Molecular and Morpho-Structural Characterization of Bioflocculant-Producing Bacterial Isolates for Sustainable Wastewater Remediation
Description
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.
Files
Steps to reproduce
1. Environmental Sampling and Microbial Isolation The process begins with the strategic collection of effluent samples from diverse industrial environments: municipal solid waste (MSW) leachate and pharmaceutical wastewater. Enrichment: Samples are inoculated into a selective production medium (KH2PO4: 2g/L; K2HPO4: 5g/L; (NH4)2SO4: 0.2g/L; MgSO4.7H2O: 0.2g/L; Yeast Extract: 0.5g/L). Purification: Individual colonies are obtained via the streak plate method on nutrient agar. Four specific Bacillus strains (ASW5, SED11, A13, S14) are identified through 16S rRNA gene sequencing. 2. Primary Flocculation Screening and Kinetics To quantify efficiency, the Kaolin Clay Suspension Assay is utilized. Assay Protocol: A 4g/L Kaolin suspension is adjusted to pH 7.0. Add 2 mL of cell-free supernatant and 1 mL of 1% CaCl2 (coagulant aid) to 100 mL of the suspension. Measurement: After rapid mixing (2 minutes) and slow stirring (5 minutes), allow 15 minutes for settling. Measure OD 550 nm using a UV-Vis spectrophotometer. Sludge Metrics: Calculate the Sludge Volume Index (SVI) by measuring the volume of settled sludge (Vss) in a graduated cylinder to assess sedimentation potential. 3. Media Optimization and Bioflocculant Extraction Nutritional and physical parameters are systematically varied to maximize yield. Optimization: Replace basal nutrients with Glucose and Peptone. Adjust cultivation conditions to the identified optima: mesophilic temperature, neutral pH, and moderate agitation speed. Extraction: Post-fermentation, centrifuge at 10,000 rpm for 20 minutes (4 degree C). Add two volumes of ice-cold ethanol to the supernatant and leave at 4 degree C for 24 hours. Purification: Collect the precipitate, re-dissolve in deionized water, and dialyze against distilled water to remove low-molecular-weight impurities before freeze-drying. 4. Biochemical and Structural Profiling The dry biopolymer undergoes rigorous chemical analysis: Composition: Quantify total carbohydrates (Phenol-Sulfuric acid), proteins (Bradford), and uronic acids (Carbazole-Sulfuric acid). Thermal and Elemental Analysis: Perform TGA from 30 degree C to 800 degree C to confirm thermal stability up to 600 degree C. Use a CHNS analyzer to determine the elemental stoichiometry and C:N ratio. Spectroscopy: Conduct 1HNMR in D2O to identify carboxyl and hydroxyl protons. Perform GC-MS following methanolysis to detect bioactive fatty acid methyl esters and alkanes. 5. High-Resolution Morphological Imaging Physical evidence of bridging and entrapment is captured via electron microscopy. FEG-SEM: Dry samples are gold-coated and scanned to visualize the porous, fibrous surface architecture. FEG-TEM: Samples are dispersed on a copper grid to examine the internal branched morphology of the biopolymer matrix.
Institutions
- The Charutar Vidya Mandal (CVM) UniversityGujarat, Vallabh Vidyanagar