Control of Lactobacillus contaminants in continuous fuel ethanol fermentations by constant or pulsed addition of penicillin G

The addition of penicillin G to combat microbial contamination in continuous fuel alcohol fermentations was performed using both continuous and pulsed addition regimes. In continuous fermentations where both Saccharomyces cerevisiae and Lactobacillus paracasei were present, the mode of addition of penicillin G determined final numbers of viable L. paracasei. When the same overall average concentration of penicillin G was added in both pulsed and continuous modes, the initial viable number of L. paracasei (8.0 x 10(9) cfu ml(-1)) decreased to a greater degree (1.02 x 10(5) cfu ml(-1) L. paracasei) when penicillin G was pulsed at 6 h frequencies at an overall average concentration of 2,475 U/l than when penicillin G was added continuously at 2,475 U/l (2.77 x 10(5) cfu ml(-1) L. paracasei). Pulsed additions over longer frequencies at 2,475 U/l were not as effective in reducing viable bacteria. Viable yeasts increased during both treatment conditions by more than 2-fold. The two addition regimes also eliminated the 40% decrease in ethanol concentration caused by the intentional bacterial infection. Although there was 3 times more bacterial death with 6 h pulsed additions compared to continuous additions of penicillin G at 2,475 U/l, there was, by that point, no practical difference in either final ethanol concentration or relative ethanol recovery.     

Oxygen derepresses deacetoxycephalosporin C synthase and increases the conversion of penicillin N to cephamycin C in Streptomyces clavuligerus.

When dissolved oxygen (DO) was maintained at saturation level during batch fermentations of Streptomyces clavuligerus (NRRL 3585), the accumulation of the intermediate penicillin N was lowered while formation of the end product cephamycin C was increased relative to fermentations without DO control. The specific activity of the penicillin ring-expansion enzyme deacetoxycephalosporin C synthase (DAOCS) was increased 2.3-fold under oxygen saturated conditions, whereas the penicillin ring-cyclizing enzyme isopenicillin N synthase (IPNS) showed only a 1.3-fold increase. Thus oxygen derepression of DAOCS appears to be an important regulatory mechanism in the conversion of penicillin N to cephamycin C in S. clavuligerus. IPNS, an early acting enzyme in cephamycin C biosynthesis, and DAOCS, which acts late in the pathway, both disappeared from cell extracts at 60 h, just prior to cessation of cephamycin production.     

Penicillin Acylase Activity of Penicillium chrysogenum

The penicillin acylase activity of Penicillium chrysogenum was studied. Washed mycelial suspensions of a high penicillin-producing and a nonproducing strain were found to be similar in respect to relative acylase activity on benzylpenicillin, 2-pentenylpenicillin, heptylpenicillin, and phenoxymethylpenicillin. The relative rates for both strains, as determined by 6-aminopenicillanic acid formation, were approximately 1.0, 2.5, 3.5, and 6.0 on the penicillins in the order given. The high producing strain formed both 6-aminopenicillanic acid and "natural" penicillins in fermentations to which no side-chain precursor had been added. Therefore, its demonstrated ability to cleave the natural penicillins, 2-pentenylpenicillin and heptylpenicillin, suggests that at least some of the 6-aminopenicillanic acid produced during such fermentations arises from the hydrolysis of the natural penicillins. At pH 8.5, the mycelial acylase activity of the nonproducing strain was about three times that at pH 6.0; at 35 C, it was about 1.5 times as active as it was at 30 C. When tested on penicillin G or V, no differences in either total or specific penicillin acylase activity were observed among mycelia harvested from cultures of the nonproducer to which penicillin G, penicillin V, or no penicillin had been added. Acetone-dried mycelium from both strains displayed acylase activity, but considerably less than that shown by viable mycelium. Culture filtrates were essentially inactive, although a very low order of activity was detected when culture filtrate from the nonproducer was treated with acetone and the acetone-precipitated material was assayed in a minimal amount of buffer.     

Enhancing penicillin fermentations by increased oxygen solubility through the addition of n-hexadecane

n-Hexadecane was added to fermentation media to increase the medium oxygen solubilities, thus enhancing oxygen transfer rates in penicillin fermentations. For shake flask fermentations, cells were found to grow faster in the flasks with n-hexadecane than those without. The addition of n-hexadecane to penicillin fermentations was shown to significantly increase cell growth and penicillin production and reduce formation of mycelial pellets. The result was attributed to the enhancement of oxygen transfer in mycelial fermentations due to the higher oxygen solubilities of fermentation media achieved by adding n-hexadecane.     

