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Production and characterization of a bioflocculant produced by Proteus mirabilis AB 932526.1 and its application in wastewater treatment and dye removal

  • Nkanyiso C. Nkosi , Albertus K. Basson , Zuzingcebo G. Ntombela , Nkosinathi G. Dlamini , Tsolanku S. Maliehe and Rajasekhar V. S. R. Pullabhotla ORCID logo EMAIL logo
Published/Copyright: March 8, 2023
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Pure and Applied Chemistry
From the journal Pure and Applied Chemistry Volume 95 Issue 3

Abstract

Microbial flocculants affect the aggregation of suspended solutes in solutions, thus, they are a viable alternative to inorganic and organic synthetic flocculants which are associated with deleterious health problems. Moreover, a potential solution for wastewater treatment. The study aimed to produce and characterize a bioflocculant from Proteus mirabilis AB 932526.1 and apply it in domestic wastewater treatment and dye removal. The bioflocculant was extracted using butanol and chloroform (5:2 v/v). Carbohydrates, proteins, and uronic acid were identified using phenol-sulphuric acid, Bradford, and Carbazole essays. The morphology, crystallinity and elemental composition of the purified bioflocculant were determined using a Scanning electron microscope (SEM), X-ray diffraction analysis and SEM energy dispersive elemental detector (SEM-EDX). The antimicrobial properties and dye removal efficiencies were evaluated. About 3.8 g/L yields of the purified bioflocculant were attained. Chemical composition analysis revealed the presence of 65 % carbohydrates, 10 % proteins, and 24 % uronic acids. The bioflocculant displayed an amorphous and crystalline structure. Bioflocculant further shows some remarkable properties as they can be able to inhibit the growth of both Gram-positive and Gram-negative microorganisms. The removal efficiencies of 85 % (COD), 82 % (BOD), and 81 % (SO42−) in domestic wastewater were achieved. Moreover, the high removal efficiency of staining dyes such as methylene blue (71 %), carbol fuchsin (81 %), safranin (83 %), methylene orange (90 %), and Congo red (90 %) were found. The produced bioflocculant can imply industrial applicability.

Keywords: Bioflocculant; dyes; Proteus mirabilis AB 932526.1; removal efficiency; VCCA-2022; wastewater

Introduction

Increasing industrialization has been regarded as a desirable choice because of its contribution to economic growth. However, it has significantly increased the rate of water pollution, particularly from industrial sources, and this become a serious environmental concern [1, 2]. The disposal of effluents without appropriate treatment could result in long-term undesired negative impacts, especially on the environment and human health [3]. Furthermore, the majority of the paper and textile industries use dyes and discharge their effluents into water bodies [4]. Dyes are frequently persistent organic compounds that generate a colour change while also contributing to the organic load and toxicity of textile industrial wastewater [5]. Various methods have been employed for the removal of pollutants from wastewater, and the most commonly used have been precipitation, adsorption, ion exchange, and filtration [6, 7]. All of these treatment technologies are important in the management of industrial effluents. However, the main disadvantages of these approaches are that they are either more expensive or produce enormous amounts of sludge [8]. The development of environmentally friendly wastewater purification technologies is consequently of academic and practical significance. Flocculation has attracted a lot of attention because of its advantages, which include effectiveness, efficiency, stability, and wide applicability [9]. Flocculation is a process that occurs when a chemical coagulant is added to water and acts to facilitate particle bonding, resulting in larger aggregates that are easier to separate [10]. Flocculants are important agents in the aggregation of colloids, cells, and suspended particles, and they are widely utilized in the production of drinking water production, wastewater treatment, fermentation processes, and food production [11]. In general, flocculants are divided into three categories: inorganic flocculants, such as aluminium sulphate and polyaluminum chloride; organic synthetic flocculants, such as polyacrylamide derivatives and polyethylene imine; and naturally occurring flocculants, such as chitosan, sodium alginate and microbial flocculants [12]. Despite the strong flocculating performance and low cost of synthetic chemical flocculants, their use has resulted in some health and environmental issues, which greatly restrict the wide applications of these flocculants [13]. Therefore, the search for biodegradable, cost-effective, and sustainable extracellular biopolymers has been on the rise globally. Bioflocculants, which are both actively secreted by microorganisms and released upon cell lysis, are a type of environmentally friendly material with the advantages of non-toxicity and biodegradability, and they have been recognized as an alternative to the use of chemical flocculants in wastewater treatment [9]. Because of all of these characteristics, the use of bioflocculants has been considered to be a potential solution to environmental pollution in recent years [14]. The main components of these bioflocculants are glycoproteins, polysaccharides, and proteins [15]. Bioflocculants are obtained in the presence of microorganisms includes bacteria, algae, fungi and actinomycetes or biodegradable macromolecular flocculants released by microorganisms [16]. The research on bioflocculants began in the 1950s when Japanese researchers found microbial culture liquid with flocculation [17]. However, the high cost of production has long been a major impediment to the widespread use of bioflocculants [18]; consequently, the search for low-cost substrates for the synthesis of bioflocculants by cultivated microorganisms is of considerable practical importance.

