Characterization of a bioflocculant
Functional groups account for adsorption sites of flocculants for colloids in suspension (Ntozonke 2015). The multiple functional groups indicate the number of adsorption sites for colloidal particles. The bioflocculant by Bacillus safensis revealed with IR spectrum to possess various functional groups such as hydroxyl signalled with an absorption peak at 3303 cm−1 carbonyl represented by a small absorption peak at 1666 cm−1 and amine groups shown overlapping with hydroxyl (O-H) at stretching peak at 3303 cm−1 which are principal functional groups found inside the microbial flocculant’ s binding sites accountable for flocculation process (Figure 1a). Thermal stability of the bioflocculant produced by B. safensis was investigated using TG analyser. TG analysis was performed to institute the devolatilization description of the produced bioflocculant. From the analysis, three phases were noticed for the produced bioflocculant. The initial phase was observed between 29 – 100 oC with around 7% (w/w) weight loss owing to water content dissipation. Another weight loss was observed between 150- 200 oC with weight loss of 15.54% accounts for moisture content loss. The last phase was observable in higher temperatures where the weight loss of 33.20% (w/w) was confirmed for the bioflocculant due to the decomposition of the bioflocculant (Figure 1b).
The surface morphological structures of the flocculants have the essential roles in the process of flocculation (Akapo et al. 2019). The SEM analysis of the bioflocculant, kaolin particles and flocculated kaolin particles was investigated and the results are shown in Figure 6.2. SEM images (Figure 6.2) show the crystal-like facet patterning framework of the microbial flocculant (Figure 2a), fine as well as scattered appearance for kaolin particles (Figure 2b) and very big flocs for flocculated kaolin particles (Figure 2c) that are easily precipitate due to gravity. SEM-EDX analysis showed the elements present in the purified bioflocculant in mass proportion (% w/t) such as C (19.0), N (1.0), O (48.8), Na (0.7), Mg (2.7), P (7.2), S (0.1), Cl (0.7), Si (5.5), Al (5.5), K (1.0) and Ca (7.3) (Ntombela et al. 2020) which are accountable for the flexibility and stability of the bioflocculant.
Flocculation properties of the microbial flocculant from B. safensis
In bioflocculation, optimization of the microbial flocculant conditions is essential to improve its flocculation efficiency. Insufficient dosage concentration of microbial flocculant fails to adequately counterbalance certain negative charges present in suspended colloids leading to poor flocculation rate (Selepe 2017). Excessive bioflocculant dosages adversely influence the sedimentation and stabilization of the flocs owing to the escalated viscidity (Okaiyeto et al. 2015). Bacillus safensis produced a bioflocculant which preferred the 0.4 mg/mL dosage concentration for its optimum flocculating activity (Figure 3a). The dosage concentrations below and above 0.4 mg/mL inhibited the flocculating activity of the bioflocculant (Ntombela et al. 2020).
Cations have a significant function in the flocculation reaction as they enhance the neutralization and stabilization rate of the functional groups in the molecular chain of the microbial flocculant and kaolin particles resulting in the improved flocculating activity (Ayangbenro and Babalola 2018). Bacillus safensis produced a bioflocculant which preferred the Ba2+ as a stimulating agent for the optimum flocculating activity among others tested (Figure 3b). pH is one of the essential parameters in the reaction mixture due to its effect on the surface charge and electrification condition of bioflocculants and suspended colloids and therefore possess a substantial effect on the flocculation efficiency (Sun et al. 2015). In Figure 3(c), the flocculation rate showed by the microbial flocculant from B. safensis was hugely influenced by the pH of the kaolin solution. Bacillus safensis produced the bioflocculant favoured by alkaline, neutral and acidic conditions for great flocculating activity greater than 70% with an optimum flocculating activity of 91% at pH 11 (Ntombela et al. 2020).
The bioflocculant exhibited thermal stability properties as it retained over 55% flocculating activity when exposed to high temperatures. The thermal behaviour of the bioflocculant is in line with the existence of functional groups such as carboxylic and hydroxyl groups within the bioflocculant that might have permitted the hydrogen bonds formation. This indicates that the bioflocculant has got the carbohydrates as a backbone and less protein content is available (Figure 3d) (Ntombela et al. 2020).
