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Bioalteration of kraft pine lignin by Bacillus megaterium isolated from compost piles
JOURNAL OF FERMENTATION AND BIOENGINEERING VO1. 68, NO. 2, 151-153. 1989
Bioalteration of Kraft Pine Lignin by Bacillus rnegaterium Isolated from Compost Piles F E R N A N D O P E R E S T E L O , 1. M I G U E L A N G E L F A L C O N , 1 M A R I A LUZ PI~REZ, z E M I L I O C O R O M I N A S ROIG, 2 AND G A B R I E L DE LA F U E N T E M A R T I N 3 Department of Microbiology and Cellular Biology, University of La Laguna, ~ Group of Microbiology, Department of Ornamentales, ICIA, 2 and IPNO, CSIC, 3 La Laguna, Tenerife, Canary Islands, Spain Received 20 October 1988/Accepted 5 June 1989 A strain of Bacillus megaterium had complex effects on kraft lignin degradation. In media with 0.1~o and 0.5 % glucose, a 50/00decrease and a 15% increase were observed respectively, in the acid-precipitable lignin fraction. No correlation was found between tyrosinase and iaccase production and lignin degradation.
Kraft lignin, a waste product of the major pulping process, is the main contributor to the color and toxicity of plant effluents. Kraft lignin differs from natural lignin in that it undergoes a variety of reactions including aryl-alkyl cleavages, strong modification of side chains, and various ill-defined condensation reactions (1). Nevertheless, this lignin preparation has been widely used, as experimental lignin, for studies of biodegradation (2-5). The ability of white-rot fungi to degrade lignin in wood has been recognized for years (6, 7). The role of brown-rot fungi and bacteria is generally considered to be a minor one, although some actinomycete strains are considered to be important in lignin degradation (8, 9). Little is known about the lignin degrading enzyme system of bacteria. Although no pathways for lignin degradation have been confirmed, monooxygenases (10), dioxygenases (11), and phenoloxidases (12, 13) seem to be the important enzymes in lignin biodegradation. Although phenoloxidases can theoretically bring about some oxidations and degradation of lignin, the extent and exact nature of their role in lignin degradation is not clear (14). The purpose of this work was to investigate the effects of the co-substrate concentration in the biodegradation of Kraft pine lignin by Bacillus megaterium. Moreover, laccase and tyrosinase production was studied in relation to lignin degradation by the above-mentioned strain. Strains were isolated from compost piles according to a modification of the Casida Jr. Method (15); compost extract was added instead of soil extract to mineral medium, as described by Odier and Monties (16). Other media used were Nutrient Agar (Difco) and Yeast Extract (Difco), supplemented with actidione (0.06 mg/ml). These strains were studied for their ability to degrade lignin on plates by the Sundmann and Nfise's test described initially for fungi (17). The extracellular laccase and intracellular tyrosinase productions were tested as described by Law and Timberlake (18), Brisou and Menantaud (19) and following the BDH Enzymes catalogue, respectively. All media were incubated at 40°C for one week. One of the strains isolated was identified using the usual identification techniques ( A P 1 2 0 E System together with other biochemical tests) following Bergey's manual of determinative bacteriology (20). It was inoculated in the mineral
