The present study was carried out to find the effective concentration and initial feeding rate of Sodium phenyl acetate and the pH of the media at which the yield is maximum, and also to find an alternative precursor for Na-PA by substituting K-PA & NH4 –PA at different concentrations and at different initial feeding time, and to check the activity of better yield in Pilot scale.The number of biotechnological production processes in the fine chemicals/pharmaceuticals sector has been steadily rising during the past decades (Schmid et al., 2001). A prominent example is the production of β-lactam antibiotics, which cover about 65% of the world market for anti-infectives.
Already since the 1940’s, penicillin G, penicillin V and cephalosporin C are biotechnologically produced by fungal fermentation. The staring material for the production of semi-synthetic antibiotics is mostly penicillin G, which is obtained by fermentation using the fungus Penicillium chrysogenum. The annual production exceeds 40,000 tons. About 75% of this amount is used for the production of 6-aminopenicillianic acid (6-APA) (Elander, 2003), the β-lactam nucleus that is used to synthesize semi-synthetic penicillins (Dissertation-URL). Standard industrial penicillin production consists of a batch cultivation followed by a fed- batch penicillin production stage. The initial batch cultivation is performed to obtain vegetative biomass as inoculum for the fed-batch production process. The media used in the batch and fed-batch processes are usually similar in composition (Hersbach et al., 1984 and Nielson, 1995).
To support substantial formation of biomass, the starting concentration of the major carbon source, e.g. glucose or sucrose, is much higher in the initial batch process. Usually a complex nitrogen source like corn steep liquor is employed. Corn steep liquor contains multiple amino acids and various aromatic amines but also large amounts of lactate and several important metal ions (Nielson, 1995). During the fed-batch stage glucose, a nitrogen source e.g. Ammonium and the side-chain precursor of choice are fed at controlled rates. In general, a medium pH of 6.5 is maintained throughout the production stage. On smaller scale glucose is often replaced by a slower metabolized sugar like lactose. Penicillin biosynthesis is regulated by environmental factors such as the phosphate, carbon, nitrogen and oxygen content of the medium (Feng et al., 1994). Penicillin biosynthesis is sensitive to nitrogen source repression
The penicillin fermentation provides a very good example of the use of fed-batch culture for the production of a secondary metabolite. The penicillin process is two-stage fermentation; an initial growth is followed by the production phase or idiophase. During the production phase glucose is fed to the fermentation at a rate, which allows a relatively high growth rate (and therefore rapid accumulation of biomass) yet maintains the oxygen demand of the culture within the aeration capacity of the equipment. If the oxygen demand of the biomass were to exceed the aeration capacity of the fermenter anaerobic conditions would result and the carbon source would be used inefficiently. During the production phase the biomass must be maintained at a relatively low growth rate and thus, the glucose is fed at a low dilution rate. Phenyl acetic acid is a precursor of the penicillin molecule but it is also toxic to the producer organism above a threshold concentration. Thus, the precursor is also fed into the fermentation continuously, thereby maintaining its concentration below the inhibitory level.
The control of the onset of secondary metabolism has been studied extensively in batch culture and to a lesser extent, in continuous culture. On the other hand, medium components have been demonstrated to repress secondary metabolism, the earliest observation being that of Saltero and Johnson in 1953 of the repressing effect of glucose on benzyl penicillin formation. Carbon sources that support high growth rates tend to support poor secondary metabolism. Phosphate sources have also been implicated in the repression of secondary metabolism. Therefore, it is essential that repressing nutrients should be avoided in media to be used for the industrial production of secondary metabolites or that the mode of operation of the fermentation maintains the potentially repressing components at sub-repressing levels.
Phenyl Acetic Acid In Penicillin Fermentation
The formation of desirable penicillin can be stimulated by the addition of precursors. Here, for the Benzyl penicillin formation, Phenyl acetic acid derivatives are added. Phenyl acetic acid (C6H5.CH2.COOH) supplies the side chain of Penicillin-G. Fortunately, Corn steep liquor, employed in the formulation of the fermentation medium, is a source of Phenyl acetic acid derivatives. High levels of PAA are, however, toxic to the mould, and so all of it cannot be added at a time.The penicillin nucleus, to which the 6-amino-penicillanic acid (6-APA) side-chain is attached is believed to be synthesized from the amino acids L-cysteine and L-valine.
