Zentralbl. Mikrobiol. 148 (1993),351-356 Gustav Fischer Verlag lena [Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia]
Oxytetracycline Formation in Blackstrap Molasses Medium by Streptomyces rimosus Oxytetracyclin-Produktion in einer "Schwarzstreifen Melasse" durch Streptomyces rimosus A. A. Abou-Zeid, J. A. Khan, K. O. Abulnaja Key words: Blackstrap-molasses-urea, KH zP0 4 , Oxytetracycline, Streptomyces rimosus
Summary Analyses of blackstrap molasses revealed that it contains many miscellaneous compounds in the form of monosaccharides such as glucose, fructose, arabinose and dissacharide such as sucrose and trisaccharid such as raffinose. It also contains some amino acids beside citric and aconitic acids and many elements such as sodium, potassium, magnesium and calcium. Utilization of urea as organic nitrogen source was more effective than (NH4lzS04 as inorganic nitrogen source for the oxytetracycline formation by Streptomyces rimosus. The suitable urea concentration was in the range of 1.5 mg/ml. The suitable KH zP0 4 concentration was also in the range of 1.5 mg/ml. Blackstrap molasses was better for the antibiotic formation than glucose as carbon source. The suitability may be attributed to its content of a balance of materials such as different sugars, minerals and amino and organic acids. Moreover, it is cheaper than other raw resources.
Zusammenfassung Analysen der "Schwarzstreifen-Melasse" zeigten, daB diese viele unterschiedliche Verbindungen enthalt, z. B. bei Monosacchariden Glucose, Fructose, Arabinose, bei Disacchariden Saccharose und bei Trisacchariden Raffinose. Sie enthalt weiterhin einige Aminosauren sowie Citronen- und Aconitsaure und mehrere Elemente wie Natrium, Kalium, Magnesium und Calcium. Die Verwertung von Harnstoff als organische N-Quelle war effektiver als die von (NH4lzS04 als anorganische Quelle in bezug auf die Oxytetracyclinproduktion durch Streptomyces rimosus. Die geeignete Hamstoffkonzentration lag im Bereich von 1,5 mg/ml, die geeignete KH zP04-Konzentration war ebenfalls 1,5 mg/ml. Die Melasse war fur die Antibiotikaproduktion geeigneter als Glucose. Dies beruht auf dem zusatzlichen Gehalt an verschiedenen Substanzen wie Zucker, Mineralstoffe, Aminosauren und organische Sauren, Dariiber hinaus ist sie billiger als andere Rohstoffquellen.
Introduction Oxytetracycline is broad-spectrum antibiotic discovered by Finlay et al. (1950). Since its discovery, many workers (Borenstajn and Wolf 1955; Zygmunt 1961, 1963; Orlova 1971, 1972; Baeva et al. 1978; Ettler and Cechner 1979; Semenova et al. 1982, 1983; Mandal et al. 1981, 1983; Makismova et al. 1982, 1988; Abou-Zeid et al. 1991) studied different factors affecting the fermentation process of oxytetracycline. One of the important factor was the ingredients of the fermentation medium utilized in the formation of the antibiotic. Blackstrap molasses is a renewable bioresource obtained from the sugar industry, the work reported in the present paper dealt with the utilization of blackstrap molasses and other ingredients for the fermentative formation of oxytetracycline by Streptomyces rimosus.
