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Antibiotics production of cellulosic waste with solid state fermentation by Streptomyces
WREC 1996
ANTIBIOTICS
PRODUCTION
OF CELLULOSIC
FERMENTATION
BY
WASTE
WITH
SOLID
STATE
STREPTOMYCES
S. S. YANG
Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 10617, Republic of China
ABSTRACT
Cellulosic waste, corncob, was used as a substrate in the production of oxytetracycline by Strepfomyces rimosus TM-55 in solid state fermentation. Oxytetracycline was detected on the fourth day, and reached its maximum on the eighth day. During cultivation, the moisture content of substrate increased as incubation being, and pH value increased slightly. Optimal conditions for oxytetracycline production were an initial pH of 5.2 to 6.3, an initial moisture content of 64 to 67%, supplemented with 20% (w/w) rice bran or 1.5 to 2.5% (NH&SO, as the sole nitrogen source, 1.0% CaCO,, 2% MgSOi7H,O, 0.5% RI-&PO.,, and 0.6 to 0.8% aspartic acid or lysine, with incubation for 8 days at 25 to 30°C. Each gram of substrate produced 10 to 11 mg of oxytetracycline.
KEYWORDS
Antibiotic
production;
solid state fermentation;
cellulosic waste; Strepiomyces; rice bran
INTRODUCTION
Solid state fermentation is characterized by low-moisture environments, often in natural, non-aseptic conditions, as in the production of Oriental fermented foods. It also has high product concentrations and relatively low energy requirements (Mudgett ef al., 1982; Yang and Yuan, 1990). Tetracyclines are board-spectrum antibiotics and are used in medicine, industry and agriculture. They are normally produced by submerged culture (Bhatnagar et al., 1988). Cellulosic materials are abundantly available globally and can be used by a number of microorganisms. In order to be economically competitive, the bioconversion of renewable cellulosic materials into antibiotics with solid state fermentation is investigated in this paper.
MATERIALS
AND METHODS
Corncob Corncob was purchased from the Farmers’ Association of Taiwan. It contains total carbohydrate, 32.5% crude flbre, 2.2% crude protein and 3.5% ash.
976
10.5% moisture,
48.9%
WREC 1996 Microorganisms Streptomyces rimosus TM-55 was provided by Dr. T. H. H. Ku (Cyanamid Taiwan Corporation,) was used for antibiotics production, Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity.
Culture Media and Conditions The basal solid medium contained (g): corncob, 100; nitrogen source [rice bran, 20; or (NH&SO,, 1.5 to 2.51; CaCO,, 1; MgS0,.7H,O, 2; and KH,PO,, 0.5. Each gram medium was completely mixed with 2.0 x 10’ spores. The initial moisture content was adjusted to 65%, and incubated at 26°C for 7 to 10 days.
Determination
of Antibiotic Activity
After fermentation, the culture mass was extracted with 4 vol. distilled water by shaking at room temperature for 5 min. Antimicrobial activity of the extract was measured by the paper disc method in Antibiotic medium 1 (Difco) at 30°C (Yang and Ling, 1989). Total oxytetracycline equivalent potency was calculated from the clear zones, using a standard curve for oxytetracycline in the range of 1 pg ml-’ to 10 mg ml-‘.
RESULTS Mechanism
of Oxytetracycline
Secretion
The time course of oxytetracycline production and substrate pH in solid state fermentation with 20% rice bran as sole nitrogen source is shown in Fig. 1. During fermentation, oxytetracycline was first detected on day 4, reached maximal yield of 6.5 mg (g substrate)’ on day 8 and decreased gradually thereafter. The moisture content of substrate increased gradually over the first 20 days and then remained approximately constant. The substrate pH value also increased from 6 to 7.2 during this incubation.
