Evaluation of the effects of the parameters involved in the purification of clavulanic acid from fermentation broth by aqueous two-phase systems.

The purification of clavulanic acid (CA), which is an important beta-lactam antibiotic produced by submerged cultivation of Streptomyces clavuligerus, was studied through the use of phosphate and polyethylene glycol-based aqueous two-phase systems. The parameters' effect on the yield and purification was evaluated through an experimental design and the preliminary results showed that the polyethylene molecular mass and tie-line length and phase volume ratio exerted the strongest effect on the yield and distribution coefficient in the range tested. In addition, the response surface methodology was used to optimize the distribution coefficient, yield, and purification factor. The optimal conditions of yield and purification factor are in the regions where polyethylene has a low molecular mass, pH close to the isoelectric point, and lower top phase volume. A 100% yield and a 1.5-fold purification factor are obtained when extracting CA by maximizing the conditions of an aqueous two-phase system.

Antagonistic properties of some microorganisms isolated from Brazilian tropical savannah plants against Staphylococcus coagulase-positive strain

Endophytic microorganisms are relatively unstudied as potential sources of novel natural products for medical and commercial exploitation. The aim of this work was to investigate some Brazilian tropical savannah trees Cassia leptophylla and Prunus spp. in order to isolate the endophytic microorganisms associated with these plants. The samples were disinfected to eliminate the epiphytic population. Colonies were diluted and displayed as drops in media and growing colonies were inactivated. Staphylococcus coagulase-positive strain was used as indicator microorganism and subjected to the antibioses test. Data showed that the microorganisms isolated from Cassia leptophylla had no inhibition against Staphylococcus. On the other hand, microorganisms isolated from Prunus spp. leaves showed antibacterial activity and inhibited Staphylococcus when cultivated in peptone agar as well as in yeast extract agar. Investigation proceeds in order to classify the isolated microorganisms presenting bioactive substance and exploit the potential of the compounds produced to inhibit the indicator bacteria. Other bioactive properties will be investigated.

Production of hyaluronic acid by streptococcus: the effects of the addition of lysozyme and aeration on the formation and the rheological properties of the product.

The effects of the addition of lysozyme and forced aeration on the rheological properties and production of hyaluronic acid by Streptococcus zooepidemicus were investigated. Lysozyme was added to the culture broth in two pulses during the exponential and stationary phases of a fermentation carried out in a rotary shaker (150 rpm), using 200 mL Erlenmeyer flasks. The effect of aeration was evaluated by feeding air into a 2.5 L fermentor at a 2 vvm rate. The effects were analyzed in terms of concentration, viscosity, viscoelasticity, and molecular weight of the hyaluronic acid produced.

Influence of dissolved oxygen and shear conditions on clavulanic acid production by Streptomyces clavuligerus.

Clavulanic acid (CA), a potent beta-lactamase inhibitor, is produced by a filamentous bacterium. Here, the effect of DO and shear, expressed as impeller tip velocity, on CA production was examined. Cultivations were performed in a 4 L fermentor with speeds of 600, 800 and 1,000 rpm and a fixed air flow rate (0.5 vvm). Also, cultivation with automatic control of dissolved oxygen, at 50% air saturation, by varying stirrer speed and using a mixture of air and O(2) (10% v/v) in the inlet gas, and a cultivation with fixed stirrer speed of 800 rpm and air flow rate of 0.5 vvm, enriched with 10% v/v O(2), were performed. Significant variations in CA titer, CA production rate and O(2) uptake-rate were observed. It was also found that the DO level has no remarkable effect on CA production once a critical level is surpassed. The most significant improvement in CA production was related to high stirrer speeds.

Continuous cephalosporin C purification: dynamic modelling and parameter validation.

A mathematical kinetic model for the adsorption and desorption of cephalosporin C on Amberlite XAD-2 resin is proposed. The model can represent Langmuir, Freundlich or linear isotherms at equilibrium. The intrinsic kinetic parameters and adsorption isotherms as well as physical parameters such as the effective diffusivity and the external mass transfer coefficient were obtained at different temperatures and ethanol concentrations. An unfavourable cephalosporin C adsorption occurred when ethanol was present in the solution. It has been shown that at 25 degrees C the ethanol, at concentrations from 1.5% to 2.5%, decreases the cephalosporin C adsorption. However, this behaviour was not observed at 10 degrees C. The kinetic model fitted the experimental data well under different conditions. The model was validated in a continuous process of cephalosporin C purification using the same resin. The model with the validated parameters is able to predict the behaviour of the reactor system. The continuous process is composed of two stirred tank reactors with adsorber recycle. The adsorption occurs in the first stage, and elution of the product takes place in the second stage with ethanol as eluent. The dynamic behaviour of the process was described using the following parameters: hydraulic residence time for the first (theta(h1)) and second stage (theta(h2)), solid residence time (theta(s)), initial concentration of CPC (C(0)), inlet ethanol concentration (C(ET0)) and kinetics parameters.

