Effects of glucose and inoculum concentrations on production of bioactive molecules by Paenibacillus polymyxa RNC-D: a statistical experimental design

The composition of culture media and the microbial cell mass influence on secondary metabolites production in fermentative procedures. The effect of two process variables, namely glucose and inoculum concentrations on biomass formation, bioactivity and surface tension of extract obtained during the cultivation of endophytic Paenibacillus polymyxa RNC-D was evaluated. Assays were performed using glucose and inoculum concentrations varying from 5.0 to 40.0 g.l−1 and from 2.5% to 5.0% (v/v), respectively, according to a 22 full factorial design. The microorganism was cultivated in orbital shaker (30 ◦C, 180 rpm) for 96 h. Cell growth was estimated by optical density (600 nm) vs dry weight calibration curve. Bioassays were performed using two-fold serially diluted extract displayed in 96-well plates. Escherichia coli ATCC 25923 and Staphylococcus aureus ATCC 25922 were used as indicator strains. The minimal inhibitory concentration (MIC) was expressed in g.ml−1. Surface tension (mN.m−1) of extracts was measured using a tensiometer. A significant (p<0.01) and positive effect of glucose and inoculum concentrations was observed on biomass formation. Bioactivity results were also affected by the two studied variables (p<0.01). The lowest MIC value of E. coli was obtained when the highest glucose and inoculum concentrations were used. Otherwise, MIC of S. aureus was increased when the maximum glucose was applied. Surface tension was affected by the two evaluated variables and also by their interaction (all of negative signal, p<0.1). The highest biomass formation (4.11 g.l−1) and the lowest MIC of E.coli (15.6g.ml−1) were attained under the highest concentrations of glucose and inoculum, while the surface tension reduction reached the maximum (20.0 mN.m−1) when using the lowest glucose and the highest inoculum concentrations. Such results can still be improved by performing additional assays for the establishment of the quadratic models, as suggested by analysis of the experimental design

Optimization of the precipitation of clavulanic acid from fermented broth using t-octylamine as intermediate

This work describes the use of clavulanic acid (CA) precipitation as the final step in the process of purification of CA from fermentation broth as an alternative to conventional methods employed traditionally. The purpose of this study was to use a stable intermediate (t-octylamine) between the conversion of CA to its salt form (potassium clavulanate), thereby enabling the resulting intermediate (amine salt of clavulanic acid) to improve the purification process and maintain the stability of the resulting potassium clavulanate. To this end, response surface methodology was employed to optimize the precipitation step. For the first reaction, five temperatures (6.6 to 23.4 ºC), concentrations of clavulanic acid in organic solvent (6.6 to 23.4 mg/mL) and t-octylamine inflow rates (0.33 to 1.17 drop/min) were selected based on a central composite rotatable design (CCRD). For the second reaction, five temperatures (11.6 to 28.4 ºC), concentrations of clavulanic acid amine salt in organic solvent (8.2 to 41.8 mg/mL) and concentrations of potassium 2-ethylhexanoate (0.2 to 1.2 molar) were also selected using CCRD. From these results, precipitation conditions were selected and applied to the purification of CA from the fermentation broth, obtaining a yield of 72.37%.23124

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

The dead core model applied to beads with immobilized cells in a fed-batch cephalosporin C production bioprocess

Viable cells immobilized in inert supports are currently studied for a wide range of bioprocesses. The intrinsic advantages of such systems over suspended cultures incite new research, including studies on fundamental aspects as well as on the industrial viability of these non-conventional processes. In aerobic culture of filamentous fungi, scale-up is hindered by oxygen mass transfer limitation through the support material and bioprocess kinetics must be studied together with mass transfer limitation. In this work, experimental and simulated data of cephalosporin C production were compared. Concentrations in the bulk fermentation medium and cellular mass profiles inside the bioparticles are focused. Immobilized cells were used in a tower bioreactor, operated in fed-batch mode. To describe the radial variation of oxygen concentration within the pellet, a dead core model was used. Despite the extremely low sugar concentrations, bioreaction rates in the pellets were limited by the dissolved oxygen concentration. Cell growth occurs only in the outer layers, a result also confirmed by scanning electron microscopy. (C) 2001 Elsevier B.V. Ltd. All rights reserved

Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor

The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina (<44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity. (C) 2004 Wiley Periodicals, Inc

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 p recess in an aerated stirred-tank bioreactor with free cells of Cepha-losporium acremonium or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch profess. 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, eel 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 experimental data and the mathematical model an optimal mass flow rate to maximize the bioprocess productivity could be proposed

Influence of glycerol and ornithine feeding on clavulanic acid production by Streptomyces clavuligerus

The influence of glycerol and ornithine feeding on clavulanic acid (CA) production by Streptomyces clavuligerus was investigated. In batch experiments, CA maximum concentration (Cp max) ranged randomly from 430 to 560 mg.L-1, with a maximum increase of 10% in relation to the control run, without ornithine. However, the maximum volumetric productivity of CA (Pp max) of 13.7 mg.L-1.h-1 was obtained with 0.66 g.L-1 of ornithine, 44.2% higher than the Pp max in the control run. In fed-batch experiments, Cp max varied within the narrow range from 1.254 to 1.405 g.L-1, 2.5 times higher than that obtained in the control run. The presence of ornithine increased the Pp max, although it influenced only slightly the Cp max. Concerning glycerol, the highest CA production of 1.6 g.L-1 was obtained in the fed-batch with glycerol and ornithine (180 and 3.7 g.L&#8722;1) in a 10-L bioreactor, showing a positive effect of ornithine and glycerol, in the proper proportion (48.6:1), on CA biosynthesis

Modelling and optimization of the cephalosporin C production bioprocess in a fed-batch bioreactor with invert sugar as substrate

Cephalosporin C production process optimization was studied based on four experiments carried out in an agitated and aerated tank fermenter operated as a fed-batch reactor. The microorganism Cephalosporium acremonium ATCC 48272 (C-10) was cultivated in a synthetic medium containing glucose as major carbon and energy source. The additional medium contained a hydrolyzed sucrose solution as the main carbon and energy source and it was added after the glucose depletion. By manipulating the supplementary feed rate, it was possible to increase antibiotic production. A mathematical model to represent the fed-batch production process was developed. It was observed that the model was applicable under different operation conditions, showing that optimization studies can be made based on this model. (C) 1999 Elsevier B.V. Ltd. All rights reserved