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Article

Isolation and Optimisation of Culture Conditions for a Marine Bioflocculant-Producing Bacterium and Application of Its Bioflocculant in Wastewater Treatment

by
Tlou Nelson Selepe
1,*,
Tsolanku Sidney Maliehe
2,
Kgabo Moganedi
2,
Peter Masoko
2 and
Vusimuzi Mulaudzi
3
1
Department of Water and Sanitation, University of Limpopo, Private Bag X1106, Polokwane 0727, South Africa
2
Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Polokwane 0727, South Africa
3
Department of Chemistry, University of Limpopo, Private Bag X1106, Polokwane 0727, South Africa
*
Author to whom correspondence should be addressed.
Int. J. Environ. Res. Public Health 2022, 19(16), 10237; https://doi.org/10.3390/ijerph191610237
Submission received: 29 June 2022 / Revised: 3 August 2022 / Accepted: 5 August 2022 / Published: 18 August 2022
(This article belongs to the Section Water Science and Technology)
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Versions Notes

Abstract

:
The application of bioflocculants has become an alternative to that of chemical flocculants in wastewater treatment due to their environmental friendliness and non-toxic effects. This study aimed at isolating a bioflocculant-producing bacterium from marine water, optimisation of its culture conditions, and investigation of the removal efficiency of its bioflocculant on pollutants in wastewater. The bacterium was identified by 16S rRNA gene analysis. Optimal carbon and nitrogen sources, inoculum size, temperature, pH, and time were determined by the one-factor-at-a-time assay. The cytotoxicity of the bioflocculant was assessed on African green monkey kidney and bovine dermis cells using a tetrazolium-based columetric (MTT) method. Its removal efficiencies on chemical oxygen demand (COD), biological oxygen demand (BOD) and sulphur were determined using the Jar test method. The bacterial isolate was identified as Ochrobactrum oryzae AB84113. A maximum flocculating activity of 92% and a yield of 3.768 g/L were obtained when a 1% (v/v) inoculum size was used in the presence of starch and yeast extract at pH 7, 30 °C, and after 72 h of cultivation. The bioflocculant demonstrated non-cytotoxic effects on bovine dermis and African green monkey kidney cells. The bioflocculant removed 98% COD, 91% BOD and 86% of Sulphur. The bioflocculant has potential for pollutant removal from industrial wastewater.

1. Introduction

Water is an essential resource for life. However, water quality is constantly deteriorating due to increased contamination from industrial activities and urbanisation [1]. Globally, approximately 80% of wastewater is not properly treated prior to its discharge into various water bodies [2]. Water pollution affects more than 1.2 billion people globally, leading to poor economic growth and food insecurity [3]. Moreover, this leads to a challenge in achieving environmental sustainability as addressed by the Millennium Development goal No.7 [4]. Thus, it is it imperative to effectively treat wastewater and water to appraise its quality.
Flocculation is a process employed in solid-liquid separation in several industrial processes such as wastewater treatment, drinking water purification, food and fermentation processes [5]. Flocculants are agents that reduce or eliminate the stability of colloids in suspensions, enabling the dispersed particles to aggregate. Generally, flocculants are grouped into inorganic (i.e., aluminium sulfate, aluminium chloride, ferric chloride and ferrous sulphate), organic synthetic flocculants (i.e., polyacrylamide derivatives and polyethyleneimine) and naturally occurring flocculants (i.e., plant based and microbial flocculants) [6]. Among the three types of flocculants, inorganic and organic synthetic flocculants are mainly used in wastewater treatment because of their availability at low cost and their high flocculating abilities [7,8]. However, these types of flocculants are harmful to the environment and pose health problems [9]. For instance, ferric salts tend to be corrosive due to their hydrolysis, which leads to acidic conditions in water [10]. Aluminium salts are also linked to Alzheimer’s disease in humans [11]. Moreover, polyacrylamide derivatives are often non-degradable in nature and exhibit a resilient biological toxicity [12]. Therefore, alternative eco-friendly and innocuous flocculants are needed.
Natural occurring flocculants such as plant-based and microbial flocculants are the preferred alternatives to inorganic and organic synthetic flocculants [13,14]. Plant-based and microbial flocculants are environmentally friendly and have low or non-toxic effects [15]. Although plant-based flocculants exhibit strong activities in wastewater treatment [14,16], generally their biosynthesis is not convenient in terms of quality and productivity due to ecological conditions and climatic change. Thus, microbial flocculants are gaining more attention because they are quickly reproducible within a short time and are weather-independent [17]. Microbial flocculants are produced by bacteria and fungi and are mainly composed of carbohydrates, proteins, and lipids [18]. Although bioflocculants are promising alternatives, the high costs of their production and the generally low yields are major bottlenecks for their industrial utilisation [15]. Thus efforts to reduce the cost of production and application include screening of high-yield producing microorganisms and optimization of their culture conditions [8].
Several bioflocculant-producing bacterial species have been reported for their significant flocculating activities (>70–90%), yield, and various additional functions in wastewater treatment [19,20]. Much research has been done on bioflocculant-producing bacteria in activated sludge and soil [21,22], but not on those from marine environments [23].
This study aimed at isolating and identifying a marine bioflocculant-producing bacterium from Mtuzini Beach in Kwa-Zulu Natal, South Africa. To maximize bioflocculant yield by the isolate, the medium composition (carbon and nitrogen sources) and culture conditions (inoculum size, temperature, initial pH of the medium and cultivation time) were optimised using the one-factor-at-a-time method. The surface morphology, functional groups, and degradation property of the bioflocculant were identified using scanning electron microscope (SEM), Fourier-transform infrared (FT-IR) spectroscopy and thermogravimetric analysis (TGA), respectively. The biosafety of the bioflocculant was assessed by evaluating its cytotoxicity on the African green monkey kidney and Bovine dermis using a tetrazolium-based columetric (MTT) method. Lastly, its ability to remove materials expressed by biological oxygen demand and chemical oxygen demand, as well as sulphur from wastewater, were determined using the Jar test method.

2. Materials and Methods

2.1. Chemicals and Production Medium

All chemicals, reagents and media used were of analytical grade and were procured from Merck Pty Limited and Sigma-Aldrich, Johannesburg, South Africa. The selective media used for isolation were marine agar (MA) and reasoner’s 2A agar (R2A). The enrichment medium was composed of 3 g beef extract, 10 g tryptone and 5 g sodium chloride. The production medium by Zhang et al. (2007) was used for evaluation of bioflocculant production. The medium was composed of glucose (20.0 g), KH2PO4 (2.0 g), K2HPO4 (5.0 g), (NH4)2SO4 (0.2 g), NaCl (0.1 g), CH4N2O (0.5 g), MgSO4 (0.2 g) and yeast extract (0.5 g). All media were prepared in filtered marine water before been sterilised by autoclaving at 121 °C for 15 min.

2.2. Sample Collection

Water samples were collected from Mtunzini Beach in KwaZulu Natal, South Africa (28°57′ S 31°45′ E). Sterile Scott bottles were used to collect water samples on three different sites and on each site water samples were collected in triplicate. The physiochemical parameters, temperature, pH, total dissolved solid, dissolved oxygen, salinity and pressure were determined in-situ using HI 98194 PH/E/DO multi-parameters. Thereafter, the water samples were put in an ice box and transported to the laboratory at the Department of Water and Sanitation at the University of Limpopo, South Africa.

