Effects of Nitrogen Sources on Cellulose and Synthetic Lignin Degradation by Phanerochaete chrysosporium

Ian D. Reid Appl. Environ. Microbiol. 1983, 45(3):838. Downloaded from http://aem.asm.org/ on May 24, 2012 by guest

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APPLIED

AND

ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p. 838-842

Vol. 45, No. 3

0099-2240/83/030838-05$02.00/0

Effects of Nitrogen Sources on Cellulose and Synthetic Lignin Degradation by Phanerochaete chrysosporiumt
IAN D. REID
National Research Council of Canada, Prairie Regional Laboratory, Saskatoon, Saskatchewan, Canada S7N

OW9

Received 3 November 1982/Accepted 15 December 1982

Phanerochaete chrysosporium degraded cellulose faster with organic nitrogen than with NH4Cl. Simple and complex nitrogen sources added at the time of inoculation to N-limited cultures of P. chrysosporium, with glucose as carbon/energy source, transiently stimulated degradation of synthetic [14C]lignin to 14Co2. The same nitrogen sources added 5 days after inoculation, when the cultures were entering secondary metabolism, delayed 14CO2 production. The various N sources affected synthetic lignin degradation in defined medium differently than lignin degradation in aspen wood.
sources

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Nutrient nitrogen has been reported to repress modified Kirk medium (D-glucose, 10 g; KH2PO4, 2 g; the degradation of synthetic lignin by Phanero- MgSO4 * 7H20, 0.5 g; CaCl2 * 2H20, 0.1 g; L-asparachaete chrysosporium (2, 3, 6, 8). In a compan- gine monohydrate, 93 mg; NH4NO3, 50 mg; trace [8], 1 ml; thiamine, 100 ion paper (11), I describe how some nitrogen element solution [Aldrich Chemical Co., ,ug; dialyzed polyacrylic acid sources stimulate, rather than inhibit, lignin deg- Wis.], 0.72 g; per liter of glass-distilled Milwaukee, water; pH radation in aspen wood by P. chrysosporium. To adjusted to 4.5 with 6 M KOH) plus various nitrogen better understand these results and to relate supplements. them to previous work, I examined the effects of The culture vessels were closed with rubber stopnitrogen supplements on growth and lignin me- pers, with cotton-plugged glass tubes for gas inlet and tabolism by P. chrysosporium in simpler sys- outlet. The experimental cultures were inoculated with tems. This paper compares the effects of various suspensions of P. chrysosporium conidia grown on N sources on the conversion of isolated cellu- potato dextrose agar (Difco Laboratories, Detroit, They lose and synthetic lignin to CO2 by P. chryso- Mich.). of 02- were incubated at 39C under an atmosphere sporium. Monosodium glutamate was purchased from MatheMATERIALS AND METHODS The culture of Phanerochaete chrysosporium Burds., PRL 2750 (= ME-446, ATCC 34541) was originally obtained from T. K. Kirk, Forest Products Laboratory, Madison, Wis. Synthetic [ring-14C]lignin ([14C]DHP) (specific radioactivity, 9.2 x 105 dpm/mg) was a gift from T. K. Kirk. DHP was added to cultures as an aqueous suspension, prepared by adding a solution in N,Ndimethylformamide to sterile water with vigorous stirring, as described by Kirk et al. (8). For studies of cellulose degradation, the cultures were grown in 60-ml glass bottles containing 500 mg of Solka Floc (Brown Co., Berlin, N.H.) moistened with 3.0 ml of water containing 7.2 mg of 2,2-dimethylsuccinic acid adjusted to pH 4.5, 4 mg of KH2PO4, 1 mg of MgSO4 * 7H20, 0.2 mg of CaC12 * 2H20, 0.2 ,ug of thiamine, 0.2 ,ul of trace element solution (9), plus various nitrogen supplements. Solka Floc is approximately 90% glucan, 8% xylan, and 2% mannan (8). For studies of synthetic lignin degradation, the cultures were grown in 125-ml Erlenmeyer flasks containing 10 ml of either 2 mM NH4 medium (13) or
t Paper no. 20797 of the National Research Council of Canada.

son, Coleman & Bell, Norwood, Ohio. Other nitrogen sources were as described in the companion paper

(11). Production of 14CO2 and total CO2 by the culture was measured as previously described (10, 14).

