APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1978, p. 201-204
0099-2240/78/0036-0201$02.00/0
Copyright i) 1978 American Society for Microbiology
Vol. 36, No. 1
Printed in U.S.A.
Effect of Municipal Refuse Metals on Cellulase Production by Thermomonospora curvata FRED STUTZENBERGER'* AND ION STERPU2 Clemson University, Clemson, South Carolina 29631,' and Research Institute for Food Industry and Chemistry, Bucharest, Romania2 Received for publication 8 February 1978
The high-concentration metals in municipal refuse compost were tested for effects on cellulase production and activity in Thermomonospora curvata. Although none altered cellulase reaction rates, both Al and Ca appeared to specifically inhibit cellulase production.
Refuse composting has been defined as the aerobic, thermophilic degradation of putrescible material in refuse by microorganisms (14). The temperature profile of the compositing mass passes through three general stages: (i) an initial stage in which the temperature is brought from ambient to about 400C by the metabolic activities of mesophilic fungi and bacteria; (ii) a stage in which the temperature peaks at 65 to 700C and the flora consist mostly of thermophilic actinomycetes and fungi; (iii) a stage of temperature decline during which the mesophiles again gain predominance. The thermophilic stage is considered the most important because most degradation of raw materials such as cellulose occurs during that time. In this stage, actinomycete populations often reach 108 colony-forming units per g of wet compost (12). One of these actinomycetes, Thermomonospora curvata, is the predominant actinomycete in mushroom compost (4) and is also a common isolate from municipal refuse compost (17). T. curvata produces both Cl and C. cellulases (15, 16) and probably plays a major role in cellulose degradation during composting. Because cellulose constitutes about one-half of the dry weight of municipal refuse compost (14), cellulase production by the indigenous thermophilic flora greatly influences the conversion rate of raw refuse to a stable product. In the large-scale composting of municipal refuse or vegetable- and fruit-canning wastes, calcium oxide or calcium sulfate is often added to the raw mass to raise the pH (initially as low as 4.5) in an attempt to speed the process (13, 14). For this and other reasons, the Ca content of the compost ranges from 1.3 to 5.5% on a dry weight basis (18). A detailed report (14) revealed that in addition to Ca, the metals Mg, Al, and Fe were also usually in high concentrations (1 to 2% of dry weight). Relatively high concentrations of Ca stimulate induction of cellulase pro-
duction in the fungus Trichoderma viride (8), and several microbial cellulases appear to be activated or stabilized by the presence of metals such as Fe (9). Consequently, we studied these common municipal refuse metals as to their influence on growth of T. curvata and its cellulase production, reaction rate, and thermal stability. The minimal medium employed for cellulase production was as follows: (NH4)2SO4, 0.1%; MgCl2 6H20, 0.01%; phosphate buffer, 0.1 M; ground surgical cotton (40 mesh, Johnson and Johnson), 0.8%. Final pH after autoclaving was 7.6 to 7.8. Sterile-filtered solutions of biotin and thiamine (Sigma Chemical Co.) were added aseptically (final concentrations of 0.5 jig/ml) to the cooled flasks. The metals used in this study were sterilized separately as solutions of their chloride salts and added aseptically to the mineral salts medium. Initial concentrations of the metals in the medium were determined by atomic absorption spectrophotometry in a Perkin-Elmer model 403 equipped with a heatedgraphite atomizer. T. curvata cultures (100 ml) were grown at 520C in 250-ml Erlenmeyer flasks on a New Brunswick gyratory shaker at 200 rpm. Under these conditions, cellulase activity reached a maximum in 5 to 6 days and then declined on further incubation. Cl and C. cellulase activities were measured as previously described (16). Enzyme activity units were calculated as micromoles of reducing sugar (as glucose) released per minute. Protein was estimated by the method of Lowry et al. (7) and by the Bio-Rad dye binding method (Bio-Rad Laboratories technical bulletin 1051, 1977). Cellulases were purified as previously described (16) except for the following modifications. Crude culture fluids were concentrated about 20-fold by ultrafiltration on an Amicon PM-10 membrane before the ammonium sulfate precipitation step. The Corning controlled-pore
201
202
APPL. ENVIRON. MICROBIOL.