A. A. Abou-Zeid et al.
Material and methods Chemical analysis of blackstrap molasses: Sugars of blackstrap molasses were determined according to the colorimetric methods of Somogyi (1945) and Nelson (1944) and total nitrogen was determined by the method of Miller and Houghton (1945). The detection of amino acids present in blackstrap molasses was carried out by the method of Whitehead (1964). Identification of sugars was carried out by the method of Bourne et al. (1970). The developing solvent mixture was n-butanol-pyridine-water (6:4:3 by volume). The spraying agent was aniline hydrogen phthalate and alkaline silver nitrate reagents (Block et al. 1955). For comparison authentic samples of different sugars were also chromatographed. Sodium, potassium, magnesium, calcium, iron, lithium, strontium, barium, aluminium, copper, nickel, cobalt and lead were determined with an atomic absorption spectrophotometer, Model 5000 (Perkin Elmer, USA). Maintenance medium: An active strain of Streptomyces rimosus producing oxytetracycline was maintained on the medium containing the following ingredients (g litre""): glucose, 10.0; peptone, 5.0; KH zP0 4 , 1.0; agar, 20.0; distilled water to 1000 ml. The initial pH of the medium was adjusted to 6.5. The medium was dispensed in 5 ml amounts into test tubes and the tubes were plugged with cotton and sterilized at 120°C for 15 min. The slants were inoculated with the experimental organism under aseptic conditions and incubated at 27°C for 10 days to obtain luxuriant growth and sporulation, and then kept in a refrigerator at 5°C. Vegetative medium: The vegetative medium used for growing the active strain of Streptomyces rimosus contained the following ingredients (g litre-I): glucose, 10.0; peptone, 9.0; yeast extract, 5.0; KH zP0 4 , 1.0; distilled water to 1000 ml. The initial pH of the medium was adjusted to 6.5. The medium was portioned into Erlenmeyer flasks (100 ml capacity), each contained 20 ml. The flasks were plugged with cotton and sterilized at 120°C for 15 min. When the flasks attained room temperature, they were inoculated with the active strain of S. rimosus maintained in slants (one slant/flask) under aseptic conditions. The inoculated flasks were inserted on a shaker (200 rpm) at 30°C for 72 h in order to obtain good growth. The growing strain was used as inoculum for the fermentation medium. Fermentation medium: The fermentation medium contained the following ingredients (g litre-I): glucose, 20.0; (NH4)zS04, 2.0; KH zP04 , 1.0; MgS0 4' 7H zO, 0.5; MnS04 '4H zO, 0.05; FeS04' 7H zO, 0.005; distilled water to 1000 ml. The initial pH of the medium was adjusted to 6.5. The medium was portioned into 250 ml Erlenmeyer flasks, each containing 50 ml. The flasks were plugged with cotton and sterilized at 120°C for 15 min. The flasks were inoculated with standard inoculum of Streptomyces rimosum. The percentage of inoculum was 1.0. The inoculated flasks were inserted on a shaker (200 rpm) at 27°C for 120 h. At the end of the fermentation period, oxytetracycline was determined according to the method of Agrawal and Dutta (1962). The final pH of the fermented medium was also determined by pH-meter (Corning Scientific Instruments, Model 12). The microbial biomass was determined after centrifugation, and it was dried at 100°C till constant weight. For further studies, (NH4hS04, KH zP0 4 and glucose of the synthetic fermentation medium were replaced by different concentrations of urea, KHzP04 and blackstrap molasses to investigate their potencies for the biosynthetic formation of the antibiotic.
Results and Discussion Chemical analysis of blackstrap molasses The results obtained (Table 1- 3) show that blackstrap molasses contains miscellaneous compounds. It contains 50 % total sugars. The major sugar is sucrose which represents 32 %. The total nitrogen is 0.88 %. It is small compared with the percentage of sugars. The ash content of blackstrap molasses is 8.0 % . Paper chromatographic analysis revealed that the main sugars of blackstrap molasses were: arabinose, fructose, glucose, raffinose and sucrose, while the amino acids were: alanine, aspartic acid, cystine, glutamic acid, glycine, histidine, leucine, lysine, methionine, proline, serine, threonine, tyrosine and valine. Aconitic, citric and malic acids were also present in blackstrap molasses. The elemental analysis of blackstrap molasses ash indicated the presence of the following elements: Na, K, Mg, Li, Sr, AI, Cu and Pb, while Fe, Ba, Ni and Co were not detected.
Oxytetracycline Formation in Molasses
Table I. Chemical constituents of blackstrap molasses Constituents
Moisture Total sugars Sucrose Reducing sugars Total nitrogen Ash
22.30 50.00 32.00 18.00 0.88 8.00
Table 2. Paper chromatographic detection of sugars, amino acids and organic acids present in blackstrap molasses
Table 3. Elemental analysis of blackstrap molasses ash
Arabinose Fructose Glucose Raffinose Sucrose
Alanine Aspartic acid Cystine Glutamic acid Glycine Histidine Leucine Lysine Methionine Proline Serine Threonine Tyrosine Valine
Aconitic acid Citric acid Malic acid
Na K Mg Ca Fe Li Sr Ba Al Cu Ni Co Pb
0.24 3.46 0.28 1.49 trace trace trace trace
- Not detected
Table 4. Influence of different urea concentrations as organic nitrogen source on the oxytetracycline (OTC) formation by Streptomyces rimosus Urea (mg/ml)
Control** 0.5 1.0 1.5 2.0 2.5 3.0 3.5
6.0 8.0 9.1 8.1 8.5 8.6 8.7 9.0
3.5 5.0 5.5 3.5 3.0 2.6 2.7 2.4
187 250 270 310 270 210 200 150
* The initial pH of the medium was 6.5. ** Control = synthetic medium containing (NH4hS04 as inorganic nitrogen source.