RESULTS

Growth of P. chrysosporium on cellulose was monitored by measuring CO2 release. Very little CO2 was produced in the absence of a nitrogen source; in the presence of the nitrogen sources, CO2 was evolved at constant rates after day 2. The amounts of CO2 produced within 14 days of incubation with various N sources are listed in Table 1. Yeast extract supported the fastest growth, followed by albumen hydrolysate. Asparagine, casein hydrolysate, albumen, and peptone supported slightly slower growth, and NH4Cl supported even slower growth. The pH values of the cultures after 14 days of growth were between 4.0 and 4.5. The effects of various nitrogen supplements added to a basal medium containing 2 mM NH4Cl on the degradation of [ring-14C]DHP to

838

VOL. 45, 1983 TABLE 1. Growth of P. chrysosporium on cellulose with various nitrogen sources CO2 per cultureb Nitrogen sourcea
(mmol)

EFFECTS OF N ON DHP CATABOLISM

839

0.12 +0.01 1.08 0.05 3.17 0.25c 3.29 0.28c Albumen ................. 3.56 0.27c Casein hydrolysate .............. 3.74 0.22c Albumen hydrolysate .............. 4.38 0.13 Yeast extract ...... ........ 5.26 0.26 a The amount of N supplied by each source was 1% of the cellulose weight. b Released during 14 days of incubation. Data are means standard error of five replicates. I These four values are not significantly different at the 95% confidence level according to the StudentNewman-Keuls test. None
.................

NH4C1 ................. Peptone ................. L-Asparagine .................

14CO2 are shown in Fig. 1. All of the N supplements caused 14CO2 to be produced earlier than in the basal medium. 14CO2 evolution from the supplemented cultures stopped within a few days, at the same time as the cessation of total CO2 release from the cultures. The cultures growing in basal medium continued to release 4CO2 for a longer period; by day 30, the yield of 14CO2 from the cultures in 2 mM NH4 medium was significantly more than the yield from the Nsupplemented cultures. Some of the differences in the final yields of 14CO2 from the cultures supplemented with the various N sources were statistically significant (Table 2). Urea supported the least lignin degradation; albumen and albumen hydrolysate supported the most. The final pH values in all treatments were between 4.0 and 4.5. The hastening of 14CO2 production from lignin by nitrogen supplements observed in this experiment contrasted with the delay of lignin degradation reported by Fenn et al. (2). In their experiments, the nitrogen supplements were added to 5-day-old cultures entering secondary metabolism. To check the possibility that the time of addition of the nitrogen supplements to the cultures had an important effect, I repeated the experiment, adding the N supplements either on day zero, as before, or on day 5. The results are shown in Fig. 2. The basal medium in this experiment was patterned after that of Kirk et al. (8), with a mixed NH4NO3-asparagine nitrogen source. The nitrogen supplements added on day zero had generally the same effect as that seen in Fig. 1: they caused an early, but short-lived, release of 14CO2 (Fig. 2A). The slowdown in 14CO2 release from the N-supplemented cultures was simultaneous with a slowdown in the release of

total CO2 from those cultures (Fig. 2C). The N supplements added on day 5 stimulated respiration (Fig. 2C) but delayed production of 14CO2 (Fig. 2B). Peptone caused a longer delay than yeast extract, albumen, and albumen hydrolysate. The cultures receiving N supplements on day 5 had final pH values between 4.2 and 4.7. The cultures which had received N supplements on day zero had pH values on day 12 of 5.5. The high final pH values in the cultures supplemented on day zero probably resulted from autolysis after the glucose was exhausted; their pH values during the phase of active ligninolysis were probably similar to those measured in the cultures supplemented on day 5. Glutamate and NH4Cl supplements were also tested in this experiment, but they caused pH changes large enough to inhibit lignin degradation. When the buffer capacity of the medium was increased with 50 mM 2,2-dimethylsuccinate, the culture pH values remained between 4.0 and 4.8. Ammonium chloride added on day 5 delayed 14CO2 production for about 1 day (Fig. 3). Asparagine and casein hydrolysate supplements inhibited 14CO2 release for about 3 days, and added glutamate was inhibitory for at least 5 days.
DISCUSSION P. chrysosporium grows readily in synthetic medium with NH4CI as the sole N source and thiamine as the only vitamin (8, 10). It will also grow, although more slowly, with NaNO3 as the
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FIG. 1. Effect of nitrogen sources on kinetics of conversion of [14C]DHP to 14CO2 by P. chrysosporium. Control cultures (0) contained 10 ml of 2 mM NH4 medium (glucose carbon source) plus 5.5 x 104 dpm of [ring-14C]DHP. Experimental cultures contained the same plus: A, 4.3 mg of NH4Cl; V, 2.4 mg of urea; O, 16.1 mg of casein hydrolysate; 0, 7.75 mg of albumen; *, enzymatic hydrolysate of 7.75 mg albumen; U, 7 mg of peptone; A, 9 mg of yeast extract. Each supplement supplied 80 iLmol of N per culture.

840

REID

APPL. ENVIRON. MICROBIOL.