Evaluation of feeding strategies in carbon-regulated secondary metabolite production through mathematical modeling

There is now growing evidence that the production of many secondary metabolic by microorganisms is subjected to carbon-catabolite regulation. Even though the exact mode of this regulation is not yet clear, an engineering analysis of the production process is still possible based upon a suitable hypothesis. By way of simulation of penicillin fermentation data obtained from the literature, a mechanistic model involving a substrate inhibition kinetics of product formation has been verified in this paper. Such a model has been found successful not only in predicting simple sugar-feeding strategy, but also a complicated computer guided strategy based upon controlling biomass growth rates in the tropo and idiophases. Using this model, for strategies for sugar feeding into penicillin fermentation have been investigated. These results show that similar penicillin productivities can be obtained using any of these strategies provided fermentations are carried out under optimal conditions corresponding to the strategy chosen. Effect of maximum oxygen transfer capacity of the fermentor under the conditions of fungal growth has been incorporated using an upper limit of biomass concentration on achievement of which the fermentations must be stopped due to serious oxygen limitations. Results of model simulations with such limits throw light upon the way in which different fermentors may behave with respect to product formation.     

Evaluation of two unstructured mathematical models for the penicillin G fedbatch fermentation

The mathematical model for the penicillin G fed-batch fermentation proposed by Heijnen et al. (1979) is compared with the model of Bajpai & Reuß (1980). Although the general structure of these models is similar, the difference in metabolic assumptions and specific growth and production kinetics results in a completely different behaviour towards product optimization. A detailed analysis of both models reveals some physical and biochemical shortcomings. It is shown that it is impossible to make a reliable estimation of the model parameters, only using experimental data of simple constant glucose feed rate fermentations with low initial substrate amount. However, it is demonstrated that some model parameters might be key factors in concluding whether or not altering the substrate feeding strategy has an important influence on the final amount of product.It is illustrated that feeding strategy optimization studies can be a tool in designing experiments for parameter estimation purposes.     

Theoretical conversion yields for penicillin synthesis.

The efficiency of conversion of the carbon-energy source to product is of primary importance in many fermentation processes. In order to assess the efficiency of a process, one must know how close the actual conversion yield is to the theoretical maximum. Theoretical conversion yields are useful, therefore, as guides in improving a process. This knowledge is particularly important today because the cost of raw materials is rapidly rising. In this study, the biochemical pathway of penicillin synthesis was used to estimate the theoretical yield of penicillin from glucose, ammonia, and sulfate. These values are compared with experimental data from the literature. An analysis of the role of glucose in the synthesis of cell mass and penicillin and in the maintenance of cells makes it possible to assess the efficiency of carbon-source utilization and to direct further advances in penicillin fermentations.     

Periodic operation of immobilized cell systems: analysis

The authors' mathematical model of transient immobilized cell growth and product formation is applied here to examine the performance of an immobilized cell system subject to periodic cycling of the rate-limiting substrate supply. The model system consists of a single hydrogel-like (porous) particle entrapping viable microorganisms. Proper nutrient cycling is shown to yield a relaxed periodic system and to virtually eliminate the leakage of biomass from the support that is commonly observed experimentally in steady (continuous nutrient supply) operation of these systems. The use of cyclic operation is evaluated by calculating the average product yield (the ratio of product formed to substrate consumed) and the average product flux from the particle (a measure of the total productivity of the system), for various cycling rates. Cycling increased the average product yield by at least a factor of three in nongrowth-related fermentations, relative to steady operation, without any significant sacrifice in average total productivity. Growth-related fermentations lost significant total productivity under most cycling conditions, while the average product yield was approximately unchanged at all cycling rates. Thus, immobilization in conjunction with periodic operation should be considered as an alternative process design for the production of nongrowth-related products such as penicillin and monoclonal antibodies.     

Fermentation seed quality analysis with self‐organising neural networks

Industrial fermentation processes operate under well defined operating conditions to attempt to minimise production variability. Variability occurs for many reasons but a long held belief is that variation in the state of the seed is highly influential. In this paper a seed stage (a batch process) of an industrial antibiotic fermentation is considered and the performance of the main production fermentations is correlated with the quality of the seed using an unsupervised Kohonen self‐organising feature map (SOM). It is shown that using only seed information poor performance in the final stage fermentations can be predicted. Data from industrial penicillin G fermenters is used to demonstrate the procedure. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 82–91, 1999.     

Solid state fermentation—An overview

Since ancient times many solid state fermentations have utilized fungi and bacteria, almost always in mixed culture. The discovery and development of penicillin led to extensive use of liquid fermentations using actinomycetes and fungi and to subsequent neglect of research on solid state fermentations and the use of mixed cultures. This paper reviews the types of solid state fermentations, equipment used, products made, as well as the advantages and disadvantages of solid state fermentations. Products resulting from old and new solid and liquid substrate fermentations are enumerated.     