2.3. Isolation of the Bioflocculant-Producing Bacteria

Two millilitres of the marine water samples were transferred into 8 mL of sterile saline water (0.85% (w/v)) and agitated for 30 s. Thereafter, serial dilutions were performed and 100 µL of the diluted and undiluted samples were spread on the surface of marine and Reasoner’s 2A (R2A) agar plates. The agars were previously prepared using filtered marine water from Mtunzini Beach and were adjusted to pH 6.8 prior to autoclaving at 115 °C for 15 min. The R2A plates were incubated for 4 days at 27 °C and the marine agar plates were incubated for 8 days at the same temperature. Colonies from both media were selected according to their appearance and morphology. The selected colonies were subculture twice on their isolated agar media.

2.4. Activation of the Isolates

Enrichment medium (see Section 2.1) was used to activate bacteria. About 50 mL of the medium was poured into different test tubes and autoclaved at 121 °C for 15 min. A loopful of the isolates was inoculated into the activation broth and incubated in a rotary shaker at 160 rpm, 37 °C for 24 h [24].

2.5. Cultivation Medium for Bioflocculant Production

Activated isolates (1 mL) were inoculated into the sterile production medium (see Section 2.1) at 27 °C for 72 h at pH 6.8 and a shaking speed of 160 rpm. After the incubation period, 2 mL of the broth cultures were withdrawn and centrifuged at 10,000 rpm for 15 min. The supernatants were used for the evaluation of flocculating activity.

2.6. Determination of Flocculating Activity of Isolates

Cell-free supernatants were used to determine flocculating activity according to Kurane et al. [25]. Briefly, 2 mL of the supernatants and 3 mL of 1% (w/v) CaCl2 were added to 100 mL of kaolin suspension (4.0 g/L, pH 7.0) in a conical flask. The mixture was shaken for a minute, poured into a measuring cylinder and allowed to stand for 5 min. Afterwards, 2 mL of the clarifying upper phase layers were carefully withdrawn and their optical densities (OD) were measured at 550 nm with a spectrophotometer (Spectro-quant, Pharo 300 Merck, Boston, MA, USA). Sterile distilled water (2 mL) was used as the control. The percentage flocculation activity (%FA) of all the supernatants was calculated according to the equation:
%FA = (Ao − A)/Ao × 100,
where Ao and A are the optical densities of the control and the test samples at 550 nm. The bacterial isolate with the highest flocculating activity was selected for identification.

2.7. Identification of the Bacterium

The selected bacterial isolate was identified using 16S rRNA gene sequence analysis. Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene fragments and sequencing of PCR products were carried out as described by Shida et al. [26]. The obtained sequences were analysed by comparing them with those retrieved from the National Center for Biotechnology Information (NCBI) databases to identify the closest bacterial species.

2.8. Optimization of the Culture Conditions

Different inoculum sizes were used to determine their effect on bioflocculant production. Flasks (100 mL) containing 50 mL of the sterile production medium were inoculated with 0.5, 1.0, 1.5 and 2 mL of the cultured broth to give 1, 2, 3 and 4% (v/v) inoculum sizes, respectively. The inoculums were then incubated for 72 h at 30 °C at a shaking speed of 160 rpm. The broth cultures were then centrifuged at 10,000 rpm for 15 min and the supernatants were analysed for flocculating activity as described previously [27]. The effect of carbon and nitrogen substrates was determined using the method of Luo et al. [28]. Glucose in the original medium was substituted with 20 g/L of the following carbon sources: fructose, sucrose, maltose, lactose, xylose, starch and molasses. Thereafter, the flocculating activity was assessed as previously stated [29]. Mixed nitrogen sources (urea, yeast extract and (NH4)2SO4) in the original production medium were also replaced with 1.2 g/L of organic (casein, peptone, urea and yeast extract) and inorganic (ammonia) nitrogen sources to determine their effect on bioflocculant production [30]. The flocculating activity was then measured as described previously. The effect of pH of the growth medium was determined according to the method of He et al. [31]. The pH of the medium was adjusted in the range of 3 to 12 using 0.1 M HCl and 0.1 M NaOH and autoclaved. Thereafter, the isolate was inoculated and incubated for 72 h in a rotary shaker at 30 °C and 160 rpm. Flocculating activity was determined thereafter. To assess the effect of temperature, sample of inoculum was pipetted into the medium and incubated at various temperatures (20, 25, 30, 35 and 40 °C). Flocculating activity was then measured [32]. The culture medium was prepared based on previously obtained optimal growth conditions. The isolate was cultured at different shaking speeds in the range of 0–220 rpm for 72 h. Thereafter, the flocculating activity was determined as previously described [33]. The effect of incubation time on bioflocculant production was evaluated. The medium was composed based on previously obtained optimal growth conditions. The broth culture (2 mL) was withdrawn, centrifuged and evaluated for flocculating activity after every 12 h over a period of 120 h. The optical density (OD) of the broth was measured at 550 nm, representing bacterial biomass, and the pH was also recorded [34].

2.9. Extraction and Purification of the Bioflocculant

Extraction and purification of bioflocculant was done using the method of Dlamini et al. [35]. The bacterium was cultured under optimum culture conditions in 1 L of the medium and the broth culture was centrifuged (8000 rpm, at 4 °C for 30 min) to obtain the cell free supernatant. One volume of sterile distilled water was poured into the supernatant and re-centrifuged to remove insoluble materials. Thereafter, two volumes of alcohol were added to the supernatant; the mixture was agitated and then allowed to precipitate for 12 h at 4 °C. Then, the precipitate was vacuum dried, and the crude product was re-dissolved in the sterile distilled water to give a solution of 1% w/v. A mixture of chloroform and n-butyl alcohol (5:2 v/v) was added to the bioflocculant solution in a ratio of 2:1 (v/v). The mixture was agitated and was allowed to stand at room temperature for 12 h. The supernatant was collected; centrifuged (8000 rpm for 30 min at 4 °C) and dialyzed for 12 h against distilled water. The dialysate was vacuum-dried to obtain the purified bioflocculant.

2.10. Characterizations of the Bioflocculant

The elemental composition, functional groups and pyrolysis profile of the bioflocculant was investigated. Surface structure of bioflocculant was investigated using a scanning electron microscope (SEM) (SIGMA VP-03-67, ZEISS Microscopy, Cambridge, UK). Elemental analysis was carried out by an elemental detector (X-Max EDS System, Oxford Instruments Inc, Oxford, UK). The functional groups of the bioflocculant were evaluated using a Fourier transform infrared spectrophotometer (PerkinElmer UATR TWO, 2000, PerkinElmer LAS, Rodgau, Germany), with a spectral range of 500–4000 cm−1 [36]. The pyrolysis property of the bioflocculant was determined using of thermo-gravimetric analyser (Model: DTG-60, Shimadzu Corporation, Tokyo, Japan). The bioflocculant was heated in the range of 22 to 800 °C at a constant rate of 10 °C per minute under a constant flow of nitrogen gas [37].

