Industrial production of tetracycline antibiotics.

Since the discovery of tetracycline in 1940s, tetracycline has been a very important component in the treatment of various bacterial infections. Tetracycline belongs to the family of antibiotics that inhibits the protein synthesis. Tetracycline prevents the attachment of aminoacyl-tRNA to the ribosomal acceptor site. Tetracycline is a broad-spectrum antibiotic which affects a wide range of Gram positive as well as Gram negative bacteria. It has antimicrobial properties, and the human body is not adversely affected, this is the reason why tetracycline has found extensive use in human therapy and also in animal infections. Chlortetracycline or aureomycin was the first member of the tetracycline family to be discovered. It was discovered by Benjamin Duggar. Chlortetracycline is a natural fermentation product of Streptomyces aureofaciens which is naturally present in soil. Oxytetracycline or terramycin was discovered around 2 years later. Oxytetracycline is a product of S. rimosus. Other tetracyclines were discovered later on, they were either naturally synthesized e.g., dimethyl chlortetracycline from S. aureofaciens, or semisynthetic approaches can be used for their production e.g., metacycline, doxycycline, etc.

1.     Strain

Streptomyces spp. are multi-cellular actinomycetes which are Gram positive have found various applications in various industries. Streptomyces aureofaciens can produce two antibiotic substances together i.e., it can produce chlortetracycline as well as tetracycline under given set of cultivational conditions. The strain of S. rimosus produces oxytetracycline and to a lesser extent tetracycline as well.

2.     Strain improvement

Strain improvement can be carried out in-order to get a higher rate of tetracycline production. The strains can be subjected to mutagenic treatment such as exposure to ultraviolet light, X- ray treatment, irradiation of gamma rays, treatment with N-methyl-N-nitro-N- nitrosoguanidine, etc. It should be noted that only morphological variability in colonies cannot be a marker for the isolation of the strains producing higher amount amounts of tetracycline.

Increased resistance of the producers to tetracycline can also increase the production of

tetracyclines. If the strain shows sufficient resistance towards its own products there will not be any end-product inhibition. Hybridization a method of genetic recombination can also be used to improve the production of tetracycline producers.

1.     Inoculum development

The basic requirement for a good production strain is a good sporulating medium. A good sporulating medium facilitates easier maintenance of the strain and also facilitates genetic improvement of the organisms. Carry-over effect showed be considered at all times high levels of inorganic phosphate does not have any adverse effects on growth, but it can have an effect on the biosynthesis of tetracycline when transferred into the fermentation stage. The inoculum development of tetracycline can be operated in continuous or semicontinuous conditions. But exceptional care needs to be taken that the strain is not degenerated as it may lead to loss of productivity. There is always a risk of operating in the continuous mode as contamination at any stage is possible. So, the inoculum stage is carried out in a batch mode to insure a low burden of contamination at the initial step.

The basic inherent characteristic features of the inoculum include that the vegetative inoculum should be in its threshold age, genetic homogeneity, metabolic reactive profile, etc.

2.     The fermentation process.

  • Fermentation medium

Tetracycline can be produced using a complex media or a synthetic medium. The medium should contain a source of energy for example carbohydrate or glyceride oils. They also act as the source of the basic structural elements for the formation of antibiotics. The medium should also have proteins as a source of nitrogen. Calcium carbonate should also be present as it serves to bind to the antibiotic in insoluble form. The raw materials such as corn-steep liquor, molasses, soyabean oil meal, etc. may contain mineral salts but if not present need to be supplemented.

Synthetic medium can also be used for the production of tetracycline, but the yields are comparatively low. These mediums are used for research purposes.

The mediums used to carry out fermentation must be adequately sterilized. The medium used for production should contain:

a.     Carbohydrate source

Tetracyclines are polyketide antibiotics and are primarily constituted of acetate units obtained from the degradation of sugars such as monosaccharides, disaccharides, or starch. These units can also be formed by degradation of fatty acids added in the medium as esters.