TABLE 2. Effect of nitrogen supplement type on yield of "CO2 from [ring-l4C]DHPa 14CO2 ( of 14C Nitrogen supplement
Urea .... ........... 8.9 Casein hydrolysate .............. 11.1 12.1 Peptone ............... 13.2 NH4Cl ............... Yeast extract .............. 14.5 Albumen ............... 16.6 Albumen hydrolysate .............. 17.0 None ............... 22.7
0.4A

0.4AB

0.6AB l.0'c 0.3BCD 0.9CD 0.7D 2.2

a Data are the means standard error of five replicates. Means followed by the same capital letter are not significantly different at the 95% level according to the Student-Newman-Keuls test. Experimental conditions are described in the legend to Fig. 1. Cultures were incubated for 30 days.

sole N source (8). The results in this paper show that organic compounds of N can support faster growth, as measured by conversion of cellulose to C02, than NH4Cl. The single amino acid L-

asparagine was as good a nitrogen source as the mixtures of amino acids in casein hydrolysate and peptone. The mixture of amino acids in an enzymatic hydrolysate of bovine serum albumen, however, supported significantly faster growth, and yeast extract stimulated growth even more. Yeast extract contains vitamins and other growth factors as well as a complex mixture of nitrogen sources; any of these components might have contributed to the rapid growth of P. chrysosporium. The differences between the nitrogen sources were not caused by pH changes. In the experiments reported here, 14Co2 was the only product of lignin degradation measured. The accompanying paper (11) demonstrates that 14C02 production can seriously underestimate lignin degradation by P. chrysosporium in aspen wood, especially in the presence of yeast extract and peptone supplements. It is difficult to accurately measure the total extent of degradation of small amounts of DHP added to cultures. Chua et al. (M. G. S. Chua, S. Choi, and T. K. Kirk,
C
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Effect of

10

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production of total CO2 (C). Dotted lines refer to cultures supplemented on day zero and solid lines to cultures supplemented on day 5. Control cultures (0) contained 10 ml of modified Kirk medium plus 4.8 x 104 dpm of [ring-14C]DHP. Experimental cultures contained the same plus: 0, 7.75 mg of albumen; *, hydrolysate of 7.75 mg albumen; *, 7 mg of peptone; 9 mg of yeast extract. Each supplement supplied 80 p, F~~~~~molof N per culture.

VOL. 45, 1983

30 _

EFFECTS OF N ON DHP CATABOLISM

841

14CO2 than in the low N control cultures. In other words, the relative quantities of 14CO2 recorded in Table 2 may not accurately reflect the relative quantities of lignin degraded in the o _ .o different treatments. / c 20 loA third possibility is that the delay in "4CO2 release observed when the nitrogen supplements were added to 5-day-old cultures was caused by diversion of "4C into products other than "4CO2, 0' rather than by inhibition of DHP degradation. Available evidence does not rigorously exclude this possibility but does suggest that the effect of N 0 or1 the nitrogen supplement was on lignin degradation. First, 10 mM NH4 does not directly inhibit conversion of water-soluble lignin degradation intermediates to CO2 by P. chrysosporium (12). lo 4 2 6 o 8 TIME (days) Second, the nitrogen sources glutamate and hisFIG. 3. Effect of nitrogen supplements on conver- tidine inhibit mycelial binding and depolymerision of [(4C]DHP to "CO2 by P. chrysosporium in zation of DHP by P. chrysosporium, as well as highly buffered medium. Cultures contained 10 ml of 4CO2 production (Chua et al., in press). modified Kirk medium plus 50 mM 2,2-dimethylsucThe data presented here confirm the findings cinate and 4.9 x 104 dpm of [ring-14C]DHP. On day 5, of Fenn et al. (2) that nitrogen added to P. control cultures (0) received 0.5 ml of water, and chrysosporium cultures entering secondary meother cultures received 0.5 ml of solutions containing: tabolism delays the onset of lignin degradation A, 4.3 mg of NH4Cl; V, 5.3 mg of L-asparagine; O, 15 mg of monosodium glutamate; O, 13.4 mg of casein and extend their result to a wider variety of hydrolysate. Each supplement supplied 80 1Lmol of N nitrogen sources. Nitrogen supplements added to cultures at the start of growth, however, per culture. temporarily stimulated lignin degradation. For the following reasons, I think that stimulation Holzforschung, in press) have demonstrated was mediated by carbohydrate limitation, as that a large fraction of the DHP cannot be described by Jeffries et al. (5). The kinetics of separated from the mycelia, and it is difficult to 14CO2 release in the N-supplemented cultures differentiate this bound DHP from more exten- resembled those in the C-limited cultures of sively degraded fragments which have been in- Jeffries et al. (5). The N supplements stimulated corporated into the fungal biomass. P. chryso- conversion of glucose to C02, and the N-supplesporium also produces water-soluble products mented cultures ran out of carbohydrate, as from DHP (12). Therefore, we must consider the evidenced by cessation of CO2 release, within 3 possibility that 14CO2 production has underesti- days of starting to produce 14CO2. Glucose mated lignin degradation in the present experi- uptake was not measured in these experiments, ments and how such underestimation might af- but previous experience (10) indicates that the 10 mM nitrogen concentration in the supplefect the interpretation of the results. One possibility is that the cessation of 0CO2 mented cultures was sufficient to cause exhausrelease was caused by the diversion of 14C from tion of the extracellular glucose on day 3 of lignin into some product other than 14CO2, rath- growth, approximately the time at which 14CO2 er than by the cessation of DHP degradation. production began. There were significant differences between This seems unlikely, because the cultures at that point were starved for carbon and energy and the effects of the various nitrogen sources on could be expected to convert any available car- DHP degradation in synthetic media and their bon compounds to CO2. Furthermore, there is effects on lignin degradation in aspen wood (11). good evidence that lignin degradation by white- DHP degradation in the presence of the nitrogen rot fungi depends on the availability of a cosub- supplements lasted only a few days, because of the limited carbohydrate supply in the medium. strate (1, 4, 7). A second possibility is that the various nitro- Differences in the yields of ' CO2 from degradagen sources may have caused differences in the tion of DHP in cultures with the various N fraction of carbon from degraded lignin that was supplements were smaller than the differences in released as CO2. Experience with aspen wood release of "4CO2 from lignin-labeled aspen (11) suggests that in the presence of albumen and wood. None of the N sources permanently prealbumen hydrolysate, and especially peptone vented DHP degradation in adequately buffered and yeast extract, a smaller fraction of the 14C cultures. In aspen wood, casein hydrolysate and from degraded lignin would be converted to asparagine permanently and stringently inhibit-