A hybrid neural network algorithm for on-line state inference that accounts for differences in inoculum of Cephalosporium acremonium in fed-batch fermentors.

One serious difficulty in modeling a fermentative process is the forecasting of the duration of the lag phase. The usual approach to model biochemical reactors relies on first-principles, unstructured mathematical models. These models are not able to take into account changes in the process response caused by different incubation times or by repeated fedbatches. To overcome this problem, we have proposed a hybrid neural network algorithm. Feedforward neural networks were used to estimate rates of cell growth, substrate consumption, and product formation from on-line measurements during cephalosporin C production. These rates were included in the mass balance equations to estimate key process variables: concentrations of cells, substrate, and product. Data from fed-batch fermentation runs in a stirred aerated bioreactor employing the microorganism Cephalosporium acremonium ATCC 48272 were used. On-line measurements strongly related to the mass and activity of the cells used. They include carbon dioxide and oxygen concentrations in the exhausted gas. Good results were obtained using this approach.

Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor.

Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess. Other alternatives for the production of beta-lactam antibiotics, such as a cephalosporin C fed-batch process in an aerated stirred-tankbioreactor with free cells of Cephalosporium acremonium, or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch process. In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium. In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations. For this study's model, modifications were introduced to take into account the influence of supplementary medium flow rate. The balance equations considered the effect of oxygen limitation inside the bioparticles. In the Monod-type rate equations, cell concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate. The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval. The simulation results showed that the proposed model fit well with the experimental data, and based on the

Kinetic studies of clavulanic acid recovery by ion exchange chromatography.

Clavulanic acid (CA) is a beta-lactamase inhibitor produced by strains of Streptomyces clavuligerus. Nowadays, the combination of CA with amoxycillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamases together with an inhibitor of these enzymes. Clavulanic acid is purified from fermentation broth by a series of steps consisting mainly of two-phase separation processes such as liquid-liquid extraction, adsorption or ion-exchange chromatography, among others. Amberlite IRA 400, a strong anion-exchange resin, has a very high adsorption capacity for CA (Mayer et al. 1997). This resin can be pre-treated with NaCl (chloride cycle), to remove selectively only those anions, which are able to displace chloride from the resin or with NaOH (hydroxyl cycle), to remove all species of anions. In order to decide the best operating conditions for CA recovery by ion-exchange resins and then to construct a model of this separation process, batch experiments were conducted using Amberlite IRA 400 in the chloride cycle. These runs were carried out in a 200 ml stirred tank, at two different initial solution pH, 6.2 and 4.0; the temperature was maintained at 10 degrees C and 20 degrees C during adsorption and 30 degrees C during the desorption step. It was possible, on the basis of these batch results, to model the separation process, including the adsorption kinetics, equilibrium data and mass transfer limitations.

Ethanol production by a flocculant yeast strain in a CSTR type fermentor with cell recycling.

Tests were performed in a continuous stirred tank reactor (CSTR), with and without cell recycling, to produce ethanol. The reactor without cell recycling produced the kinetic model of ethanol production, whereas the reactor with cell recycling allowed for a study of process stability. The Levenspiel kinetic model was adopted; however, in the case of fermentation with cell recycling, the coefficient of cell death was added. It was observed that cellular viability varied greatly throughout the fermenting process and that microaeration is of fundamental importance in maintaining the stability of the process.

Comparison between experimental and theoretical values of effectiveness factor in cephalosporin C production process with immobilized cells.

Cells of Cephalosporium acremonium ATCC 48272 immobilized in calcium alginate beads were utilized for cephalosporin C production and the results were compared with those obtained with free cells. The experiments were performed with synthetic medium containing glucose and sucrose as carbon and energy sources. Experimental effectiveness factor values were obtained at various cell and dissolved-oxygen concentrations, considering Monod kinetics for the respiration rate, and were compared with the values calculated with zero-order kinetics in spherical bioparticle. The results showed that the assumption of oxygen limitation by diffusion in the bioparticle was correct, and that cephalosporin C production with immobilized cells is perfectly viable, although a slightly lower rate than that obtained in the free cell process was observed.

Phenomenological and neural-network modeling of cephalosporin C production bioprocess.

Cephalosporin C production process with Cephalosporium acremonium ATCC 48272 in synthetic medium was investigated and the experimental results allowed the development of a mathematical model describing the process behavior. The model was able to explain fairly well the diauxic phenomenon, higher growth rate during the glucose-consumption phase, and the production occurring only in the sucrose-consumption phase. Moreover, the process was simulated utilizing the neural-networks technique. Two feed-forward neural-networks with one hidden layer were employed. Both models, phenomenological and neural-networks based, satisfactorily describe the bioprocess. The difficulties in determining kinetic parameters are avoided when neural networks are utilized.