NOTES
glass beads were replaced with Bio-Gel P-200 polyacrylamide beads (Bio-Rad Laboratories) in a Glenco column (1.5 by 28 cm) run at room temperature, collecting 3-ml fractions. The eluting buffer was 0.01 M phosphate (pH 7.5), with 0.1 M NaCl and 0.02% NaN3 as a preservative. Polyvalent cations such as A13+ produce marked changes in the electrokinetic potential of proteins (2). Such changes may influence both thermal stability and activity of extracellular enzymes. A13+ (10-5 to 10-3 M) in T. curvata cultures inhibited cellulase production without a proportional decrease in extracellular protein accumulation (Fig. 1). Cellulase activity in cultures with 1 mM A13+ was decreased to about half that of the controls. However, A13+ had no detectable effect on the therrnal stability (at either 52 or 65°C) of either crude or purified cellulase. Because Mg, Fe, and Ca are considered macronutrient elements in microbial metabolism, we tested effects of these metals on the growth of T. curvata under conditions where cellulase production was not essential (cellulose was replaced by 0.5% cellobiose). The results were then compared with those obtained under conditions where cellulase production was essential for growth (cotton fiber cellulose was the sole carbon and energy source). These three metals differed markedly as to their comparative effects on growth versus cellulase production. Mg concentrations of 1o-4 to 10-2 M provided a broad optimum range both for growth on cellobiose and for cellulase production on cellulose.
There was no detectable growth or cellulase production below 5 x 106 M or above 5 x 10-2 M Mg. Fe has very low solubility at neutral pH in the presence of oxygen because it forms a highly insoluble ferric hydroxide (1). In our studies, precipitation of Fe compounds was evident in the medium at concentrations above 10-5 M. Microscopic examination of cultures revealed that the mycelial strands of T. curvata adhered to the surfaces of the precipitate. Cellulase and protein production were decreased about 50% at 103 M Fe and completely inhibited at 2 x 10'M. Ca had the most unexpected effect on T. curvata. In cellobiose-mineral salts medium, where cellulase production is poorly induced, increasing the Ca concentration from 1.8 x 10-5 M (the trace Ca contamination in the medium) to 2 x 10'3 M (the limit of solubility in the medium) resulted in stimulation of growth (Fig. 2). However, in cellulose-mineral salts medium, the optimum Ca concentration for cellulase production was in the range of 0.1 to 0.6 mM. Higher concentrations up to 1.8 mM inhibited extracellular cellulase and protein production (Fig. 3). This finding contrasted with earlier studies (3, 8) on fungi and actinomycetes which employed Ca concentrations of 2 to 3 mM as optimum for cellulase production in similar mineral salts media. The effect of metals on T. curvata indicates
f I.-
,0
O 000 1
-J0
o
so.
> ooff-f U *g 100
/~~~~/
w
u
0
o~~0z X0SO ' -o-C, X --Cx r ** X
I
o-so ^
o
[AI'J (mol-s/Iit.r ! FIG. 1. Effect of Al on cellulase and soluble-protein production by T. curvata. Each point is the average of three independent experiments.
I
no1
I
I
io2
MOLAR CA CONC. FIG. 2. Effect of Ca concentration on cell yield of T. curvata cultures in cellobiose-mineral salts medium. Dry cell weight measurements were made after 48 h of incubation under conditions described in the text. Each point is the average of values from duplicate flasks.
VOL. 36, 1978
NOTES
203
100-
2 I
zt
-I
0~~ u
a
0.5_
J50
oo x
_
I 0O u0N
10
~0 10~~~s
1o~1 0
MOLAR CA CONC. FIG. 3. Effect of Ca concentration on production of cell-free C, (0), C. (A), and protein (O) by T. curvata in cellulose-mineral salts medium. Data are expressed as the peak values attained at each Ca concentration during a 6-day incubation. Each point is the average of values from three separate experiments.
that the high metal content of municipal solid waste might be detrimental to the rate of cellulase production in the composting process. In particular, the addition of more Ca as lime or limestone would seem to be of no advantage despite the transient pH increase because such addition also results in loss of nitrogen and a lower-quality compost (14). If a pH rise of composting material is required, the addition of urea (which has been used to advantage in fruit waste compost [13]) would be more desirable. The inhibitory effect of Ca at millimolar concentrations seemed directed at cellulase production rather than growth; therefore, we tested the possibility that Ca decreased the thermal stability of the cellulase in the culture fluids to cause inactivation soon after release from the cells. However, the cellulase was completely stable at 520C for at least 30 h in culture fluids with no added Ca or with Ca concentrations of 0.1 to 1.0 mM. Furthermore, at 650C (the temperature of compost during peak heating) Ca protected the cellulase against thermal inactivation (Fig. 4). This relative degree of protection was observed in both crude and purified cellulase preparations. All four metals were also tested at 0.1 and 1.0 mM concentrations as to their effects on cellulase reaction rates. No significant changes
1
2 3 HOURS
4
5
FIG. 4. Effect of Ca on the thermal stability of cellulase in culture fluids at pH 7.5 and 65°C. The initial activity of each sample before heating was assigned an arbitrary value of 1.0. Symbols: C, control (E); C, with I mM Ca (0); C, control (0); C, with 1 mM Ca (A). Each point is the average of values from two separate experiments.
