Evaluation of separation alternatives of clavulanic acid produced in Streptomyces clavuligerus cultures

Abstract

ABSTRACT : Clavulanic acid (CA) is a soft β-lactam antibiotic with a strong inhibitory effect on β-lactamase enzymes, which confers resistance to bacteria against several known broad-spectrum antibiotics. CA is produced by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. The production of CA at industrial scale is traditionally carried out in agitated tank bioreactors, where the mode of operation has proven to be a determining factor for obtaining high product yields.

The selection of CA separation processes is based on the physicochemical properties of the product and fermentation broth. Separation is generally carried out by solid-liquid separation operations, liquid-liquid extraction, and precipitation in the form of clavulanate. Although the metabolic restrictions to reach high CA titers are the first difficulty in the CA production process, downstream processing is compromised by the degradation of CA molecule in aqueous solution presumably by following a hydrolysis reaction mechanism catalyzed by both, acid and alkaline media.

In this research work, the extraction of CA from the fermentation broth of S. clavuligerus cultures was carried out at laboratory scale. For this, two separation operations were selected: liquid-liquid extraction and adsorption. For the case of liquid-liquid extraction, two experimental designs were developed aimed at finding the best conditions of the process; for the first design, temperature and pH of the broth were selected as the controlled variables, and CA extraction yield as the response variable. With the best conditions found, a second experimental design was developed; the control variables were the ratio between the aqueous phase/extraction agent (ethyl acetate), and the CA load in the fermentation broth. For the case of adsorption, the ion exchange resin IRA 400 was selected. For this process the same experimental design, used in the previous strategy, was carried out, for comparison purposes. The optimal contact time between phases (20 min) and the ratio of the pretreatment solvent (ether) for the purification process (ratio of 0.6) were also studied experimentally. Samples (300 L) from the aqueous phase were taken for analytical purposes. The obtained samples were derivatized with imidazole for 30 min and subsequently filtered at 0.20 m. Finally, the samples were placed into vials for subsequent HPLC analysis. CA loss was minimized at 10 °C and pH 2.0. The adsorption was favored by increasing the adsorbent to liquid ratio. Thus, the highest separation was attained in the range of 40-45% solid/liquid ratio, adsorbing a mean value of 47.7% of the CA present in the broth. Ethyl acetate was selected as the best alternative for extraction considering its low price and good performance. Further, high solvent to aqueous ratio (>2.0) allowed to extract up to 80% of the CA in the fermentation broth. The use of low volumes of organic solvent (ratios 0.2-0.6) led to 40% less CA extracted from the broth. Both liquid-liquid extraction and adsorption seem to be appropriate separation techniques for CA.