2.11. Biosafety of the Bioflocculant

Biosafety of the bioflocculant was evaluated by determining its cytotoxicity on African green monkey kidney (Vero) and Bovine dermis using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were grown to a confluency of 80% in 25 cm3 flasks using complete culture medium (CCM: minimum essential medium (MEM) supplemented with 5% foetal calf serum and 0.1% gentamicin) and harvested. They were re-suspended in growth medium at a concentration of 5 × 104 cells mL−1 and incubated for 24 h at 37 °C in 5% CO2 using a 96 well plate. Thereafter, the CCM was removed, and the cells were treated with the bioflocculant at different concentrations (50–200 µg/µL). Cells treated with 0.1% dimethyl sulfoxide (DMSO) served as the negative control and doxorubicin as the positive control. The cells were re-incubated at 37 °C in 5% CO2 for 24 h. After incubation, the medium was removed and supplemented with fresh CCM (100 µL). Thereafter, 30 µL of MTT reagent (5 mg/mL in phosphate buffered saline (PBS) was poured into the wells and incubated at 37 °C in 5% CO2 for 4 h. The MTT solution was aspirated from the wells and the formazan particles were dissolved in 50 µL of DMSO. MTT reduction was measured by reading the optical density (OD) of the samples at 540 nm using a micro plate reader (Varioskan Flash 3001, Thermo Fisher Scientific, Vantaa, Finland). Percentage cell inhibition (%CI) was assessed using the formula:
%CI = (Ao − A)/Ao × 100,
where Ao and A represent the OD readings of untreated samples and treated samples, respectively, at 540 nm. The inhibitory concentration of 50% (IC50) values was calculated from the GraphPad Prism (V6.1) using linear regression method [38].

2.12. Effect Dosages, Cations and pH on the Flocculating Activity

The effect of bioflocculant concentrations of flocculating activity was determined according to Maliehe et al. [39]. Two milliliters of the different concentrations in the range of 0.2–1.0 mg/mL (w/v) were used. Thereafter, the flocculating activity was evaluated as previously stated. The method of Tsilo et al. [40] was used to determine the influence of cations on flocculation. Metal ions included 1% of NaCl, LiCl, KCl, MnCl2, MgCl2 and FeCl3. The metal ions replaced the CaCl2 solution (3 mL, 1%) that was previously used in the flocculation assay and flocculating activity was measured. The pH of the kaolin clay solution (4 g/L) was varied between 3–12 using 0.1 M HCl and 0.1 M NaCl. Thereafter, the obtained optimum concentration was poured into a mixture of kaolin solution and 1% of the cation. Afterwards, the flocculating activity was determined at each pH level as described previously [41].

2.13. Application of the Bioflocculant in Wastewater Treatment

In the application process, the pure bioflocculant was used to treat wastewater from the Erwat Wastewater Treatment Plant from East Rand, Gauteng, South Africa. The pH of the wastewater was adjusted to 7 using 0.1 M HCl and 0.1 M NaCl. The wastewater was then treated with the bioflocculant. Inorganic flocculants, such as aluminium sulphate and ferric chloride, served as controls. The removal efficiencies of the flocculants on chemical oxygen demand (COD), biological oxygen demand (BOD) and sulphur were measured using appropriate test kits following the manufacturers’ instructions. Removal efficiency (RE) was expressed in percentage as:
%RE = (Co − C)/Co × 100,
whereby Co and C represent the values obtained before and after treatment, respectively, of the wastewater by the flocculants [7].

2.14. Statistical Analysis

All experiments were performed in triplicate and data expressed as mean values. Standard deviations were calculated. A one-way analysis of variance (ANOVA) was conducted using Graph Pad Prism™ version 6, GraphPad Software, San Diego, CA, USA. A significance difference level of p ˂ 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Physiochemical Parameters of Water

Geographic location and environmental conditions are important factors that influence the distribution and activity of marine bioflocculant-producing bacteria. Thus, measurement of physiochemical parameters is important when isolating microorganisms. The physiochemical parameters of seawater were measured using the HI 98194 PH/E/DO multi-parameter. The temperature was 23.24 ± 0.49 °C, pH 7.13 ± 0.05, TDS 23.55 ± 5.0 mg/L, DO 42.41 ± 5.5 mg/L, salinity (30.81 ± 7.14 ppt), pressure (759.56 ± 0.5 mmHg), and specific conductivity was 47.09 ± 10.1 mS/cm.

3.2. Isolation and Identification of the Bioflocculant-Producing Bacterium

A total of 31 isolates were isolated on two media with 15 isolates obtained on marine agar medium and 16 isolates on R2A medium. Among the 31 isolates, nine demonstrated flocculating activities higher than 50%, with Isolate MTZ08 revealing the highest flocculating activity of 68% against kaolin particles in solution. MTZ08 appeared non-pigmented, shiny to milky white, with a convex shape, circular and smooth. The bacterium was selected and identified using 16S rRNA gene analysis. The bacterium revealed 99% homology to Ochrobactrum oryzae AB84113. The genus Ochrobactrum is comprised of Gram-negative bacteria belonging to alpha-proteobacteria and Brucellaceae family [42]. The literature shows its application in the bioremediation of polychlorinated biphenyls and petroleum hydrocarbon [43,44]. Ochrobactrum sp. DGVK1 has been effectively used to remove N,Ndimethylformamide from wastewater and hexadecane from soils [45,46]. Ochrobactrum oryzae has been used to degrade wheat straw for production of biofuel [47] and has also been utilised in the removal of nitrogen [48].

3.3. Optimisation of Medium Composition and Culture Conditions

The literature has previously highlighted the effects of medium composition and culture conditions on bioflocculant production [49]. To maximize bioflocculant yield by the isolate, the medium composition (carbon and nitrogen sources) and culture conditions (inoculum size, temperature, initial pH of the medium and cultivation time) were optimised. The volume of the bacterial inoculum inoculated into the medium has an important role in bioflocculant production. The maximum flocculating activity of 86% was obtained at an inoculum size of 3% (v/v). However, there was no significant difference (p > 0.05) between the activity of 86% by the inoculum size of 3% and the activity of 83% achieved by 2% (v/v) inoculum size (Figure 1). Hence, an inoculum size of 2% was preferred for the following experiments. The flocculating activity was low at 1% inoculum size due to the fact that the isolate might have had an extended lag phase, consequently delaying production of the bioflocculant. Moreover, an inoculum size of 4% might have caused excess overlapping of the bacterial niche, leading to inhibition of bioflocculant production. The literature shows that high flocculating activities are often obtained at low inoculum size ranging from 1 to 5%. Makapela et al. [50] recorded high bioflocculant yield at an inoculum size of 4%, whereas Luo et al. [28] reported optimum bioflocculant production at 1% inoculum.
Carbon source plays a vital role in the production of bioflocculants [51]. The effect of carbon sources on the bioflocculant by O. oryzae AB841138 was assessed, and the maximum flocculating activity of 92% was obtained when starch was used as the carbon source, followed by glucose (86%) and fructose (81 ± 9.1%). Sucrose yielded the least flocculating activity (43%) (Table 1). It was concluded that O. oryzae AB841138 was able to effectively assimilate starch for its growth energy and bioflocculant production. Likewise, Klebsiella sp. and Sorangium cellulosum had preference for starch for optimal bioflocculant production [27,52]. Therefore, in this study, starch was utilised for subsequent investigations.
Table 1 illustrates the effect of nitrogen sources on bioflocculant production by O. oryzae AB841138. All nitrogen sources enhanced bioflocculant production, yielding flocculating activities of 85% and above. The peak flocculating activity of 92% was obtained when yeast extract was used. This indicates that O. oryzae can use yeast extract to produce bioflocculant efficiently. Similar results were found by Deng et al. [53], who reported that Bacillus mucilaginosus is capable of utilising yeast extract to produce bioflocculant with good flocculating activity. Yeast extract was used as the nitrogen source in the following experiments in this study.
The effect of temperature on bioflocculant production by the selected isolate was determined. There was a significant increase in flocculating activity with an increase in the cultivation temperature from 20 to 30 °C. The highest flocculating activity of 93% was observed when the bacterium was grown at 30 °C, and the least activity of 77% obtained when the bacteria was cultured at 20 °C (Table 1). Profound flocculating activity implied that O. oryzae is a mesophile whose enzymatic reactions function effectively at 30 °C for biosynthesis of the bioflocculant. The significant (p < 0.05) decrease in flocculating activity observed at temperatures above 30 °C may be due to a decrease in enzyme activity resulting from denaturation. The low flocculating activities at low temperatures (<30 °C) might be due to slow bacterial growth rate, leading to poor production of the bioflocculant. Similar trends were noticed for Klebsiella mobilis by Wang et al. [54]. However, our observations are contrary to the findings of Giri et al. [55], who reported high yields when Bacillus subtilis F9 was cultivated at 40 °C. The results confirm that different bacterial strains prefer different temperatures for optimum bioflocculant production.
The relationship between initial pH of the culture medium and bioflocculant production by O. oryzae AB841138 was evaluated. The optimum initial pH of the medium was in the range of 5–7 with flocculating activities above 70%. The peak flocculating activity of 93% was observed at a pH of 7, while the lowest flocculating activity was obtained at a pH of 3. (Figure 2). The initial pH of the medium influences the electrical charges on bacteria strains and their oxidation–reduction potential, which, in turn, can affect nutrient absorption and enzyme activities within the bacterial cells, thus affecting bioflocculant production [56]. Therefore, it was concluded that O. oryzae AB841138 is a neutrophilic bacterium with the ability to produce bioflocculant effectively in the pH range of 5–7. Nocardiopsis aegyptia sp. nov and Arthrobacter sp. Raats are examples of netrophilics that revealed high flocculating activity when the initial pH of the media was adjusted to 7 [57,58].
Different microorganisms prefer different incubation times for effective production of bioflocculants [58]. In this study, the flocculating activity increased with an increase in the incubation period up to 72 h. The flocculating activity was at a peak (94%) with an incubation time of 72 h (Figure 3). This meant that maximum bioflocculant production occurred during the late exponential stage or early stationary phase of growth. Similar findings have been obtained in other studies [59,60].