medium into 1 l Erlenmeyer flasks (300 ml/flask), and incubated in a reciprocal shaker (Kottermann, FGR) at 37°C for 7 d. Lignin (Indulin AT, Westvaco Co., SC, USA), dissolved in 0.1 M N a O H and sterilized by filtration, was added to the medium at 0.1°/60, and glucose was added to the first flask (0.05%), the second flask (0.1%) and the third flask (0.5%o). In the fourth flask lignin (0.1%) was the sole carbon source and in the fifth and sixth flasks glucose (0.1% and 0.5%, respectively) was the sole carbon source. The same media with 2%o Bacto agar (Difco) were used for counts of bacterial colonies. The experiments were done in triplicate and uninoculated media were used as controls. Lignin degradation was estimated spectrophotometrically in a Hitachi 100-80 spectrophotometer according to Ulmer et al. (4). The changes in the molecular weight distribution of lignin were measured by gel permeation chromatography on a Sephadex LH-20 column (1 × 55 cm) as recommended by Connors et al. (21), following the procedure described by Ulmer et al. (4). The strains were collected from compost piles at various depths and at different stages in the composting process. One of these strains was identified as Bacillus megaterium, because it showed the following positive characteristics: Gram stain; catalase; fl-galactosidase; use of citrate; motility; hydrolysis of gelatin and starch; formation of acid from glucose; and growth in 7.5%o NaC1, and in aerobiosis. On the other hand, it was negative for the oxidase reaction; arginine dihydrolase; lysine decarboxylase; formation of indole; Voges-Proskauer test; and growth in anaerobiosis. The cells were rods with a central endospore. Several Bacillus strains have been reported to attack acidolysis lignin (22), and pine wood (23). Robinson and Crawford (24) have also shown that a Bacillus strain was able to convert p4C]-(side chain)-lignin of spruce into 14COz. Growth studies of B. megaterium in the different media indicated above were done. The results are shown in Fig. 1. An inhibitory effect on the growth of B. megaterium was found when 0.1% kraft lignin was used as the sole carbon and energy source (Fig. lf). It is reported that some low-molecular-weight fragments, present in kraft lignin, may inhibit the growth of certain microorganisms (25). In our case, TLC of the supernatant showed the presence of phenolic monomers from the begin-
* Corresponding author. 151
152
PERESTELO ET AL.
J. Fee-MEYer.B[OENC;.,
Ot I¢: O~ 0
e
• 24
f 4.8
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96
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168
FIG. 1. Viable bacterial counts in culture media containing different glucose concentrations: (a) 0.5~,,~ glucose; (b) 0.594 glucose and 0.1~0 kraft lignin; (c) 0.1~6 glucose; (d) 0 . l ~ glucose and 0.19/00 kraft lngnin; (f) 0.1 ~ kraft lignin. In the latter experiment a control (e), was done by incubating cell suspensions in phosphate buffer (100 raM; pH 7). Viable counts were determined as described in the text. Each value is the mean of four determinations. ning o f the culture, which could be the cause of the observed inhibition. Nevertheless, it did not hinder the attack on the Kraft lignin by B. rnegaterium in the presence of 0.1 and 0.5%0 glucose (Fig. lc and b). Measurement of acidprecipitable lignin showed a 5 ~ decrease and a 15%0 increase in media with 0.1 o/0 and 0.5% glucose, respectively, after 7 d o f incubation. Longer incubation periods (28 d) did not improve the extent o f lignin alteration. No alteration in the acid-precipitable lignin was detected using 0.050/oo glucose, which could be due to the low cellular yield. Inhibitory effects o f glucose concentration on lignin degradation have been reported (2, 26). The changes o f the total Kraft lignin UV-Vis spectra in the incubation o f B. megaterium are presented in Fig. 2. A 9 ~ decrease and a 2 7 ~ increase at 280 nm were observed after incubation o f B. megaterium with 0.1°/00 and 0 . 5 ~ glucose, respectively. The decrease in absorbance at 280 nm o f both total lignin and acid-precipitable lignin has been attributed to lignin degradation (27), and the increase related to the oxidation of the ¢~-carbons o f the side chains to carbonyl groups (2) and to the polymerization of lignin caused by bacterial action (27). Such polymerization was also observed by gel permeation c h r o m a t o g r a p h y where a 3794 increase o f high-molecular-weight products was detected, with the concomitant decrease o f the lowermolecular-weight. No significant alteration of the total lignin elution profile was observed in cultures with 0.1% glucose. H u t t e r m a n n et al. (28) have suggested that laccase is responsible for the polymerization of lignins and lignosulfonates in cultures o f the fungus F o m e s annosus. Some workers conclude that phenoloxidases are essential in fungal lignin decay (12). We have studied, in our case,