were observed, at either concentration of any metal, in either the C, or C. assays with purified cellulase. Studies are continuing as to why Ca concentrations which stimulate growth on cellobiose are inhibitory to cellulase production on cellulose. One possibility is that millimolar Ca concentrations stabilize an extracellular protease which degrades the cellulase. Some microbial proteases require 2 mM Ca for stability (11, 20), and a relationship between protease production and cellulase inactivation has been suggested for Trichoderma (10). Protease production has been reported for T. viridis (19); if such is the case in T. curvata, it could explain the decline of cellulase activity in older cultures despite its thermal stability. This research was supported by grant no. 13538L from the U.S. Army Research Office. We are indebted to the international Research and Exchanges Board for the sponsorship of I.S. LITERATURE CITED 1. Brock, T. D. 1970. Biology of microorganisms, p. 145. Prentice-Hall, Inc., Englewood Cliffs, N.J. 2. Dixon, M., and E. C. Webb. 1964. Enzymes, p. 429. Academic Press Inc., New York. 3. Enger, M. D., and B. P. Sleeper. 1965. Multiple cellulase system from Streptomyces antibioticus. J. Bacteriol. 89:23-27.
204
NOTES
4. Fergus, C. L. 1964. Thermophilic and thermotolerant molds and actinomycetes of mushroom compost during peak heating. Mycologia 56:267-284. 5. Hagerty, D. J., J. L. Pavoni, and J. E. Heer. 1973. Solid waste management. Van Nostrand Reinhold Co., New York. 6. Jeris, J., and R. Regan. 1973. Controlling environmental parameters for optimum composting. Compost Sci. 14:16-22. 7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1953. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. Mandels, M., and E. T. Reese. 1957. Induction of cellulase in Trichoderrma viride as influenced by carbon sources and metals. J. Bacteriol. 73:269-278. 9. Mandels, M., and E. T. Reese. 1963. Inhibition of cellulases and fi-glucosidases, p. 115-157. In E. T. Reese (ed.). Advances in enzymic hydrolysis of cellulose and related materials. Pergamon Press, Oxford. 10. Mandels, M., D. Steinberg, and R. E. Andreotti. 1975. Growth and cellulase production by Trichoderma. Symposium on Enzymatic Hydrolysis of Cellulose. Aulanko, Finland. 11. Matsubara, H. 1970. Purification and assay of thermolysin. Methods Enzymol. 19:642-651. 12. Poincelot, R. P. 1974. A scientific examination of the
APPL. ENVIRON. MICROBIOL. principles and practice of composting. Compost Sci. 15:24-31. 13. Rose, W., J. Chapman, S. Roseid, A. Katsuyama, U. Porter, and W. Mercer. 1965. Composting fruit and vegetable refuse. Compost Sci. 6:13-25. 14. Satriana, M. J. 1974. Large scale composting, p. 167. Noyes Data Corp., Park Ridge, N.J. 15. Stutzenberger, F. J. 1971. Cellulase production by Thermomonospora curvata isolated from municipal solid waste compost. Appl. Microbiol. 22:147-152. 16. Stutzenberger, F. J. 1972. Cellulolytic activity of Thermomonospora curvata: optimal assay conditions, partial purification, and product of the cellulase. Appl. Microbiol. 24:83-90. 17. Stutzenberger, F. J., A. J. Kaufman, and R. D. Lossin. 1970. Cellulolytic acitvity in municipal solid waste composting. Can. J. Microbiol. 16:553-560. 18. Toth, S. J. 1968. Chemical composition of seven garbage composts produced in the United States. Compost Sci. 9:27-28. 19. Upton, M. E., and W. M. Fogarty. 1977. Production and purification of thermostable amylase and protease of Thermomonospora viridis. Appl. Environ. Microbiol. 33:59-64. 20. Yasunobu, K. T., and J. McConn. 1970. Bacillus subtilis neutral protease. Methods Enzymol. 19:569-575.