3.4. Extraction and Purification of Bioflocculant

The bioflocculant was obtained as a white powder after vacuum drying, and weighed 3.768 g. The bioflocculant yield was much higher than the 1.15 g/L recovered from Bacillus firmus [56] and the 1.36 g/L yield from Proteus mirabilis [22]. However, the yield was much lower than those reported by Natarajan [61], in which Bacillus firmus and Bacillus licheniformis produced 10 and 16.55 g/L. Therefore, there is a need to improve this strain to produce a higher yield.

3.5. Characterization of the Purified Bioflocculant

Figure 4 shows the surface structure of the bioflocculant. The bioflocculant had a hexagonal shape.
The elemental composition of the bioflocculant was investigated and the results are displayed in Table 2. The main elemental constituents were carbon (45.6%), oxygen (43.3%) and nitrogen (2.5%). Potassium and sulphur were present in the least amounts at 0.2%. The elemental composition of the bioflocculant was perceived to provide it with structural flexibility and stability [51]. The results show some similarities with the elemental composition of bioflocculant MBF-UFH from Halomonas sp. Okoh, i.e., carbon (17.21%), oxygen (40.04%) and nitrogen (6.66%) [62].
FTIR analysis was undertaken to ascertain and characterise the functional groups of the purified bioflocculant; the results are displayed in Figure 5. The FTIR spectrum showed a weak peak at 3491 cm−1 for an amino group (NH2), and a broad absorption peak at 3262 cm−1 representative of a hydroxyl group [63]. The absorption peak at 1645 cm−1 is due to the vibration of a carbonyl group in amide functionality [64]. The peaks between 1000–1200 cm−1 are due to the vibrations of an ester linkage and are generally characteristics of sugar derivatives [54]. The efficacy of bioflocculants strongly depends on their functional groups that serve as binding sites for colloidal particles in solutions [65]. Therefore, the observed functional groups enhance flocculation by serving as binding sites for kaolin particles. Moreover, the FTIR spectrum suggested the bioflocculant to possess polysaccharides and proteins. The observed results are comparable with findings from other studies [64,66,67].
Thermogravimetric analysis (TGA) was used to determine the pyrolysis property of the bioflocculant. Figure 6 shows different profiles the bioflocculant. The bioflocculant lost 2% of its weight within the temperature range of 50 and 100 °C due to loss of moisture content [7]. When it was heated at 200 °C, the bioflocculant lost 22% of its total weight, and 35% at 450 °C. The weight loss at 200 and 460 °C could be related to degradation of the bioflocculant [60]. The bioflocculant maintained approximately 62.8% of its weight after being heated at 800 °C. The high yield obtained after being heated at 800 °C showed the thermal stability of the bioflocculant. The results in this study were similar to those of Maliehe et al. [39] in which the bioflocculant was found to be it thermally stable.

3.6. Biosafety of the Bioflocculant from O. oryzae AB841138

A tetrazolium-based calorimetric method was used to determine the cytotoxic effect of the bioflocculant on bovine dermis and African green monkey kidney (Vero) cells. The bioflocculant had a mean inhibition concentration (IC50) of 180 µg/mL against bovine dermis and > 500 µg/mL on African green monkey kidney cells. The toxicity threshold level for bioflocculants is considered significant when the IC50 < 30 µg/mL [68]. Therefore, the results suggested the bioflocculant to be non-cytotoxic. Moreover, the results affirmed the probable safe use of the bioflocculant in different applications. The results were in accordance with those obtained by Sharma et al. [69] in which the exopolymer from Acinetobacter haemolyticus showed no toxic effect on sheep blood cells.

3.7. Effect of Dosage, Cations and pH on Flocculating Activity

The effects of dosage size, cations and pH on the flocculating activity of the bioflocculant from O. oryzae AB841138 were evaluated. The bioflocculant achieved a maximum flocculating activity of 95% at a dosage size of 6 mg/mL. However, there was no statistical difference (p > 0.05) between the flocculating activity obtained at the dosage size of 0.6 mg/mL and that of 0.2 mg/mL, which achieved 92% activity (Figure 7). Therefore, the preferred dosage size was 0.2 mg/mL and was used in the following experiments. Dosage concentration is a crucial factor in the determination of the effective flocculation. Excess dosage concentration results in inhibition of the interaction between bioflocculant and kaolin clay in solution, leading to a decrease in flocculation [63]. Thus, the dosage size of 0.2 mg/mL was optimum in efficiently flocculating kaolin particles. These results are in agreement with those reported by Maliehe et al. [33].
The bioflocculant was capable of high flocculating activities of above 70% within a wide pH range of 3 to 12. At pH 5, the bioflocculant showed the highest activity of 90% (Table 3). The effectiveness of the bioflocculant under a wide pH range is economical and suitable for industrial applications [7]. The findings agree with those obtained by Zhang et al. [27], in which a bioflocculant from Ruditapes philippinarum, demonstrated high flocculating efficiency over a wide range of pH (pH 3–11).
The impact of metal ions on flocculating activity of the bioflocculant was evaluated. Monovalent and divalent cations enhanced the flocculating efficiency of the bioflocculant, producing flocculating activities greater than or equal to 88%. The peak activity of 97% was observed when NaCl and MgCl were utilized. However, when a trivalent cation (Fe3+) was used, the flocculating activity drastically decreased (p < 0.05) (Table 3). Ferric chloride tends to lower the pH of water during its application as a flocculant [70]. Although the pH was adjusted to 7 prior to the application of Fe3+, the pH might have been lowered when Fe3+ was added, affecting bioflocculation. It should be noted that a high flocculating activity of 93% was observed in the absence of cations, indicating that the bioflocculant is cation independent, thus making it cost-effective. Similar results were reported for bioflocculants from Klebsiella pneumoniae and Aspergillus flavus, resulting in high flocculating activities in the absence of cations [71,72].