Proteins and carbohydrates can be added in a single raw material like corn flour. The most common and preferred carbohydrate source is starch. Other sources include glucose, sucrose, potato, spent potato washing, yam, millet, dextrose, fructose, maltose, etc.

Fats can also be used as a carbon source. The building units are obtained only after β- oxidation of fatty acids. Antibiotics are produced only after the glucose in the medium is completely exhausted and the utilization of fatty acids as carbon source is started. Fats also serve as a defoaming agent. Antioxidants are added in the medium to prevent formation of oxidative products of fats.

b.     Nitrogen source

Corn steep liquor, cottonseed meal or flour, soyabean meal, peanut flour, beef extract, yeast extract, etc. can be used as a source of organic nitrogen. Ammonium salts are a source of easily utilizable nitrogen source. They are added at the start of fermentation or are added subsequently in small doses. They also regulate the pH of the medium. 

c.     Carbon to nitrogen ratio

The carbon to nitrogen ratio needs to be maintained to about 25 for the best results. The nitrogen shortage reduces the yield more as compared to the reduction due to carbon shortage.

Phosphate source

Phosphate influences the production of many antibiotics are it is an important growth factor, but tetracycline fermentation is highly sensitive to the presence of phosphate.

Excess of phosphate depresses the level of anhydro-tetracycline oxygenase, which is the penultimate enzyme in the production of tetracycline. Thus, reducing the amount of tetracycline produced. Thus, the optimum amount of phosphate should be added depending on the organisms used.

a.     Other elements

Other elements like calcium carbonates helps to control the pH of the fermentation medium. Ca2+ and Mg2+ present form an insoluble tetracycline salt which reduces the level of free antibiotics in the fermentation medium.

b.     pH and temperature

The optimum pH is between 5-7 and optimum temperature is 30-35ºC depending on the organisms used for fermentation. 

c.     Stimulators

As S. aureofaciens produce chlortetracycline and tetracycline together, the production of tetracycline can be increased as well as the suppression of chlortetracycline can be achieved by elimination of chloride ions in the medium or even by addition of compounds that inhibit the incorporation of chloride ions from the medium known as antimetabolites. These compounds can include inorganic bromide salts which are a competitive inhibitor against chloride ions. Even nitrogen and sulfur containing compounds can be used. When chlorine is incorporated into tetracycline molecules antimetabolites act as non-competitive inhibitors. These molecules act by binding to the copper ions which cause the chlorination as the enzyme-coenzyme chelation. Thus, if these antimetabolites are added to the fermentation medium the ratio shifts towards the production of tetracycline even in presence of high concentration of chloride ions. But at concentrations this effect is exhibited by antimetabolites is toxic to the antibiotic production and the yield is drastically reduced. Benzyl thiocyanate along with 2- mercaptobenzthiazole synergistically shifts the ratio towards the production of tetracycline.

·       Bioengineering

The bioreactor is normally made up of stainless-steel along with SS connecting pipes, two-or three ways gate valves, SS pumps to provide sterile air supply. There should also be recording

gadgets which keep round-the-clock monitoring during the entire fermentation process. The physical and physiological parameters need to be controlled automatically such as pH regulation, supplementation with sterile nutrients, etc.

For the tetracycline production the fermentation tank must secure highest possible oxygen saturation and the fermentation medium must be adequately stirred. Tetracycline producers are very sensitive towards oxygen, even a short interruption of oxygen in the medium at the start of fermentation can lead to decrease in the final product.

The fermentation can be carried out by using continuous submerged cultivation or by using solid substrate fermentation.

·       Continuous submerged cultivation

Continuous submerged cultivation has an aim of isolation of secondary metabolites. The production process essentially consists of three distinct and vital stages.

Stage 1: The growth phase

During this phase there is fast utilization of nutrients incorporated in the medium. The cell mass increases. During this phase the secondary mycelium is found to modulate the specific phosphate ions present in the medium. But the ‘production type of the mycelium’ is rarely produced in the presence of large amount of phosphate ions.