0099-2240/78/0036-0201$02.00/0
Copyright i) 1978 American Society for Microbiology
Vol. 36, No. 1
Printed in U.S.A.
Effect of Municipal Refuse Metals on Cellulase Production by Thermomonospora curvata FRED STUTZENBERGER'* AND ION STERPU2 Clemson University, Clemson, South Carolina 29631,' and Research Institute for Food Industry and Chemistry, Bucharest, Romania2 Received for publication 8 February 1978
The high-concentration metals in municipal refuse compost were tested for effects on cellulase production and activity in Thermomonospora curvata. Although none altered cellulase reaction rates, both Al and Ca appeared to specifically inhibit cellulase production.
Refuse composting has been defined as the aerobic, thermophilic degradation of putrescible material in refuse by microorganisms (14). The temperature profile of the compositing mass passes through three general stages: (i) an initial stage in which the temperature is brought from ambient to about 400C by the metabolic activities of mesophilic fungi and bacteria; (ii) a stage in which the temperature peaks at 65 to 700C and the flora consist mostly of thermophilic actinomycetes and fungi; (iii) a stage of temperature decline during which the mesophiles again gain predominance. The thermophilic stage is considered the most important because most degradation of raw materials such as cellulose occurs during that time. In this stage, actinomycete populations often reach 108 colony-forming units per g of wet compost (12). One of these actinomycetes, Thermomonospora curvata, is the predominant actinomycete in mushroom compost (4) and is also a common isolate from municipal refuse compost (17). T. curvata produces both Cl and C. cellulases (15, 16) and probably plays a major role in cellulose degradation during composting. Because cellulose constitutes about one-half of the dry weight of municipal refuse compost (14), cellulase production by the indigenous thermophilic flora greatly influences the conversion rate of raw refuse to a stable product. In the large-scale composting of municipal refuse or vegetable- and fruit-canning wastes, calcium oxide or calcium sulfate is often added to the raw mass to raise the pH (initially as low as 4.5) in an attempt to speed the process (13, 14). For this and other reasons, the Ca content of the compost ranges from 1.3 to 5.5% on a dry weight basis (18). A detailed report (14) revealed that in addition to Ca, the metals Mg, Al, and Fe were also usually in high concentrations (1 to 2% of dry weight). Relatively high concentrations of Ca stimulate induction of cellulase pro-
duction in the fungus Trichoderma viride (8), and several microbial cellulases appear to be activated or stabilized by the presence of metals such as Fe (9). Consequently, we studied these common municipal refuse metals as to their influence on growth of T. curvata and its cellulase production, reaction rate, and thermal stability. The minimal medium employed for cellulase production was as follows: (NH4)2SO4, 0.1%; MgCl2 6H20, 0.01%; phosphate buffer, 0.1 M; ground surgical cotton (40 mesh, Johnson and Johnson), 0.8%. Final pH after autoclaving was 7.6 to 7.8. Sterile-filtered solutions of biotin and thiamine (Sigma Chemical Co.) were added aseptically (final concentrations of 0.5 jig/ml) to the cooled flasks. The metals used in this study were sterilized separately as solutions of their chloride salts and added aseptically to the mineral salts medium. Initial concentrations of the metals in the medium were determined by atomic absorption spectrophotometry in a Perkin-Elmer model 403 equipped with a heatedgraphite atomizer. T. curvata cultures (100 ml) were grown at 520C in 250-ml Erlenmeyer flasks on a New Brunswick gyratory shaker at 200 rpm. Under these conditions, cellulase activity reached a maximum in 5 to 6 days and then declined on further incubation. Cl and C. cellulase activities were measured as previously described (16). Enzyme activity units were calculated as micromoles of reducing sugar (as glucose) released per minute. Protein was estimated by the method of Lowry et al. (7) and by the Bio-Rad dye binding method (Bio-Rad Laboratories technical bulletin 1051, 1977). Cellulases were purified as previously described (16) except for the following modifications. Crude culture fluids were concentrated about 20-fold by ultrafiltration on an Amicon PM-10 membrane before the ammonium sulfate precipitation step. The Corning controlled-pore
201
202
APPL. ENVIRON. MICROBIOL.
