2.5 with the onset of secondary metabolism, as evidenced by the
0.6 M H2SO4 (approximately 128 ml) and deionized water production of extracellular peroxidases (Fig. 1B). Typically,
was added to bring the mixture to a volume of 1.5 liters. This MNP activity was first detected in the extracellular culture
bleached kraft lignin solution was concentrated under vac- fluid between days 2 and 3 of incubation, increased to a
uum, using a rotary evaporator, to approximately 200 ml. maximum on day 4, and declined to low levels by day 11
The pH was adjusted to 4.5 with 1.0 M NaOH, and the (Fig. 1B). LIP activity, on the other hand, first appeared
solution was stored at 4°C until used. The effluent generated between days 4 and 5, reached a maximum between days 6
with this protocol was considered typical of the effluent from and 7, and then rapidly declined. This decline in LIP activity
an industrial kraft bleach plant (19). After concentration, the during secondary metabolism was reported recently to be
effluent contained 56,000 to 69,000 CU. This concentrate due to degradation of LIP proteins by a protease induced
was added to cultures of P. chrysosporium to give a final under starvation conditions (8). The observed loss of perox-
concentration of 3,000 CU. Typically, industrial BPEs con- idase activity at days 8 to 11 also corresponded to the
tain 3,000 to 10,000 CU. In some experiments, BPE from a depletion of glucose in the culture fluid (Fig. 1A and B).
U.S. pulp mill was substituted for synthetic BPE (see The pattern of extracellular LIP and MNP production was
below). also studied by using SDS-PAGE electrophoresis. The MNP
Enzyme and analytical assays. Enzyme activities were protein band (Mr, 46,000) appeared first (between days 2 and
measured in control cultures, which were identical to exper- 3), whereas the LIP protein bands were not seen clearly until
imental cultures except that no BPE was added. MNP day 5 (Fig. 1C). On days 5 to 9 multiple MNP and LIP
activity was measured as described by Kuwahara et al. (16). protein bands were evident in the culture fluid (Fig. 1C). Fast
The sample volume was 10 to 40 RI, the pH was 4.5, and the protein liquid chromatography (FPLC) data (not shown), in
reaction time was 4 min. A unit of MNP activity was defined agreement with previous results (7), indicated that in ace-
as 1 ,umol of phenol red oxidized per liter per min, using an tate-buffered cultures the predominant MNP protein pro-
extinction coefficient of 4,460 M-1 cm-1 as determined in duced was H4 and the major LIP proteins produced were H2
our laboratory. LIP activity was measured by the procedure and H6.
of Tien and Kirk (28) at pH 2.5. Glucose was measured as Rapid decolorization of BPE was observed on days 3
reducing sugar by the dinitrosalicylic acid method, using through 7 (Fig. 1D). It was of interest that rapid decoloriza-
D-glucose as the standard (22). For determining mycelial dry tion occurred on days 3 and 4 when no LIP activity was
weight, cultures were vacuum filtered through tared GF/C- detectable. The rate of decolorization reached a maximum
grade filter paper, rinsed with 100 ml of distilled deionized between days 4 and 5 when MNP activity also reached its2370 MICHEL ET AL. APPL. ENVIRON. MICROBIOL.
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NVOL. 57, 1991 DECOLORIZATION OF BPE BY P. CHRYSOSPORIUM PEROXIDASES 2371
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FIG. 2. Decolorization of synthetic (O) and industrial (*) BPEs
by nitrogen-limited, agitated cultures of P. chrysosporium BKM-
F1767. BPE was added to a final concentration of 3,000 CU.
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 in the culture fluid. An uninoculated
control culture failed to show decolorization. Incubation of
BPE with autoclaved day 6 cultures also failed to show
decolorization activity. No significant decolorization of BPE
by filter-sterilized culture fluid, which contained both LIPs
and MNPs, was observed. Apparent first-order rate con-
stants for the data in Fig. 1D were calculated. The rates are
highest (K = 0.75 day on days 5 to 7 when both MNP and
LIP are present. However, relatively high rate constants of
0.35, 0.45, and 0.70 day-1, respectively, were observed with
3-, 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 (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.
