Isolation, screening, identification, and optimization of microorganisms with bioflocculant-production potential from kombucha tea scobies and its application in wastewater treatment

A thesis submitted to the Faculty of Science, Agriculture and Engineering in fulfilment of the requirements for the Degree of Master of Science in the Department of Biochemistry and Microbiology at the University of Zululand, South Africa, 2021

Microorganisms excrete metabolites during growth. Extracellular polysaccharides such as, glycoproteins, proteins, and nucleic acids contributes to the synthesis of bioflocculants. Through formation of bridges between suspended particles in a solution, bioflocculants assist with increase in flocculation, leading to particles precipitation. It is noteworthy that when impurities are flocculated into flocs, they tend to settle down and can easily be removed. In this study, the ability of the yeast Pichia kudriavzevii from Kombucha teas Scoby to produce a bioflocculant was investigated. A Kombucha tea Scoby was brought from Pine Town, KwaZulu-Natal Province of South Africa, and bioflocculant-producing microorganisms were isolated from the Scoby. Dilutions of the samples were made and cultivated in nutirents agar plates to obtain pure cultures. Pure cultures were screened for bioflocculant-production potential against kaolin clay suspension (4 g/L) as the test material. The isolate with better bioflocculant production potential was selected for the bioflocculant production. The isolate showed the highest flocculating activity of 84.93% against kaolin clay suspension. The identification of the organism using 16S rRNA showed the organism to have 99% similarities with the yeast Pichia kudriavzevii with accession number MH545928.1. The strain was capable of producing a bioflocculant under the optimal production conditions of 1% (v/v) inoculum size, glucose (92%) as carbon source, peptone (94%) as a source of nitrogen, at a temperature of 35 ºC (97%). At an initial pH 7 of the medium, the maximum flocculating activity (91%) was achieved and the shaking speed of 140 rpm (96%) resulted into an optimum production of a bioflocculant by P. kudriavzevii. A bioflocculant yield of 2.836 g was produced from 1 L fermentation broth after 60 hours of incubation at 35 ºC. The colour of a produced bioflocculant was milky-white and in a powdered form. The purified bioflocculant obtained had the highest flocculating activity of 80% at a dosage size of 0.4 mg/mL against kaolin suspension. The purified bioflocculant was cation-dependent with Al3+ (72%) as the most favourable cation. The purified bioflocculant was able to retain about 70% flocculating activity when exposed to 121 º C temperatures for 15 minutes, which confirmed the thermostability of the bioflocculant. After the Fourier-transform infrared (FT-IR) analysis of the purified bioflocculant, it was revealed that hydroxyl, carboxyl, amine, thiocynates, alkynes, furan functional groups are present in the molecular chain of a bioflocculant and are responsible for its best flocculation ability. The chemical composition of the purified bioflocculant showed the presence of sugar (69%), protein (11%), and uronic acid (16%) with carbohydrates as main component and responsible for its thermal stability characteristics. A cumulus-like structure of the bioflocculant was revealed using a scanning electron microscope (SEM) and the weight fractions from the elemental analysis of the purified bioflocculant were C, N, O, Na, Mg, Al, P, S, Cl, K, Ca, which accounts for 16.92: 1.03: 43:76: 0.18: 0.40: 0.80: 14.44: 1.48: 0.31: 0.34: 20.35 (%wt), respectively. The XRD analysis of the purified bioflocculant showed that the bioflocculant have bigger particles with diffraction peaks at 10º and 40º indicating the crystallinity of the purified bioflocculant. The produced bioflocculant is highly effective at 5 g/L salt concentration with flocculating activity of 81%. Increasing the salt concentration inhibibed the flocculating activity. The produced bioflocculant showed highest removal efficiencies compared to the conventional chemical flocculants (Fe3+ and alum) used in the study. The purified bioflocculant exhibited a remarkable removal efficiency in both domestic and industrial (coal mine) wastewater for COD, BOD, phosphorus, sulfate, nitrate, and total nitrogen with removal efficiency of 49% and 43% (COD), 79% and 64% (BOD), 46% and 48% (phosphorus), 79% and 73% (sulphate), 61% and 71% (nitrate), and 50% (total nitrogen), respectively. The bioflocculant also revealed strong dye removal ability with the removal efficiency of 81% (Congo red), 81% (nigrosine), 73% (methylene blue), and 74% (safranin). In conclusion, this bioflocculant from P. kudriavzevii seems to have a potential in the removal of different impurities from various wastewater especially in the domestic and industrial wastewater.