May 12, 1992 - C. DAVID BOYLE,lt* BRADLEY R. KROPP,l* AEND IAN D. REID2§. Centre de Recherche en Biologie Forestiere, Faculte de Foresterie et de ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p. 3217-3224
Vol. 58, No. 10
0099-2240/92/103217-08$02.00/0
Copyright X 1992, American Society for Microbiology
Solubilization and Mineralization of Lignin by White Rot Fungi C. DAVID BOYLE,lt* BRADLEY R. KROPP,l* AEND IAN D. REID2§ Centre de Recherche en Biologie Forestiere, Faculte de Foresterie et de Geomatique, Universite Laval, Sainte-Foy, Quebec, Canada GlK 7P4,1 and Biotechnology Research Council, National Research Council of Canada, Montreal, Quebec, Canada H4P 2R22 Received 12 May 1992/Accepted 22 July 1992
The white rot fungi Lentinula edodes, Phanerochaete chrysosporium, Pleurotus sajor-caju, Flammulina velutipes, and SchizophyUlum commune were grown in liquid media containing '4C-lignin-labelied wood, and the formation of water-soluble '4C-labelled products and '4Co2, the growth of the fungi, and the activities of extracellular lignin peroxidase, manganese peroxidase, and laccase were measured. Conditions that affect the rate of lignin degradation were imposed, and both long-term (0- to 16-day) and short-term (0- to 72-h) effects on the production of the two types of product and on the activities of the enzymes were monitored. The production of 14C02-labelled products from the aqueous ones was also investigated. The short-term studies showed that the different conditions had different effects on the production of the two products and on the activities of the enzymes. Nitrogen sources inhibited the production of both products by all species when differences in growth could be discounted. Medium pH and manganese affected lignin degradation by the different species differently. With P. chrysosporium, the results were consistent, with lignin peroxidase playing a role in lignin solubilization and manganese peroxidase being important in subsequent CO2 production. When white rot fungi degrade lignin, both water-soluble products and CO2 are formed, the former probably being precursors for the latter (11, 20). There is also evidence that formation of the two is subject to differing metabolic regulation (1, 10, 20, 21). If the CO2 is produced from the soluble compounds, conditions that specifically decrease its production should lead to accumulation of the soluble compounds. These may or may not inhibit further solubilization by, e.g., feedback inhibition of the responsible enzymes. In contrast, conditions that inhibit processes leading to solubilization would also inhibit CO2 production, although the inhibition might be delayed. The process by which fungi degrade lignin is oxidative, probably involving enzymes such as lignin peroxidases (LiP), manganese peroxidases (MnP), and laccases (7-9, 14). Some support for the involvement of these enzymes comes from comparing the effects of different conditions on their activities with the effects on the degradative process itself. For example, with Phanerochaete chrysosporium, nutrient nitrogen represses the formation of LiP and MnP and it also inhibits lignin degradation (14). Manganese (Mn2+) increases MnP activity but decreases that of LiP. It also decreases lignin degradation, consistent with LiP playing a rate-limiting role in the process (2, 18). It is not known, however, if the LiP, the MnP, or the laccase functions in the initial attack on the lignin or during later stages of degradation. Most studies of lignin degradation have been conducted with P. chrysosporium, but the process might differ among different species. For example, nitrogen does not consistently inhibit degradation by Pleurotus sp. or by Lentinula
edodes. Similarly, the optimal pH for degradation may differ among different species (5, 10, 21). These differences might indicate that the fungi have different mechanisms for degrading the lignin or they could result from the effects of the conditions on fungal growth. Altered growth could, in turn, alter the medium (e.g., carbohydrate concentration or pH), which might affect lignin degradation. The effects of altered medium composition can be minimized by frequently replacing it. Fungal growth can be measured, but the measurements do not quantitatively reflect all aspects of growth, so valid corrections for growth differences cannot be made. The confounding effects of growth can, however, be minimized by conducting experiments of short duration. This work was carried out to determine whether various conditions that affect the overall degradation of lignin differ in their effects on the production of soluble products and CO2. Since some of the conditions chosen differentially affect extracellular LiP, MnP, and laccase activities, it was hoped that insight might be gained into the functions of these enzymes in the formation of the products. In longer-term (0to 16-day) studies, the effects of the conditions on growth were monitored by measuring fluorescein-diacetate-hydrolyzing activity (FDA), which is a sensitive indicator of growth-related metabolic activity in fungi (4). The media were frequently replaced. Shorter-term (0- to 72-h) kinetic studies were also conducted to show possible precursorproduct relationships between the two products and to show whether there were physiological differences, not attributable to growth or altered medium composition, between the effects of nutrient nitrogen and pH on lignin degradation by different species. The effect of some of the conditions on the direct production of CO2 from the water-soluble compounds was also studied.