Optimization of batch fermentation processes. I. Development of mathematical models for batch penicillin fermentations

Two kinds of mathematical models have been developed for batch penicillin fermentations: (1) general models, based on averaged, nondimensionalized cell and penicillin synthesis curves from plant, scale fermentors and (2) particular models developed from specific sets of experimental data from two sources. Parameter-temperature functions used with the general models were assumed to have general shapes which could apply to many fermentations, i.e., they were based on the familiar temperature response of enzyme-catalyzed reactions. Parameter-temperature functions for the particular models were determined from experimental data for batch runs at various temperatures.     

Flow-injection-chemiluminescence method for the determination of penicillin G potassium.

The degradation product of penicillin G potassium can react with potassium permanganate in acidic medium and produce chemiluminescence, which is greatly enhanced by formaldehyde. The optimum conditions for this chemiluminescent reaction were studied in detail using a flow-injection system. The experiments indicated that under optimum conditions, the chemiluminescence intensity was linearly related to the concentration of penicillin G potassium within the range 1.0 x 10(-7)-1.0 x 10(-5) g/mL, with a detection limit (3sigma) of 7 x 10(-8) g/mL. The relative standard deviation was 1.0% for 4.0 x 10(-7) g/mL penicillin G potassium solution (n = 11). This method has the advantages of simple operation, fast response and high sensitivity. The method was successfully applied to the analysis of penicillin G potassium in raw medicines.     

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.     

ENHANCED PRODUCTION OF 6-AMINOPENICILLANIC ACID IN AQUEOUS METHYL ISOBUTYL KETONE SYSTEM WITH IMMOBILIZED PENICILLIN G ACYLASE

Enzymatic hydrolysis of penicillin G for production of 6-amino-penicillanic-acid (6-APA) was achieved by using penicillin G acylase as catalyst in an aqueous-methylisobutyl ketone (MIBK) system. The optimization was carried out and it was found that the best conversion was improved 10% more than the aqueous system, which was obtained at the conditions: initial pH 8.0, 5.0% (W/V) substrate (penicillin G), and temperature at 35°C, and the ratio of aqueous and organic phase was 3:1. The stability of the biocatalyst was studied at the operational conditions. After 5 cycles of semi-batch reactions, the residual activity of penicillin G acylase was 69.2% of the initial activity. There was no apparent loss of the yield of product. This process has a potential application in the industrial scale production of 6-APA because it simplifies the process effectively.     

Hydrolysis of penicillin G by combination of immobilized penicillin acylase and electrodialysis

Phenylacetic acid, as inhibitory product, was formed from a hydrolysis of penicillin G by immobilized penicillin acylase. In this article, electrodialysis was applied to remove phenylacetic acid continuously from the reaction mixture and to enhance an efficiency of the reaction. When 268 and 537 mM of penicillin G solution were used as the substrate, the concentration of phenylacetic acid in the reaction mixture could be maintained at less than 81 and 126 mM, respectively, and eventually, 86% and 88% of phenylacetic acid produced were removed from the reaction mixture at the end of the hydrolysis, respectively. Times required to reach 96% and 94.8% conversion from 268 and 537 mM of initial penicillin G could be reduced to 65% and 64% respectively, by means of electrodialysis; while 3.0% and 4.3% of initial penicillin G of 268 and 537 mM were permeated out of the reaction chamber during the hydrolysis, respectively. However, a loss of penicillin G by permeation could be reduced from 4.3% to 3.4% by a repeated addition of penicillin G.     

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.     

Assay of 6-oxopiperidine-2-carboxylic acid in fermentations of Penicillium chrysogenum PQ-96

Summary 6-Oxopiperidine-2-carboxylic acid (OCA; cyclic -aminoadipic acid) was assayed in fermentations of Penicillium chrysogenum PQ-96 by isotachophoresis and HPLC. From 0.36 mg ml^–1 to 2.41 mg ml^–1 OCA was accumulated in fermentations during penicillin G biosynthesis. Offprint requests to: W. Kurzkowski     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase

Bioconversion of penicillin G in PEG 20000/dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with an intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7% (w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U ml(-1), pH 7.8, temperature 37 degrees C, reaction time 40 min. Five repeated batches could be performed in these conditions. Conversions ratios between 0.9-0.99 mol of 6-aminopenicillanic acid (6-APA) per mol of penicillin G, were obtained and volumetric productivity was 3.6-4.6 micromol min(-1) ml(-1). In addition the product 6-APA could be directly crystallized from the top phase with a purity of 96%.