6
7
= =
6
60 0
4
6
16
I6
20
64
66
Time (day) Fig. 1. Time course of oxytetracycline A - A Oxytetracycline
production in solid state fermentation ?? - fl Moisture content 977
of corncob. 0 - o Substrate pH
WFEC 1996 Culture Conditions The effect of initial moisture content of substrate on oxytetracycline production is summaried in Table 1. Oxytetracycline production increased with an increase initial moisture content ranging from 40 to 65%. When the initial moisture content was less than 45%, oxytetracycline production was low as the substrate was too dry to cell growth and antibiotic production. At initial moisture content 65%, water activity of the substrate was 0.96, and bulk densities on dry and wet weight bases were 0.09 and 0.28 g cm-‘, respectively. Each gram of dry substrate produced 6.8 mg of total oxytetracycline equivalent potency. At initial moisture content 75%, total oxytetracycline equivalent potency was only 1.2 mg (g substrate)‘. The optimal initial pH value for oxytetracycline production ranged between 5.2 and 6.3, and the optimal incubation temperature was between 25 and 30°C. Oxytetracycline production decreased significantly when the incubation temperature was higher than 40°C. Table 1. Effect fermentation
of
initial
moisture
content
on
oxytetracycline
production
in
solid
state
Moisture Content (%) Total oxytetracycline PH _______________________________________ _______ _______ ____________---_Equivalent Potency Initial
Final
Initial
Final
[mg (g substrate)‘]
40.0 45.0 50.0 56.4 59.5 65.3 69.9 75.0
52.5 56.0 60.2 61.0 63.5 67.9 73.7 76.1
6.1 6.1 6.1 6.1 6.1 6.2 6.1 6.2
6.3 6.5 6.6 6.6 6.8 6.8 6.9 6.7
0.2 1.2 2.7 5.6 6.8 5.9 2.4
0.1
_________________________________________~_______~~~______~~_______~~______~~~~_____~~______ The culture medium was corncob 100 g, rice bran 20 g, MgS0,.7H,O 2 g, CaCO, 1 g, and I&PO, 1 g. Each gram of medium was mixed with 2.0 x 10’ spores of S. rimosus TM-55 and incubated at 26°C for 8 days. The data were the average of triplications.
Nitrogen Source Corncob contains 48.9% carbohydrate and 2.2% crude protein. The C/N ratio of about 60 was too high for cell growth and oxytetracycline production. Therefore, a nitrogen source supplement was necessary. All nitrogen sources enhanced oxytetracycline production. (NH,),SO, was the best inorganic nitrogen source. Each gram of dry substrate produced 5.9 mg total oxytetracycline equivalent potency. NH&I was the next (3.9 mg), then urea (3.5 mg), and NH,NO, (3.3 mg). Oxytetracycline production had a high value with 1.5 to 2.5% of (NH&SO,, and decreased when the concentration of (NH&GO, was higher than 5.0% or less than 1.0%. Each gram of dry substrate produced 5.2 to 5.9 mg total oxytetracycline equivalent potency for 1.5 to 2.5% of (NH&SO, supplementation. The utilization of agricultural waste, an organic nitrogen source was used to replace the inorganic nitrogen source. Rice bran at 20% yield the best enhancement. Each gram of dry substrate produced 6.7 mg total oxytetracycline equivalent potency. Wheat bran was the next (6.0 mg), then molasses (3.2 mg) and peanut meal residue (0.5 mg). Oxytetracycline production had high value with 20 to 60% of rice bran. Combination of rice bran 20% and (NH,),SO, 2% slightly stimulated oxytetracycline production. Each gram of dry substrate produced 7.4 mg total oxytetracycline equivalent potency.
978
WC
1996
Addition of Other Chemicals KH,PO, regulated the substrate pH and stimulated the oxytetracycline production. 0.5% had the highest potency. Addition of 1% CaCO, or 2% oyster shell powder, and 2.0 to 2.5% MgS0,.7Hz0 had the maximal production: 8.6 mg of oxytetracycline. Supplementation of 0.6% of lysine, aspartic acid, production 40 to 105%, whereas methionine, asparagine, and histidine stimulated oxytetracycline tryptophane, tyrosine, threoine, cystine, serine, phenylalanine, proline, valine, and isoleucine inhibited oxytetracycline production 17 to 87%. Each gram of dry substrate produced 11 mg total oxytetracycline equivalent potency for 0.6% of lysine addition.