In a previous study, different bacterial strains were screened for bioflocculant production. Among five selected strains, the strain named PM-7 was chosen for the entire study because of its highest flocculating activity compared with other strains, which was identified as Proteus mirabilis AB 932526.1 [19]. A series of experiments were conducted to investigate bioflocculant production and flocculation performance. P. mirabilis is a Gram-negative bacterium, a facultative anaerobe, belonging to the Enterobacteriaceae family. It is commonly found in abundance in the environment, particularly in soil and wastewater, because it decomposes organic matter. Proteus sp. has been reported to produce bioflocculant from different wastewaters [20]. However, no previous studies describing the bioflocculant have been produced by P. mirabilis AB 932526.1. The purpose of this study was to extract the bioflocculant from P. mirabilis AB 932526.1 using a solvent extraction method, to characterize the synthesized bioflocculant using different standard methods, and to apply it in treating domestic wastewater and dye removal in comparison with other chemical flocculants.

Materials and methods

Chemicals and production medium

The media, reagents, and chemicals used to obtain bioflocculant-producing bacterium were all purchased from Sigma-Aldrich (St. Louis MO, USA). The selective medium used for bioflocculant producing-bacterium 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. Nkosi et al. [19] method was followed for the evaluation of bioflocculant production. The culture medium composes of 20 g glucose, 0.5 g urea, 0.5 g yeast extract, 0.2 g (NH4)2 SO4, 2 g KH2 PO4, 5 g K2 HPO4, 0.1 g NaCl and 0.2 g MgSO4·7H2O were prepared. All mediums were prepared in filtered activated sludge water before being sterilized by autoclaving at 121 °C for 15 min.

Source of bacterium and activation of the isolates

Proteus mirabilis AB 932526.1 was previously isolated from activated sludge effluent in KwaDlangezwa, KwaZulu-Natal Province, South Africa, identified using internal transcribed spacer (ITS) rRNA gene sequence analysis and preserved in 20 % glycerol broth at – 80 °C, (ChemLabs, Johannesburg, South Africa), in the Department of Biochemistry and Microbiology Laboratory at the University of Zululand, Republic of South Africa. For the activation of the isolates, the enrichment medium (see Section “Chemicals and production medium”) was prepared at 28 °C for 72 h at pH 6 and a shaking speed of 160 rpm. After incubation time, 2 mL of broth cultures were taken and centrifuged at 10 000 rpm for 30 min. The supernatant was used to determine flocculating activity.

Extraction and purification of the bioflocculant

Extraction and purification of bioflocculant produced by P. mirabilis AB 932526.1 was carried out as described by Ugbenyen et al. [21]. The bacterium was cultured under optimal culture conditions in a litre of filtered activated sludge water. To obtain the cell-free supernatant the broth culture was centrifuged at 4000 rpm for 15 min at 4 °C. To isolate EPS, one volume of distilled water was added to the supernatant phase and re-centrifuged to remove insoluble substances. The cell-free supernatant was mixed with 1000 mL of distilled water, vigorously agitated, and centrifuged again. Thereafter, two volumes of ice-cold ethanol (95 %) were added to the culture supernatant; agitated properly, and stored in a cold cabinet at 4 °C for 12 h for the bioflocculant to precipitate. The precipitate was vacuum dried, and the crude product was re-dissolved in 100 mL of sterile distilled water to form a solution of 1 % (w/v). For the purification of bioflocculant, a mixture of chloroform and 1-butanol (5:2 v/v) (100 mL) was added to the solution, shaken vigorously, and stored in a separation hopper for 12 h at room temperature. Thereafter, the superior phase was collected; centrifuged at 4000 rpm for 15 min at 4 °C and dialysed using distilled water. The dialysate was vacuum-dried to obtain the purified bioflocculant. The weight of the dried purified bioflocculant was measured, expressed in g L−1 culture, and used for subsequent assay.