The results shown in Table 4 represent utilization of different urea concentrations instead of (NH4hS04 as organic nitrogen source in the oxytetracycline formation by Streptomyces rimosus. The results indicated that urea was good nitrogen source for the antibiotic formation. The antibiotic yield was increased with the increase of urea concentration reaching its
A . A . Abou-Ze id et al.
maximum at 1.5 mg/ml, above which a decrease in the antibiotic was recorded. The final pH of the fermented medium was increased with the increase of urea concentration . The increase of pH value of the fermented medium was attributed to the liberation of ammonia during its utilization as nitrogen source. The increase of pH value led to decreases in the microbial biomass and antibiotic output. Table 5. Influence of different KHzP0 4 concentrations on the oxytetracycli ne (OTC) formation by Streptomyces rimosus
1.0 1.5 2.0 3.0
Dry biomass (mg/ml)
6.7 8.0 7.5 6.5 6.4 6.5
2.5 3.5 5.7 5.5 5.0 4.5
110 250 320 340 300 280
* The initial pH of the medium was 6.5. ** The synthetic medium was depleted from KH zP0 4 •
The results obtained (Table 5), show that the antibiotic yield was also increased with the increase of KH zP04 concentration reaching its maximum at 1.5 mg/ml, above which a decrease in the antibiotic output was recorded. When the synthetic medium was depleted from KH zP04 the antibiotic was sharply decreased, but the antibiotic was produced due to the presence of phosphorus as impurities in the different ingredients of the fermentation medium. When the medium was supplemented with phosphorus in the form of KH zP04 , the antibiotic yield was increased due to the importance of phosphate in the phosphorylation reactions carried out during the biosynthetic formation of oxytetracycline. Table 6. Influence of different blackstrap molasses concentrations replac ing glucose on the oxytetracycl ine (OTC) formation by Streptomyces rimosus Blackstrap molasses sugars (mg/m!)
Dry biomass (mg/ml)
10 15 20 25 30 35 40
8.3 8.4 7.7 7.5 7.8 7.2 7.0
2.5 3.5 4 .0 4 .3 4.5 5.7 6.0
260 370 450 660 520 490 480
* The initial pH of the medium was 6.5 .
The results presented in Table 6 show that the antibiotic yield was increased with the increase of blackstrap sugar concentration reaching its maximum at 25 mg/ml, above which a decrease in the antibiotic titre was recorded . The decrease may be correlated to the presence of minerals of blackstrap molasses. Blackstrap molasses not only supplied the fermentation
Oxytetracycline Formation in Molasses
process with carbon source , but also it supplied the medium with nitrogen source in the form of amino acids and other nitrogen sources . From the analytical point of view , blackstrap molasses contained miscellaneous compounds, especially sugars in the form of sucrose , glucose , fructose, raffinose and arabinose which were utilized by the experimental organism, and amino acids namely alanine, aspartic acid , cystine , glutamic acid, glycine , histidine, leucine , lysine , methionine, proline, serine , threonine , tyrosine and valine. The presence ofthese amino acids in the medium initiated the organism to give high titres ofthe antibiotics (Zygmunt 1963 and AbouZeid et al. 1991). Blackstrap molasses could supply the fermentation process with several elements such as sodium , potassium , magnesium and calcium . From the economical point of view, when blackstrap molasses was used in the fermentative production of oxytetracycline, it could be cheaper than the other substrates . The value of blackstrap molasses lies in its content of a balance of materials (minerals, sugars, amino and organic acids).
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Whitehead, R. G.: Rapid determination of plasma amino acids in subclinical K washioker. Lancet. 1 (1946), 250. Zygmunt, W. A.: Oxytetracycline formation by Streptomyces rimosus in chemically defined medium. Appl. Microbiol. 9 (1961), 502. Zygmunt, W. A.: Stimulation of oxytetracycline formation by N-acetyl derivatives of certain amino acids. Nature, 198 (1963), 289. Authors' address: Abou-Zeid A. Abou-Zeid, Jalaleldin A. Khan, Khalid O. Abulnaja, Biochemistry Department, Faculty of Science, P. O. Box No. 9028, King Abdulaziz University, Jeddah-2l413, Saudi Arabia.