Production, culture optimisation and biosafety assessment of microbial bioflocculants

Identifying high bioflocculant-producing strains is essential for enhancing the efficiency and economic feasibility of large-scale wastewater treatment and related biotechnological processes. Therefore, this study focused on the isolation of potent bioflocculant-producers and enhancement of their yields through optimisation of culture conditions. The bacteria and fungi were isolated using conventional methods and identified by the 16 S rRNA and internal transcribed spacer (ITS) analysis. The significant factors (pH, temperature, agitation and nitrogen source) were optimised using the response surface methodology by Box Behnken Design (BBD). Fourier Transform Infrared Spectroscopy (FTIR) was employed to assess the functional group composition of the bioflocculants. Furthermore, the cytotoxic effect of the bioflocculants was assessed on MCF-7 (breast cancer cell line) and HepG2 (human liver cancer cell line) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The selected isolates were identified as Klebsiella pneumoniae and Meyerozyma guilliermondii. K. pneumoniae produced the highest bioflocculant yield (8.60 g/L) under optimal conditions of pH 10, 20 °C, and 150 rpm whereas M. guilliermondii yielded 5.20 g/L under pH 4, 20 °C, and a 1 g/L mixed nitrogen source comprising of urea, yeast extract, and ammonium sulfate. FTIR spectra revealed hydroxyl, amine, and alkene groups as key functional groups responsible for flocculation. Moreover, the bioflocculant from K. pneumoniae (Kp1) exhibited median inhibitory concentration (IC₅₀) values of 6.97 µg/mL for breast cancer cells (MCF-7) and 38.6 µg/mL for HEPG2 whereas that from M. guilliermondii (Mg1) displayed IC₅₀ values of 6.97 µg/mL for MCF-7 and 45.5 µg/mL for HEPG2.