Penicillium chrysogenum is a filamentous fungus, which is used for commercial production of penicillin’s. It is able to synthesize penicillin’s with specific hydrophobic side chains when the appropriate precursor is fed to the production medium. Here, for the Benzyl penicillin formation, Phenyl acetic acid derivatives are added. Phenyl acetic acid (C6H5.CH2.COOH) supplies the side chain of Penicillin-G. The Project is carried out for the following purposes in three different phases.
To find out the effective concentration and initial feeding time of Na-PA and pH of media at which the penicillin-G production is maximum in Liquid fermentation cultures.
To find out an alternative precursor for Na-PA which is used as a regular one by substituting K-PA & NH4 -PA as precursor at different concentrations and at different initial feeding time.
To check out the activity profile of the better yielded cultures in pilot scale.
In phase –I Experiments the best penicillin yields with Sodium Phenyl acetate are obtained with concentrations ranging from 8-12 mg/ml and with 1-day old culture in a normal pH of media. In phase –II Experiments the cultures containing potassium phenyl acetate gives low penicillin yield when compared to sodium phenyl acetate (Table: 06) whereas ammonium phenyl acetate gives significant increase in yield. In pilot scale, comparison of the lab scale results that gives higher productivity with the pilot scale is beneficary irrespective of the operating conditions in the pilot scale.
Effect of Media Composition on the Penicillin Production
The conventional penicillin fermentation medium is composed of corn steep liquor, glucose, lactose, minerals, oil, and precursor. The penicillin activity was not affected, due to the addition of carbonate 0-1% or whale oil 0.5% instead of 1%. Also the omission of cupric, magnesium, manganese, zinc sulphates, and acetic acid did not affect the penicillin activity, while the omission of ammonium nitrate and potassium dihydrogen phosphate decreased the penicillin activity in the medium. The penicillin activity of a medium containing 2% calcium super phosphate was higher than that of the control medium, containing 0.4% potassium dihydrogen phosphate and 1% calcium carbonate. Instead of adding the precursor twice, after 0 and 48 h, the addition of phenyl acetamide in the amount of 0.2%, at the start, did not affect the activity, while addition of phenyl acetic acid (0.2%), at the start, decreased the penicillin activity. The omission of the precursors in the medium decreased the penicillin activity measured microbiologically, however, 6-aminopenicillanic acid content was relatively higher compared with that of the control medium, containing precursor. These results were confirmed, using iodometric assay and paper chromatographic analysis
Effect of Growth Rate on the Synthesis of Penicillium
The production of penicillin by Penicillium spp., like the synthesis of other secondary metabolites, is thought to be dissociated from the growth of the organism. The antibiotic is produced at its maximal rate in batch cultures after an initial period of rapid growth of the mold. The initial period of rapid growth and a second phase in which little or no growth occurs are considered necessary to the accumulation of high concentrations of penicillin. From an analysis of batch culture data, Jarvis and Johnson (1947) concluded that the specific rate of production of penicillin (units of penicillin per milligram of mycelial nitrogen per hour) was at its highest when the organism growth rate was close to zero.
Maxon (1955) and Gaden (1959) presented penicillin fermentation as an example of a class of microbial processes in which growth and product formations are temporally separated. However, the restriction of penicillin synthesis to the period after the rapid growth of the organism may not reflect a dependency of penicillin biosynthesis on slow or no growth. In most research work, batch cultivation with a variety of carbon and nitrogen sources is practiced. It is difficult to obtain meaningful information from such processes, because numerous, inter-related parameters such as pH, growth rate, and nutritional status vary simultaneously.
Penicillium chrysogenum is an important industrial organism due to its capacity to produce penicillin, which is still one of the main commercial antibiotics. Standard industrial penicillin production consists of a batch cultivation followed by a fed-batch penicillin production stage. Penicillin biosynthesis is affected by the addition of phenyl acetic acid. The present study is carried out to find out the effective concentration and initial feeding time of NaPA and pH of media at which the penicillin-G production is maximum in liquid fermentation cultures, to find out an alternative precursor for Na-PA which is used as a regular one by substituting K-PA & NH4-PA as precursor at different concentrations and at different initial feeding time, to check out the activity profile of better yielded cultures in pilot scale.
The toxicity of sodium phenyl acetate is associated with the initial acidity of the culture media. The toxicity should be sufficiently over come by adding the sodium phenyl acetate prior to inoculation and by raising the pH of the culture media .The optimum concentration of sodium phenyl acetate is not clearly determined, but it appears to the between 8mg and 12mg /ml of culture medium preferably 10mg/ml-Batch 2(Activity: 27987 IU/ml). The optimum initial feeding of sodium phenyl acetate is 24-hour from the time of inoculation.