Synthesis and characterization of zinc nanoparticles using consortium of bioflocculant-producingmicroorganisms isolated from uMlalazi catchment, KZNand their application for wastewater treatment

Thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy (PhD) in Biochemistry in the Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, at the University of Zululand, 2021.

Flocculation is a simple and effective mechanical purification technology of eliminating colloidal particles, suspended solid substances, detritus, and microbial cells in wastewater through precipitation and formation of large flocs which are easily removable. Bioflocculants are macromolecules secreted by microorganisms duringgrowth. Bioflocculants are safe, biodegradable, innocuous, and produce nosecondary pollutants in the ecosystem which fuels their interest in both scientific and biotechnological fields, compared to the frequently used chemical flocculants. This study focussed mainly on the production, characterization and application of bioflocculants from individual marine bacteria and their consortium. The study alsofocused on the use of the produced bioflocculant from consortium as capping and stabilizing agent inthe ‘green’ synthesis of zinc oxide nanoparticles and their characterization as well as the applications.The medium composition and culture conditions of previously isolated and identifiedbioflocculant-producing bacteria form uMlalazi catchment, Mthunzini area, KwaZulu-Natal, South Africa, such as BacillussafensisKX94275.1and Bacillussp.KC782848.1and their consortium were optimized. The bioflocculants were produced through solventextraction and purification techniques. Bioflocculants physicochemical analysis were attained by scanning electron microscopy (SEM),energy dispersive spectroscopy (EDX),Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analyzer(TGA). Spectrophotometrically, the pH and thermal stabilities of bioflocculants were assessed. The cytotoxicity, solubilityand antimicrobial activity effects werealso investigated. The purified bioflocculants were also assessed for their removal potential spectrophotometricallyon biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen, aluminium, sulphur, phosphate, calcium, and sulphide from variouswastewater samples from Tendele coal mine wastewater treatmentplant (Somkhele area, Mtubatuba, KwaZulu-Natal, RSA), domestic wastewater (Vulindlela treatment plant, KwaDlangezwa, KZN, RSA) and Mzingazi river water (Richards Bay area, KZN, RSA). Their dye removal ability wasalso determined from various dye solutions. Inoculumsizes for optimal production of bioflocculants were 1% (v/v) (Bacillussafensis, 5% (v/v) (Bacillussp.) and 4% (v/v) for their consortium. Carbohydrates such as glucose (for both individualstrains) and starch (for consortium) were used for theiroptimumproduction.Both strains were favoured by the presence of urea, yeast extract and ammonium sulphate(combined)as theirnitrogen sources in their production media and their consortium preferred only Page | xxxiiammonium sulphate as the nitrogen sourcefor optimumbioflocculants production. Thebacteria produced their bioflocculants optimally at 30-40 oC and at 120-165 rpm in the presence of cations as stimulating agents. The consortium produced a bioflocculant (8.50g/L) at pH 3 that is much higher when compared to individual strains which produced 2.10g/L (Bacillussafensis)at pH 6and 1.522 g/L (Bacillussp.)at pH 4. The produced bioflocculants are anionic glycoproteins in nature and mainly composed of carbohydrates. Thecation-dependent bioflocculants are effective at low dosage concentrationsof 0.4 mg/mL (pure strains) and 0.8 mg/mL (consortium). Elements including N, C, O, P, Al, S,K, Cl and Na were revealed in the produced bioflocculants. The FT-IR spectra revealed functional groups such as hydroxyl, amide, amino, carboxyl and saccharides to be present in all bioflocculants.The bioflocculants showed thermal and wide pH stabilityproperties and solubility in distilled water. No antimicrobial activity effects have been demonstrated by the purified bioflocculants and exhibited a margin of safety as they revealed insignificant cytotoxicity effects. All bioflocculants showed great potential in the removal of dye from different dye solutions in the presence of