* Corresponding author. t Present address: The Research and Productivity Council, 921 College Hill Rd., Fredericton, New Brunswick, Canada E3B 6Z9. t Present address: Biology UMC 5305, Utah State University, Logan, UT 84322. § Present address: Pulp and Paper Research Institute of Canada, Pointe-Claire, Quebec, Canada H9R 3J9.
MATERIALS AND METHODS Cultures. L. edodes (Berk.) Pegler was obtained from the American Type Culture Collection (ATCC 48971). P. chry3217
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BOYLE ET AL.
sosponium Burds. (strain BKM-F-1767), Pleurotus sajorcaju Fr.:, and Flammulina velutipes (Curt.:Fr.) Singer were the same cultures as those used previously (4). Schizophyllum commune Fr.: Fr. is deposited as QFB 536 C at the Laurentian Forestry Centre, Ste. Foy, Quebec, Canada. Stock cultures were maintained at 4°C on slants of nutrient-rich medium made as described previously (4). At 4-month intervals, working cultures were started on plates containing the same medium. Inocula for the experiments were made by cutting 1-cm-diameter discs from the edge of 2-week-old colonies and preincubating these, hyphae side
up, for 48 h on water agar. Culture media. The media were modifications of the low-N medium described previously (4), except that the 2,2-dimethylsuccinic acid buffer concentration was 50 mM and Casamino Acids and manganese were omitted. Media 1 and 2 (low-N media) contained 0.1 g of casein enzymatic hydrolysate (Sigma type 2) per liter. Media 3 and 4 (high-N media) contained 0.5 g of glutamic acid and 0.5 g of NH4tartrate in addition to the casein hydrolysate. MnSO4. 7H20 was added to the low-Mn (media 1 and 3) and high-Mn (media 2 and 4) media to give 0.4 and 40 ppm of Mn, respectively. The pH was adjusted with NaOH to the values indicated in the tables, and the media were filter sterilized before use. Production of '4C-lignin-labelled wood. L-[U-_4C]phenylalanine (50 ,uCi, 554.8 mCi/mmol) was deaminated by aseptically incubating it at 25°C for 5 days with 0.6 U of dialyzed, filter-sterilized phenylalanine ammonia lyase (Sigma) (19). After acidification with HCl, the solution was extracted three times with CHCl3, and the extracts were combined and evaporated to dryness. The radioactive product cochromatographed with trans-cinnamic acid on a silica-gel thin-layerchromatography plate developed with benzene-acetone-acetic acid (100:5:2). No other labelled products were detected. The 14C-cinnamic acid (43 ,uCi) was dissolved in 1 ml of 10 mM phosphate buffer (pH 7.0) and fed to a cut branch of birch (Betula papynfera) (about 5 mm in diameter and 1 m in length) (6). After 3 days of incubation with daytime illumination and nighttime darkness, the branch was dried at room temperature and debarked. The wood was ground in a Wiley mill (no. 20 mesh), extracted in a Soxhlet extractor for 12 h with water and then for 12 h with ethanol-benzene (1:1), and then dried. The specific radioactivity (measured by combustion) was 15,790 dpm/mg. This preparation was mixed 1:9 (wt/wt) with the birch-maple wood chip mixture described previously (4) after this preparation had been milled to the same grade. This mixture was used as the 14C-lignin-labelled wood. Unlabelled wood contained only the milled birchmaple wood chip mixture. Culture conditions. Samples (120 mg) of the labelled (189,480 dpm) or unlabelled wood were placed into glass 55-ml screw-top test tubes (25 by 150 mm) with 1 ml of distilled water. The tubes were autoclaved for 15 min at 121°C, and after 24 h, 4 ml of the specified medium was added. The tubes were inoculated with fungal discs and closed with serum stoppers (Suba Seal no. 37; Aldrich). The tubes were flushed with at least 30 volumes of sterile 02 through hypodermic needles. The tubes were incubated on their sides at 30°C without shaking. The wood and nutrient solutions formed a shallow layer extending from one end of the tube to the other. The fungi grew on the surface of this layer, with hyphae contacting all of the wood particles 3 to 6 days after inoculation, depending on the species and the medium. Every fourth day, the media and gases were aseptically withdrawn and replaced as described below.
APPL. ENvIRON. MICROBIOL.
