AGM 312 Microbial Biotechnology (1+1) Page 37 E xp er im en t 8 Production of antibiotics Date: Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, absence of secondary metabolites does not result in immediate death, but rather in a long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites often play an important role in defense against herbivory and other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs. Antibiotics (Greek word - antiviotika) also called antibacterials , are a type of antimicrobial drug used in the treatment and prevention of bacterial infections. They may either kill or inhibit the growth of bacteria. The term antibiotic was first used in 1942 by Selman Waksman and his collaborators in journal articles to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. Streptomycin is derived from the bacterium Streptomyces griseus . Streptomycin has three constituents, namely; N-methyl L-glucosamine, Streptose, and streptavidin. The present-day streptomycin producers are the mutants of Streptomyces derived for higher yields with yield as high as 25,000 units per ml. The industrial production of streptomycin is carried out using submerged fermentation processes. The fermentation media consists of glucose, starch, dextrin, soy meal, steep corn liquor, sodium sulfate. The streptomycin fermentation requires high aeration and agitation. The fermentation is carried out at 28-30ºC with pH maintained at 7.6-8 for good productivity. The fermentation lasts for 5-7 days with the yield of 1-3 g/L of the fermentation broth. Objective To mass multiply streptomycin producing organism Streptomyces griseus . Materials Required: Commercial production of streptomycin contains media with soybean meal, glucose, and sodium chloride. Kenknight’s Broth: Dextrose 1.0 g KH 2 PO 4 0.1 g NaNO 3 0.1 g
AGM 312 Microbial Biotechnology (1+1) Page 38 KCl 0.1g MgSO 4 0.1g Distilled water 1000 ml Procedure: The streptomycin fermentation proceeds through three phases. Fermentation : In the first phase, the organism produces proteases that digest the soybean meal and release ammonia and carbohydrates. These are utilized for increasing biomass. Glucose is slowly utilized, and the net production of streptomycin is low during this phase. The pH of the medium rises from 6.7 or 6.8 to 7.5 or higher. This phase lasts for 24h. The next phase is the idiophase or the stationary phase, during which maximum streptomycin (secondary metabolite) is produced. It ranges from 24h to 6-7 days. Rapid utilization of ammonia and glucose occurs with no mycelial growth, and pH during this phase remains relatively constant at 7.6 to 9.0. In the last phase (death phase) the sugars have been completely depleted in the medium, and streptomycin production ceases completely. The ammonia released due to the cell lysis raises medium pH. The fermentation broth is generally harvested before the last phase begins. Recovery of Streptomycin On completion of fermentation, the mycelium is separated from the broth by filtration. The remaining liquid is then percolated through cation exchange resin columns where streptomycin gets adsorbed and is finally eluted by washing with buffer as streptomycin sulfate. Further impurities are removed by treating it with sodium hypochlorite, EDTA, and activated carbon. The purified streptomycin sulfate solution is concentrated under vacuum and dried aseptically.
AGM 312 Microbial Biotechnology (1+1) Page 39 Flow chart for Streptomycin production Work Done: PHASE I • Initial fermentation phase - little streptomycin production • Rapid growth with production of mycelial biomass • Proteolytic enzyme activity of S. griseus relases ammonia and raised pH of media to 7.5 PHASE II • Additional production of mycelium occurs • Streptomycin accumulates in the medium • pH remains fairly constant ranging between 7.6 to 8.0 PHASE III • Final fermentation phase • Streptomycin production ceases and bacterial cells begin to lyse. • Ammonia from lysed cells increase the pH
AGM 312 Microbial Biotechnology (1+1) Page 40 Review Questions 1. Who discovered streptomycin? 2. What is the mode of action of streptomycin? 3. Write the range of the target organisms of streptomycin? 4. What are the other methods for testing the antibiotic activity of microorganisms? 5. What is MIC? 6. List the common solvents used for extraction in downstream processing.
AGM 312 Microbial Biotechnology (1+1) Page 41 7. Differentiate upstream and downstream processes. 8. Why is down streaming important? Reference Najafpour, Ghasem. (2007). Production of Antibiotics. In: Biochemical Engineering and Biotechnology. DOI: 10.1016/B978-044452845-2/50011-2.