Characteristics of the purified bioflocculant

Chemical composition analysis

The phenol-sulphuric acid method was employed to measure the total polysaccharides content of the purified bioflocculant with d-glucose used to prepare the standard curve [19]. Briefly, 0.2 g of the purified bioflocculant was poured into a beaker containing 100 mL of autoclaved distilled water. About 0.2 mL of phenol was added into the solution together with 1.0 mL of sulphuric acid. The mixture was allowed to wait for 10 min at room temperature, and vigorously shaken for a minute. Thereafter, absorbance was measured using a spectrophotometer (Tensor 27, Bruker Unic-7230, Shanghai Lianhua Company, Shanghai, China) at a wavelength of 490 nm. The protein content of the purified bioflocculant was determined using Bradford assay with bovine serum albumin (BSA) used as the standard curve. Briefly, about 20 µL of each solution was pipetted into a 96-well plate after which 180 µL was added into dilutions [22]. The mixture was left to stand at room temperature for 2 min. Thereafter the solution was read at 595 nm using a spectrophotometer (Tensor 27, Bruker Unic-7230, Shanghai Lianhua Company, Shanghai, China). The carbazole-sulphuric acid method using carbazole reagent was performed in a test tube by adding 50–400 mol of uronic acids in 0.4 mL of water. About 40 µL of 4 mol/L sulfamic acid-potassium sulfamates was added to the sample followed by 2.4 mL of concentrated H2SO4. The solution was mixed gently and place at room temperature for 30 min. About 100 µL (0.1 % w/v of carbazole in EtOH) of the carbazole reagent was added. The tube was placed in a boiling water bath for 20 min, followed by cooling in an ice-water bath until room temperature was reached [23]. The light absorbance of the solution was measured at 525 nm using a spectrophotometer (Unic-7230, Shanghai Lianhua Company, Shanghai, China). The standard curve was prepared using d-Gluconoric acid.

Morphological, diffraction, and elemental analysis of the bioflocculant

Scanning electron microscopy (SEM) (SEM-Sipma-VP03-67, Zeiss, and P-Sigma, Germany) was employed to evaluate the surface morphology and intensity of the purified bioflocculant. A 0.5 g/L of bioflocculant was placed on a silicon-coated slide and fixed for 1 min using a spin coater at 1000 rpm. The fine powder of the bioflocculant was analysed using the X-ray diffractometer. This was accomplished by stacking the bioflocculant into the flat aluminium sample holder and a rotating anode with a bioflocculant at the X-ray source operated at 40 kV and 40 mA. The data were obtained between 20 and 80° 2θ with a Bruker D8 advance diffractometer [24]. Elemental composition was determined using a Thermo Fisher (Nova Nano SEM) coupled to an energy-dispersive X-ray (EDX) detector.

Flocculation characteristics of a bioflocculant produced by P. mirabilis AB 932526.1

Effect of flocculating dosage of purified bioflocculant on flocculation

This was done according to the protocol of Sivasankar et al. [25]. Different concentrations ranging from 0.2 to 1.0 mg/mL of the purified bioflocculant were prepared and their flocculating activities against 4 g/L kaolin clay suspension were measured. In 250 mL beakers, 3.0 mL of 1 % (w/v) CaCl2 was added to the various concentrations of the purified bioflocculant and mixed with 100 mL of kaolin clay suspension. The solution was rapidly mixed at 160 rpm for 2 min, flocculated gradually at 40 rpm for 2 min, and sedimented for 5 min. After sedimentation, 2 mL of the upper clarifying phase was gently withdrawn to measure the flocculating activity at 550 nm with a spectrophotometer (Unic-7230, Shanghai Lianhua Company, Shanghai, China) [26]. The concentration dosage that gave the best flocculating activity was used for the subsequent experiment.