Stage 2: Production phase

The maximum amount of tetracycline is produced in this phase. The growth rate of the organism decreases and almost ceases by this time.

Stage 3 : The final stage

In this stage the production of tetracycline is the lowest and the mycelium undergoes fragmentation and also the process of cleavage starts.

The washing out of microorganisms from the cultivation vessel is done after the physiological state of overproduction has been reached. This allows the growth of new cells. The culture cannot be limited in nutrients, so a continuous flow rate of nutrients needs to be maintained.

The two- stage continuous cultivation is advantageous when the product of specific growth rate and concentration of organisms are higher in the first stage than the second stage.

·       Solid substrate fermentation.

Though continuous submerged fermentation is mostly used for production of tetracycline. There are alternative methods that reduce the amount of agricultural waste as well as save energy. Another advantage of using solid substrate fermentation is that, there is on autolysis of the cells due to prolonged incubation as in the case of submerged fermentation.

Tetracycline can also be produced by using sweet potato reside by Streptomyces viridifaciens ATCC 11989. Using a suitable solid medium which was inoculated with 1 ×108 conidia per ml and incubated at 26ºC. The conidia germinated on the second day, mycelial grew on the third day and on the sixth day the stationary phase was reached. Maximal potency was seen on the sixth day. 

Tetracycline can also be produced by using agricultural waste such as peanut shell, corncob, corn pomace, cassava peels, etc. by using solid substrate fermentation. A suitable media was inoculated with 1 ×108 conidia per ml and incubated at 28-31ºC. Tetracycline production started on day 3 and reached the maximum level on day 5.

1.     Extraction

Tetracycline is obtained from the clear filtrate by sterilized SS- plate type filter press or Pdobielniak counter current extractor. The filtrate is first treated with activated carbon.

There are various methods for extraction of tetracycline from the fermentation broth filtrate like chromatographic techniques which include ion exchange chromatography in particular, also liquid-liquid or liquid-solid extractions, crystallization, reverse micellar extraction or a combination of these methods are used for extraction and purification of antibiotics. The most commonly used technique is liquid-liquid extraction which uses organic solvents like ethyl acetate, acetonitrile and TCA. This technique however has few drawbacks. Organic compounds used are volatile and also hazardous to human health.

Aqueous two-phase system (ATPS) is a low cost, gentler and biocompatible alternative available. A new novel ATPS which is composed ionic liquids + water + organic/ inorganic salts, amino acids, polymers, or carbohydrates are used. This novel technique can extract tetracycline directly from the fermentation broth with an extraction efficiency of higher than 80%. Some of these systems are formed when two mutually incompatible substances are used, though both are soluble in water. At a critical concentration there is a spontaneous phase separation and macroscopic biphasic system is formed.

The final purified form of tetracycline that is precipitated or crystallized is carefully dried under vacuo.

1.     Contamination

Even though tetracycline is a broad-spectrum antibiotic, it is susceptible to bacterial contamination like all other antibiotics. The economic losses due to bacterial contamination is immense. The effect can range from a complete failure to only a moderate decrease in the yield. The moderately damaged yield can be salvaged but when there is a problem in processing beyond the fermentation stage such as clogged filters, etc. the product cannot be salvaged in any possible way. Contamination could take place due to the production medium used, the raw material used in its preparation, the technology process itself, type of nutrient medium used, the inoculum used for the process, etc.

2.     Immobilized cells

S. aureofaciens can be immobilized in calcium alginate. The germinated spores form a thin cell layer below the surface of alginate beads. Immobilized cells show a sevenfold increase in production compared to free cells.

Conclusion

There has been a widespread use of tetracycline and there is an alarming rise in the number of resistant organisms. There are other problems also like toxicity, poor absorption and molecular instability. In the past 3 decades there has been a lot of efforts put in to modify the structure of tetracycline and the attempt to improve their properties. There is numerous studies carried out in this field and the development of third generation of tetracyclines have been developed.