280 '320 3;0 4~)0 440 Wavelength (nm) FIG. 2. Absorption spectra of total kraft lignin, in 0. l M NaOH, attacked by Bacillus megaterium after 7 d of incubation in media containing 0.1°~ lignin and different glucose concentrations: (a) control culture; (b) 0.10g glucose; (c) 0.5 P4 glucose. phenoloxidases p r o d u c t i o n over 168 h o f incubation o f B. rnegaterium in different culture media. As shown in Fig. 3, the highest levels of laccase and tyrosinase were detected when the strain was grown in media supplemented with 0 . 5 ~ glucose, no lignin degradation being detected under these conditions. The maximal levels of laccase and tyrosinase activity ( 1 4 U / m l and 1.4 U / m l , respectively) were recorded after 48 h of growth. No laccase activity was detected when the cultures (0.1.90/ and 0 . 5 ~ , glucose), were done in the presence o f thioglycollate (2 raM), and inhibitor of laccase enzyme, nor was any alteration observed in the UV-Vis spectrum of either the total lignin or the acid-precipitable one. In conclusion, our results show that the co-substrate is important in lignin alteration by B. tnegaterium. In media with 0 . 1 ~ and 0 . 5 ~ glucose, a 5% decrease and a 15P~6 increase were observed, respectively, in the acid-precipitable lignin fraction. On the other hand, the laccase enzyme may be involved in both the degradation and polymerization processes. The authors with to express their sincere thanks to the Gobierno Autonomo de Canarias for financial support. We also thank Ana Rodr~guez for help in preparation of the manuscript, lndulin AT was kindly supplied by Westvaco Polychemicals (USA). REFERENCES 1. Marton, J,: Reactions in alkaline pulping, p. 639-689. In
Sarkanen, K. V. and Ludwig, C. H. (ed.}, Lignins: ocurrence, formation, structure, and reactions. Wiley-lmerscience, New York (1971). 2, Forney, L.J. and Reddy, C. A.: Racterial degradalion of Krat~ lignin. Dev. lnd. Microbiol., 20, 163-175 (1979). 3. lanshekar, H., Brown, C., Haltmeier, Th., Leisola, M., and
VOL. 68, 1989
NOTES
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FIG. 3. Courses of laccase (o) and tyrosinase (A) production by B. megaterium, in three different media containing 0.1% kraft lignin: (a) 0.05 °6 glucose; (b) 0.1% glucose; (c) relationship between culture growth ( ) and development of laccase and tyrosinase activity in cultures supplemented with 0.5°4 glucose. Fiechter, A.: Bioalteration of Kraft pine lignin by Phanerochaete chrysosporium. Arch. Microbiol., 132, 14-21 (1982). 4. Ulmer, D. C., Leisola, M. S. A., Sehmidt, B. H., and Fiechter, A.: Rapid degradation of isolated lignins by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 45, 1795-1801 (1983). 5. Goycoolea, M., Seelenfreund, D., Rntlimann, C., Gonz~ilez, B., and Vieufia, R.: Monitoring bacterial consumption of low molecular weight lignin derivatives by high performanced liquid chromatography. Enzyme Microb. Technol., 8, 213-216 (1986). 6. Kirk, T.K.: Degradation and conversion of lignocelluloses, p. 266-289. In Smith, J . E . , Berry, D. R., and Kristiansen, B. (ed.), The filamentous fungi. Edward Arnold, London (1983). 7. Kirk, T.K. and Farreli, R.L.: Enzymatic combustion: the microbial degradation of lignin. Ann. Rev. Microbiol., 41,465505 (1987). 8. Crawford, D. L.: Lignocellulose decomposition by selected Streptomyces strains. Appl. Environ. Microbiol., 35, 1041-t045 (1978). 