The flocculating activity was calculated using the following equation:

(1) F l o c c u l a t i n g   a c t i v i t y   ( % ) = [ ( B − A ) / B ] × 100

where A and B are the respective absorbance of the control and sample experiment measured at 550 nm.

Effect of metal ions on bioflocculant activity

The effects of various cations on the flocculation activity of P. mirabilis AB 932526.1 were investigated [24]. In the standard method, about 3 mL of 1 % CaCl2 (w/v) solution was substituted by dissimilar metal ions such as Li, K, Na, BaCl2, MgCl2, MnCl2, FeCl3 and AlCl3. Experimental control was prepared by adding free-cell supernatant in a kaolin solution with no cation being added. The effect of each cation on flocculating activity was then measured using a kaolin solution as described in “Effect of flocculating dosage of purified bioflocculant on flocculation” section.

Effect of MnCl2 concentration on flocculation

The most preferable cation (MnCl2) for the bioflocculant to achieve the highest flocculating efficiency was subjected to obtain the optimum amount of manganese chloride concentration required for flocculating efficiency. Before testing the flocculating efficiency against kaolin solution (4 g/L), MnCl2 concentrations ranging from 0.25 to 1.5 g/mL were prepared and used.

Antimicrobial activity test of the bioflocculant

Test bacteria Escherichia coli (ATCC 25922) and Bacillus pumilus (ATCC 7065) were first resuscitated by inoculation into the sterile nutrient broth and incubated at 37 °C for 12 h. Thereafter, 1 mL from each culture was inoculated into separate test tubes containing 9 mL of sterile nutrient broth and incubated at 37 °C overnight. The fresh sterile nutrient broth was further used to adjust the turbidity of all the organisms to attain an absorbance of 0.8, which is within the McFarland accepted standard (1 × 108 colony forming units per millimeters [CFU/mL]). The antibacterial activity of the microbial bioflocculant was evaluated in terms of the minimum inhibitory concentration (MIC) using a rapid Mueller Hinton broth micro-dilution method with p-iodonitrotetrazolium violet solution (0.2 mg/mL) as an indicator [27]. Ciprofloxacin was used as a positive control, while dimethyl sulphoxide (DMSO) (10 %) was used as a negative control [28].

Application of the bioflocculant in domestic wastewater treatment

Wastewater samples were collected from the Vulindlela Wastewater Treatment Plant in KwaDlangezwa, KwaZulu-Natal, Republic of South Africa. Parameters such as biochemical oxygen demand (BOD), sulphate (SO42−), and chemical oxygen demand (COD) were measured before and after treatment with the bioflocculant to determine removal efficiencies [29]. In a 250 mL conical flask, 3 mL of 1 % (w/v) MnCl2 solution and 2 mL of 0.4 mg/mL bioflocculant solution were mixed with 100 mL of wastewater sample. The mixtures were shaken at 200 rpm for 3 min, then at 40 rpm for 5 min. For sedimentation, the flasks were left at room temperature for 10 min. Conventional flocculants such as alum and ferric chloride (0.4 mg/mL) were used as a comparison. Following that, the removal efficiency percentage (RE %) of the bioflocculant on BOD, S, and COD was determined by measuring optical densities at 680 nm. The RE % was calculated using the formula below [30];

(2) R E % = [ ( C o − C ) / C o ] × 100

where Co and C are the values before and after the flocculation process measured at 680 nm respectively [31].

Application of the bioflocculant in dye removal

The method by Maliehe et al. [32], was adopted for dye removal using the bioflocculant. The different dye solutions such as methylene blue, Congo red, nigrosine, and safranin (4 g/L) were prepared. In 100 mL of each dye solution, 2 mL of 0.4 mg/mL bioflocculant solution and 3 mL of 1 % (w/v) MnCl2 solution were added. The mixtures were agitated at 200 rpm for 3 min before being reduced to 40 rpm for 5 min. The control solution was the solution without the bioflocculant. The RE % was calculated using the same equation as previously stated. See “Application of the bioflocculant in domestic wastewater treatment” section.

