Bioscience Research - Innovative Scientific Information & Services ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Available online freely at www.isisn.org
Bioscience Research
Print ISSN: 1811-9506 Online ISSN: 2218-3973
Journal by Innovative Scientific Information & Services Network
RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2020 18(1): 188-207. OPEN ACCESS
Removal efficiency of a thermostable and non-toxic
bioflocculant produced by a consortium of two
marine bacteria
Ntombela Zuzingcebo Goldern1, Basson Albertus Kotze1, Madoroba Evelyn1,
Pullabhotla Viswanadha Srirama Rajasekhar2 and Moganavelli Singh3
1Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand, P/ Bag X
1001, KwaDlangezwa, 3886, South Africa
2Department of Chemistry, Faculty of Science and Agriculture, University of Zululand, P/ Bag X 1001,
KwaDlangezwa, 3886, South Africa
3School of Life Science, University of KwaZulu-Natal, Private Bag X 54001 Durban, 4001, South Africa
*Correspondence: ntombelaZ@unizulu.ac.za Received 20-08-2020, Revised: 04-1-2021, Accepted: 05-01-2021 e-Published:
10-01-2021
The dispensing of inadequately treated wastewater into water bodies has far reaching consequences,
resulting in negative impact on both human beings and aquatic environment. Thus, in this study we
assessed the removal efficiency of the bioflocculant from a consortium of two marine bacterial strains of
Bacillus safensis KX94275 and Bacillus sp. KC782848 from uMlalazi catchment, Mthunzini area, South
Africa. The produced polysaccharide and heat-stable bioflocculant with hydroxyl, amino, carboxyl and
carbonyl functional groups was found to be non-toxic with 96% human embryonic kidney (HEK 293)
cells survival and 78% of MFC7 cells survival after treatment with the highest concentration (100 µg/µL)
of a bioflocculant. The granular-like structure bioflocculant with elements such as C: O: Na: Al: S: K: Ca
of 18.46: 39.44: 5.32: 0.26: 13.39: 20.33: 2.80 (w/t %, in mass proportions) was revealed with a
degradation temperature (Td) of 150 oC. The bioflocculant is only soluble in water and no antimicrobial
activity has been exhibited by the bioflocculant on both Gram-positive and Gram-negative bacteria. The
cation-dependant bioflocculant (Ba2+) removed more than 85% dye from different dye solutions at
neutral pH, such as methylene blue (92%), Congo red (94%), malachite green (87%) and safranin O
(93%), using 0.8 mg/mL optimum dosage concentration. The removal efficiencies of 86%, 87% and 39%
of chemical oxygen demand (COD), biological oxygen demand (BOD) and total nitrogen, respectively
were obtained, with a flocculation rate of 95% for Tendele Coal mine wastewater. Thus, the bioflocculant
shows potential application in the wastewater treatment.
Keywords: Bioflocculant, flocculating activity, cytotoxicity, removal efficiency.
INTRODUCTION organic matter, depletion of water resources and
Access to pure water is one of the essential water shortage. This is triggered by an increase in
needs for human life to create healthy conditions human population and anthropogenic activities
for humans (Sayadi et al. 2016). However, there is that produces waste water (Tazaki et al. 2015). It
a general crisis of water pollution, due to industrial is therefore imperative to use water resources as
discharge of wastewater containing different sparingly as possible as well as to use, recycle
pollutants including suspended particles, sludge, and re-use resources. Water contamination
heavy metals, aromatic compounds, dyes and resulting from inorganic and organic contaminantsNtombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
has become a major concern of this era applied in varied industrial processes including
(Shahadat et al. 2017). Numerous perilous treating starch wastewater, drinking wastewater
organic and inorganic contaminants such as dyes and in downstream processes (Salehizadeh and
from textile industries and secondary phenol Shojaosaditi, 2001) to separate suspended
compounds from different industrial fields have colloidal substances from wastewater through
been noted as a global crisis (Dilling and Lemons, bridging, charge neutralization and colloid
2011). entrapment (Lee et al. 2014). Flocculants are
Wastewater contaminated with dye is one of accessible in various forms including
the major environmental concern due to the conventionally used (such as organic synthetic
number of industrial fields (paper and pulp, textile, flocculants and inorganic flocculants) and
leather and food) producing such wastewater comparatively recent biological flocculants, (e.g.
(Bisht and Lal, 2019). Most dyes were reported to bioflocculants) (Zhang et al. 2010; Lee et al.
be mutagenic, carcinogenic and tetratogenic to 2014).
human beings, microorganisms and aquatic Conventionally used flocculants have been
environment (Aljeboree et al. 2017) and they are reported to be inexpensive and possess high
also known to be intractable to microbial flocculation efficiency compared to comparatively
degradation (Yang et al. 2013). Textile industrial used flocculants, but reports show that they
fields are using and discharging large amount of impose health risks and environmental hazards
dye wastewater and other suspended pollutants. (Renault et al. 2009; Arafa et al. 2014). Synthetic
Textile industries have a tendency of producing a flocculants are neurotoxic and carcinogenic, their
large quantity of effluvium with dangerous by-products are toxic, non-biodegradable and they
materials during dyeing process with about 4 - 5% produce lot of sludge (Serdar et al. 2011; Ahmad
range of solid substances (Bisht and Lal, 2019). et al. 2015, Sanagi et al. 2016). Due to these
The unused effluvium produced by the system is drawbacks, attention has been shifted to the
released as wastewater that contains biochemical naturally occurring bioflocculants as an
oxygen demand (BOD), colour, chemical oxygen alternative.