Measurement of lignin degradation, growth (FDA), and production of extracellular enzymes. At 4, 8, 12, and 16 days after inoculation, the media were withdrawn from the cultures by use of a sterile syringe, care being taken to minimize agitation. Samples of these media and 5-ml gas samples, withdrawn by use of a syringe equipped with a gas sampling valve, were analyzed for radioactivity. Four milliliters of fresh, filter-sterilized medium was introduced by syringe, the tubes were reflushed with 02, and the incubation was continued. On day 16, enzyme activities and the FDA of the cultures were also measured. In some experiments, the conditions were changed at day 12, and subsequent short-term (0- to 72-h) effects on 4C production and enzyme activities were monitored. The media of some cultures were replaced with the same media (controls), while others received media with different pH values or Mn or N concentrations. Some cultures received media containing 0.1% KCN. The gases were replaced with 02, N2, or 02 containing 10% CO as indicated in the tables. At intervals, 1-ml medium samples and 5-ml gas samples for 14C analysis were aseptically withdrawn by syringe and replaced with equal volumes of fresh medium and gas. The rates of '4C appearance in the two phases were calculated by subtracting values obtained at the preceding sampling time. After the last sampling, the pH values and the enzyme activities were measured. The effect of pH on lignin degradation was assessed in a similar experiment. On days 0, 4, and 8, the cultures received medium 1 (P. chrysosporium) or 2 (L. edodes and P. sajor-caju) at pH 4.5. On day 12, the media were replaced with media adjusted to the pHs indicated in the tables. After 72 h, aqueous- and gas-phase samples were withdrawn and analyzed for radioactivity. Production of '4Co2 from the 14C-labelled water-soluble compounds. P. chrysosponum and S. commune were grown on unlabelled wood with pH 3.5 medium 1 and pH 4.5 medium 2, respectively, as described above. On day 12, the medium in the P. chrysosporium cultures was replaced with medium 1 or 3 and the tubes were flushed with 02 or N2. The medium in the S. commune cultures was replaced with fresh pH 4.5 medium 2, and the tubes were flushed with 02. After 48 h, a 3-ml sample of the filter-sterilized aqueous phase, taken from P. chrysosponum cultures after growth for 12 days on 14C-labelled wood with medium 1, was injected. This volume contained a total of 5,280 dpm of 14C. After 48 h, gas samples were withdrawn from the cultures and analyzed for radioactivity. In another experiment, P. chrysosporium was grown on either labelled or unlabelled wood, with changes of medium 1 (pH 3.5) and flushing with 02 on every fourth day until day 12. The media were then withdrawn and filter sterilized (pore size, 0.2 ,um). A portion of the labelled liquid was boiled for 2 min (to denature enzymes). Three milliliters of the labelled or unlabelled solution, as specified, was then reinjected into the cultures. Three milliliters of the labelled or boiledlabelled solution was also injected into tubes containing sterile unlabelled wood. At intervals, 5-ml gas samples were withdrawn and analyzed for radioactivity. 14C measurement. The 5-ml gas samples were injected into serum-stoppered scintillation vials containing 1 ml of hyamine hydroxide (1 M in methanol; Amersham). The vials were gently shaken, and after 1 h, the stoppers were removed, 12 ml of Cytoscint counting fluid (ICN Biomedicals, Irvine, Calif.) was added, and radioactivity was measured by using an LKB Wallace 1217 Rackbeta liquid scintillation counter. Culture liquid samples were acidified with 10 M
VOL.
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LIGNIN DEGRADATION BY WHITE ROT FUNGI
58,1992
TABLE 1. Degradation of '4C-labelled lignin, growth (FDA), and enzyme production by fungi in various mediaa Enzyme activities'
Lignin degradation
Species P. chrysosporium
P. sajor-caju
L. edodes
F. velutipes
S. commune
Medium and conditions
1, pH 3.5, low Mn, low N 2, pH 3.5, high Mn, low N 3, pH 3.5, low Mn, high N 1, pH 5.5, low Mn, low N 2, pH 5.5, high Mn, low N 3, pH 5.5, low Mn, high N 1, pH 3.5, low Mn, low N 2, pH 4.5, high Mn, low N 4, pH 4.5, high Mn, low N
14C recov-
Ratec at 4-8
Ratec at 8-12
Ratec at 12-16
ered as:
days
days
days
Soluble
CO2 Soluble
CO2 Soluble
CO2 Soluble
CO2 Soluble
CO2 Soluble
CO2 Soluble
CO2 Soluble
CO2 Soluble
CO2
1.12 1.49 0.67 1.24 0.96 1.12
(0.32) (0.43) (0.17) (0.27) (0.46) (0.14)
1.62 (0.27) b
FDA
MnP
Laccase
0.08 (0.03) a
16 (10) a
ND
81 (7) a
ND
35 (9) B
ND
2.01 1.43 0.97 a AB 1.14 0.71 a AB 0.57
(0.32) (0.24) (0.17) (0.27) (0.08) (0.03)