Statistical analysis

All data were collected in triplicates with mean and standard deviation values determined where differences were considered significant at 0.05 at confidence level (p > 0.05) using Graph Pad Prism™ version 6. The data were analysed using One-way variance (ANOVA).

Results and discussions

Physico-chemical analysis of a bioflocculant and its characterization

Characterisation of the bioflocculant produced from P. mirabilis AB 932526.1 was carried out using advanced techniques [33]. About 3.8 g of the purified bioflocculant was obtained from 1 L of fermented broth within 72 h of incubation. Compared with reports from several other microorganisms, the yield of this bacterium can be regarded as promising. For instant, the yield produced by P. mirabilis AB 932526.1 was higher compared to bioflocculants previously extracted from Virgibacullus sp. Rob [26], Bacillus firmus [34] and Enterobacter clocoae [30] which shower the yields of 2.43, 1.36, and 2.27 g/L respectively. However, the study by He et al. [35] reported a higher yield of 4.52 g/L for bioflocculant produced by Halomonas sp. V3 as compared to bioflocculant by P. mirabilis AB 932526.1.

The chemical composition of the purified bioflocculant confirmed the existence of a glycoprotein molecule containing 65 % total carbohydrates, 24 % uronic acid, and 10 % total proteins (w/w) content respectively in varying quantities (Table 2). Moreover, the produced bioflocculant confirmed its heat stability since displayed a high concentration of carbohydrates [19]. Similar results were shown in various bioflocculants reported by Lungmann et al. [36], Okaiyeto [37] and Sivasankar et al. [25] (Table 1).

Table 1:

Chemical compositions of the purified bioflocculant.

Samples Percentage (%)
Carbohydrates 65.25 %
Uronic acids 23.91 %
Proteins 10.42 %

Figure 1 shows the SEM results of the purified bioflocculant. The morphology of the bioflocculant is amorphous in structure with a bigger cell size.

Fig. 1: SEM image showing the bioflocculant structure.
Fig. 1:

SEM image showing the bioflocculant structure.

The XRD analysis was undertaken to ascertain the peak of the bioflocculant. XRD showed intense peaks at 31° and 69° which indicate the presence of crystalline in nature, while the characteristics of tiny diffraction were observed from 10 to 30°. The tiny diffraction peaks might be due to the lack of bulkier groups within the biopolymer and the presence of intermolecular hydrogen bonds [38]. Comparable results were observed by Ngema [39] (Fig. 2).

Fig. 2: X-ray diffractogram of the purified bioflocculant.
Fig. 2:

X-ray diffractogram of the purified bioflocculant.

The elemental composition of the bioflocculant was examined and the results are shown in Fig. 3. The key elemental affirmed are carbon (22 %), nitrogen (3), oxygen (47 %), sodium (0.54), magnesium (5), phosphorous (7.46), molybdenum (3), potassium, and calcium (12 %). The presence of these elements in the bioflocculant confirmed the structural flexibility and stability [19]. Similar elements were observed in the work of Zhu et al. [40].

Fig. 3: Elementary analysis of the bioflocculant.
Fig. 3:

Elementary analysis of the bioflocculant.

Dosage of the bioflocculant

The effect of the dosage of the extracted biopolymer on flocculation was determined. The biopolymer demonstrated the optimum flocculation (81.9 %) at 0.4 mg/mL and revealed the lowest activity (49.4 %) at 1.0 mg/mL. The decline in the flocculating activity with the increase in dosage was due to the formation of the high viscosity, which hindered the interaction between the effective structures of the biopolymer and kaolin [16]. The reflective activity at low dosage size of the bioflocculant implicit economic advantage. The results from this study contrasted those reported by Akapo [41] where the flocculating activity of the bioflocculant produced by Bacillus atrophaeus required a dosage of 0.4 mg/mL. However, the observations of the study opposed with those observed by Okaiyeto [37] where the dosage size of 12 mg/L revealed the maximum flocculation for the bioflocculant produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6.

