Evaluation of the flocculation potential and characterisation of a bioflocculant produced by activated sludge bacteria from Vulindlela Wastewater Treatment Plant

A dissertation submitted to the Department of Biochemistry and Microbiology in fulfilment of the requirements for the Degree of Master of Science (Microbiology) in the Faculty of Science and Agriculture at the University of Zululand, 2021.

Biofflocculants are flocculating agents produced by microorganisms during their phase of growth as secondary metabolites. These bioflocculants play an important role in wastewater treatment and industrial applications. This study aimed to isolate, screen and identify bioflocculant producing-bacterium isolated from Vulindlela wastewater treatment plant (VWWTP). The production conditions for bioflocculant were optimized for improved flocculating activities and production yield. After evaluation and purification the bioflocculant was characterized and used in wastewater treatment and dye removal from different dye soluctions. The bacterium was isolated using nutrient agar plates prepared with sterile activated sludge and screened for bioflocculant production. The bacterial identification was done through 16S rRNA gene sequencing and the selected isolate has got about 98% similarities with Proteus mirabilis. The nucleotide sequences were deposited in the Gene bank as Proteus mirabilis with accession number AB 932526.1. The optimum production of bioflocculant was achieved using 3% (v/v): [1.5 (mL)] inoculum size with 90% flocculating activity, sucrose (95%) and yeast extract (91%) as a sole carbon and nitrogen sources, respectively. The addition of BaCl2 enhanced the flocculating activity (95%) at a pH 3 - 6 with maximum flocculating activity at an initial pH 6 (83%), at optimum temperature of 30 ºC (96%), shaking speed of 120 rpm (98%) and the fermentation time of 72 hrs (92%). After extraction and purification, a yield of 3.8 g/L of the purified bioflocculant was obtained. The highest flocculating activity of 76% with kaolin clay solution (4 g/L) was achieved at 0.4 mg/mL in the presence of divalent cation (Ba2+) as stimulating agent. Chemical composition analysis showed that a purified bioflocculant is mainly composed of carbohydrates (64%), protein (10%) and uronic acid (24%). Fourier transform infrared (FT-IR) analysis revealed stronger intensity of the carboxyl, hydroxyl and amino group as the main fuctional groups of the purified bioflocculant responsible for its high flocculation rate. XDR analysis indicates the lack of crystallinity and inter-molecular hydrogen bonds. The elemental analysis carried out with scanning electron microscope (SEM) equipped with elemental analyser showed that the bioflocculant Proteus mirabilis AB 932526.1 revealed the presence of elements such as N, C, O, Na, Mg, P, Mo, K, and Ca. The bioflocculant was more efficient in acidic to neutral pH ranges with the maximum flocculating activity of 97% at pH 6. xv | P a g e The bioflocculant retained more than 55% flocculating activity at 121 ºC revealing its thermal stability potential. It shows great potential in removing COD, BOD, sulphide, nitrogen, and phosphorus in wastewater treatment. More than 70% removal efficiency was demonstrated by