demand (COD), turbidity, and high level of toxic Bioflocculants are macromolecule polymers
chemicals. These discharges from the process that are secreted by microorganisms as they grow
are usually released to water bodies (rivers, sea, (Zhuang et al. 2012). The main role of these
lakes, etc.) without being treated properly and extracellular polysaccharides (EPS) that are
become the environmental nuisance (Verma et al. secreted by microorganisms, as they grow are
2012). Moreover, these contaminants present in important, in wastewater treatment because they
water bodies are not easily treated and removed, remove colloidal particles from solution during the
which is a necessity, before being discharged. process of bioflocculation (Chen and Stewart,
Therefore, it is of most importance to develop 2002). These EPSs are made up of different
techniques which are efficacious in uprooting of substances including but not limited to proteins,
these pollutants and colloidal particles from water carbohydrates, nucleic acids, lipids, and ironic
and wastewater (Liu et al. 2018). acids (More et al. 2014; Nouha et al. 2018).
For the treatment of industrial effluents, a Compared to conventionally used flocculants,
variety of techniques and equipment have been microbial flocculants are biodegradable, non-toxic
utilized including coagulation / flocculation, and lack secondary pollution which makes them
membrane filtration, ion-exchange, activated alternative flocculants in wastewater treatment
carbon, electrochemical and photo-catalytic (Sun et al. 2012). However, the progress in
degradation, incineration and adsorption-oxidation research on bioflocculants production has been
(Yang et al. 2013; Wu et al. 2015; Ab Kadir et al. dented by low production yield by microorganism
2017.) and efficiency by microorganisms, high production
Amongst the afore-mentioned processes, costs, easily degradable active components, and
flocculation/coagulation has been a trusted and low stability (Yin et al. 2014; Dlangamandla et al.
vital part of the wastewater treatment (Shih et al. 2016). Nonetheless, researchers have recently
2001). Flocculants are substances with synthetic focused their attention into exploring the utilization
or natural origin used for sedimentation: their main of low-cost substances which are rich in carbon
role is to bring about separation of solids and and nitrogen sources together with the
liquid through the process of flocculation in optimization of fermentation parameters to
industrial plants. Flocculants are manipulative in increase their yields and removal efficiency. Xia et
the flocculation process and have been greatly al. (2018) suggested the use of mixed culture of
Bioscience Research, 2020 volume 18(1): 188-207 189Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
microorganisms in consortia as opposed to medium and incubated for 72 h at 30 °C with an
individual strain to improve bioflocculant yields. agitation speed of 165 rpm. The fermented
Zhu et al. (2004) and Zhang et al. (2007) also medium was then used as the standard sample.
emphasized the combination of strains of To make-up the bacterial consortium, 1 mL of the
microorganisms in consortia as an option to standard inoculum of bacterial strains were both
produce high yields of bioflocculants with better cultivated into the same 50 mL of the freshly
flocculating activity. prepared fermentation medium and incubated at
In this study, the bioflocculant produced from 30 °C at the shaking speed of 165 rpm for 72 h.
the consortium of two bacterial strains of Bacillus After 72 h, two millilitres of fermented broth was
safensis KX694275 and Bacillus sp. KC782848 centrifuged at 8000 × g for 15 min at 4 °C and the
was characterized and applied in decolourization cell-free solution was used for the determination
of solutions, used in the removal of turbidity, of flocculating activity (FA) (Zhang et al. 2007).
chemical oxygen demand (COD), biochemical
oxygen demand (BOD) and total nitrogen in Determination of flocculating activity
comparison with commercially used flocculants. To investigate the flocculating activity, the
Its solubility effect on different solvents, method of Akapo et al. (2019) was used. The
antimicrobial activity assay and cytotoxicity effect concentration of 4 g/L of kaolin solution was used
were assessed. as the test material. About 4 g of kaolin powder
was dissolved in 1000 mL of distilled water. Kaolin
MATERIALS AND METHODS solution of 100 mL was dispensed into 250 mL
conical flask and mixed with 2 mL of cell- free
Source of bacteria supernatant and 3 mL of 1% (w/v) calcium
The bacteria were isolated from the sediments chloride solution. The conical flask containing the
and water samples collected from Mthunzini mixture was agitated thoroughly for 60 sec and
catchment in the province of KwaZulu-Natal, then the solution was transferred into 100 mL
South Africa. The isolates were identified as graduated measuring cylinder. The mixture was
Bacillus safensis KX94275 and Bacillus sp. left at room temperature to settle for 5 min. To
KC82848 and maintained in 20% glycerol at −80 prepare the control, 2 mL cell-free supernatant
°C refrigeration in the Department of Biochemistry was replaced with freshly prepared fermentation
and Microbiology at the University of Zululand. medium. The optical density (OD) of the top liquid
was measured using UV-Vis spectrophotometer at
Sample Preparation 550 nm. The flocculating activity was estimated as
The sample was prepared according to the follows:
description of Nwodo et al. (2012). A production Flocculating Activity (%) = [(A –B)/A)] ×100.
medium made up of starch (10 g), ammonium Where A is the optical density of kaolin
sulphate (0.2 g), KH2PO4 (1 g), K2HPO4 (2.5 g), solution at 550 nm and B is optical density of a
NaCl (0.05 g) and MgSO4 (0.1 g) was prepared in treated kaolin solution at 550 nm (Kurane et al.