DISCUSSION Oxytetracycline in Streptomyces is a secondary metabolite, synthesizes and secretes in the late log phase or in the stationary phase. In submerged fermentation, antibiotic activity sharply decreased after prolonged incubation due to cell autolysis (Yang and Ling, 1989). During solid state fermentation, moisture content of substrate increased, which might be due to the production of metabolic water of Streptomyces. Supplementation with 1.5 to 2.5% of (NH,),SO, stimulated oxytetracycline production, whereas 5.0% was inhibition. High concentration of nitrogen source also inhibited l3-lactam production in Cephalosporium acremonium (Shen et al., 1987) and spiramycin production in S. ambofaciens (Ahmed et al., 1987). Combination nitrogen sources of rice bran and ammonium sulfate slightly enhanced oxytetracycline production of corncob in this study, as also observed in sweet potato residue with S. rimosus (Yang and Yuan, 1990). The product of solid state fermentation was more stable than that input was also less (Yang and Ling, 1989). The product has the stored without losing activity significantly. Hence, it is concluded fermentation of renewable cellulosic materials as an economic pharmaceuticals and agricultural chemicals.
in submerged culture, and the energy advantage that it can be temporarily that it is feasible to apply solid state alternative in producing value-added
ACKNOWLEDGEMENTS The author thanks Dr. W. J. Swei for her experimental assistance and the National of the Republic of China for financial support (NSC 83-0406-E002-003).
Science
Council
REFERENCES Ahmed, L., P. Germain and G. Lefebvre (1987). Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus. Appl. Microbial. Biotechnol., 26, 130-135. Bhatnagar, R. K., J. L. Doull and L. C. Vining (1988). Role of the carbon source in regulating chloramphenicol production by Streptomyces venezulae: studies in batch and continuous cultures. Can. J. Microbial., 34, 1217-1233. Mudgett, R. E., J. Nash and R. Rufner (1982). Controlled gas environments in solid state fermentations, Develop. Indust. Microbial., 23, 397-405. Shen, Y. Q., J. Heim, N. A. Solomon, S. Wolfe and A. L. Demain (1984). Repression of beta-lactam production in Cephalosporium acremonium by nitrogen sources, J. Antibiotics, 5, 503-5 11. Yang, S. S. and M. Y. Ling (1989). Tetracycline production with sweet potato residue by solid state fermentation. Biotechnol. Bioeng., 33, 1021-1028. Yang, S. S. and S. S. Yuan (1990). Oxytetracycline production by Streptomyces rimosus in solid state fermentation of sweet potato residue. World J. Microbial. Biotechnol., 6, 236-244. 979
ANTIBIOTICS
PRODUCTION
OF CELLULOSIC
FERMENTATION
BY
WASTE
WITH
SOLID
STATE
STREPTOMYCES
S. S. YANG
Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 10617, Republic of China
ABSTRACT
Cellulosic waste, corncob, was used as a substrate in the production of oxytetracycline by Strepfomyces rimosus TM-55 in solid state fermentation. Oxytetracycline was detected on the fourth day, and reached its maximum on the eighth day. During cultivation, the moisture content of substrate increased as incubation being, and pH value increased slightly. Optimal conditions for oxytetracycline production were an initial pH of 5.2 to 6.3, an initial moisture content of 64 to 67%, supplemented with 20% (w/w) rice bran or 1.5 to 2.5% (NH&SO, as the sole nitrogen source, 1.0% CaCO,, 2% MgSOi7H,O, 0.5% RI-&PO.,, and 0.6 to 0.8% aspartic acid or lysine, with incubation for 8 days at 25 to 30°C. Each gram of substrate produced 10 to 11 mg of oxytetracycline.
KEYWORDS
Antibiotic
production;
solid state fermentation;
cellulosic waste; Strepiomyces; rice bran
INTRODUCTION
Solid state fermentation is characterized by low-moisture environments, often in natural, non-aseptic conditions, as in the production of Oriental fermented foods. It also has high product concentrations and relatively low energy requirements (Mudgett ef al., 1982; Yang and Yuan, 1990). Tetracyclines are board-spectrum antibiotics and are used in medicine, industry and agriculture. They are normally produced by submerged culture (Bhatnagar et al., 1988). Cellulosic materials are abundantly available globally and can be used by a number of microorganisms. In order to be economically competitive, the bioconversion of renewable cellulosic materials into antibiotics with solid state fermentation is investigated in this paper.
MATERIALS
AND METHODS
Corncob Corncob was purchased from the Farmers’ Association of Taiwan. It contains total carbohydrate, 32.5% crude flbre, 2.2% crude protein and 3.5% ash.