c B a AB a AB
1.09 1.25 0.73 0.92
(0.17) (0.18) (0.11) (0.14)
0.11 (0.01) a a AB 1.14 (0.12) b a AB
0.15 0.13 0.16 0.17 0.22 0.21 0.00 0.02
(0.10) (0.09) (0.03) (0.06) (0.10) (0.16) (0.00) (0.00)
ab AB ab AB ab AB a A
0.28 0.13 0.27 0.24 0.17 0.22 0.01 0.01
(0.30) (0.13) (0.18) (0.06) (0.08) (0.07) (0.00) (0.00)
ab AB ab AB ab B ab A
0.31 0.21 0.06 0.10
0.13 (0.03) a (0.18) b (0.10) 0.95 (0.01) b (0.02) a (0.10) AB
a
0.12 (0.10) ab 0.12 (0.16) AB 0.14 (0.03) ab 0.17 (0.02) AB 0.08 (0.03) ab 0.12(0.03) AB 0.02 (0.08) ab 0.03 (0.01) A
__d
0.04 (0.04) a 0.00 (0.00) A 0.23 (0.10) ab 0.08 (0.06) AB
0.23 (0.09) ab 12 (4) a
2.8 (3.8) a
0.13 (0.07) a
41 (8) b
78.2 (51) b
1.12 (0.11) c
22 (14) a 10.4 (6.8) a
0.41 (0.20) b
17 (13) a
6.2 (7.3) a
22 (6) a
0a
15 (3) a
3.3 (2.6) b
1, pH 4.5, low Mn, low N
0.01 (0.01) a 0.02 (0.02) A
0.02 (0.03) a 0.02 (0.03) A
0.02 (0.01) a 0.01 (0.01) A
0.29 (0.16)
CO2
1, pH 4.5, low Mn, low N
Soluble
0.06 (0.01) a 0.01 (0.00) A
0.04 (0.01) a 0.00 (0.00) A
0.03 90.01) a
0.27 (0.04)
CO2
Soluble
a Results within each fungal treatment that differ significantly at P > 0.05 are followed by different letters. Uppercase letters are used for CO2 production, and lowercase ones are used for soluble product formation and enzyme activities. I FDA measured as nanomoles of substrate converted minute-' milliliter-'. The measurements were made on day 16. ND, not detected. c Values are the percentages recovered of the total 14C-lignin supplied per 24 h as either soluble products or as CO2. They are means of triplicate cultures, with standard deviations shown in parentheses. d _ not measured. -,
lactic acid (to eliminate carbonates) and centrifuged (12,000 x g, 10 min), and 0.9-ml samples were counted in 12 ml of Cytoscint. Quench corrections were made by using the external-standard channels ratio method. Background corrections were made by subtracting values for samples taken from uninoculated tubes containing labelled wood treated in the same way as that of the inoculated treatments. Measurement of enzyme activities. Culture fluid samples were centrifuged (12,000 x g, 10 min), and LiP, MnP, and laccase activities in the supernatant were measured. LiP and MnP activities were measured as described previously (references 22 and 17, respectively). Laccase activity was measured by using a modification of the methods of Szklarz et al. (22) by measuring the increase in optical density at 525 nm after the addition of syringaldazine to samples that had been preincubated with 100 U of catalase (from Aspergillus niger; Sigma) at room temperature for 15 min. Measurement of FDA. Ten milliliters of sterile 50 mM Tris buffer (pH 7.5) was added to the cultures, and the cultures were blended for 10 s at a setting of 5 with a Polytron homogenizer (type PT 10/35; Brinkman, Rexdale, Ontario, Canada) equipped with a 1.1-cm-diameter head. The rate at which 1-ml samples of this slurry hydrolyzed fluorescein diacetate was measured as previously described (4). Statistical analysis. Significant differences between results were identified by either one-way or two-way analysis of variance using the Student-Newman-Keuls' test.
RESULTS Interspecies variation in the effects of medium composition on enzyme production and lignin degradation. In the longerterm assays, lignin degradation by P. chrysosporium was always much faster than that by the other species, and it was
generally faster in low-Mn, low-N medium than in high-Mn or high-N medium (Table 1). The variability was high, but the trends were consistent between sampling periods. The total of soluble 14C and "4CO2 released by day 16 was significantly higher in medium 1 than in medium 2 or 3 (P = 0.014). The percentage of the 14C activity recovered as 14C02 was higher on high-Mn medium 2 than on medium 1 or 3 in the first two sampling periods (P < 0.001). Laccase activity was not detected. MnP activity was highest on the high-Mn medium 2. Cultures on medium 2 became brown or black at the later sampling times, probably reflecting MnO2 formation. LiP activity was assayed but not detected in any of the cultures. The FDA values were similar on media 1 and 2 but much higher on high-N medium 3. Visual inspection also showed similar amounts of growth in media 1 and 2 but a much greater amount of growth in medium 3. In contrast, lignin degradation by P. sajor-caju was not markedly affected by either the Mn or N content of the medium, although rates of degradation were generally higher
and less variable in medium 2. In the pH 3.5 medium, degradation was usually slower. The fraction of the activity recovered as CO2 did not show any consistent relationship to
3220
BOYLE ET AL.