The effectively used of metal ion on the purified bioflocculant

The effect of metal ions on flocculation was evaluated. The subsequent addition of Mn2+, Mg2+, and Fe3+ into the bioflocculant solution resulted in an improvement in the flocculating activity while adding no metal ions in the bioflocculant solution led to a decrease in the flocculating performance. Mn2+ gave the highest flocculating activity of 98.4 %; however, the flocculating activity was statistically equal to that obtained when Mg2+ was used (Table 2). This implied that any of the two metal ions (Mn2+ and Mg2+) could be used during the application of this bioflocculant as they effectively neutralised and stabilised the charges of the functional groups of the kaolin and the bioflocculant molecules, consequently leading to an improved flocculation process. Similar results were observed in a study conducted by Okaiyeto [37] which resulted in high flocculation activities when Mn2+ was used.

Table 2:

Effect of dosage and cations on the bioflocculant.

Dosage (mg/mL) FA (%) ± SD Metal ions FA (%) ± SD
0.2 57.1 ± 5.78a Na+ 63.3 ± 0.15a
0.4 81.9 ± 1.96b Li+ 65.3 ± 0.13a
0.6 80.8 ± 6.31b,c K+ 64.0 ± 0.21a
0.8 65.5 ± 1.75a Ca2+ 70.2 ± 0.11b
1.0 49.4 ± 1.62a,d Ba2+ 77.2 ± 0.42c
Mn2+ 98.4 ± 0.13d
Mg2+ 97.3 ± 0.14d
Fe3+ 90.2 ± 0.16e
Control 43.3 ± 0.10f

  1. FA % indicates flocculating activity while SD indicates standard deviation. Different letters (a, b, c, d, e, and f) indicate statistical significance at (p < 0.05).

The influence of MnCl2 concentration on bioflocculant

It is important to identify the highest cation concentration to produce flocculant particles because this reduces costs while increasing activity. The flocculating activity of the bioflocculant was tested using varying doses of MnCl2. It was observed that the flocculating activity of the cations began to increase as the cation’s concentration increased until reached the optimum cation concentration of 1.0 mg/mL. Further increase in cations concentration resulted in a decline in flocculating activity (Table 3).

Table 3:

The effect of MnCl2 concentrations of flocculation.

MnCl2 (g/L) FA (%) ± SD
0.25 62.00 ± 1.25a
0.5 73.14 ± 3.43a
0.75 77.32 ± 6.20b
1.0 98.30 ± 0.04b
1.25 68.15 ± 3.05b
1.5 58.22 ± 1.04c

  1. Different letters (a, b, and c) denotes statistical significance at (p < 0.05).

Antibacterial effect of the bioflocculant

The bioflocculant was evaluated for its antimicrobial effect in comparison with ciprofloxacin, which was used as a control for the experiment (Table 4). The bioflocculant showed moderate properties for inhibiting both the Gram-positive and Gram-negative organisms. Gram-negative bacteria are generally more resistant to most antibacterial agents than Grapositive strains due to their outer membrane, which prevents some antibacterial agents from penetrating the bacteria and exerting their action. The scenario was observed in this study whereby B. pumilus (Gram-positive) was more sensitive to the antimicrobial agents’ comparison to E. coli (Gram-negative). In conclusion, the bioflocculant can be used in wastewater treatment to remove pathogens.

Table 4:

MIC values of the bioflocculant against the test bacteria.

Bacterial strains Bioflocculant (mg/mL) Ciprofloxacin (µg/mL)
E. coli (ATCC 25922) 3.125 0.015
B. pumilus (ATCC 7065) 1.560 0.015

Purified bioflocculant application

Domestic wastewater treatment

The application of microbial flocculants for the removal of pollutants such as BOD, COD, and SO42− in domestic was determined in comparison to conventional flocculants. The effectiveness of wastewater treatment is most important in terms of reduction in the biological oxygen demand (BOD) and chemical oxygen demand (COD). The high levels of BOD and COD in wastewaters do not sustain aquatic life and lead to foul odours and anaerobic conditions, which consequently result in death [42]. Okaiyeto et al. [43], reported similar results with bioflocculant-produced by MBF-UFH in comparison with polyacrylamide. Furthermore, Patil et al. [44] found better removal efficiencies for COD and BOD in comparison with traditional flocculants on the bioflocculant produced from Azobacter indicus. Therefore, it can be concluded that the bioflocculant from P. mirabilis AB 932526.1 holds a promising future to replace in-use chemical flocculants in wastewater treatment (Table 5).

