Extraction And Crystallization Of Clavulanic Acid From Fermentation Broths

Clavulanic acid (CA) is a potent (3-lactamase inhibitor and its application in conjunction with β-lactam antibiotics such as penicillin has been a successful prescription in countering the problem of resistance to antibiotics. CA is prepared through microbial fermentation, and the subsequent purification process is complex because of high cell density and protein content in fermentation broths. The removal of cells and proteins by membrane filtration consumes much time and energy, and a large amount of acid waste water is generated in the subsequent organic solvent extraction. At the same time, the two-step crystallization process with amine of clavulanic acid as an intermediateis complex leads to the high production cost, low yield and serious environmental pollution. It is known that CA in the separation process is chemically unstable like other β-lactam compounds. In this paper, salting-out extraction of CA directly from fermentation broths and preparation of potassium clavulanate by one step crystallization were investigated. It is a new attempt for preparation of alkali metal salt of clavulanic acid. Also, the stability of CA in the organic phase of salting-out extraction systems (SOES) and ethyl acetate decoloring solution was investigated.Firstly, from different SOESs’investigation, the ethanol/phosphate SOES was considered to be more favorable. The effect of salt, ethanol and product concentration on the partition of clavulanic acid were investigated. At the appropriate condition (ethanol (20%)/K2HPO4 (15%) and KH2PO4 (10%), w/w), the system formed phase completely through the low speed centrifugation, and the recovery yield of CA reached above 96%. Additionally,99.6% of cells and 64.7% of proteins could be simultaneously removed from the fermentation broths.Secondly, the stability of CA in the organic phase of SOES and ethyl acetate decoloring solution was explored. The experimental results showed that CA degradation followed a pseudo-first-order kinetics. Temperature, water content and impurities are all important factors which influence the stability of CA. The kinetics and thermodynamics parameters implied that the more ethanol content in SOES, the higher the stability of CA was, and the lower water content in ethyl acetate decoloring solution, the higher the stability of CA was. At the same time, it inferred that CA degradation was a normally non-spontaneous process in ideal CA crystal without impurity because the process was endothermic and entropy reduction. Finally, the preparation of potassium clavulanate by one step crystallization was studied. Reaction strategies, types of cosolvent and water content in cosolvent were investigated, and the results showed a better chose that anhydrous methanol was used to dissolve potassium 2-ethylhexanoate into ethyl acetate decoloring solution drop by drop. The influences of anhydrous methanol dosage and the molar ratio of potassium 2-ethylhexanoate to CA on crystallization were also studied. The optimal yield of CA reached to 66.57% and the impurity content was accorded with pharmacopoeia standards.To sum up, salting-out extraction of CA directly from the fermented broths, and preparation of potassium clavulanate by one-step crystallization are potential, and have a good application prospect. Further research should be carried out in the future...