APPL. ENvIRON. MICROBIOL. TABLE 2. Effect of pH on lignin degradation by various species Lignin degradationa at pH:
Species
P. chrysosporium L. edodes P. sajor-caju
3
3.5
4.5
5.5
6.5
1.23 (0.44) 0.55 (0.13) 0.32 (0.09)
0.63 (0.22) 0.53 (0.15) 0.31 (0.09)
0.51 (0.11) 0.33 (0.06) 0.31 (0.11)
0.44 (0.11) 0.34 (0.14) 0.49 (0.23)
0.37 (0.02) 0.20 (0.06) 0.27 (0.08)
a Values are the percentages recovered of the total 14C-lignin supplied per 24 h as soluble products and standard errors shown in parentheses. Results within a fungal treatment do not differ at P 2 0.05.
the medium. Laccase and MnP activities were much higher the high-Mn medium 2. Cultures on this medium also became dark brown or black at the later sampling times. Medium 2 was used as the reference medium in subsequent experiments with this species. FDA values were much higher on the high-N medium 3, as was the apparent amount of growth, while FDA values and apparent growth were similar on the other two media. Degradation by L. edodes varied greatly between sampling times. Between days 4 and 8, degradation was generally highest in the high-N medium, but it was lower in the high-N medium between days 12 and 16. MnP activity was similar on the two media, while laccase was the only activity detected on the high-N medium. FDA activity was much higher on the high-N medium. L. edodes did not grow well in the tubes, possibly because of the high oxygen concentration, but the amount of mycelium was greater in the highthan in the low-N medium at the end of the incubation period. Degradation by F. velutipes and S. commune was very low and variable. MnP, LiP, and laccase were not measured. FDA values were comparable to those of the other species when they were also grown in low-N media, and visual inspection showed similar amounts of mycelium. The pH of the medium had different short-term effects on on
CO2. They are means of triplicate cultures, with the
lignin degradation by the different species (Table 2). Although variability was high, there was a clear trend for degradation by P. chrysosporium and L. edodes to be highest at the lower pH values while that by P. sajor-caju was maximal at pH 5.5. The inhibitory effects of low pH on lignin degradation by P. sajor-caju was substantiated in subsequent experiments (see Table 4). The pH had no visible effect on the amount of mycelium in the tubes at the end of the experiment. Short-term effects of incubation conditions on lignin solubilization and mineralization. High-Mn conditions had little effect on the total lignin degradation rate by P. chrysosponum at the 48-h sampling time, but they caused a decrease in the accumulation of water-soluble 14C and an increase in the production of 14C02 (Table 3). At the later (48- to 72-h) sampling time, high-Mn conditions caused a decrease in total degradation, but the fraction of product recovered as 14C02 remained high. In contrast, the change to high-N conditions initially decreased the production of both products to about the same extent. During the 48- to 72-h period, label disappeared from the aqueous phase (negative rate of production), while 14C02 production continued slowly. Exposure to high N, anoxia, or KCN decreased both the solubilization and the mineralization of lignin during the first 48 h. After a longer incubation period, the N2 atmo-
TABLE 3. Production of 14C02 and 14C-labelled soluble compounds and production of enzymes by P. chrysosporium after transfer to various conditionsa Medium and conditions
Enzyme activities'
Lignin degradation 14
Ratec at 0-48 h
Ratec at 48-72 h
CO2
0.34 (0.12) b 0.69 (0.07) B
Soluble
CO2 Soluble
C recovered as:
MnP
LiP
pH
0.37 (0.14) B 0.43 (0.06) BC
25 (13) a
7.0 (13) b
3.9
0.24 (0.11) ab 0.83 (0.25) B
0.04 (0.07) a 0.15 (0.08) AB
101 (12) b
0 (0) a
3.8
0.11 (0.03) ab 0.24 (0.07) a
-0.05 (0.03) a 0.15 (0.28) AB
13 (11) a
0 (0) a
3.8
CO2 Soluble
0.08 (0.02) a 0.28 (0.03) A
-0.08 (0.08) a 0.10 (0.01) AB
19 (15) a
0 (0) a
4.0
CO2 1, +0.1% KCN
Soluble CO2
0.11 (0.07) ab 0.25 (0.03) A
0.02 (0.05) a -0.12 (0.04) A
7 (3) a
0 (0) a
4.2
1, +10% CO
Soluble CO2
0.53 (0.16) c 0.76 (0.25) B
0.64 (0.12) c 0.62 (0.32) C
22 (2) a
0 (0) a
4.0
1 (control), low Mn, low N
2, high Mn, high N
3, low Mn, high N 1, N2 atmosphere
Soluble
a Results within each column that differ significantly at P . 0.05 are followed by different letters. Uppercase letters are used for CO2, and lowercase ones are used for soluble products and for enzyme activities. b Values are means for triplicate cultures, with the standard deviations shown in parentheses, and are expressed as nanomoles of substrate converted minute-' milliliter of culture supernatant-1. Measurements were made 72 to 74 h after transfer to the various conditions. c Values are the percentages recovered of the total 14C-lignin supplied per 24 h as either soluble products or as CO2. They are means of triplicate cultures with the standard deviations shown in parentheses.