3.8. Removal Efficiency of the Bioflocculant

The bioflocculant and other flocculants (aluminium sulphate and ferric chloride) were compared in the treatment of wastewater from the Erwat Treatment Plant. The bioflocculant demonstrated 98% removal efficiency of COD, whereas aluminium sulphate and FeCl3 showed 97% and 98% efficiency, respectively (Table 4). Moreover, the bioflocculant effectively reduced BOD and sulphur concentrations by 91 and 86%, respectively. Aluminium sulphate had removal efficiencies of 82% and 90% for the removal of BOD and sulphur, while FeCl3 had 86% removal efficiency for BOD and 90% for sulphur. In general, the removal efficiencies of the bioflocculant were statistically similar to those observed when aluminium sulphate and FeCl3 were utilised in all parameters. This implies that the bioflocculant can be used as an alternative to chemical flocculants in wastewater treatment. Kaur et al. [73] presented similar results in which the bioflocculant effectively removed impurities from leachate.

4. Conclusions

The most promising bioflocculant-producing strain was identified as O. oryzae AB841138. The bioflocculant from O. oryzae AB841138 exhibited a maximum flocculating activity of 92% and a yield of 3.768 g/L when cultured optimally at pH 7, with an inoculum size of 1% (v/v) and when fructose and yeast extract were used as carbon and nitrogen sources, after cultivation at 30 °C for 72 h. The bioflocculant had a hexagonal structure with diverse functional groups such as hydroxyl, carboxyl, amine, and amide. The purified bioflocculant was found to be thermally stable and effective at a low dosage rate of 0.6 mg/mL. Moreover, the bioflocculant demonstrated negligible cytotoxicity on both bovine dermis and African green monkey kidney cells, indicative of its biosafety. It also had excellent removal efficiencies for tested pollutants in wastewater. These properties suggest potential applicability of the bioflocculant from O. oryzae AB841138 in wastewater treatment. For further studies involving cheaper substrates, the mode of action of the bioflocculant and its effectiveness in other wastewater effluents, should be undertaken.

