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%.

The present work describes the study of the growth and the cytotoxic and antitumor activities of the extracts of the marine microorganisms Streptomyces acrymicini and Streptomyces cebimarensis, the latter a new strain. Both microorganisms were collected from coastal marine sediments of the north coast of São Paulo state. Growth was performed in a shaker and in a bioreactor using Gym medium and the broths of both microorganisms were extracted with ethyl acetate and n-butanol. Three extracts, two organic and one aqueous, from each microorganism were obtained and tested for cytotoxic and antitumor activity using the SF-295 (Central Nervous System), HCT-8 (Colon) cell lines, and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The growth methods were compared and show that, although the shaker presented reasonable results, the bioreactor represents the best choice for growth of these microorganisms. The biological activity of the different extracts was evaluated and it was demonstrated that the growth methodology may influence the secondary metabolite production and the biological activity.

A study was made of the extraction of clavulanic acid (CA) from a fermented medium and its concentration and purification. The fermented broth was obtained by cultivation of Streptomyces clavuligerus in complex medium containing glycerol as the Carbon and energy source, and soybean protein isolate as the Nitrogen source. The extraction steps proposed here were evaluated from the standpoint of Yield, purification factor and concentration factor. The extractions were carried out with ethyl acetate, butyl acetate, 2-butanol, n-butanol and methyl isobutyl ketone. The distribution coefficient was related to the solvent properties and it was found that distribution coefficient is directly proportional to the product of the dielectric constant, with solubility. It was observed that extraction with butanol leads to a higher distribution coefficient than ethyl acetate; however, the higher solubility of water in butanol impairs the final precipitation of clavulanic acid. The optimal condition with ethyl acetate occurs at pH 1.63, yielding an extraction factor of 35.6% with a distribution coefficient of 0.73.

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−1) in a 10-L bioreactor, showing a positive effect of ornithine and glycerol, in the proper proportion (48.6:1), on CA biosynthesis.

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.

Clavulanic acid (CA) is a potent inhibitor of beta-lactamases, enzymes that are responsible for the hydrolysis of beta-lactam antibiotics. It is a secondary metabolite produced by the filamentous aerobic bacterium Streptomyces clavuligerus in submerged cultivations. In the present work clavulanic acid production in batch, fed-batch and continuous bioreactors was studied with the objective of increasing productivity. The operating conditions: temperature, aeration and agitation, were the same in all cases, 28º C, 0.5 vvm and 800 rpm, respectively. The CA concentration obtained in the fed-batch culture, 404 mg L-1, was ca twice the value obtained in the batch culture, 194 mg L-1, while 293 mg L-1 was obtained in the continuous culture. The highest productivity was obtained in the continuous cultivation, 10.6 mg L-1 h-1, as compared with 8.8 mg L-1 h-1 in the fed-batch process and 3.5 mg L-1 h-1 in the batch process, suggesting that continuous culture of Streptomyces clavuligerus is a promising strategy for clavulanic acid production.

This paper reports on the use of the gas balance and dynamic methods to obtain an estimate of the volumetric oxygen transfer coefficient (kLa) in a conventional reactor during the growth phase of the microorganism Cephalosporium acremonium. A new way of calculating kLa by the dynamic method employing an electrode with a slow response, is proposed. The calculated values of kLa were used in the training of a feedforward neural network, for which the inputs were the parameter measurements of the related variables. The neural network technique proved effective, predicting values of kLa accurately from input data not used during the training phase. In contrast, the gas balance method was shown to be less useful. This could be attributed to the poor data obtained with the apparatus used to measure the oxygen in the exhaust gas, explained by the low rate of oxygen consumption by the microorganism.