VOL. 58, 1992
LIGNIN DEGRADATION BY WHITE ROT FUNGI
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TABLE 4. Production of "4CO2 and "4C-labelled soluble compounds by P. sajor-caju after transfer to various conditions
Lignin degradation Medium and conditions
2 (Control), high Mn, low N, initial pH of 5.5
4, high Mn, high N
2, N2 atmosphere 2, pH of 3.5
pH
CO2
0.17 (0.05) a 0.12 (0.03) A
Rate0 at 48-72 h 0.27 (0.04) c 0.14 (0.03) B
Soluble
0.17 (0.09) a 0.13 (0.02) A
0.15 (0.04) b 0.02 (0.10) A
5.4
CO2 Soluble
0.17 (0.10) a 0.12 (0.02) A
0.06 (0.07) a 0.03 (0.03) A
5.4
CO2 Soluble
0.04 (0.05) a 0.11 (0.02) A
0.01 (0.01) a 0.07 (0.05) A
3.9
14C recovered as: Soluble
Rate' at 0-48 h
CO2
5.4
0 Values are the percentages recovered of the total 14C-lignin supplied per 24 h as either soluble products or as CO2. They are means of triplicate cultures, with the standard deviations shown in parentheses. Results in each column that differ at P > 0.05 are followed by different letters. Uppercase letters are used for CO2 production, and lowercase ones are used for soluble product formation.
sphere, like the high-N medium, had its most inhibitory effects on the production of soluble compounds (the rates being negative), while exposure to KCN resulted in the loss of 1 CO2. The addition of 10% CO increased both lignin solubilization and, to a lesser extent, mineralization. LiP activity was detected only in medium 1, and MnP activity was highest in the high-Mn medium 2. The pH remained within 0.2 unit of the initial value of 4.0 in all cases. With P. sajor-caju, a low pH generally decreased lignin solubilization and, to a lesser extent, mineralization in the 48-h assay. Although these results were not statistically significant, they were confirmed in the 72-h assay (Table 4). Both the high-N medium and the N2 atmosphere caused a decrease in the total degradation by 72 h, the high-N medium inhibiting 14C02 production the most and the N2 atmosphere inhibiting the production of both 14C02 and soluble products to about the same extent. Only MnP activity was detected in these cultures. This was low, variable, and did not show treatment effects, so it is not presented. The pH decreased by 0.1 unit during the course of the experiment in all cases, except for the pH 3.5 treatment where it increased by 0.4 unit. This increase might have been due to dilution by residual pH 5.5 solution. Production of '4C02 from 14C-labelled water-soluble lignin degradation products. When labelled aqueous phase was added to P. chrysosporium cultures on unlabelled wood chips, the most 14C02 was produced by cultures on medium 1 under an atmosphere. Both the high-N medium and, to a greater extent, the N2 atmosphere caused a decrease in the 14C02 production (Table 5). S. commune also produced some 14C02. The rate of production was very low with both species, with only a small part of the 5,280 dpm that was added being metabolized to CO2 within 48 h. 14C02 production rates remained approximately constant for at least 120 h, while production rates in aseptic controls were negligible. When labelled or unlabelled aqueous phase was added to P. chrysosponum cultures on either labelled or unlabelled wood, 14C02 production was much higher on the labelled wood (Fig. 1). The rates of 14C02 production by these cultures dropped rapidly during the first few hours of the experiment for all of the treatments (Fig. 1, inset graph). For the cultures on the unlabelled wood that received the labelled aqueous phase, the rates continued to drop in subsequent sampling periods, while they remained essentially constant for the cultures on the labelled chips. The highest initial (0- to 2-h) rate was with cultures on the labelled wood 02
that received the labelled aqueous phase. The other cultures had initial rates that were 60 to 70% as high. When labelled aqueous phase was added to sterile media, 14C02 production was detected only in the earliest sampling period. 4C02 was not produced when boiled aqueous phase was added. DISCUSSION Lignin degradation by various species. Only the species that have been shown to be active lignin degraders in other studies degraded the labelled substrate at substantial rates. The use of labelled cinnamic acid for synthesis of the 14C-labelled lignin makes it unlikely that nonlignin substances (e.g., protein) in the wood contained significant amounts of 14C. With P. chrysosporium, as much as half of the supplied 14C activity was recovered by day 16, and about half of this was in the gas phase (presumably C02). This indicates that the fungus had completely oxidized substantial amounts of the carbon atoms of the aromatic rings of the lignin. Degradation rates by P. sajor-caju and L. edodes were considerably lower but were still much higher than that by F. velutipes or S. commune. Effects of various conditions on total lignin degradation and on fungal growth. In the longer-term experiments, some of the treatments had significant effects on fungal growth as indicated by inspection and, more objectively, by FDA. Although FDA could not be used to correct lignin degradation rates for differences in fungal growth, it was a sensitive means for showing the effects of the various treatments on TABLE 5. Production of 14C02 from '4C-labelled water-soluble lignin degradation products by P. chrysosponium in different conditions and by S. commune Fungus
chrysosporium
P.