We then compared the ability of P. chrysosporium to
decolorize synthetic BPE versus BPE collected from a U.S.
pulp mill. Our results showed that both the rates and extents
of decolorization of the synthetic BPE and pulp mill BPE
were very similar (Fig. 2).
Effect of varying MNP and LIP levels on BPE decoloriza-
tion. It has been well documented that high levels of nitrogen
in the medium repress LIP and MNP production in cultures
of P. chrysosporium (10, 15). More recently, manganese
levels in the medium were shown to have a dramatic effect
on the levels of production of MNPs and LIPs (2, 5, 25).
High levels (100 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 (2, 5, 25).
To determine the relative contribution of LIPs versus
MNPs to BPE decolorization, we added various concentra-
tions of Mn(II) to nitrogen-limited (2.4 mM) cultures of P.
chrysosporium to manipulate LIP and MNP levels and to
determine the effect of these variations on BPE decoloriza-
tion. Three levels of Mn(II) were added to cultures: 0, 12,
and 100 ppm, corresponding to low, basal, and high Mn(II)
levels (Fig. 3A and B). Basal-Mn(II) cultures exhibited high
MNP and LIP activity, reaching maxima on days 4 to 5 and
6 to 7, respectively. Low-Mn(II) cultures exhibited relatively
low MNP activity compared with high-Mn(II) cultures and
reached a maximum on day 4. In high-Mn(II) cultures LIP2372 MICHEL ET AL. APPL. ENVIRON. MICROBIOL.
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a v-VOL. 57, 1991 DECOLORIZATION OF BPE BY P. CHRYSOSPORIUM PEROXIDASES 2373
TABLE 1. Apparent first-order decolorization rate of kraft BPE
by P. chrysosporium as affected by nitrogen and Mn(II)
levels in the mediuma
Nitrogen Mn(II) concn Predominant Apparent rate
level (mM) (ppm) peroxidase(s) constant (K)
level(mM) (ppm) ~~~~~(dayl1)b
24 12 None 0.01
2.4 0 LIP 0.10
2.4 12 LIP, MNP 0.70
2.4 100 MNP 0.65
a P. chrysosporium was grown in nitrogen-limited medium (7) with various
levels of nitrogen and Mn(II). Other conditions were as described in the
legend to Fig. 2.
b K denotes first-order rate constant. Differences in rate constants of 0.07
day-1 are considered significant.
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activity was not detected, but MNP activity was greater than
that in basal-Mn(II) cultures. In high-nitrogen (24 mM)
cultures containing 12 ppm of Mn(II), neither MNP nor LIP
activity was detected (Fig. 3A and B) and no LIP and MNP
bands were seen on the SDS-PAGE gel (Fig. 3C). SDS-
PAGE analysis of the culture fluid from high-manganese [100
ppm of Mn(II)] and basal-manganese [12 ppm of Mn(II)]
cultures showed a single intense protein band (Fig. 3C)
which appears to be an MNP protein on the basis of 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 corre-
sponding to LIP and MNP proteins were seen.
When BPE was added to 4-day-old basal- and high-Mn(II)
cultures, decolorization proceeded at a high initial rate (Fig.
3D). A mass balance analysis 4 days after the addition of
BPE showed approximately 8% decolorization in high-nitro-
gen (nitrogen-sufficient) cultures containing 12 ppm of
Mn(II), 27% decolorization in cultures without added
Mn(II), 75% decolorization in high-Mn(II) cultures, and 85%
decolorization in basal-Mn(II) cultures. The apparent first-
order rate constants (Table 1) for decolorization of BPE in
cultures containing different levels of nitrogen and Mn(II)
showed that, when LIP and MNP were not present, little
decolorization was observed. The absence of LIPs as in the
high-manganese (100 ppm) cultures had a minimal effect on
the decolorization rate, whereas decreasing the activity of
MNPs as in the low-manganese culture had a pronounced
effect on the decolorization rate even when the LIPs were
present.
