Molecular and Morpho-Structural Characterization of Bioflocculant-Producing Bacterial Isolates for Sustainable Wastewater Remediation

This study investigates the isolation, screening, and characterization of bioflocculant-producing bacterial strains from diverse wastewater environments, including municipal solid waste and pharmaceutical effluent sources. Out of the total isolates screened, four promising strains- Bacillus thuringiensis (ASW5), Bacillus paranthracis (SED11), Bacillus halotolerans (A13), and Bacillus velezensis (S14)-demonstrated superior flocculating activity and were subjected to comprehensive physicochemical and biochemical profiling. The initial selection was based on quantitative flocculating efficiency, determined via optical density reduction at 550 nm, followed by evaluation of sludge volume index (SVI) and settling time. These parameters revealed that selected strains achieved over 85% flocculating activity within 15 minutes and showed significant sedimentation efficiency, indicating strong potential for sludge minimization and water clarity improvement. Optimization of culture conditions was performed using various carbon and nitrogen sources, pH, temperature, inoculum size, and agitation speed, revealing glucose and peptone as the most effective nutritional substrates. Further biochemical investigations using phenol-sulfuric acid, Bradford, and carbazole-sulfuric acid methods confirmed that the extracted bioflocculants were composed predominantly of extracellular polysaccharides, proteins, and uronic acids, thereby underlining their anionic nature and bridging capacity. Elemental analysis through CHNS profiling, conducted on dry powder samples, revealed high carbon and nitrogen content with C/N ratios indicating favorable stoichiometry for flocculation processes. Thermogravimetric analysis (TGA) demonstrated high thermal stability of the bioflocculants up to 600°C, affirming their structural robustness. Gas chromatography-mass spectrometry (GC-MS) identified several bioactive constituents including fatty acid methyl esters, esters, and alkanes with potential flocculating, emulsifying, and antimicrobial properties. Proton nuclear magnetic resonance (^1H NMR) spectra of the purified extracts further elucidated functional group signatures, supporting the presence of hydroxyl, amine, and carboxylic functional groups critical for metal ion bridging and particle aggregation. High-resolution imaging via field emission gun scanning electron microscopy (FEG-SEM) and transmission electron microscopy (FEG-TEM) confirmed the porous, fibrous, and branched morphology of the biopolymer matrix, providing physical evidence of its particle entrapment capability. Collectively, these findings highlight the multifunctionality of the selected bioflocculant-producing strains and emphasize their potential for sustainable wastewater treatment applications. This study thus paves the way for further scaling-up and pilot-scale evaluation in diverse industrial wastewater treatment settings, contributing toward green biotechnological advancement and circular bioeconomy.