Green synthesis and characterisation of metal-based nanoparticles using Kombucha tea SCOBY yeast-based bioflocculant and their application in wastewater treatment | Green synthesis and metal-based nanoparticles

A Thesis submitted to the Faculty of Science, Agriculture and Engineering in fulfilment of the requirements for the Degree of Doctor of Philosophy of Science in Microbiology in the Department of Biochemistry and Microbiology at the University of Zululand, South Africa [2024].

Flocculation is a mechanical purification used in wastewater treatment, involving the precipitation of larger flocs to eliminate microbial cells and suspended particles. Microorganisms naturally secrete bioflocculants during growth, offering safer, environmentally friendly alternative to chemical flocculants. This study focuses on a bioflocculant from a yeast strain in Kombucha tea SCOBY, highlighting its production, characterization, and application. Additionally, the bioflocculant explored as a stabilizing agent in the green synthesis of copper, silver, and iron nanoparticles, with potential applications in wastewater treatment. The approach prioritizes safety, lack of secondary pollution, and biodegradability in environmental contexts. In the production of the bioflocculant, the optimal medium composition and culture condition for the bioflocculant-producing yeast, previously isolated from Kombucha tea SCOBY from GreenHeart Organics, Durban, KwaZulu Natal, South Africa, were determined through process optimization. Solvent extraction and purification techniques were employed to produce the bioflocculant. This bioflocculant was then used in synthesizing copper, iron, and silver nanoparticles. Characterization involved various analytical techniques such as FT-IR, X-ray diffraction, SEM-EDX, UV-Vis spectroscopy, TEM, and TGA. The flocculating activity of both the bioflocculant and the bio-synthesized nanoparticles was investigated, with subsequent optimization of dosage size, temperature, pH, metal ions, and shaking speed. Additionally, the study assessed the antimicrobial activity, cytotoxicity, and dye removal capabilities of the bioflocculant and bio-synthesized copper, iron, and silver nanoparticles. Spectrophotometric evaluation was conducted on the removal potential of these components for parameters including BOD, COD, total nitrogen, phosphorus, and sulphate, using wastewater samples from Tendele coal mine and Vulindlela wastewater treatment plants in South Africa. Through the analysis of the internal transcribed spacer (ITS) region sequences was used for fungi identification. The fungus was identified as Pichia kudriavzevii MH545928.1. Under optimal conditions, including a 1% (v/v) inoculum size, glucose and peptone as nutrient sources, a temperature of 35 ⁰C, pH 7, and a shaking speed of 140 rpm for 60 h, the fungus produced a bioflocculant with a remarkable flocculating activity of 99.1% and the yield of 2.836 g/L. Scanning electron microscopy (SEM) analysis unveiled a cumulus-like structure, while Fourier transform infrared spectroscopy (FT-IR) indicated the presence of hydroxyl, carboxyl, amine, and thiocyanate functional groups which is essential in the flocculation process. X-ray diffraction (XRD) analysis revealed the bioflocculant exhibited large particles with distinct diffraction peaks at 10⁰ and 40⁰, suggesting its crystalline nature. The results obtained suggest that Pichia kudriavzevii MH545928.1 holds promising potential for industrial applications as a producer of bioflocculants. The produced bioflocculant demonstrated removal efficiencies of 73% for BOD, 49% for COD, and 47% for phosphate (P). The obtained bioflocculant, with a concentration of 2.836 g/L, exhibited composition comprising carbohydrates (69%), protein (11%), and uronic acid (16%). The elemental composition analysis of the bioflocculant revealed the presence of the following elements: carbon (C) at 16.92%wt, nitrogen (N) at 1.03%wt, oxygen (O) at 43.76%wt, sodium (Na) at 0.18%wt, magnesium (Mg) at 0.40%wt, aluminium (Al) at 0.80%wt, phosphorus (P) at 14.44%wt, sulfur (S) at 1.48%wt, chlorine (Cl) at 0.31%wt, potassium (K) at 0.34%wt, and calcium (Ca) at 20.35%wt. In coal mine wastewater, the bioflocculant demonstrated removal efficiencies of 43% for chemical oxygen demand (COD), 64% for biochemical oxygen demand (BOD), 73% for phosphate (P), and 50% for nitrogen (N). Comparing the bioflocculant to traditional flocculants, it was comparable in terms of dye removal with over 72% removal efficiency across all tested dyes. The biosafety profile of the bioflocculant showed survival effect of over 80% HEK cell lines with 25 μg/μL concentration. It was noted that on both Gram-positive and Gram-negative microorganisms, the bioflocculant did not show to be effective. The viability of colour change from blue to light blue in the formation signifies the success in the synthesis of CuNPs. The peaks observed in the characteristics of the bioflocculant-treated CuNPs were at 3482 cm-1 (-OH), 3261 cm-1, 1640 cm-1, 1059 cm-1, 580 cm-1, and 519 cm-1 (Cu-O). These peaks indicate the presence of functional groups including hydroxyl, amine, and copper oxide bonds. The UV-Vis analysis of the as-synthesized CuNPs demonstrated the presence of surface plasmon resonance (SPR) within the absorbance range of 500 - 600 nm, with peak maxima observed at 535 nm and 560 nm. The XRD pattern exhibited prominent planes such as (200) and (220) at 2θ values of 40° and 51°, respectively. Furthermore, the particle size of the CuNPs, determined using the Debye-Scherrer equation, was found to be 30 nm. Transmission electron microscopy analysis confirmed the presence of spherical shaped CuNPs with an average size of 20 nm. EDX analysis showed the presence of copper (Cu) in the as-synthesized CuNPs, indicating successful biosynthesis as it was not detected in the bioflocculant used during synthesis. The biosynthesized CuNPs were thermal stable retaining above 70% flocculating activity when subjected to TG analysis. The optimal dosage for the