NOTES
glass beads were replaced with Bio-Gel P-200 polyacrylamide beads (Bio-Rad Laboratories) in a Glenco column (1.5 by 28 cm) run at room temperature, collecting 3-ml fractions. The eluting buffer was 0.01 M phosphate (pH 7.5), with 0.1 M NaCl and 0.02% NaN3 as a preservative. Polyvalent cations such as A13+ produce marked changes in the electrokinetic potential of proteins (2). Such changes may influence both thermal stability and activity of extracellular enzymes. A13+ (10-5 to 10-3 M) in T. curvata cultures inhibited cellulase production without a proportional decrease in extracellular protein accumulation (Fig. 1). Cellulase activity in cultures with 1 mM A13+ was decreased to about half that of the controls. However, A13+ had no detectable effect on the therrnal stability (at either 52 or 65°C) of either crude or purified cellulase. Because Mg, Fe, and Ca are considered macronutrient elements in microbial metabolism, we tested effects of these metals on the growth of T. curvata under conditions where cellulase production was not essential (cellulose was replaced by 0.5% cellobiose). The results were then compared with those obtained under conditions where cellulase production was essential for growth (cotton fiber cellulose was the sole carbon and energy source). These three metals differed markedly as to their comparative effects on growth versus cellulase production. Mg concentrations of 1o-4 to 10-2 M provided a broad optimum range both for growth on cellobiose and for cellulase production on cellulose.
There was no detectable growth or cellulase production below 5 x 106 M or above 5 x 10-2 M Mg. Fe has very low solubility at neutral pH in the presence of oxygen because it forms a highly insoluble ferric hydroxide (1). In our studies, precipitation of Fe compounds was evident in the medium at concentrations above 10-5 M. Microscopic examination of cultures revealed that the mycelial strands of T. curvata adhered to the surfaces of the precipitate. Cellulase and protein production were decreased about 50% at 103 M Fe and completely inhibited at 2 x 10'M. Ca had the most unexpected effect on T. curvata. In cellobiose-mineral salts medium, where cellulase production is poorly induced, increasing the Ca concentration from 1.8 x 10-5 M (the trace Ca contamination in the medium) to 2 x 10'3 M (the limit of solubility in the medium) resulted in stimulation of growth (Fig. 2). However, in cellulose-mineral salts medium, the optimum Ca concentration for cellulase production was in the range of 0.1 to 0.6 mM. Higher concentrations up to 1.8 mM inhibited extracellular cellulase and protein production (Fig. 3). This finding contrasted with earlier studies (3, 8) on fungi and actinomycetes which employed Ca concentrations of 2 to 3 mM as optimum for cellulase production in similar mineral salts media. The effect of metals on T. curvata indicates
f I.-
,0
O 000 1
-J0
o
so.
> ooff-f U *g 100
/~~~~/
w
u
0
o~~0z X0SO ' -o-C, X --Cx r ** X
I
o-so ^
o
[AI'J (mol-s/Iit.r ! FIG. 1. Effect of Al on cellulase and soluble-protein production by T. curvata. Each point is the average of three independent experiments.
I
no1
I
I
io2
MOLAR CA CONC. FIG. 2. Effect of Ca concentration on cell yield of T. curvata cultures in cellobiose-mineral salts medium. Dry cell weight measurements were made after 48 h of incubation under conditions described in the text. Each point is the average of values from duplicate flasks.
VOL. 36, 1978
NOTES
203
100-
2 I
zt
-I
0~~ u
a
0.5_
J50
oo x
_
I 0O u0N
10
~0 10~~~s
1o~1 0
MOLAR CA CONC. FIG. 3. Effect of Ca concentration on production of cell-free C, (0), C. (A), and protein (O) by T. curvata in cellulose-mineral salts medium. Data are expressed as the peak values attained at each Ca concentration during a 6-day incubation. Each point is the average of values from three separate experiments.
that the high metal content of municipal solid waste might be detrimental to the rate of cellulase production in the composting process. In particular, the addition of more Ca as lime or limestone would seem to be of no advantage despite the transient pH increase because such addition also results in loss of nitrogen and a lower-quality compost (14). If a pH rise of composting material is required, the addition of urea (which has been used to advantage in fruit waste compost [13]) would be more desirable. The inhibitory effect of Ca at millimolar concentrations seemed directed at cellulase production rather than growth; therefore, we tested the possibility that Ca decreased the thermal stability of the cellulase in the culture fluids to cause inactivation soon after release from the cells. However, the cellulase was completely stable at 520C for at least 30 h in culture fluids with no added Ca or with Ca concentrations of 0.1 to 1.0 mM. Furthermore, at 650C (the temperature of compost during peak heating) Ca protected the cellulase against thermal inactivation (Fig. 4). This relative degree of protection was observed in both crude and purified cellulase preparations. All four metals were also tested at 0.1 and 1.0 mM concentrations as to their effects on cellulase reaction rates. No significant changes
1
2 3 HOURS
4
5
FIG. 4. Effect of Ca on the thermal stability of cellulase in culture fluids at pH 7.5 and 65°C. The initial activity of each sample before heating was assigned an arbitrary value of 1.0. Symbols: C, control (E); C, with I mM Ca (0); C, control (0); C, with 1 mM Ca (A). Each point is the average of values from two separate experiments.