Author Contributions

Conceptualization, T.N.S. and P.M.; formal analysis, V.M.; investigation, T.S.M.; supervision, T.N.S.; Writing-original draft, T.S.M.; Writing-review and editing, K.M.; project administration, P.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to acknowledge the staff in the Department of Biochemistry, Microbiology and Biotechnology and the Department of Water and Sanitation, at the University of Limpopo for their outstanding support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Gayathiri, E.; Prakash, P.; Selvam, K.; Awasthi, M.K.; Gobinath, R.; Karri, R.R.; Ragunathan, M.G.; Jayanthi, J.; Mani, V.; Poudineh, M.A. Plant microbe based remediation approaches in dye removal: A review. Bioengineered 2022, 13, 7798–7828. [Google Scholar] [CrossRef]
  2. Devda, V.; Chaudhary, K.; Varjani, S.; Pathak, B.; Patel, A.K.; Singhania, R.R.; Taherzadeh, M.J.; Ngo, H.H.; Wong, J.W.; Guo, W. Recovery of resources from industrial wastewater employing electrochemical technologies: Status, advancements and perspectives. Bioengineered 2021, 12, 4697–4718. [Google Scholar] [CrossRef] [PubMed]
  3. Ma, F.; Zheng, L.; Chi, Y. Applications of biological flocculants (BFs) for coagulation treatment in water purification: Turbidity elimination. Chem. Biochem. Eng. Q. 2008, 22, 321–326. [Google Scholar]
  4. Programme, W.W.A.; UN-Water. Water in a Changing World; Wiley: New York, NY, USA, 2009. [Google Scholar]
  5. Maruyama, H.; Seki, H. Evaluation of flocculation performance of polysaccharide-protamine complex flocculant by flocculation model. Biochem. Eng. J. 2022, 180, 108356. [Google Scholar] [CrossRef]
  6. Akinola, O.; Adebayo, E. Screening and characterization of bioflocculant producing bacteria isolated from domestic waste water in Bowen University, Iwo, Osun State, Nigeria. Niger. J. Biotechnol. 2018, 35, 159–166. [Google Scholar] [CrossRef]
  7. Okaiyeto, K.; Nwodo, U.U.; Mabinya, L.V.; Okoli, A.S.; Okoh, A.I. Evaluation of flocculating performance of a thermostable bioflocculant produced by marine Bacillus sp. Environ. Technol. 2016, 37, 1829–1842. [Google Scholar] [CrossRef]
  8. Salehizadeh, H.; Yan, N. Recent advances in extracellular biopolymer flocculants. Biotechnol. Adv. 2014, 32, 1506–1522. [Google Scholar] [CrossRef]
  9. Agunbiade, M.; Pohl, C.; Ashafa, O. Bioflocculant production from Streptomyces platensis and its potential for river and waste water treatment. Braz. J. Microbiol. 2018, 49, 731–741. [Google Scholar] [CrossRef]
  10. Maliehe, T.; Basson, A.; Singh, M. Wastewater treatment by a novel bioflocculant from a consortium of Bacillus pumilus JX860616 and Bacillus subtilis CSM5. Biosci. Res. 2020, 17, 1610–1625. [Google Scholar]
  11. Zaki, S.A.; Elkady, M.F.; Farag, S.; Abd-El-Haleem, D. Characterization and flocculation properties of a carbohydrate bioflocculant from a newly isolated Bacillus velezensis 40B. J. Environ. Biol. 2013, 34, 51–58. [Google Scholar]
  12. Ajao, V.; Fokkink, R.; Leermakers, F.; Bruning, H.; Rijnaarts, H.; Temmink, H. Bioflocculants from wastewater: Insights into adsorption affinity, flocculation mechanisms and mixed particle flocculation based on biopolymer size-fractionation. J. Colloid Interface Sci. 2021, 581, 533–544. [Google Scholar] [CrossRef] [PubMed]
  13. Kurniawan, S.B.; Abdullah, S.R.S.; Imron, M.F.; Ahmad, A.; Mohd Said, N.S.; Mohd Rahim, N.F.; Mohammad Alnawajha, M.; Abu Hasan, H.; Othman, A.R.; Purwanti, I.F. Potential of valuable materials recovery from aquaculture wastewater: An introduction to resource reclamation. Aquac. Res. 2021, 52, 2954–2962. [Google Scholar] [CrossRef]
  14. Zafar, L.; Khan, A.; Kamran, U.; Park, S.-J.; Bhatti, H.N. Eucalyptus (camaldulensis) bark-based composites for efficient Basic Blue 41 dye biosorption from aqueous stream: Kinetics, isothermal, and thermodynamic studies. Surf. Interfaces 2022, 31, 101897. [Google Scholar] [CrossRef]
  15. Mu, J.; Cui, X.; Shao, M.; Wang, Y.; Yang, Q.; Yang, G.; Zheng, L. Microbial origin of bioflocculation components within a promising natural bioflocculant resource of Ruditapes philippinarum conglutination mud from an aquaculture farm in Zhoushan, China. PLoS ONE 2019, 14, e0217679. [Google Scholar] [CrossRef] [PubMed]
  16. Grenda, K.; Gamelas, J.A.; Arnold, J.; Pellizzer, L.; Cayre, O.J.; Rasteiro, M.G. Evaluation of anionic eco-friendly flocculants prepared from Eucalyptus pulps with diverse lignin contents for application in effluent treatment. Polymers 2020, 13, 25. [Google Scholar] [CrossRef]
  17. Nxumalo, C.I.; Ngidi, L.S.; Shandu, J.S.E.; Maliehe, T.S. Isolation of endophytic bacteria from the leaves of Anredera cordifolia CIX1 for metabolites and their biological activities. BMC Complement. Med. Ther. 2020, 20, 300. [Google Scholar] [CrossRef]
  18. Kurniawan, S.B.; Imron, M.F.; Chik, C.E.N.C.E.; Owodunni, A.A.; Ahmad, A.; Alnawajha, M.M.; Rahim, N.F.M.; Said, N.S.M.; Abdullah, S.R.S.; Kasan, N.A. What compound inside biocoagulants/bioflocculants is contributing the most to the coagulation and flocculation processes? Sci. Total Environ. 2022, 806, 150902. [Google Scholar] [CrossRef]
  19. Ben Rebah, F.; Mnif, W.; Siddeeg, S.M. Microbial flocculants as an alternative to synthetic polymers for wastewater treatment: A review. Symmetry 2018, 10, 556. [Google Scholar] [CrossRef]
  20. Bisht, V.; Lal, B. Exploration of performance kinetics and mechanism of action of a potential novel bioflocculant BF-VB2 on clay and dye wastewater flocculation. Front. Microbiol. 2019, 10, 1288. [Google Scholar] [CrossRef]
  21. Yin, Y.-J.; Tian, Z.-M.; Tang, W.; Li, L.; Song, L.-Y.; McElmurry, S.P. Production and characterization of high efficiency bioflocculant isolated from Klebsiella sp. ZZ-3. Bioresour. Technol. 2014, 171, 336–342. [Google Scholar] [CrossRef]
  22. Arafa, R.A.; El-Rouby, M.N.; Abass, H.A.; El-Khier, Z. Bioflocculants produced by bacterial isolates from Egyptian soil 1-characterization and application of extracellular bioflocculants and nanoparticles for treatment of river Nile water. J. Pharm. Biol. Sci. 2014, 9, 103–114. [Google Scholar] [CrossRef]
  23. Mabinya, L.V.; Cosa, S.; Mkwetshana, N.; Okoh, A.I. Halomonas sp. OKOH—A marine bacterium isolated from the bottom sediment of Algoa Bay—Produces a polysaccharide bioflocculant: Partial characterization and biochemical analysis of its properties. Molecules 2011, 16, 4358–4370. [Google Scholar] [CrossRef] [PubMed]
  24. Tsilo, P.H.; Basson, A.K.; Ntombela, Z.G.; Maliehe, T.S.; Pullabhotla, R.V. Isolation and optimization of culture conditions of a bioflocculant-producing fungi from Kombucha tea SCOBY. Microbiol. Res. 2021, 12, 950–966. [Google Scholar] [CrossRef]
  25. Kurane, R.; Hatamochi, K.; Kakuno, T.; Kiyohara, M.; Hirano, M.; Taniguchi, Y. Production of a bioflocculant by Rhodococcus erythropolis S-1 grown on alcohols. Biosci. Biotechnol. Biochem. 1994, 58, 428–429. [Google Scholar] [CrossRef]
  26. Shida, O.; Takagi, H.; Kadowaki, K.; Nakamura, L.K.; Komagata, K. Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int. J. Syst. Evol. Microbiol. 1997, 47, 289–298. [Google Scholar] [CrossRef]
  27. Zhang, Z.-Q.; Bo, L.; Xia, S.-Q.; Wang, X.-J.; Yang, A.-M. Production and application of a novel bioflocculant by multiple-microorganism consortia using brewery wastewater as carbon source. J. Environ. Sci. 2007, 19, 667–673. [Google Scholar] [CrossRef]
  28. Luo, Z.; Chen, L.; Chen, C.; Zhang, W.; Liu, M.; Han, Y.; Zhou, J. Production and characteristics of a bioflocculant by Klebsiella pneumoniae YZ-6 isolated from human saliva. Appl. Biochem. Biotechnol. 2014, 172, 1282–1292. [Google Scholar] [CrossRef] [PubMed]