S. commune
Medium and conditions
production "'CO2 (dpm 24 h-'
1, pH 3.5, low Mn, low N 3, low Mn, high N 1, N2 atmosphere
23 (2) a 11 (1) b 3 (1) c
culture -a)
2, pH 4.5; high Mn, low N
7
(1) d
Values are means, with the standard deviations shown in parentheses (n = 4), calculated from measurements made 1 h and 2 days after addition of a
aqueous phase containing 5,280 dpm of "'C. Results followed letters differ at P 2 0.05.
by different
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BOYLE ET AL.
6000 100
rCr
;-4
0
0 FIG.
1.
l.la 40 80 Hours after treatment established
120
'4C02 production by P. chiysosporium on either '4C-lignin-labelled or unlabelled wood, after replacement of the aqueous phase
with various solutions: inoculated labelled wood with the aqueous phase from a labelled wood culture aqueous phase
(A\);
(C1);
labelled wood with unlabelled
unlabelled wood with labelled aqueous phase (0). '4C02 production by sterile unlabelled wood with labelled aqueous
phase (+) and sterile unlabelled wood with boiled-labelled aqueous phase (@) is also presented. Points are means with standard error bars for triplicate cultures. The inset graph shows the rate of 14C02 production during each time interval for the same treatments.
growth. With P. chrysosporium, lignin degradation was decreased by nitrogen, regardless of whether growth increases were considered. With P. sajor-caju and L. edodes, nitrogen had variable effects on degradation, but growth increased in both cases. The FDA in conjunction with visual inspection showed that the failure of F. velutipes and S. commune to degrade significant amounts of the lignin was not due to poor growth. In the short-term studies where growth differences and growth-related alterations in media composition were minimized, lignin degradation by all species tested was markedly inhibited by nitrogen. It would be interesting to extend this approach to include other species whose lignin-degrading activities appear to be insensitive to nitrogen, such as the unidentified species NRRL 6464, which was isolated from a high-N environment (10), and the P. chrysosporium mutant, which was selected for the insensitivity of its lignin-degrading system to high levels of N (24). The optimal pH for lignin degradation differed between species even when effects of pH on growth were either considered (FDA) or minimized (short-term studies). Kirk et al. (15) found that lignin degradation by P. chrysosporium decreased at pHs below 4, this being accompanied by decreased growth. The results of the short-term assays here showed that lignin degradation by P. chrysosporium and L. edodes tended to increase as the pH decreased to 3, which was the lowest pH tested. Interestingly, LiP also shows optimal activity at pH 3 (23). In contrast, lignin degradation by P. sajor-caju was lower at a low pH. Although neither LiP nor veratryl alcohol oxidases were detected with this species in this work, it is interesting that veratryl alcohol
oxidases show a maximal activity at around pH 5.5 (3) that is consistent with a role for these enzymes in lignin degradation by this species. Effects of various conditions on formation on soluble and gaseous lignin degradation products. Changing the incubation conditions sometimes changed the relative amounts of 14CO2 and soluble products recovered, confirming that different processes are rate limiting for the formation of the two. Some factors decreased soluble product formation first, while others inhibited CO2 formation first. In either case, qualitatively similar changes eventually occurred in the rate of formation of the other product. This could reflect precursor-product relationships between the two and/or the operation of regulatory mechanisms (e.g., feedback inhibition) coordinating the two processes. The total lignin degradation and LiP activity of P. chrysosponum were highest in medium 1 and the MnP activity of it was low, as found previously by Perez and Jeffries (18). MnP apparently does not limit total lignin degradation (the formation of CO2 from lignin) by P. chrysosponum. In the short-term studies presented here, Mn decreased the production of soluble products first, consistent with these being precursors for the CO2. Both the percent product recovered as 14C02 and the MnP activity increased with high levels of Mn. High levels of N cause decreases in both MnP and LiP activities and the formation of both lignin degradation products. These findings are consistent with LiP being important in the solubilization process and with MnP being important in the subsequent formation of CO2. The differences in the effects of the N2 atmosphere, KCN, and CO were surprising since all three inhibit cellular respi-
VOL. 58, 1992