were observed, at either concentration of any metal, in either the C, or C. assays with purified cellulase. Studies are continuing as to why Ca concentrations which stimulate growth on cellobiose are inhibitory to cellulase production on cellulose. One possibility is that millimolar Ca concentrations stabilize an extracellular protease which degrades the cellulase. Some microbial proteases require 2 mM Ca for stability (11, 20), and a relationship between protease production and cellulase inactivation has been suggested for Trichoderma (10). Protease production has been reported for T. viridis (19); if such is the case in T. curvata, it could explain the decline of cellulase activity in older cultures despite its thermal stability. This research was supported by grant no. 13538L from the U.S. Army Research Office. We are indebted to the international Research and Exchanges Board for the sponsorship of I.S. LITERATURE CITED 1. Brock, T. D. 1970. Biology of microorganisms, p. 145. Prentice-Hall, Inc., Englewood Cliffs, N.J. 2. Dixon, M., and E. C. Webb. 1964. Enzymes, p. 429. Academic Press Inc., New York. 3. Enger, M. D., and B. P. Sleeper. 1965. Multiple cellulase system from Streptomyces antibioticus. J. Bacteriol. 89:23-27.
204
NOTES
4. Fergus, C. L. 1964. Thermophilic and thermotolerant molds and actinomycetes of mushroom compost during peak heating. Mycologia 56:267-284. 5. Hagerty, D. J., J. L. Pavoni, and J. E. Heer. 1973. Solid waste management. Van Nostrand Reinhold Co., New York. 6. Jeris, J., and R. Regan. 1973. Controlling environmental parameters for optimum composting. Compost Sci. 14:16-22. 7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1953. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. Mandels, M., and E. T. Reese. 1957. Induction of cellulase in Trichoderrma viride as influenced by carbon sources and metals. J. Bacteriol. 73:269-278. 9. Mandels, M., and E. T. Reese. 1963. Inhibition of cellulases and fi-glucosidases, p. 115-157. In E. T. Reese (ed.). Advances in enzymic hydrolysis of cellulose and related materials. Pergamon Press, Oxford. 10. Mandels, M., D. Steinberg, and R. E. Andreotti. 1975. Growth and cellulase production by Trichoderma. Symposium on Enzymatic Hydrolysis of Cellulose. Aulanko, Finland. 11. Matsubara, H. 1970. Purification and assay of thermolysin. Methods Enzymol. 19:642-651. 12. Poincelot, R. P. 1974. A scientific examination of the
APPL. ENVIRON. MICROBIOL. principles and practice of composting. Compost Sci. 15:24-31. 13. Rose, W., J. Chapman, S. Roseid, A. Katsuyama, U. Porter, and W. Mercer. 1965. Composting fruit and vegetable refuse. Compost Sci. 6:13-25. 14. Satriana, M. J. 1974. Large scale composting, p. 167. Noyes Data Corp., Park Ridge, N.J. 15. Stutzenberger, F. J. 1971. Cellulase production by Thermomonospora curvata isolated from municipal solid waste compost. Appl. Microbiol. 22:147-152. 16. Stutzenberger, F. J. 1972. Cellulolytic activity of Thermomonospora curvata: optimal assay conditions, partial purification, and product of the cellulase. Appl. Microbiol. 24:83-90. 17. Stutzenberger, F. J., A. J. Kaufman, and R. D. Lossin. 1970. Cellulolytic acitvity in municipal solid waste composting. Can. J. Microbiol. 16:553-560. 18. Toth, S. J. 1968. Chemical composition of seven garbage composts produced in the United States. Compost Sci. 9:27-28. 19. Upton, M. E., and W. M. Fogarty. 1977. Production and purification of thermostable amylase and protease of Thermomonospora viridis. Appl. Environ. Microbiol. 33:59-64. 20. Yasunobu, K. T., and J. McConn. 1970. Bacillus subtilis neutral protease. Methods Enzymol. 19:569-575.