  29. Ugbenyen, A.M.; Simonis, J.J.; Basson, A.K. Screening for bioflocculant-producing bacteria from the marine environment of Sodwana Bay, South Africa. Ann. Sci. Technol. 2018, 3, 16–20. [Google Scholar] [CrossRef]
  30. Abdel-Aziz, S.M.; Hamed, H.A.; Mouafi, F.E.; Abdelwahed, N.A. Extracellular metabolites produced by a novel strain, Bacillus alvei NRC-14:3. Synthesis of a bioflocculant that has chitosan-like structure. Life Sci. J. 2011, 8, 883–890. [Google Scholar]
  31. He, P.; Geng, L.; Wang, Z.; Mao, D.; Wang, J.; Xu, C. Fermentation optimization, characterization and bioactivity of exopolysaccharides from Funalia trogii. Carbohydr. Polym. 2012, 89, 17–23. [Google Scholar] [CrossRef]
  32. Xia, S.; Zhang, Z.; Wang, X.; Yang, A.; Chen, L.; Zhao, J.; Leonard, D.; Jaffrezic-Renault, N. Production and characterization of a bioflocculant by Proteus mirabilis TJ-1. Bioresour. Technol. 2008, 99, 6520–6527. [Google Scholar] [CrossRef] [PubMed]
  33. Maliehe, T.; Simonis, J.; Basson, A.; Reve, M.; Ngema, S.; Xaba, P. Production, characterisation and flocculation mechanism of bioflocculant TMT-1 from marine Bacillus pumilus JX860616. Afr. J. Biotechnol. 2016, 15, 2352–2367. [Google Scholar]
  34. Ugbenyen, A.; Cosa, S.; Mabinya, L.; Okoh, A. Bioflocculant production by Bacillus sp. Gilbert isolated from a marine environment in South Africa. Appl. Biochem. Microbiol. 2014, 50, 49–54. [Google Scholar] [CrossRef]
  35. Dlamini, N.G. Biosynthesis of Copper Nanoparticles Using a Bioflocculant from Alcaligenis faecalis, Characterization and Its Application; University of Zululand: Richards Bay, South Africa, 2017. [Google Scholar]
  36. Ngema, S.; Basson, A.; Maliehe, T. Synthesis, characterization and application of polyacrylamide grafted bioflocculant. Phys. Chem. Earth Parts A/B/C 2020, 115, 102821. [Google Scholar] [CrossRef]
  37. Dlamini, N.G.; Basson, A.K.; Pullabhotla, V. Biosynthesis and characterization of copper nanoparticles using a bioflocculant extracted from Alcaligenis faecalis HCB2. Adv. Sci. Eng. Med. 2019, 11, 1064–1070. [Google Scholar] [CrossRef]
  38. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  39. Maliehe, T.S.; Basson, A.K.; Dlamini, N.G. Removal of pollutants in mine wastewater by a non-cytotoxic polymeric bioflocculant from Alcaligenes faecalis HCB2. Int. J. Environ. Res. Public Health 2019, 16, 4001. [Google Scholar] [CrossRef]
  40. Tsilo, P.H.; Basson, A.K.; Ntombela, Z.G.; Maliehe, T.S.; Pullabhotla, V.R. Production and characterization of a bioflocculant from Pichia kudriavzevii MH545928. 1 and Its Application in Wastewater Treatment. Int. J. Environ. Res. Public Health 2022, 19, 3148. [Google Scholar] [CrossRef]
  41. Masuku, S.K. Synthesis and Application of a Grafted Flocculant Produced from a Chemical Combination of a Bioflocculant TKT and Acrylamide (AM). Ph.D. Thesis, University of Zululand, Richards Bay, South Africa, 2019. [Google Scholar]
  42. Gohil, K.; Rajput, V.; Dharne, M. Pan-genomics of Ochrobactrum species from clinical and environmental origins reveals distinct populations and possible links. Genomics 2020, 112, 3003–3012. [Google Scholar] [CrossRef]
  43. Murínová, S.; Dercová, K. Potential use of newly isolated bacterial strain Ochrobactrum anthropi in bioremediation of polychlorinated biphenyls. Water Air Soil Pollut. 2014, 225, 1980. [Google Scholar] [CrossRef]
  44. Bezza, F.A.; Beukes, M.; Chirwa, E.M.N. Application of biosurfactant produced by Ochrobactrum intermedium CN3 for enhancing petroleum sludge bioremediation. Process. Biochem. 2015, 50, 1911–1922. [Google Scholar] [CrossRef]
  45. Kumar, S.S.; Kumar, M.S.; Siddavattam, D.; Karegoudar, T. Generation of continuous packed bed reactor with PVA–Alginate blend immobilized Ochrobactrum sp. DGVK1 cells for effective removal of N,N-dimethylformamide from industrial effluents. J. Hazard. Mater. 2012, 199, 58–63. [Google Scholar] [CrossRef] [PubMed]
  46. Samaei, M.R.; Mortazavi, S.B.; Bakhshi, B.; Jafari, A.J.; Shamsedini, N.; Mehrazmay, H.; Ansarizadeh, M. Investigating the effects of combined bio-enhancement and bio-stimulation on the cleaning of hexadecane-contaminated soils. J. Environ. Chem. Eng. 2022, 10, 106914. [Google Scholar] [CrossRef]
  47. Tsegaye, B.; Balomajumder, C.; Roy, P. Biodegradation of wheat straw by Ochrobactrum oryzae BMP03 and Bacillus sp. BMP01 bacteria to enhance biofuel production by increasing total reducing sugars yield. Environ. Sci. Pollut. Res. 2018, 25, 30585–30596. [Google Scholar] [CrossRef]
  48. Loan, L.T.; Phuong, T.V.; Diep, C.N. Isolation and identification of bioflocculant-producing bacteria, heterotrophic nitrogen removal bacteria and poly-phosphate bacteria in wastewater from “My Tho rice noodle” factories, Tien Giang province, Vietnam. Int. J. Innov. Eng. Technol. 2018, 10, 185–198. [Google Scholar]
  49. Shahadat, M.; Teng, T.T.; Rafatullah, M.; Shaikh, Z.; Sreekrishnan, T.; Ali, S.W. Bacterial bioflocculants: A review of recent advances and perspectives. Chem. Eng. J. 2017, 328, 1139–1152. [Google Scholar] [CrossRef]
  50. Makapela, B.; Okaiyeto, K.; Ntozonke, N.; Nwodo, U.U.; Green, E.; Mabinya, L.V.; Okoh, A.I. Assessment of Bacillus pumilus isolated from fresh water milieu for bioflocculant production. Appl. Sci. 2016, 6, 211. [Google Scholar] [CrossRef]
  51. Deng, S.; Yu, G.; Ting, Y.P. Production of a bioflocculant by Aspergillus parasiticus and its application in dye removal. Colloids Surf. B Biointerfaces 2005, 44, 179–186. [Google Scholar] [CrossRef]
  52. Sheng, Y.; Zhang, Q.; Sheng, Y.; Li, C.; Wang, H. Screening and flocculating properties of bioflocculant-producing microorganisms. J. Univ. Sci. Technol. Beijing Miner. Metall. Mater. 2006, 13, 289–292. [Google Scholar] [CrossRef]
  53. Deng, S.; Bai, R.; Hu, X.; Luo, Q. Characteristics of a bioflocculant produced by Bacillus mucilaginosus and its use in starch wastewater treatment. Appl. Microbiol. Biotechnol. 2003, 60, 588–593. [Google Scholar] [CrossRef]
  54. Wang, S.-G.; Gong, W.-X.; Liu, X.-W.; Tian, L.; Yue, Q.-Y.; Gao, B.-Y. Production of a novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater. Biochem. Eng. J. 2007, 36, 81–86. [Google Scholar] [CrossRef]
  55. Giri, S.S.; Harshiny, M.; Sen, S.S.; Sukumaran, V.; Park, S.C. Production and characterization of a thermostable bioflocculant from Bacillus subtilis F9, isolated from wastewater sludge. Ecotoxicol. Environ. Saf. 2015, 121, 45–50. [Google Scholar] [CrossRef] [PubMed]
  56. Salehizadeh, H.; Shojaosadati, S. Isolation and characterisation a bioflocculant produced by Bacillus firmus. Biotechnol. Lett. 2002, 24, 35–40. [Google Scholar] [CrossRef]
  57. Mabrouk, M.E. Production of bioflocculant by the marine actinomycete Nocardiopsis aegyptia sp. nov. Life Sci. J. 2014, 11, 27–35. [Google Scholar]
  58. Mabinya, L.V.; Cosa, S.; Nwodo, U.; Okoh, A.I. Studies on bioflocculant production by Arthrobacter sp. Raats, a freshwater bacteria isolated from Tyume River, South Africa. Int. J. Mol. Sci. 2012, 13, 1054–1065. [Google Scholar] [CrossRef]
  59. Gong, W.-X.; Wang, S.-G.; Sun, X.-F.; Liu, X.-W.; Yue, Q.-Y.; Gao, B.-Y. Bioflocculant production by culture of Serratia ficaria and its application in wastewater treatment. Bioresour. Technol. 2008, 99, 4668–4674. [Google Scholar] [CrossRef]
  60. Nkosi, N.C.; Basson, A.K.; Ntombela, Z.G.; Maliehe, T.S.; Pullabhotla, R.V. Isolation, identification and characterization of bioflocculant-producing bacteria from activated sludge of Vulindlela Wastewater Treatment Plant. Appl. Microbiol. 2021, 1, 586–606. [Google Scholar] [CrossRef]