ration, and it was assumed that respiratory processes were ultimately responsible for the 14C02 production. The differences in the specific effects of these factors could reflect differences between the mechanisms by which they inhibit enzymes. For example, CN- reacts with enzymes containing ferric iron while CO reacts with enzymes containing iron in a more reduced state (25). It is not clear, however, how this would result in increased lignin degradation with the CO. This should be investigated further. The disappearance of the soluble products in some treatments could have been due to either continued production of 14C02 from the soluble compounds after their production had ceased or, alternatively, to polymerization reactions, both of which decrease the solubilities of the compounds. Laccase, LiP, and probably many other enzymes can catalyze polymerization of lignin-related compounds (12, 13). The disappearance of the 4CO2 was probably due to fixation into either soluble or insoluble compounds by the fungus and not to carbonate formation, since the medium pH was low and the samples were acidified before measurement. With P. sajor-caju, the conditions took longer to affect the degradation rates and did not always have the same effects as they did with P. chrysosponium. Some of the differences were not significant since variability was high with this fungus. Others, such as the effect of the pH 3.5 medium, underline that differences exist in the mechanisms and control of lignin degradation by different fungi. With this fungus, the high-nitrogen conditions inhibited the formation of4 C02 more than they inhibited the solubilization reactions. This conflicts with the results of Freer and Detroy (10) and of Ander et al. (1). However, their experiments extended for relatively long periods, so effects due to changes in fungal growth could not be discounted. Production of 14Co2 directly from water-soluble compounds. The low rate at which P. chrysosporium produced 14C02 from the added 14C-labelled aqueous-phase compounds and the decreased rates of production in the presence of high N or a nitrogen atmosphere were similar to the results of Reid and Deschamps (21) and showed that processes leading to 14C02 production, like those leading to lignin solubilization, can be inhibited by high-nutrient nitrogen and low oxygen availability. S. commune also produced some 14C02 from the labelled soluble compounds, indicating that the low lignin-degrading activity of this species resulted primarily from its inability to solubilize lignin. In both species, rates of 14C02 production from the added labelled soluble compounds were low, possibly because of slow diffusion of the labelled compounds to the site of degradation. The results suggest that only a small fraction of the 14C02 measured with the cultures on the labelled wood was produced by enzymes in the medium. It is more likely that both soluble and gaseous lignin degradation products were produced in close association with the hyphae. The rate of 14Co2 production from the labelled soluble compounds by cultures on the unlabelled wood decreased continuously with incubation time. A part of the decrease probably resulted from competition between the continually forming, unlabelled, soluble lignin degradation products and the labelled products that were added to the media. Extrapolation of the rates back to the time at which the labelled compounds were added minimizes the effects of this dilution, giving a better estimate of the percentage of the 14C02 derived from soluble precursors. This method gives a measure of about 70%. However, interpretation of the results is complicated by the initial drop that also occurred with the cultures on the labelled wood. The higher initial rate of
LIGNIN DEGRADATION BY WHITE ROT FUNGI
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"4C02 production by cultures on labelled chips receiving labelled, as opposed to unlabelled, aqueous phase also indicates that a considerable portion of the "4CO2 results
from metabolism of labelled aqueous-phase compounds. Shorter sampling intervals and more replicates would have to be used to obtain an accurate estimate of the fraction of the "4CO2 produced from soluble precursors. The measurable but low initial "'CO2 production when labelled aqueous phase was added to sterile medium, in contrast to the absence of production when boiled aqueous phase was added, indicated that some "'CO2 was produced by enzymes added with the aqueous phase. This "'CO2 production soon stopped, possibly because of enzyme inactivation. 14CO2 production by the medium might have been higher if the medium had not been changed so often, permitting the buildup of higher concentrations of extracellular enzymes. Conclusions. This work shows that lignin degradation can be regulated by a variety of conditions independent from the effects of these conditions on fungal growth. The results confirm that degradation processes leading to soluble products and to CO2 are distinct, being regulated separately. At the same time, they indicate that metabolic controls that coordinate the two processes exist. The specific effects that some factors (e.g., Mn, pH) have differ, depending on the species. However, nutrient nitrogen appears to inhibit lignin degradation by all species, although this inhibition can easily be masked by its pronounced stimulation of growth. ACKNOWLEDGMENT This work was funded by the National Science and Research Council of Canada.
Engineering
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