Applicability of β-lactamase entrapped agarose discs for removal of doripenem antibiotic: reusability and scale-up studies†

Abstract

To alleviate escalating antibiotic pollution in the environment, there is a pressing need for sustainable antibiotic remediation techniques. Considering this, the present study focuses on entrapping β-lactamase from Bacillus tropicus EMB20 within an agarose matrix, subsequently employing it for the bioremediation of doripenem (a carbapenem antibiotic) and other β-lactam antibiotics. The agarose discs containing entrapped lactamase efficiently hydrolysed 50 mg L⁻¹ of doripenem within 30 min of batch mode treatment. The toxicity of the antibiotic hydrolysed products was assessed using MTT assay and confocal microscopy, revealing their non-toxic nature to the antibiotic-sensitive cells of E. coli BL21 (DE3). These discs were successfully recovered and reused for up to 5 cycles with an efficiency rate of 72%. Furthermore, the discs demonstrated effectiveness in hydrolysing a mixture of antibiotics, including doripenem, meropenem, and amoxicillin, removing 100%, 96.4%, and 71.5% of each antibiotic after 30 min of treatment. This enzymatic treatment process was upscaled using a continuous mode fixed-bed column bioreactor (FBCR) packed with layers of lactamase-entrapped agarose discs and sand gravels. Remarkably, a mixture of doripenem, amoxicillin, and meropenem (each at 50 mg L⁻¹) was completely removed after a retention time of 20 min in the FBCR. This setup proved to be reusable for up to 5 cycles. Overall, the study emphasises the potential of utilising these β-lactamase-entrapped agarose discs as an effective remediation tool to control antibiotic pollution from the environment.