9. McCarthy, A. a. and Broda, P.: Screening of lignin-degrading actinomycetes and characterization of their activity against (~4C) lignin labelled wheat lignocellulose. J. Gen. Microbiol., 130, 2905-2913 (1984). 10. Tien, M. and Kirk, T.K.: Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization, and catalytic properties of a unique H202-requiring oxygenase. Proc. Natl. Acad. Sci. USA, 81, 2280-2284 (1984). 11. Higuchi, T.: Biodegradation of lignin: biochemistry and potential applications. Experientia, 38, 15%166 (1982). 12 Ander, P. and Eriksson, K. E.: The importance of phenol-oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum. Arch. Microbiol., 109, 1-8 (1976). 13. lshihara, T.: The role of laccase in lignin biodegradation, p. 1731. In Kirk, T. K., Higuchi, T., and Chang, H. H. (ed.), Lignin biodegradation: microbiology, chemistry, and potential applications, vol. 2. CRC Press, Boca Raton (1980). 14. Buswell, J. A. and Odier, E.: Lignin biodegradation. CRC Crit. Rev. Biotechnol., 6, 1-60 (1987). 15. Casida, Jr., L. E.: Methods for the isolation and estimation of activity of soil bacteria, p. 97-122. In Gray, T. R. G. and Parkinson, D. (ed.), The ecology of soil bacteria. Liverpool University Press,
Liverpool (1968). 16. Odier, E. and Monties, B.: Biodegradation de la lignine de b16 par Xanthomonas 23. Ann. Inst. Pasteur Microbiol., 129 A, 361-377 (1978). 17. Sundmann, V. and N~ise, L.: A simple plate test for direct visualisation of biological lignin degradation. Papper Och. Tr~i., 2, 67-71 (1971). 18. Law, D . J . and Timberlake, W.E.: Development regulation of laccase levels in Aspergillus nidulans. J. Bacteriol., 144, 509-517 (1980). 19. Brisou, J. and Menantand, J.: Metabolisme des produits cycliques et aromatiques, p. 241. In Masson et Cie (ed.), Techniques d'enzimologie bacterienne. Paris ( 1971 ). 20. Buchanan, R. E. and Gibbons, N. E. (ed.): Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore (1974). 21. Connors, W. J.: Gel chromatography and association complexes of lignin. Holzforschung, 34, 80-85 (1980). 22. Odier, E. and Monties, B.: Activit6 ligninolytique "in vitro" de bacteries isol6es de paille de b16 en decomposition. C.R. Acad. Sci. Paris. S6rie D, T., 284, 2175-2178 (1977). 23. Schmidt, O. and Baueh, J.: Lignin in woody tissues after chemical pretreatment and bacterial attack. Wood Sci. Technol., 14, 229-239 (1980). 24. Robinson, L. E. and Crawford, R. L.: Degradation of ~4C-labelled lignins by Bacillus megaterium. FEMS Microbiol. Lett., 4, 301302 (1978). 25. Zemek, J., Valent, M., Podov~i, M., Kosikov~i, B., and Joniak, D.: Antimicrobial properties of aromatic compounds of plant origin. Folia Microbiol., 32, 421-425 (1987). 26. Leisola, M. S. A., Ulmer, D. C., and Fieehter, A.: Factors affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium. Arch. Microbiol., 137, 171-175 (1984). 27. Janshekar, H., Haltmeier, Th., and Brown, C.: Fungal degradation of pine and straw alkali lignins. Eur. J. Appl. Microbiol. Biotechnol., 14, 174-181 (1982). 28. Hiittermann, A., Herehe, C., and Haars, A.: Polymerization of water-insoluble lignins by Fomes annosus. Holzforschung, 34, 64-66 (1980).
Bioalteration of Kraft Pine Lignin by Bacillus rnegaterium Isolated from Compost Piles F E R N A N D O P E R E S T E L O , 1. M I G U E L A N G E L F A L C O N , 1 M A R I A LUZ PI~REZ, z E M I L I O C O R O M I N A S ROIG, 2 AND G A B R I E L DE LA F U E N T E M A R T I N 3 Department of Microbiology and Cellular Biology, University of La Laguna, ~ Group of Microbiology, Department of Ornamentales, ICIA, 2 and IPNO, CSIC, 3 La Laguna, Tenerife, Canary Islands, Spain Received 20 October 1988/Accepted 5 June 1989 A strain of Bacillus megaterium had complex effects on kraft lignin degradation. In media with 0.1~o and 0.5 % glucose, a 50/00decrease and a 15% increase were observed respectively, in the acid-precipitable lignin fraction. No correlation was found between tyrosinase and iaccase production and lignin degradation.