BPE decolorization by mutant cultures. To investigate the
importance of extracellular peroxidases in BPE decoloriza-
tion further, two mutant strains (per and lip) and two WT
strains (ME-446 and BKM-F1767) of P. chrysosporium were
used. The per mutant lacks the ability to produce extracel-
lular peroxidases on the basis of several lines of evidence.
First, no MNP or LIP activity is detectable in culture fluid of
a per mutant grown in low-nitrogen basal medium, whereas
under identical conditions both MNPs and LIPs are pro-
duced in large amounts by the WT. When concentrated
culture fluid from the per mutant cultures was fractionated
by SDS-PAGE, no LIP or MNP bands were evident. In the
same gel, however, concentrated culture fluid from 4-day-
old cultures of the lip mutant and the WT showed a single
band (corresponding to MNP). Moreover, FPLC analysis of
the concentrated extracellular fluid from the per mutant
cultures showed no LIP or MNP peaks. In addition, we have
found no physiological difference between the per mutant2374 MICHEL ET AL. APPL. ENVIRON. MICROBIOL.
studies. Furthermore, although Paice and Jurasek (24) have
shown that horseradish peroxidase can catalyze BPE decol-
orization, the role of LIPs and MNPs in the decolorization
process has never been clearly established. Our results
60
indicate that the extracellular peroxidases of P. chrysos-
Residual porium play a key role in BPE decolorization by this
Color (%) organism. Little or no BPE decolorization was seen when P.
40
chrysosporium was grown in high-nitrogen medium, which
blocks production of both LIPs and MNPs (Fig. 3A and B),
20 suggesting that these peroxidases are required for decolori-
zation activity. This is independently supported by the
experiment with the per mutant, which lacks the ability to
0 1 2 3 4 5 produce LIPs and MNPs but produces H202 comparable to
Days the WT (4). The fact that the per mutant shows negligible
FIG. 4. Decolorization of BPE by P. chrysosporium WT strains BPE decolorization (Fig. 4) is consistent with the idea that
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ME-446 (*) and BKM-F1767 (-) and two mutants, per (A) and lip extracellular peroxidases play a key role in BPE decoloriza-
(C1), of ME-446. BPE (3,000 CU) was added to each of the cultures tion.
on the peak day of MNP activity, and the extent of BPE decolori- A determination of the relative contribution of LIPs ver-
zation was monitored each day for the next 5 days. Values shown sus MNPs to BPE decolorization has been an important
are averages of duplicate cultures in two separate experiments.
focus in this investigation. Several lines of evidence indi-
cated that the MNPs play a predominant role in BPE
decolorization by P. chrysosporium. First, a high degree of
and the WT (aside from a lack of MNP and LIP production). BPE decolorization was observed in the high-Mn(II) WT
The per mutant consumed glucose at the same rate as the cultures and in the lip mutant (3) cultures, in which high
WT, produced the same amount of cell biomass, and formed levels of MNP activity, but no LIP activity, were seen.
mycelial pellets similar in size to those of the WT. The lip Second, very low levels of BPE decolo-rization were ob-
mutant has been characterized previously (3).- It produces served in WT cultures grown without Mn(II), in which high
MNPs and H202 but lacks the ability to produce the LIPs. levels of LIP and very low levels of MNP activity were seen
The WT strains ME-446 and BKM-F1767 produce both LIPs (Fig. 3D). Third, BPE decolorization activity temporally
and MNPs. paralleled the appearance of MNP rather than LIP activity in
BPE was added to the per mutant, lip mutant, and WT WT cultures containing 12 ppm of Mn(II). For example,
cultures on the day of peak MNP activity (day 4 of incuba- relatively high rates of BPE degradation are observed on
tion), and the extent of decolorization was monitored daily days 3 and 4 of incubation in basal nitrogen-limited medium,
for the next 5 days (Fig. 4). The presence (or absence) of at which time little or no LIP activity is seen. These results
extracellular peroxidases was determined by the LIP and lead us to conclude that MNPs play a more important role
MNP assays as well as by SDS-PAGE of the concentrated than LIPs in decolorizing BPE.