Table 5:

Application of flocculants in the removal of pollutants in domestic wastewater.

Types of flocculants Removal efficiency (%)
Water quality BOD COD S
Bioflocculant Before treatment 183 mg/L 407 mg/L 4.00 mg/L
After treatment 28.13 mg/L 75 mg/L 0.76 mg/L
Removal efficiency 85a 82a 81a
Alum Before treatment 183 mg/L 407 mg/L 4.00 mg/L
After treatment 47.12 mg/L 111 mg/L 1.57 mg/L
Removal efficiency 74a 73a 61b
FeCl3 Before treatment 183 mg/L 407 mg/L 4.00 mg/L
After treatment 31.15 mg/L 77 mg/L 1.10 mg/L
Removal efficiency 83a 81a 72b

  1. Different letters (a and b) denote statistical significance at (p < 0.05).

The application of a bioflocculant to remove dye solutions

The potential of dye removal using a bioflocculant was investigated. The dye removal efficiency of the bioflocculant was evaluated using three cationic dyes (safranin, methylene blue, and carbol fuchsin) and two anionic dyes (congo red and methylene orange). The bioflocculant’s ability to decolourize varied depending on the dye used. As shown in Fig. 3, the bioflocculant possesses huge potential for removing stains in all tested dye solutions. The bioflocculant revealed the removal ability of 83, 81 and 71 % against safranin, carbol fuchsin and methylene blue. Congo red and methylene blue attained a 90 % removal rate. This demonstrates that the bioflocculant was effective because of the revealed removal efficiency of above 70 % for all dye solutions tested. A study by Deng et al. [45], showed the same results where the bioflocculant produced by Aspergillus parasiticus, effectively removed selected dyes with an average flocculating efficiency of 92 %. A bioflocculant produced by Alteromonas sp. was established to be able to remove Congo red, and methyl blue with flocculating efficiencies of 98.5 %, and 72.3 %, respectively [46]. Therefore, the bioflocculant produced by P. mirabilis AB 932526.1 could be used in the treatment of wastewater containing dyes (Fig. 4).

Fig. 4: Dye removal efficiencies of the bioflocculant. Different letters (a, b, c and d) signify statistical significance at (p < 0.05).
Fig. 4:

Dye removal efficiencies of the bioflocculant. Different letters (a, b, c and d) signify statistical significance at (p < 0.05).

Conclusions

Proteus mirabilis AB 932526.1 was found to be a good microbial flocculant for bioflocculant production. After purification, the yielded purified bioflocculant was 3.8 g/L, which was better than in previous studies. The bioflocculant was found to be soluble in water but insoluble in all organic solvents tested. It’s a glycoprotein molecule made up of carbohydrates, proteins, and uronic acid. The X-ray diffraction patterns revealed different peaks which is an indication that the bioflocculant is crystalline. The morphological study proved the bioflocculant to be an amorphous structure. When evaluated for antimicrobial activity against both Gram-negative and Gram-positive bacteria, bioflocculant was able to inhibit all the tested strains at the lowest concentration of 1.56 mg/mL was found. Bioflocculant possesses great properties for pollutant removal in domestic wastewater and dye removal in comparison to conventional flocculants. From the consequence of the findings of this study, an investigation of the possibility of using agricultural wastes such as sugarcane waste products for the production of bioflocculant should be made. Furthermore, molecular methods on bacterial strains should be evaluated to increase the yield of bioflocculant. Further analysis of bioflocculant that includes genes implication, proton essay, molecular weight and determination of shelf-life of bioflocculant should be done. Additionally, the bioflocculant could be used as a capping agent in synthesizing nanoparticles to deal with yield production crisis as little amount of nanoparticles is used which could be useful in the wastewater treatment process or industrially.

Corresponding author: Rajasekhar V. S. R. Pullabhotla, Department of Chemistry, Faculty of Science, Agriculture and Engineering, University of Zululand, P/Bag X 1001, KwaDlangezwa 3886, South Africa, Phone: +27-359-026-155, e-mail:

Article note: A collection of invited papers based on presentations at the Virtual Conference on Chemistry and its Applications 2022 (VCCA-2022) held on-line, 8–12 August 2022.