Kraft lignin, a waste product of the major pulping process, is the main contributor to the color and toxicity of plant effluents. Kraft lignin differs from natural lignin in that it undergoes a variety of reactions including aryl-alkyl cleavages, strong modification of side chains, and various ill-defined condensation reactions (1). Nevertheless, this lignin preparation has been widely used, as experimental lignin, for studies of biodegradation (2-5). The ability of white-rot fungi to degrade lignin in wood has been recognized for years (6, 7). The role of brown-rot fungi and bacteria is generally considered to be a minor one, although some actinomycete strains are considered to be important in lignin degradation (8, 9). Little is known about the lignin degrading enzyme system of bacteria. Although no pathways for lignin degradation have been confirmed, monooxygenases (10), dioxygenases (11), and phenoloxidases (12, 13) seem to be the important enzymes in lignin biodegradation. Although phenoloxidases can theoretically bring about some oxidations and degradation of lignin, the extent and exact nature of their role in lignin degradation is not clear (14). The purpose of this work was to investigate the effects of the co-substrate concentration in the biodegradation of Kraft pine lignin by Bacillus megaterium. Moreover, laccase and tyrosinase production was studied in relation to lignin degradation by the above-mentioned strain. Strains were isolated from compost piles according to a modification of the Casida Jr. Method (15); compost extract was added instead of soil extract to mineral medium, as described by Odier and Monties (16). Other media used were Nutrient Agar (Difco) and Yeast Extract (Difco), supplemented with actidione (0.06 mg/ml). These strains were studied for their ability to degrade lignin on plates by the Sundmann and Nfise's test described initially for fungi (17). The extracellular laccase and intracellular tyrosinase productions were tested as described by Law and Timberlake (18), Brisou and Menantaud (19) and following the BDH Enzymes catalogue, respectively. All media were incubated at 40°C for one week. One of the strains isolated was identified using the usual identification techniques ( A P 1 2 0 E System together with other biochemical tests) following Bergey's manual of determinative bacteriology (20). It was inoculated in the mineral
medium into 1 l Erlenmeyer flasks (300 ml/flask), and incubated in a reciprocal shaker (Kottermann, FGR) at 37°C for 7 d. Lignin (Indulin AT, Westvaco Co., SC, USA), dissolved in 0.1 M N a O H and sterilized by filtration, was added to the medium at 0.1°/60, and glucose was added to the first flask (0.05%), the second flask (0.1%) and the third flask (0.5%o). In the fourth flask lignin (0.1%) was the sole carbon source and in the fifth and sixth flasks glucose (0.1% and 0.5%, respectively) was the sole carbon source. The same media with 2%o Bacto agar (Difco) were used for counts of bacterial colonies. The experiments were done in triplicate and uninoculated media were used as controls. Lignin degradation was estimated spectrophotometrically in a Hitachi 100-80 spectrophotometer according to Ulmer et al. (4). The changes in the molecular weight distribution of lignin were measured by gel permeation chromatography on a Sephadex LH-20 column (1 × 55 cm) as recommended by Connors et al. (21), following the procedure described by Ulmer et al. (4). The strains were collected from compost piles at various depths and at different stages in the composting process. One of these strains was identified as Bacillus megaterium, because it showed the following positive characteristics: Gram stain; catalase; fl-galactosidase; use of citrate; motility; hydrolysis of gelatin and starch; formation of acid from glucose; and growth in 7.5%o NaC1, and in aerobiosis. On the other hand, it was negative for the oxidase reaction; arginine dihydrolase; lysine decarboxylase; formation of indole; Voges-Proskauer test; and growth in anaerobiosis. The cells were rods with a central endospore. Several Bacillus strains have been reported to attack acidolysis lignin (22), and pine wood (23). Robinson and Crawford (24) have also shown that a Bacillus strain was able to convert p4C]-(side chain)-lignin of spruce into 14COz. Growth studies of B. megaterium in the different media indicated above were done. The results are shown in Fig. 1. An inhibitory effect on the growth of B. megaterium was found when 0.1% kraft lignin was used as the sole carbon and energy source (Fig. lf). It is reported that some low-molecular-weight fragments, present in kraft lignin, may inhibit the growth of certain microorganisms (25). In our case, TLC of the supernatant showed the presence of phenolic monomers from the begin-
* Corresponding author. 151
152
PERESTELO ET AL.