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APPL. ENVIRON. MICROBIOL.

sporium. Appl. Environ. Microbiol. 42:290-296. 6. Keyser, P., T. K. Kirk, and J. G. Zeikus. 1978. Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J. Bacteriol. 135:790-797. 7. Kirk, T. K., W. J. Connors, and J. G. Zeikus. 1976. Requirement for a growth substrate during lignin decomposition by two wood-rotting fungi. Appl. Environ. Microbiol. 32:192-194. 8. Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, and J. G. Zeikus. 1978. Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117:277-285. 9. Reid, I. D. 1975. Growth of Tremella mesenterica haplonts with various nitrogen sources. Can. J. Bot. 53:73-77. 10. Reid, I. D. 1979. The influence of nutrient balance on lignin degradation by the white-rot fungus Phanerochaete chrysosporium. Can. J. Bot. 57:2050-2058. 11. Reid, I. D. 1983. Effects of nitrogen supplements on degradation of aspen wood lignin and carbohydrate components by Phanerochaete chrysosporium. AppI. Environ. Microbiol. 45:830-837. 12. Reid, I. D., G. D. Abrams, and J. M. Pepper. 1982. Water-soluble products from the degradation of aspen lignin by Phanerochaete chrysosporium. Can. J. Bot. 60:23572364. 13. Reid, I. D., and K. A. Seifert. 1980. Lignin degradation by Phanerochaete chrysosporium in hyperbaric oxygen. Can. J. Microbiol. 26:1168-1171. 14. Reid, I. D., and K. A. Seifert. 1982. Effect of an atmosphere of oxygen on growth, respiration, and lignin degradation by white-rot fungi. Can. J. Bot. 60:252-260.

ed lignin degradation. These differences are difficult to explain with our present knowledge and are reminders that results obtained under simple laboratory conditions, although valuable, cannot be uncritically generalized to more complex situations.
ACKNOWLEDGMENTS I thank C. S. Mallard for capable technical assistance and T. K. Kirk for generously supplying [14C]DHP.

LITERATURE CITED 1. Ander, P., and K.-E. Eriksson. 1975. Influence of carbohydrates on lignin degradation by the white-rot fungus Sporotrichum pulverulentum. Sven. Papperstidn. 78:643652. 2. Fenn, P., S. Choi, and T. K. Kirk. 1981. Ligninolytic activity of Phanerochaete chrysosporium: physiology of suppression by NH4' and L-glutamate. Arch. Microbiol. 130:66-71. 3. Fenn, P., and T. K. Kirk. 1981. Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium. Arch. Microbiol. 130:59-65. 4. Hirol, T., and K.-E. Eriksson. 1976. Microbiological degradation of lignin. Part 1. Influence of cellulose on the degradation of lignins by the white-rot fungus Pleurotus ostreatus. Sven. Papperstidn. 79:157-161. 5. Jeffries, T. W., S. Choi, and T. K. Kirk. 1981. Nutritional regulation of lignin degradation by Phanerochaete chryso-

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