extracellular fluid on the day of BPE addition. The MNP The results of this study indicating a minor role for LIPs
activities in the ME-446, BKM-F1767, and lip mutant cul- and a major role for MNPs are an interesting contrast to
tures were 3,550, 2,200, and 2,050 U/liter, respectively, at previous results which showed that the LIPs play a major
the time of BPE addition. The per mutant, which lacks the role in the degradation of synthetic 14C-lignin to 14CO2,
ability to produce both MNPs and LIPs, exhibited negligible whereas the MNPs play a minor role in this process (3, 25).
decolorization of BPE (Fig. 4), while the WT strains exhib- Boominathan et al. (3) showed that the lip mutant of P.
ited rapid decolorization of BIPE. The lip mutant (which chrysosporium, which lacks the ability to produce LIPs but
produces MNPs only) showed about 80% of the decoloriza- produces a full complement of MNPs, exhibits only about
tion activity exhibited by ME-446. These results, consistent 16% of the ligninolytic activity of the WT, indicating that
with the other data presented above, indicate that the MNPs play a minor role in lignin degradation by P. chryso-
extracellular peroxidases are important for BPE decoloriza- sporium. These conclusions were further supported by the
tion and that the MNPs play a predominant role in BPE results of Perez and Jeffries (25). They showed that high
decolorization by P. chrysosporium. levels of MNP activity but no LIP activity were seen when
P. chrysosporium was grown in nitrogen-limited medium in
DISCUSSION the presence of 39.8 ppm of Mn(II). These cultures exhibited
only about 10 to 11% of the lignin degradation as compared
A number of previous studies have shown that P. chrys- with that exhibited by the same organisms in low-N medium
osporium as well as some other white-rot fungi rapidly containing 0.35 ppm of Mn(II), which supports the produc-
decolorize BPE. Sundman et al. (27) showed that BPE from tion of high levels of LIP but negligible levels of MNP
the first alkali extraction stage after chlorination (El effluent) activity. These apparent differences in contributions of LIPs
was decomposed to low-molecular-weight colorless prod- versus MNPs to lignin degradation versus BPE degradation
ucts. P. chrysosporium was shown to degrade significant are quite interesting and unexpected. The observed differ-
amounts of '4C-labeled bleached kraft lignin to 1'CO2 (4, 19). ences in activities may reflect the differences in the two
Campbell (6) and Eaton et al. (9) developed the MyCoR substrates (i.e., lignin and BPE). BPE is predominantly
process, which utilizes P. chrysosporium immobilized on soluble, chlorinated, and partially degraded lignin as op-
partially submerged rotating disks to decolorize industrial posed to dehydrogenation polymerizate, the structure of
BPE. Yin et al. (30) examined the kinetics of BPE decolor- which more closely resembles the native lignin polymer.
ization in the MyCoR process and indicated that BPE In conclusion, the results of this study indicate that
decolorization consisted of three distinct stages. The LIP or extracellular peroxidases are important for BPE decoloriza-
MNP activities were not reported in any of these previous tion by P. chrysosporium and that MNPs play a predominantVOL. 57, 1991 DECOLORIZATION OF BPE BY P. CHRYSOSPORIUM PEROXIDASES 2375
role and LIPs play a relatively minor role in this decoloriza- 13. Kelley, R. L., and C. A. Reddy. 1986. Identification of glucose
tion process. oxidase activity as the primary source of hydrogen peroxide
production in ligninolytic cultures of Phanerochaete chrysospo-
rium. Arch. Microbiol. 144:248-253.
ACKNOWLEDGMENTS 14. Kim, K. J., and C. A. Reddy. Unpublished data.
We are grateful for the support of this work by the Research 15. Kirk, T. K., and R. L. Farrell. 1987. Enzymatic "combustion":
Excellence Fund from the State of Michigan; the Agricultural the microbial degradation of lignin. Annu. Rev. Microbiol.
Experiment Station, Michigan State University; the Center for 41:465-505.
Microbial Ecology-an NSF Science and Technology Center; and 16. Kuwahara, M., J. K. Glenn, M. A. Morgan, and M. H. Gold.
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