J. Fee-MEYer.B[OENC;.,
Ot I¢: O~ 0
e
• 24
f 4.8
"/2
96
l 120
I 144
I
168
FIG. 1. Viable bacterial counts in culture media containing different glucose concentrations: (a) 0.5~,,~ glucose; (b) 0.594 glucose and 0.1~0 kraft lignin; (c) 0.1~6 glucose; (d) 0 . l ~ glucose and 0.19/00 kraft lngnin; (f) 0.1 ~ kraft lignin. In the latter experiment a control (e), was done by incubating cell suspensions in phosphate buffer (100 raM; pH 7). Viable counts were determined as described in the text. Each value is the mean of four determinations. ning o f the culture, which could be the cause of the observed inhibition. Nevertheless, it did not hinder the attack on the Kraft lignin by B. rnegaterium in the presence of 0.1 and 0.5%0 glucose (Fig. lc and b). Measurement of acidprecipitable lignin showed a 5 ~ decrease and a 15%0 increase in media with 0.1 o/0 and 0.5% glucose, respectively, after 7 d o f incubation. Longer incubation periods (28 d) did not improve the extent o f lignin alteration. No alteration in the acid-precipitable lignin was detected using 0.050/oo glucose, which could be due to the low cellular yield. Inhibitory effects o f glucose concentration on lignin degradation have been reported (2, 26). The changes o f the total Kraft lignin UV-Vis spectra in the incubation o f B. megaterium are presented in Fig. 2. A 9 ~ decrease and a 2 7 ~ increase at 280 nm were observed after incubation o f B. megaterium with 0.1°/00 and 0 . 5 ~ glucose, respectively. The decrease in absorbance at 280 nm o f both total lignin and acid-precipitable lignin has been attributed to lignin degradation (27), and the increase related to the oxidation of the ¢~-carbons o f the side chains to carbonyl groups (2) and to the polymerization of lignin caused by bacterial action (27). Such polymerization was also observed by gel permeation c h r o m a t o g r a p h y where a 3794 increase o f high-molecular-weight products was detected, with the concomitant decrease o f the lowermolecular-weight. No significant alteration of the total lignin elution profile was observed in cultures with 0.1% glucose. H u t t e r m a n n et al. (28) have suggested that laccase is responsible for the polymerization of lignins and lignosulfonates in cultures o f the fungus F o m e s annosus. Some workers conclude that phenoloxidases are essential in fungal lignin decay (12). We have studied, in our case,
280 '320 3;0 4~)0 440 Wavelength (nm) FIG. 2. Absorption spectra of total kraft lignin, in 0. l M NaOH, attacked by Bacillus megaterium after 7 d of incubation in media containing 0.1°~ lignin and different glucose concentrations: (a) control culture; (b) 0.10g glucose; (c) 0.5 P4 glucose. phenoloxidases p r o d u c t i o n over 168 h o f incubation o f B. rnegaterium in different culture media. As shown in Fig. 3, the highest levels of laccase and tyrosinase were detected when the strain was grown in media supplemented with 0 . 5 ~ glucose, no lignin degradation being detected under these conditions. The maximal levels of laccase and tyrosinase activity ( 1 4 U / m l and 1.4 U / m l , respectively) were recorded after 48 h of growth. No laccase activity was detected when the cultures (0.1.90/ and 0 . 5 ~ , glucose), were done in the presence o f thioglycollate (2 raM), and inhibitor of laccase enzyme, nor was any alteration observed in the UV-Vis spectrum of either the total lignin or the acid-precipitable one. In conclusion, our results show that the co-substrate is important in lignin alteration by B. tnegaterium. In media with 0 . 1 ~ and 0 . 5 ~ glucose, a 5% decrease and a 15P~6 increase were observed, respectively, in the acid-precipitable lignin fraction. On the other hand, the laccase enzyme may be involved in both the degradation and polymerization processes. The authors with to express their sincere thanks to the Gobierno Autonomo de Canarias for financial support. We also thank Ana Rodr~guez for help in preparation of the manuscript, lndulin AT was kindly supplied by Westvaco Polychemicals (USA). REFERENCES 1. Marton, J,: Reactions in alkaline pulping, p. 639-689. In
Sarkanen, K. V. and Ludwig, C. H. (ed.}, Lignins: ocurrence, formation, structure, and reactions. Wiley-lmerscience, New York (1971). 2, Forney, L.J. and Reddy, C. A.: Racterial degradalion of Krat~ lignin. Dev. lnd. Microbiol., 20, 163-175 (1979). 3. lanshekar, H., Brown, C., Haltmeier, Th., Leisola, M., and
VOL. 68, 1989
NOTES
153
~8 1,,
t
:,
.