  61. Natarajan, K. Production and characterization of bioflocculants for mineral processing applications. Int. J. Miner. Process. 2015, 137, 15–25. [Google Scholar]
  62. Okaiyeto, K.; Nwodo, U.U.; Mabinya, L.V.; Okoh, A.I. Characterization and flocculating properties of a biopolymer produced by Halomonas sp. Okoh. Water Environ. Res. 2015, 87, 298–303. [Google Scholar] [CrossRef]
  63. Li, Z.; Chen, R.-W.; Lei, H.-Y.; Shan, Z.; Bai, T.; Yu, Q.; Li, H.-L. Characterization and flocculating properties of a novel bioflocculant produced by Bacillus circulans. World J. Microbiol. Biotechnol. 2009, 25, 745–752. [Google Scholar] [CrossRef]
  64. Cosa, S.; Mabinya, L.V.; Olaniran, A.O.; Okoh, O.O.; Bernard, K.; Deyzel, S.; Okoh, A.I. Bioflocculant production by Virgibacillus sp. Rob isolated from the bottom sediment of Algoa Bay in the Eastern Cape, South Africa. Molecules 2011, 16, 2431–2442. [Google Scholar] [CrossRef]
  65. Ajao, V.O. Flocculants from Wastewater; Wageningen University and Research: Wageningen, The Netherlands, 2020. [Google Scholar]
  66. Elkady, M.; Farag, S.; Zaki, S.; Abu-Elreesh, G.; Abd-El-Haleem, D. Bacillus mojavensis strain 32A, a bioflocculant-producing bacterium isolated from an Egyptian salt production pond. Bioresour. Technol. 2011, 102, 8143–8151. [Google Scholar] [CrossRef] [PubMed]
  67. Guo, J.; Yu, J.; Xin, X.; Zou, C.; Cheng, Q.; Yang, H.; Nengzi, L. Characterization and flocculation mechanism of a bioflocculant from hydrolyzate of rice stover. Bioresour. Technol. 2015, 177, 393–397. [Google Scholar] [CrossRef] [PubMed]
  68. Pathak, M.; Devi, A.; Bhattacharyya, K.; Sarma, H.; Subudhi, S.; Lal, B. Production of a non-cytotoxic bioflocculant by a bacterium utilizing a petroleum hydrocarbon source and its application in heavy metal removal. RSC Adv. 2015, 5, 66037–66046. [Google Scholar] [CrossRef]
  69. Sharma, V.; Kaur, T.; Bridle, H.; Ghosh, M. Antimicrobial efficacy and safety of mucoadhesive exopolymer produced by Acinetobacter haemolyticus. Int. J. Biol. Macromol. 2017, 94, 187–193. [Google Scholar] [CrossRef]
  70. Hu, Y.; Gu, Z.; He, J.; Li, Q. Novel strategy for controlling colloidal instability during the flocculation pretreatment of landfill leachate. Chemosphere 2022, 287, 132051. [Google Scholar] [CrossRef]
  71. Aljuboori, A.H.R.; Uemura, Y.; Osman, N.B.; Yusup, S. Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source. Bioresour. Technol. 2014, 171, 66–70. [Google Scholar] [CrossRef]
  72. Zhao, H.; Liu, H.; Zhou, J. Characterization of a bioflocculant MBF-5 by Klebsiella pneumoniae and its application in Acanthamoeba cysts removal. Bioresour. Technol. 2013, 137, 226–232. [Google Scholar] [CrossRef]
  73. Kaur, R.; Roy, D.; Yellapu, S.K.; Tyagi, R.D.; Drogui, P.; Surampalli, R.Y. Enhanced composting leachate treatment using extracellular polymeric substances as bioflocculant. J. Environ. Eng. 2019, 145, 04019075. [Google Scholar] [CrossRef]
Ijerph 19 10237 g001 550
Figure 1. Effect of inoculum size on bioflocculant production. The letters (a,b) denote significant differences (p < 0.05).
Figure 1. Effect of inoculum size on bioflocculant production. The letters (a,b) denote significant differences (p < 0.05).
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Figure 2. Effect of pH on bioflocculant production. The letters (a, b, c, d) denote significant differences (p < 0.05).
Figure 2. Effect of pH on bioflocculant production. The letters (a, b, c, d) denote significant differences (p < 0.05).
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Figure 3. The impact of incubation time on bioflocculant production. The letters (a, b, c) denote significant differences (p < 0.05).
Figure 3. The impact of incubation time on bioflocculant production. The letters (a, b, c) denote significant differences (p < 0.05).
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Figure 4. SEM image showing bioflocculant structure.
Figure 4. SEM image showing bioflocculant structure.
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Figure 5. FTIR spectrum of the purified bioflocculant.
Figure 5. FTIR spectrum of the purified bioflocculant.
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Figure 6. TGA spectrum of the bioflocculant from O. oryzae AB841138.
Figure 6. TGA spectrum of the bioflocculant from O. oryzae AB841138.
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Figure 7. Impact of dosage size on flocculating activity. The letter (a) denotes insignificant differences (p > 0.05).
Figure 7. Impact of dosage size on flocculating activity. The letter (a) denotes insignificant differences (p > 0.05).
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Table
Table 1. Effect carbon and nitrogen sources and temperature on bioflocculant production.
Table 1. Effect carbon and nitrogen sources and temperature on bioflocculant production.
Carbon SourcesFA(%) ± SDNitrogen SourcesFA(%) ± SDTemperature (°C)FA(%) ± SD
Sucrose81 ± 2.1 aCasein89 ± 0.5 a2077 ± 0.1 a
Molasses70 ± 2.2 aPeptone88 ± 2.2 a2584 ± 0.2 b
Lactose66 ± 4.6 bAmmonia91 ± 4.7 a3093 ± 0.4 c
Xylose76 ± 1.2 aUrea85 ± 3.2 a3582 ± 0.6 b
Sucrose43 ± 0.6 cYeast extract92 ± 3.8 a4080 ± 0.1 b
Glucose86 ± 7.1 a
Starch92 ± 1.7 a
Flocculating activity, SD—Standard deviation FA with different letters (a, b, c) are significantly different (p < 0.05).
Table
Table 2. Percentage elemental composition of the bioflocculant.
Table 2. Percentage elemental composition of the bioflocculant.
Element% (w/w)
Potassium0.2
Oxygen43.3
Magnesium1.7
Sodium2.3
Sulphur0.2
Nitrogen2.5
Chlorine1.3
Calcium0.8
Carbon45.6
Phosphorus1.9
Table
Table 3. Effect of pH and cations on flocculating activity.
Table 3. Effect of pH and cations on flocculating activity.
pHFA(%) ± SDCationsFA(%) ± SD
374 ± 0.2 aControl93 ± 3.5 a
479 ± 0.9 aLi+88 ± 3.5 a
590 ± 4.5 bNa+97 ± 2.7 a
689 ± 0.2 aK+89 ± 3.8 a
785 ± 0.4 bBa2+96 ± 4.4 a
885 ± 1.0 bMn2+90±11.3 a
984 ± 1.3 bMg2+97 ± 1.5 a
1081 ± 1.7 a,bCa2+96 ± 2.1 a
1180 ± 1.3 a,bFe3+47 ± 1.0 b
1275 ± 0.4 a
FA denotes flocculating activity, SD denotes standard deviation and the letters (a, b) show statistical differences (p < 0.05).
Table
Table 4. Removal efficiency of the bioflocculant from O. oryzae AB841138.
Table 4. Removal efficiency of the bioflocculant from O. oryzae AB841138.
FlocculantsCOD (%)BOD (%)Sulphur
Bioflocculant98 ± 2.0 a91 ± 1.4 a86 ± 0.5 a
Aluminium sulphate97 ± 0.1 a82 ± 3.1 a90 ± 1.2 a
FeCl398 ± 1.8 a86 ± 0.5 a90 ± 0.1 a
The letter (a) illustrates statistical insignificance (p > 0.05).
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Selepe, T.N.; Maliehe, T.S.; Moganedi, K.; Masoko, P.; Mulaudzi, V. Isolation and Optimisation of Culture Conditions for a Marine Bioflocculant-Producing Bacterium and Application of Its Bioflocculant in Wastewater Treatment. Int. J. Environ. Res. Public Health 2022, 19, 10237. https://doi.org/10.3390/ijerph191610237

AMA Style

Selepe TN, Maliehe TS, Moganedi K, Masoko P, Mulaudzi V. Isolation and Optimisation of Culture Conditions for a Marine Bioflocculant-Producing Bacterium and Application of Its Bioflocculant in Wastewater Treatment. International Journal of Environmental Research and Public Health. 2022; 19(16):10237. https://doi.org/10.3390/ijerph191610237

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Selepe, Tlou Nelson, Tsolanku Sidney Maliehe, Kgabo Moganedi, Peter Masoko, and Vusimuzi Mulaudzi. 2022. "Isolation and Optimisation of Culture Conditions for a Marine Bioflocculant-Producing Bacterium and Application of Its Bioflocculant in Wastewater Treatment" International Journal of Environmental Research and Public Health 19, no. 16: 10237. https://doi.org/10.3390/ijerph191610237

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