Solubility assay of the bioflocculant
Bioflocculant compounds are not the same in the stability of charged, polar and hydrophobic constituents they possess on their outer membranes. The bioflocculant produced by Bacillus safensis was assessed for its solubility effect towards various dissolvent including methyl alcohol (methanol), hexane, ethyl acetate, ethanol, distilled water, benzene and dimethyl ketone (acetone). Only the distilled water was able to dissolve the bioflocculant completely among other tested dissolvent. The availability of carbohydrates component in the molecular chain has a significant character in the solubility effect of the bioflocculant. The microbial flocculants having polysaccharides as the dominant components are more of hydrophilic fraction compared to hydrophobicity nature of the bioflocculants with proteins as major components (More et al. 2014). The bioflocculant in this study was revealed to be predominately a polysaccharide (Ntombela et al. 2020), thus, the bioflocculant possesses charged and polar groups that are simply dissolved by water particles and eventually making the bioflocculant dissolvable and have a strong affinity of water.
Solubility of the bioflocculants could be explained based on the protein and carbohydrates content which is related to their stability towards different solvents treated. According to Maliehe et al. (2016), bioflocculants are likely to completely dissolve in water and in acidic and basic media. This bioflocculant has been revealed through FT-IR spectrum to have hydroxyl groups in its structure (Ntombela et al. 2020), which has been linked with strong attraction forces between bioflocculant molecules, resulting in the development of very strong crystalline solids leading to the occurrence of the rigid hydrogen bonding. Other solvents than water were incapable to dissociate these forces and the bioflocculant failed to dissolve in all of the organic solvents tested. Therefore, for the bioflocculant to completely dissolve in water or aqueous medium it must have OH- functional groups in their molecular chain in order to form hydrogen bonding with water molecules (Okaiyeto et al. 2015). This behaviour shown by the bioflocculant from B. safensis to dissolve in distilled water or aqueous solution only has been documented by numerous authors including Zaki et al. (2011) and Bisht and Lal (2019).
Antimicrobial activity assay of a bioflocculant
Bacillus subtilis, Escherichia coli, Bacillus cereus and Klebsiella pneumoniae are the microorganisms used in the assessment of the antimicrobial activity potential of the bioflocculant in comparison with the antibiotic (Ciprofloxacin). MIC tests conducted revealed that microorganisms investigated managed to grow optimally in the presence of the bioflocculant, but their growth was inhibited when the Ciprofloxacin was used. Only the antibiotic used has an inhibitory effect on all tested microorganisms and the bioflocculant was observed to exhibit no inhibitory properties over all tested microorganisms. Bioflocculants are thought to remove microbes with flocs during the bioflocculation process where the microorganisms settle down together with the flocs being formed as opposed to block their cell multiplication (Ntombela et al. 2021a). Although bioflocculants have been documented to block the growth of microorganisms, there is less information regarding their mechanism of action on the removal of pathogens in wastewater has been documented. Bacteria can attach to the suspended particles or kaolin particles and eventually collected together with the flake-like substances generated. Ciprofloxacin (positive control) inhibited the growth of all tested bacteria with the lowest concentrations of 6.25 mg/mL (Escherichia coli), 3.125 mg/mL (Bacillus cereus & Bacillus subtilis) and 1.56 mg/mL (Klebsiella pneumoniae). The smallest dosage amount of 1.56 mg/mL was more than enough to inhibit the microbial growth for Klebsiella pneumonia bacterium. Some researchers have reported various bioflocculants to eliminate microorganisms from wastewater. For example, Klebsiella pneumoniae produced a microbial flocculant capable of removing the Acanthamoeba cysts present in contaminated water (Zhao et al. 2013) and Dlamini et al. (2020a) reported the bioflocculant passivated in Fe@Cu core-shell nanoparticles to remove both Gram-negative and Gram-positive microorganisms from wastewater.