976
10.5% moisture,
48.9%
WREC 1996 Microorganisms Streptomyces rimosus TM-55 was provided by Dr. T. H. H. Ku (Cyanamid Taiwan Corporation,) was used for antibiotics production, Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity.
Culture Media and Conditions The basal solid medium contained (g): corncob, 100; nitrogen source [rice bran, 20; or (NH&SO,, 1.5 to 2.51; CaCO,, 1; MgS0,.7H,O, 2; and KH,PO,, 0.5. Each gram medium was completely mixed with 2.0 x 10’ spores. The initial moisture content was adjusted to 65%, and incubated at 26°C for 7 to 10 days.
Determination
of Antibiotic Activity
After fermentation, the culture mass was extracted with 4 vol. distilled water by shaking at room temperature for 5 min. Antimicrobial activity of the extract was measured by the paper disc method in Antibiotic medium 1 (Difco) at 30°C (Yang and Ling, 1989). Total oxytetracycline equivalent potency was calculated from the clear zones, using a standard curve for oxytetracycline in the range of 1 pg ml-’ to 10 mg ml-‘.
RESULTS Mechanism
of Oxytetracycline
Secretion
The time course of oxytetracycline production and substrate pH in solid state fermentation with 20% rice bran as sole nitrogen source is shown in Fig. 1. During fermentation, oxytetracycline was first detected on day 4, reached maximal yield of 6.5 mg (g substrate)’ on day 8 and decreased gradually thereafter. The moisture content of substrate increased gradually over the first 20 days and then remained approximately constant. The substrate pH value also increased from 6 to 7.2 during this incubation.
6
7
= =
6
60 0
4
6
16
I6
20
64
66
Time (day) Fig. 1. Time course of oxytetracycline A - A Oxytetracycline
production in solid state fermentation ?? - fl Moisture content 977
of corncob. 0 - o Substrate pH
WFEC 1996 Culture Conditions The effect of initial moisture content of substrate on oxytetracycline production is summaried in Table 1. Oxytetracycline production increased with an increase initial moisture content ranging from 40 to 65%. When the initial moisture content was less than 45%, oxytetracycline production was low as the substrate was too dry to cell growth and antibiotic production. At initial moisture content 65%, water activity of the substrate was 0.96, and bulk densities on dry and wet weight bases were 0.09 and 0.28 g cm-‘, respectively. Each gram of dry substrate produced 6.8 mg of total oxytetracycline equivalent potency. At initial moisture content 75%, total oxytetracycline equivalent potency was only 1.2 mg (g substrate)‘. The optimal initial pH value for oxytetracycline production ranged between 5.2 and 6.3, and the optimal incubation temperature was between 25 and 30°C. Oxytetracycline production decreased significantly when the incubation temperature was higher than 40°C. Table 1. Effect fermentation
of
initial
moisture
content
on
oxytetracycline
production
in
solid
state
Moisture Content (%) Total oxytetracycline PH _______________________________________ _______ _______ ____________---_Equivalent Potency Initial
Final
Initial
Final
[mg (g substrate)‘]
40.0 45.0 50.0 56.4 59.5 65.3 69.9 75.0
52.5 56.0 60.2 61.0 63.5 67.9 73.7 76.1
6.1 6.1 6.1 6.1 6.1 6.2 6.1 6.2
6.3 6.5 6.6 6.6 6.8 6.8 6.9 6.7
0.2 1.2 2.7 5.6 6.8 5.9 2.4
0.1
_________________________________________~_______~~~______~~_______~~______~~~~_____~~______ The culture medium was corncob 100 g, rice bran 20 g, MgS0,.7H,O 2 g, CaCO, 1 g, and I&PO, 1 g. Each gram of medium was mixed with 2.0 x 10’ spores of S. rimosus TM-55 and incubated at 26°C for 8 days. The data were the average of triplications.