AGM 312 Microbial Biotechnology (1+1) Page 42 E xp er im en t 9 Assay of amylase and antibiotics Date: Down streaming refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals such as antibiotics, hormones, etc. Among the three steps of any fermentation process viz., upstreaming, fermentation, and down streaming, down streaming is the most critical step involving the recovery of the product and decides the economy of the final product. Down streaming involves the following four steps: 1. Removal of insoluble is the first step and involves the capture of the product as a solute in a particulate-free liquid. Typical operations are flocculation, floatation, filtration, centrifugation, sedimentation, and precipitation. 2. Product isolation is the removal of those components whose properties vary markedly from that of the desired product. Eg. Solvent extraction, adsorption, ultrafiltration, and precipitation. 3. Product purification is the removal of those contaminants that resemble the product very closely in physical and chemical properties. Eg. Chromatography, crystallization, or fractional precipitation. 4. Product polishing describes the final processing step and ends with the packaging of the product in a form that is stable, transportable in a convenient way. ASSAY OF AMYLASE Objective To extract amylase enzyme and antibiotic streptomycin and test its activity. Principle: The reducing sugars produced by the action of α and β amylase react with dinitrosalicylic acid and reduce it to a brown colored product, nitroaminosalicylic acid. Materials: 1. Sodium acetate buffer, 0.1 M pH 4.7 2. Starch 1% solution (Prepare a fresh solution by dissolving 1 g starch in 100 ml acetate buffer, if necessary) 3. Dinitro salicylic Acid reagent (Dissolve by stirring 1g dinitrosalicylic acid, 200 mg crystalline phenol, and 50 mg sodium 42sulfite in 100 ml 1% NaOH. Store at 4ºC. 4. 40 % Rochelle salt solution
AGM 312 Microbial Biotechnology (1+1) Page 43 5. Maltose solution: Dissolve 50 mg maltose in 50 ml distilled water in a standard flask and store it in a refrigerator. 6. Extraction of Amylase Extract the sample material with 5-10 volumes of ice-cold 10mM calcium chloride solution overnight at 4ºC or for 3 h at room temperature. Centrifuge the extract at 54,000g at 4 ºC for 20 min. Procedure: Pipette out 1 ml of starch solution and 1ml of the adequately diluted enzyme in a test tube Incubate it at 27ºC for 15 min Stop the reaction by the addition of 2 ml of dintrosalicyclic acid reagent Heat the solution in a boiling water-bath for 5 min. While the tubes are warm, add 1 ml potassium sodium tartrate solution Then cool it in running tap water Make up the volume to 10ml by addition of 6 ml water Read the absorbance at 560 nm. Terminate the reaction at zero time in the control tubes. Prepare a standard graph with 0-100 µg maltose. Calculation: A unit of α or β amylase is expressed as mg maltose produced during 5 min incubation with 1% starch. Observation: Examine the plates for the starch hydrolysis around the line of growth of each organism, ie., the color change of the medium. Estimate the amylase production. Record the results in a table and discuss. II. Streptomycin assay Materials required 1. 10 days old culture of Streptomyces sp. grown in Ken knight‟s broth 2. Test organisms viz., Bacillus, E.coli and Xanthomonas (24 h old) 3. Solvent (Diethyl ether/ diethyl acetate) 4. Separating funnel 5. Sterile Petri plates 6. Micropipette (20-200 µl) 7. Sterile distilled water 8. Standard antibiotic (Streptomycin) 9. Ken knight‟s broth 10. Sterile cork borer
AGM 312 Microbial Biotechnology (1+1) Page 44 PROCEDURE: a. Extraction of antibiotic Prepare Ken knight‟s broth, sterilize, inoculate with Streptomyces sp. and incubate for 10 days (till it attains stationary phase to ensure antibiotic production) Filter the culture broth to separate the filtrate from the organism Take the filtrate containing the antibiotic (Streptomycin) in a separating funnel Add an equal volume of the solvent Diethyl ether (1:1) Mix them thoroughly by vigorous shaking for 15 -20 minutes. This is to extract and bring the antibiotic present in the filtrate completely into the solvent. Leave the funnel for about 5-10 min to separate the solvent (upper layer) and the solute phase (lower layer). Drain the solute phase at the bottom of the funnel and retain the solvent phase containing the antibiotic for testing its efficiency against different bacterial cultures. b. Testing the efficiency of the extracted antibiotic (Agar Well Diffusion Assay) Prepare sterile nutrient broth in three separate flasks and inoculate the test organisms viz., Bacillus, E.coli, and Xanthomonas Allow the organisms to grow for 24 h till they attain a log phase. Prepare sterile nutrient agar medium in three separate conical flasks. To seed the medium, inoculate the 24 h grown test cultures @ 1% separately into each flask containing the medium at bearable warmth condition. Swirl the medium for uniform mixing of the cultures. Pour the medium seeded with the test cultures immediately into sterile Petri plates, and allow for solidification. Label the respective plates. In each of the plates, make four small wells equidistant from one another using a sterile cork borer Add 20 µl of the extracted antibiotic into the first and second wells; 20 µl of standard antibiotic (streptomycin 50 ppm) into the 3 rd well (positive control) and 20 µl of sterile distilled water into the 4 th well which serves as the negative control. Incubate for about 2 days Observe the plates for the formation of inhibition zones produced around each well and measure the zone of inhibition. Record the results and interpret.
AGM 312 Microbial Biotechnology (1+1) Page 45 Work Done: Reference W. W. Windish and N. S. Mhatre, “Microbial amylases,” in Advances in Applied Microbiology, W. U. Wayne, Ed., pp. 273 – 304, Academic Press, New York, NY, USA, 1965. Kabay A. (1971). Rapid quantitative microbiological assay of antibiotics and chemical preservatives of a nonantibiotic nature. Applied microbiology , 22 (5), 752 – 755.