Cytotoxicity effect on HEK 293 and MFC 7 cell-lines
Cytotoxicity experiments of microbial flocculant on the HEK 293 and MFC 7 cell lines were carried out using MTT assay (Moodley and Singh 2019) and the resulted are shown in Figure 4. Although bioflocculants have been reported to be non-toxic by numerous researchers but there is a necessity to assess their cytotoxicity prior to their utilization for biosafety reasons as some bioflocculants may exhibit toxic effect (Maliehe et al. 2019). In this study, the bioflocculant revealed a margin of safety with above 95% viability of normal cells (HEK 293) exhibited in all evaluated bioflocculant concentrations. At the lowest bioflocculant concentration (25 µg/µL), no cell inhibition has been observed with an average of 100% cell survival and 96% viability when the cells were subjected to the maximum dosage of 100 µg/µL (Figure 4a). Cancerous cells (MFC 7) showed no cell inhibition as 100% cell survival has been observed at the lowest concentration (25 µg/ µL) used (Figure 4b). About 90% cell survival was obtained when the maximum bioflocculant dosage was used which is little bit less compared to the smallest bioflocculant dosage concentration. Therefore, the bioflocculant produced by B. safensis has demonstrated a good safety property that could be safe for application in different industrial reactions. Other authors also reported the microorganisms to produce non-toxic bioflocculants including Sharma et al. (2017) reported the toxic-free bioflocculant produced by Acinetobactor haemolyticus against sheep blood cells and in the in-vivo study on rats, no toxicity effects were reported. Maliehe et al. (2020) also reported a non-toxic microbial flocculant tested on HEK 293 cell line.
Dye removal by bioflocculant
Wastewater that contains dyes is a major concern globally, for industries that produce these wastewater, including paper and pulp, food, textile and leather. These dyes have negative effects towards human beings, microorganisms and aquatic-dependent matters (Yang et al. 2013). Aljeboree et al. (2017) reported that dyes are tetragenic, carcinogenic, mutagenic and occasionally recalcitrant to microbial degradation. Fabric industrial fields are known to generate large volumes of effluvium containing toxic materials, which eventually entering water bodies when not treated. Therefore, the microbial flocculant produced by Bacillus safensis was also investigated in this study, for its ability to remove dyes from different solutions and findings are shown in Figure 4c. More than 80% of the dye removal potential was revealed by the microbial flocculant in the entire dyes tested. In congo red, high removal efficiency (94%) was observed, followed by 93% for basic fuchsine, 90% methylene blue, and the lowest removal efficiency of 87% for crystal violet. Other bioflocculants were also reported to remove dyes from wastewater including the bioflocculant produced by Bacillus sp. (Ntombela et al. 2021b) and the microbial strains Xn11 and Xn7 produced the microbial flocculant reported to successfully remove colour from carbol fuchsine medium with more than 90% removal rate and less than 40% removal ability shown for reactive black dye from the solution (Zhang et al. 2012).
Wastewater treatment using a bioflocculant
Table 1 and Table 2 show the ability of the bioflocculant from Bacillus safensis to remove various contaminants available in domestic (Vulindlela Township, KwaZulu-Natal) and coal mine wastewater (Tendele coal mine, Mtubatuba, KwaZulu-Natal) in comparison with alum and ferric chloride. Excessive amount of pollutants such as COD and BOD does not support aquatic life (Verma et al. 2012). Nutrients in excess including nitrogen, phosphate and sulphur in water promote eutrophication. Nitrates and nitrogen have been considered as the huge threat in aquatic life as they lead to eutrophophication and eventually influence the cost of the availability of potable water. The availability of phosphate in water encourages the growth of marine plants and plankton, which avail food for fish. This increase in growth may increase the population growth of fish and enhance overall water quality. But the excessive amount of phosphate may result in wild growth of plants and algal in water leading to the eutrophication or excessive fertilization of receiving waters. Over fertilization may lead to the decay of vegetation and quality of life owing to lowered dissolved oxygen standards. High levels of phosphate may lead to phosphate toxicity in both humans and animals (Komaba and Fukagawa 2016). Therefore, it is of importance to remove them from water. The application of bioflocculant for the removal of these pollutants from industrial wastewater and domestic wastewater was investigated in comparison with traditional flocculants. The bioflocculant had a better removal efficiency of COD (48%) and BOD (68%) present in domestic wastewater compared to traditional flocculants (Table 1). The produced bioflocculant had effectively removed total nitrogen, sulphide, phosphate and turbidity from domestic wastewater with removal efficiencies of 69%, 71%, 61% and 91%, respectively. The removal rate for the tested pollutants by bioflocculant is very comparable and even better than that of harmful chemical flocculants used in the experiments.