.
.
.
916
120
144
~
'; is
m
',,7, 'r
oI.
24
48
72
96
120
144
168
Time
24
48
72
(h)
168
9 on
~
e-
o"°tl l l o
~ 24
48
72
96
120
144
Time
(h)
168
FIG. 3. Courses of laccase (o) and tyrosinase (A) production by B. megaterium, in three different media containing 0.1% kraft lignin: (a) 0.05 °6 glucose; (b) 0.1% glucose; (c) relationship between culture growth ( ) and development of laccase and tyrosinase activity in cultures supplemented with 0.5°4 glucose. Fiechter, A.: Bioalteration of Kraft pine lignin by Phanerochaete chrysosporium. Arch. Microbiol., 132, 14-21 (1982). 4. Ulmer, D. C., Leisola, M. S. A., Sehmidt, B. H., and Fiechter, A.: Rapid degradation of isolated lignins by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 45, 1795-1801 (1983). 5. Goycoolea, M., Seelenfreund, D., Rntlimann, C., Gonz~ilez, B., and Vieufia, R.: Monitoring bacterial consumption of low molecular weight lignin derivatives by high performanced liquid chromatography. Enzyme Microb. Technol., 8, 213-216 (1986). 6. Kirk, T.K.: Degradation and conversion of lignocelluloses, p. 266-289. In Smith, J . E . , Berry, D. R., and Kristiansen, B. (ed.), The filamentous fungi. Edward Arnold, London (1983). 7. Kirk, T.K. and Farreli, R.L.: Enzymatic combustion: the microbial degradation of lignin. Ann. Rev. Microbiol., 41,465505 (1987). 8. Crawford, D. L.: Lignocellulose decomposition by selected Streptomyces strains. Appl. Environ. Microbiol., 35, 1041-t045 (1978). 9. McCarthy, A. a. and Broda, P.: Screening of lignin-degrading actinomycetes and characterization of their activity against (~4C) lignin labelled wheat lignocellulose. J. Gen. Microbiol., 130, 2905-2913 (1984). 10. Tien, M. and Kirk, T.K.: Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization, and catalytic properties of a unique H202-requiring oxygenase. Proc. Natl. Acad. Sci. USA, 81, 2280-2284 (1984). 11. Higuchi, T.: Biodegradation of lignin: biochemistry and potential applications. Experientia, 38, 15%166 (1982). 12 Ander, P. and Eriksson, K. E.: The importance of phenol-oxidase activity in lignin degradation by the white-rot fungus Sporotrichum pulverulentum. Arch. Microbiol., 109, 1-8 (1976). 13. lshihara, T.: The role of laccase in lignin biodegradation, p. 1731. In Kirk, T. K., Higuchi, T., and Chang, H. H. (ed.), Lignin biodegradation: microbiology, chemistry, and potential applications, vol. 2. CRC Press, Boca Raton (1980). 14. Buswell, J. A. and Odier, E.: Lignin biodegradation. CRC Crit. Rev. Biotechnol., 6, 1-60 (1987). 15. Casida, Jr., L. E.: Methods for the isolation and estimation of activity of soil bacteria, p. 97-122. In Gray, T. R. G. and Parkinson, D. (ed.), The ecology of soil bacteria. Liverpool University Press,
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