stimulators. Their removal ability for various pollutants in wastewater were comparatively effectiveto traditional flocculants. Study findings suggest the use of bacterial consortium forthe enhanced bioflocculant yields and the potential of bioflocculants to be used as a replacement of conventional chemical flocculants.Nanotechnology has offered agreat opportunity in dealing with environmental concerns includingwater purificationfor potable water.Through the utilization of nanoparticles,pathogenic microorganisms, organic and inorganic toxic materials pollutingmost of the organic water, ground water and wastewater from different sourcescan be eliminated. This studyalso focussed on the biosynthesis of zinc oxide(ZnO)nanoparticles using the produced bioflocculant from marine bacterial consortium(Bacillussafensisand Bacillussp.)and their application in wastewater treatment. The biosynthesized ZnOnanoparticles were characterized using the analytical techniques such X-ray diffraction (XRD), UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FT-IR), thermogravimatic analysis (TGA), Transmission electron microscopy (TEM), and scanning electron microscopy (SEM) equipped with elemental analyzer. The absorption peaks around 300-330 nm which confirms the synthesis of ZnO nanoparticles revealed through UV-Vis spectroscopy. SEM image elucidated the spherical morphologyand agglomerated ZnO nanoparticles. Thebioflocculant-passivated ZnO nanoparticles were further investigated for their cytotoxicity effects usinghuman embryonic kidney (HEK 293) and breast cancer (MFC 7) cell lines and their antimicrobial activity effects on both Gram-negative and Page | xxxiiiGram-positive microorganisms in comparison with Ciprofloxacin and pure bioflocculant were also assessed. The produced nanomaterials were optimized for the improved flocculation efficiency. The investigations were also carried out for their potential removal efficiency of dyes from different solutions as well as their removal efficiencies of various pollutants in different wastewater samples (coal mine and river water) were also assessed. The as-synthesized ZnO nanoparticlesrevealed to have elements such as O, Al, C, P, and Zn. Oxygen contributed about 30.01% wt and zinc had39.15% wt contributionin total %wt. The X-ray diffractograms revealedthe diffraction peaks for biosynthesized nanoparticlesequated to ZnO and no other impurities were observed. The average crystallite size of the particles was estimated to be 24.66 nm. FT-IR spectroscopy reveals the presence of –NH2, –OH and Zn-O(at low wavenumber)functional groupswhich confirms the synthesisof ZnOnanoparticles using bioflocculant. The optimumflocculating activity of ZnO nanoparticles (88%) was obtained with the lowest concentration (0.2 mg/mL)favoured by the presence of LiCl as a stimulating agentcompared to optimum dosage of 0.8 mg/mL for bioflocculant used fortheirsynthesis. More than 70% flocculatingactivity was retained by ZnO nanoparticles after exposure for 30 min at 100 oC and at 121 oC (autoclaved) for 15 minconfirming its thermal stability property.The as-synthesized ZnO nanoparticlesexhibited significant cytotoxicityeffects in HEK 293 and MFC 7 cell lines in a concentration-dependent manner. Strong antimicrobial effecthas been demonstrated by ZnO nanoparticleson both Gram-negative and Gram-positive microorganisms. Great removal ability has been shown by the nanoparticles on dyes removal with an average removal efficiency greater than 90% of staining dyesusedsuch as congo red, malachite green, methylene blue and safranin. The high removal efficiencies of nutrients including phosphate, aluminium, calcium,nitrate, and total nitrogen and great potential in reducing both biological oxygen demand (BOD) and chemical oxygen demand (COD) has been demonstrated by the bioflocculant-passivated ZnO nanoparticlescompared to traditional flocculants.In conclusion, the bioflocculant-synthesized ZnO nanoparticles have the potential ofreplacing the in-use traditional flocculants in wastewater treatmentowing to their biocompatibility and eco-friendlycharacteristics.The littleamount(ZnO nanoparticles) required for flocculation of wastewater seemsto be a solution to the low yields bottleneck of bioflocculantsresulting in their limitations in term of applications industrially.