The microbial flocculant potential to remove various pollutants from coalmine effluvium was also assessed and compared with commercial flocculants (Table 2). The bioflocculant showed better removal efficiencies of COD (93%), BOD (99%), total nitrogen (68%), sulphide (83%) and phosphate (59%) and the flocculating activity of 95% as opposed to less than 65% (COD and BOD), an average 61% (total nitrogen) and less than 80% (sulphide) for both chemical flocculants used. In general, the ability of the bioflocculant to remove pollutants was attributed to its surface structure, chemical components and functional groups. The effectiveness illustrated by the bioflocculant implied that it has potential to be used in wastewater treatment especially in industrial wastewater in replacing the currently predominant traditional flocculants. Similar results were also reported where the bioflocculant from Bacillus sp. was capable of efficiently reducing various pollutants in wastewater better than the traditional flocculants (Ntombela et al. 2021b). Maliehe et al. (2020) also reported the bioflocculant from the consortium to remove the pollutants in wastewater better that chemical flocculants. The bioflocculant from mixed culture of Bacillus safensis and Bacillus sp. showed better removal rate of different pollutants in wastewater samples (Ntombela et al. 2021a).
Table 1
Removal of pollutants from domestic wastewater by a bioflocculant
Flocculants | Quality of water | BOD (mg/L) | COD (mg/L) | Nitrogen (mg/L) | Sulphide (mg/L) | Phosphate (mg/L) | Flocculation efficiency @ OD680 nm |
Bioflocculant | Untreated | 38 | 404 | 0.137 | 0.85 | 3.38 | 0.395 |
Treated | 12 | 210 | 0.043 | 0.25 | 1.33 | 0.035 |
Flocculation rate (%) | 68 | 48 | 69 | 71 | 61 | 91 |
Iron(III)chloride | Untreated | 38 | 404 | 0.137 | 0.85 | 3.38 | 0.395 |
Treated | 24 | 251 | 0.059 | 0.27 | 1.06 | 0.089 |
Flocculation rate (%) | 37 | 38 | 57 | 68 | 69 | 78 |
Alum | Untreated | 38 | 404 | 0.137 | 0.85 | 3.38 | 0.395 |
Treated | 25 | 231 | 0.057 | 0.35 | 0.99 | 0.083 |
Flocculation rate (%) | 34 | 43 | 58 | 59 | 71 | 79 |
NB: Values are means of triplicates data.
Table 2
Removal efficiency of pollutants in coalmine wash water
Flocculating agents | Quality of water | BOD (mg/L) | COD (mg/L) | Nitrogen (mg/L) | Sulphide (mg/L) | Phosphate (mg/L) | Flocculation rate @ 680 nm |
Bioflocculant | Untreated | 58 | 1557 | 7.2 | 0.90 | 2.00 | 1.936 |
Treated | 0.74 | 116 | 2.30 | 0.15 | 0.83 | 0.098 |
Flocculation rate (%) | 99 | 93 | 68 | 83 | 59 | 95 |
Iron(III)chloride | Untreated | 58 | 1557 | 7.2 | 0.90 | 2.00 | 1.936 |
Treated | 27 | 150 | 2.9 | 0.19 | 0.50 | 0.139 |
Flocculation rate (%) | 53 | 90 | 60 | 79 | 75 | 93 |
Alum | Untreated | 58 | 1557 | 7.2 | 0.90 | 2.0 | 1.936 |
Treated | 21 | 276 | 2.82 | 0.27 | 0.38 | 0.278 |
Flocculation rate (%) | 64 | 82 | 61 | 70 | 81 | 85 |
NB: Values are means of triplicates data. |