Funding source: National Research Foundation

Award Identifier / Grant number: Incentive Fund Grant (103691)

Award Identifier / Grant number: Rated Researchers Grant (112145)

Acknowledgments

The authors would like to acknowledge the staff and postgraduate students (Bioflocculation Group) in the Department of Biochemistry and Microbiology for their outstanding support.

  1. Research funding: This research was funded by National Research Foundation (NRF, South Africa), grant number 103691 and Research Developmental Grant for Rated Researchers (112145).

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Published Online: 2023-03-08
Published in Print: 2023-03-28

© 2023 IUPAC & De Gruyter. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. For more information, please visit: http://creativecommons.org/licenses/by-nc-nd/4.0/

Articles in the same Issue

  1. Frontmatter
  2. In this issue
  3. Editorial
  4. Obituary for Professor Hugh Burrows, Scientific Editor of Pure and Applied Chemistry
  5. Preface
  6. The virtual conference on chemistry and its applications, VCCA-2022, 8–12 August 2022
  7. Conference papers
  8. Production and characterization of a bioflocculant produced by Proteus mirabilis AB 932526.1 and its application in wastewater treatment and dye removal
  9. Palladium-catalyzed activation of HnA–AHn bonds (AHn = CH3, NH2, OH, F)
  10. Mechanistic aspect for the atom transfer radical polymerization of itaconimide monomers with methyl methacrylate: a computational study
  11. A new freely-downloadable hands-on density-functional theory workbook using a freely-downloadable version of deMon2k
  12. Liquid phase selective oxidation of cyclohexane using gamma alumina doped manganese catalysts and ozone: an insight into reaction mechanism
  13. Exploring alkali metal cation⋯hydrogen interaction in the formation half sandwich complexes with cycloalkanes: a DFT approach
  14. Expanding the Australia Group’s chemical weapons precursors control list with a family-based approach
  15. Effect of solvent inclusion on the structures and solid-state fluorescence of coordination compounds of naphthalimide derivatives and metal halides
  16. Peripheral inflammation is associated with alterations in brain biochemistry and mood: evidence from in vivo proton magnetic resonance spectroscopy study
  17. A framework for integrating safety and environmental impact in the conceptual design of chemical processes
  18. Recent applications of mechanochemistry in synthetic organic chemistry
https://doi.org/10.1515/pac-2022-1002
Keywords for this article
Bioflocculant; dyes; Proteus mirabilis AB 932526.1; removal efficiency; VCCA-2022; wastewater

Articles in the same Issue

  1. Frontmatter
  2. In this issue
  3. Editorial
  4. Obituary for Professor Hugh Burrows, Scientific Editor of Pure and Applied Chemistry
  5. Preface
  6. The virtual conference on chemistry and its applications, VCCA-2022, 8–12 August 2022
  7. Conference papers
  8. Production and characterization of a bioflocculant produced by Proteus mirabilis AB 932526.1 and its application in wastewater treatment and dye removal
  9. Palladium-catalyzed activation of HnA–AHn bonds (AHn = CH3, NH2, OH, F)
  10. Mechanistic aspect for the atom transfer radical polymerization of itaconimide monomers with methyl methacrylate: a computational study
  11. A new freely-downloadable hands-on density-functional theory workbook using a freely-downloadable version of deMon2k
  12. Liquid phase selective oxidation of cyclohexane using gamma alumina doped manganese catalysts and ozone: an insight into reaction mechanism
  13. Exploring alkali metal cation⋯hydrogen interaction in the formation half sandwich complexes with cycloalkanes: a DFT approach
  14. Expanding the Australia Group’s chemical weapons precursors control list with a family-based approach
  15. Effect of solvent inclusion on the structures and solid-state fluorescence of coordination compounds of naphthalimide derivatives and metal halides
  16. Peripheral inflammation is associated with alterations in brain biochemistry and mood: evidence from in vivo proton magnetic resonance spectroscopy study
  17. A framework for integrating safety and environmental impact in the conceptual design of chemical processes
  18. Recent applications of mechanochemistry in synthetic organic chemistry
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