biosynthesized CuNPs was determined to be 0.2 mg/mL, resulting in a flocculating activity of 93%. Through the optimization of various factors such as metal ions, pH, temperature, and shaking speed, flocculating activities of 93% (Ca), 94%, 93% (at 70°C), and 94% were achieved, respectively. CuNPs were effective on dye removal with over 58% removal efficiency. They were also effective on inhibiting both Gram-positive and Gram-negative microorganisms, while biosafety showed they could retain over 58% cell viability at a concentration of 200 μg/μL. CuNPs promise a potential application in an industrial wastewater treatment and dye removal and shows a good biosafety effect. Successful biosynthesis of FeNPs was confirmed by the presence of various functional groups observed in the FT-IR spectra, including hydroxyl, halogen (C-Br), carbonyl, alkanes (C-H), and Fe-O functional groups. TEM analysis demonstrated that the iron nanoparticles generated exhibited dimensions ranging from 2.6 to 6.2 nm. The UV-vis spectra displayed a peak at approximately 210, 265, and 330 nm for the newly fabricated FeNPs, providing confirmation of their formation. The EDX spectrum of the biosynthesized FeNPs indicated the presence of iron nanoparticles at a weight percentage of 0.82 %wt in addition to the elements present in the bioflocculant used in their fabrication. The FeNPs synthesized were observed to be hexagonal in shape and exhibited agglomeration, with dimensions ranging from 18 to 50 nm revealed through SEM analysis. The thermal stability of FeNPs observed through TG analysis, revealed that their thermal stable and have retained more than 60% of their initial weight at elevated temperatures. The optimal dosage for achieving the highest flocculating activity was determined to be 0.6 mg/mL for the nanoparticles. The FeNPs exhibited remarkable performance by retaining over 70% of their flocculating activity even at a temperature of 100 ⁰C. Notably, the biosynthesized FeNPs displayed the highest flocculating activity of 97% under specific conditions, including a shaking speed of 180 rpm, Fe3+ ions as the cation, and a pH level of 6. The biosynthesized Fe nanoparticles exhibited concentration-dependent cytotoxicity on HEK 293 cell lines, with the highest concentration (100 μg/μL) resulting in 34% cell survival. These nanoparticles also demonstrated potent antimicrobial properties against various Gram-positive and Gram-negative bacteria. Furthermore, they displayed high efficiency in removing dyes, with a minimum removal rate of 65% and a maximum removal rate of 93% for safranine. The Fe nanoparticles also showed remarkable effectiveness in removing different pollutants from wastewater including COD and BOD. In comparison to conventional flocculants and the bioflocculant, the biosynthesized Fe nanoparticles showed significant potential in reducing both chemical oxygen demand (COD) and biological oxygen demand (BOD) in treated wastewater samples. Making the better optimization for water treatment to replace the in use conventional flocculants. In the biosynthesis of silver NPs using a bioflocculant the SEM images unveiled the presence of smooth, close to spherical particles with an average size of 7 to 12 nm. EDX analysis revealed the presence of silver as an element, constituting 61.93% of the weight, alongside other elements found in the bioflocculant which confirm the synthesis of AgNPs from a bioflocculant. FT-IR spectra analysis indicated the existence of various functional groups, including carboxyl, polyphenols, aromatics, anhydrate, and aliphatic primary amine, amine, and halo compounds. The presence of amine groups suggested the successful synthesis of AgNPs, as they facilitated the reduction of Ag+ to Ag0. TEM imaging revealed spherical nanoparticles with sizes ranging from 7 to 12 nm. TG analysis demonstrated that AgNPs exhibited greater thermal stability, retaining over 85% of their mass at high temperatures, while the bioflocculant used for their fabrication retained 60% of its weight under similar conditions. UV-vis spectra exhibited a Surface Plasmon Resonance (SPR) band around 450 nm for the Ag nanoparticles, and X-ray diffraction analysis indicated a crystallite size of 19 nm obtained through Scherrer equation. Utilizing the bioflocculant as a capping and stabilizing agent in the biosynthesis of AgNPs, offers an ecologically safe technique that yields synthesis rates comparable to chemical methods but with faster results. The optimization process revealed that the AgNPs exhibited optimal flocculation efficiency at a dosage of 0.2 mg/mL, with the flocculating activity of 94%. The biosynthesized nanoparticles demonstrated a preference for a shaking speed of 140 rpm, resulting in a flocculating activity of 94%. Furthermore, when a pH of 7 was employed in the presence of Ca2+ as the metal ion, the optimum flocculating activity of 98% was obtained. The biosynthesized AgNPs using a bioflocculant are thermal stable as they retained more than 85% flocculating activity at 100 ⁰C. The concentration-dependent cytotoxic effects of the biosynthesized AgNPs on HEK 293 cell lines were observed and showed over 68% cell viability at a concentration of 25 μg/μL and 42% cell viability at 100 μg/μL. The AgNPs also demonstrated strong antimicrobial efficacy against various Gram-positive and Gram-negative bacteria. Additionally, the biosynthesized AgNPs exhibited efficient removal of dyes, with the removal efficiency over 75% and with a maximum removal efficiency of 96% observed for methylene blue. Furthermore, these nanoparticles proved highly effective in removing a diverse range of pollutants from wastewater, surpassing traditional flocculants in removing both BOD (92% removal efficiency) and COD (86% removal efficiency) among other pollutants. In conclusion, the Cu, Fe, and Ag nanoparticles synthesized using the bioflocculant exhibit promising potential as a substitute for traditional flocculants in wastewater treatment and dye removal due to their biocompatibility and environmentally friendly properties.