Evaluation of flocculating potentials and characterization of bioflocculants produced by three bacterial isolates from Algoa bay, South Africa

Abstract

Flocculation has been widely adopted as one of the most effective methods to remove colloidal particles in water or wastewater treatment. Synthetic flocculants are conventionally used because of their high flocculating efficiency and cost-effectiveness. However, they have been reported to have hazardous properties and implicated in some serious health problems including senile dementia and neuro-toxicity, as well as being recalcitrant in the environment. Consequently, efforts are being geared away from the use of synthetic flocculants in water and wastewater treatment. Hence, the need for safe and eco-friendly flocculants has become imperative. Compared with synthetic flocculants, bioflocculants have special advantages such as safety, biodegradability and harmlessness to the environment and humans; attributes which make them potential alternatives in water treatment, downstream as well as fermentation processes. In the current study, the potentials of bacterial isolates recovered from Algoa Bay in the Eastern Cape Province of South Africa for bioflocculant production were investigated. The bacterial isolates were identified by polymerase chain reaction (PCR) as belonging to the Bacillus genus. The analysis of 16S ribosomal deoxyribonucleic acid (rDNA) nucleotide sequence of isolate M72 showed 99% similarity to Bacillus toyonensis strain BCT-7112 and was deposited in the GenBank as Bacillus toyonensis strain AEMREG6 with accession number KP406731. Likewise, the 16S rDNA nucleotide sequences of isolates M69 and M67 showed 98% sequence similarity to Bacillus licheniformis strain W7 and Bacillus algicola strain QD43 respectively; and M67 isolate was subsequently deposited in the GenBank as Bacillus sp. AEMREG7 with accession number KF933697.1. The results of the nutritional requirements and fermentation conditions revealed that optimum inoculum size for REG-6 production was 4% (v/v), while 5% (v/v) and 3% (v/v) were most favourable for MBF-W7 and MBF-UFH production respectively. Glucose was the best carbon source for the production of bioflocculants (REG-6 and MBF-UFH) by Bacillus toyonensis AEMREG6 and Bacillus sp. AEMREG7 respectively, while maltose supported optimum bioflocculant (MBF-W7) production by Bacillus specie. Inorganic nitrogen (NH4NO3) was the favoured nitrogen source for both REG-6 and MBF-W7 production, while mixed nitrogen sources [yeast extract + urea + (NH4)2SO4] supported the maximum production of MBF-UFH. The initial medium pH for REG-6 was 5, while MBF-W7 and MBF-UFH were both maximally produced at the initial pH of 6. After a 96 h cultivation period under optimal culture conditions, 3.2 g of purified REG-6 with a maximum flocculating activity of 77% was recovered from 1 L fermented broth of Bacillus toyonensis AEMREG6. Yields of 3.8 g and 1.6 g pure bioflocculants with the respective highest flocculating activities of 94.9% and 83.2% were also obtained from 1 L, 72 h-fermented broths of Bacillus licheniformis and Bacillus sp. AEMREG7 respectively. Furthermore, all the three bioflocculants (REG-6, MBF-W7 and MBF-UFH), displayed thermal stability within the temperature range of 50 to 100 oC, with strong flocculating activities of over 80% against kaolin suspension over a wide range of pH range (3–11) and relatively low dosage requirements of 0.1-03 mg/ml in the presence of divalent cations in the treatment of kaolin clay suspension and Thyme River waters. Chemical composition analyses of the bioflocculants showed them to be glycoproteins with a predominantly polysaccharide backbones as shown by the following carbohydrate/protein (w/w) ratios: 77.8%:11.5% (REG-6); 73.7%:6.2% (MBF-W7) and 76%:14% (MBF-UFH). Fourier transform infrared spectroscopy (FTIR) revealed the presence of hydroxyl, carboxyl and amide groups which are preferred for effective flocculation. Scanning electron microscopy (SEM) images of the purified bioflocculants showed that they have an irregular, coarse-grained structure connected in netted textures; it also revealed how the bioflocculants connected the scattered kaolin particles firmly together to form bigger flocs which subsequently precipitated out of suspension as a result of gravity. MBF-W7 showed good turbidity removal potential (86.9%) and chemical oxygen demand (COD) reduction efficiency (75.3%) of Thyume River waters. MBF-UFH showed higher flocculating activity for kaolin clay suspension compared to synthetic flocculants (aluminium chloride and iron chloride). The results obtained from this study suggested that the bioflocculants (REG-6, MBF-W7 and MBF-UFH) produced by these bacterial isolates have great potentials to serve as an alternatives to hazardous synthetic flocculants conventionally utilized in various industrial processes including water/wastewater treatment, and stand as attractive candidates for further research and development for industrial-scale application.