Nitrogen Source Corncob contains 48.9% carbohydrate and 2.2% crude protein. The C/N ratio of about 60 was too high for cell growth and oxytetracycline production. Therefore, a nitrogen source supplement was necessary. All nitrogen sources enhanced oxytetracycline production. (NH,),SO, was the best inorganic nitrogen source. Each gram of dry substrate produced 5.9 mg total oxytetracycline equivalent potency. NH&I was the next (3.9 mg), then urea (3.5 mg), and NH,NO, (3.3 mg). Oxytetracycline production had a high value with 1.5 to 2.5% of (NH&SO,, and decreased when the concentration of (NH&GO, was higher than 5.0% or less than 1.0%. Each gram of dry substrate produced 5.2 to 5.9 mg total oxytetracycline equivalent potency for 1.5 to 2.5% of (NH&SO, supplementation. The utilization of agricultural waste, an organic nitrogen source was used to replace the inorganic nitrogen source. Rice bran at 20% yield the best enhancement. Each gram of dry substrate produced 6.7 mg total oxytetracycline equivalent potency. Wheat bran was the next (6.0 mg), then molasses (3.2 mg) and peanut meal residue (0.5 mg). Oxytetracycline production had high value with 20 to 60% of rice bran. Combination of rice bran 20% and (NH,),SO, 2% slightly stimulated oxytetracycline production. Each gram of dry substrate produced 7.4 mg total oxytetracycline equivalent potency.
978
WC
1996
Addition of Other Chemicals KH,PO, regulated the substrate pH and stimulated the oxytetracycline production. 0.5% had the highest potency. Addition of 1% CaCO, or 2% oyster shell powder, and 2.0 to 2.5% MgS0,.7Hz0 had the maximal production: 8.6 mg of oxytetracycline. Supplementation of 0.6% of lysine, aspartic acid, production 40 to 105%, whereas methionine, asparagine, and histidine stimulated oxytetracycline tryptophane, tyrosine, threoine, cystine, serine, phenylalanine, proline, valine, and isoleucine inhibited oxytetracycline production 17 to 87%. Each gram of dry substrate produced 11 mg total oxytetracycline equivalent potency for 0.6% of lysine addition.
DISCUSSION Oxytetracycline in Streptomyces is a secondary metabolite, synthesizes and secretes in the late log phase or in the stationary phase. In submerged fermentation, antibiotic activity sharply decreased after prolonged incubation due to cell autolysis (Yang and Ling, 1989). During solid state fermentation, moisture content of substrate increased, which might be due to the production of metabolic water of Streptomyces. Supplementation with 1.5 to 2.5% of (NH,),SO, stimulated oxytetracycline production, whereas 5.0% was inhibition. High concentration of nitrogen source also inhibited l3-lactam production in Cephalosporium acremonium (Shen et al., 1987) and spiramycin production in S. ambofaciens (Ahmed et al., 1987). Combination nitrogen sources of rice bran and ammonium sulfate slightly enhanced oxytetracycline production of corncob in this study, as also observed in sweet potato residue with S. rimosus (Yang and Yuan, 1990). The product of solid state fermentation was more stable than that input was also less (Yang and Ling, 1989). The product has the stored without losing activity significantly. Hence, it is concluded fermentation of renewable cellulosic materials as an economic pharmaceuticals and agricultural chemicals.
in submerged culture, and the energy advantage that it can be temporarily that it is feasible to apply solid state alternative in producing value-added
ACKNOWLEDGEMENTS The author thanks Dr. W. J. Swei for her experimental assistance and the National of the Republic of China for financial support (NSC 83-0406-E002-003).
Science
Council
REFERENCES Ahmed, L., P. Germain and G. Lefebvre (1987). Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus. Appl. Microbial. Biotechnol., 26, 130-135. Bhatnagar, R. K., J. L. Doull and L. C. Vining (1988). Role of the carbon source in regulating chloramphenicol production by Streptomyces venezulae: studies in batch and continuous cultures. Can. J. Microbial., 34, 1217-1233. Mudgett, R. E., J. Nash and R. Rufner (1982). Controlled gas environments in solid state fermentations, Develop. Indust. Microbial., 23, 397-405. Shen, Y. Q., J. Heim, N. A. Solomon, S. Wolfe and A. L. Demain (1984). Repression of beta-lactam production in Cephalosporium acremonium by nitrogen sources, J. Antibiotics, 5, 503-5 11. Yang, S. S. and M. Y. Ling (1989). Tetracycline production with sweet potato residue by solid state fermentation. Biotechnol. Bioeng., 33, 1021-1028. Yang, S. S. and S. S. Yuan (1990). Oxytetracycline production by Streptomyces rimosus in solid state fermentation of sweet potato residue. World J. Microbial. Biotechnol., 6, 236-244. 979