500 mL with filtered natural sea water and 1994).
adjusted to pH 3. About 50 mL of production
medium was transferred to 250 mL conical flask Extraction and purification of the bioflocculant
and autoclaved at 121 oC for 15 min. One loop full The extraction and purification of the
of bacterial colony was then inoculated into the bioflocculant from the bioflocculant-rich broth were
sterile production medium and incubated at 30 °C done in accordance with the methods of Chen et
in a shaker incubator with a speed of 160 rpm for al.(2003), with minor modifications. The
72 h. Two milliletres of broth culture were bioflocculant-rich broth was centrifuged at 8000
centrifuged at 8000 x g for 15 min at 4 °C to rpm at 4 °C for 15 min to remove bacterial cells. A
remove bacterial cells. The cell-free culture volume of deionised water was mixed with upper
supernatant was utilized for the assessment of phase liquid and re-centrifuged at 8000 rpm for 15
flocculating efficiency. min, at 4 °C. About 2000 mL of ice-cold ethanol
were added to the supernatant. The solution was
Bacterial consortium shaken vigorously and left to stand at 4 °C for 12
Both B. safensis KX694275 and Bacillus sp. h. The precipitate was vacuum-dried to obtain
KC782848 were used to construct bacterial crude bioflocculant. The crude product was
consortium. A loopful of colony from individual dissolved into 100 mL distilled water to yield a
strain was inoculated into 50 mL of the production solution (1% w/v). A volume of a mixture of
Bioscience Research, 2020 volume 18(1): 188-207 190Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
chloroform and n-butyl alcohol (5:2 v/v) was et al. (2016) was followed, with minor alterations.
added, agitated and left to stand at room Bioflocculant solutions of various concentrations
temperature for 12 h. The supernatant was then were prepared and used to investigate the
centrifuge (8000 rpm for 15 min at 4 °C) and optimum bioflocculant dosage. Concentrations of
vacuum-dried in order to obtain the purified 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL were used. From
bioflocculant. each bioflocculant solution concentrations
prepared, 2 mL was mixed with 100 mL of kaolin
Characterization of a purified bioflocculant suspension in water (0.4% w/v) containing 3 mL of
1% (w/v) CaCl2 in 250 mL flasks. The solution was
Scanning electron microscope (SEM) analysis agitated thoroughly, transferred into 100 mL
The surface morphological structure of the graduated cylinder and left to settle for 5 min at
purified bioflocculant as well as the elements room temperature. The clear upper phase of the
present in the bioflocculant was determined using supernatant was used for determination of
scanning electron microscope equipped with an flocculating activity. The flocculating activity was
energy-dispersive X-ray analyser (EDX) (JEOL measured at 550 nm as previously described.
JSM-6100). A Tungsten (W) filament and
accelerator voltage of 12 kV was used to take Effect of cations on flocculating activity
images with an emission current of 100 A. To investigate the effect of cations, 3 mL of
Bioflocculant powder was placed on copper stubs 1% (w/v) cation and 2 mL bioflocculant were
with double-sided carbon tape and carbon coated mixed with 100 mL kaolin solution. CaCl2 solution
using the JEOL vacuum evaporator. The samples (1% w/v) used as a stimulating agent in a
were then analysed and EDX was performed standard procedure was replaced by various
using JEOL JSM 6100 SEM with Bruker Quantax metal salt solutions including sodium chloride
Esprit software. The elemental composition of a (NaCl), potassium chloride (KCl), lithium chloride
purified bioflocculant in percentage weight was (LiCl), barium chloride (BaCl2), magnesium
recorded. chloride (MgCl2) and iron chloride (FeCl3). For
each salt solution, flocculating activity was
Fourier transform infrared spectroscopy (FT- determined and the standard was prepared by
IR) analysis replacing metal ion solution with equal amount of
To determine the presence of different distilled water. The mixture was shaken vigorously
functional groups in the bioflocculant, the Bruker for 60 sec, left to stand for 5 min and the optical
Tensor 27 Fourier transform infrared density (OD) was determined using a
spectrophotometer (Bruker, Gauteng, South spectrophotometer at 550 nm (Nie et al. 2011).
Africa) with standard ATR cell was used. Before
every analysis, the surface of the cell wall was The pH stability of a bioflocculant
wiped with absolute alcohol. The pressure was The pH stability of a purified bioflocculant was
adjusted to 90 gauge for proper contact between determined according to Okaiyeto et al. (2013).
the surfaces. The pH of kaolin solutions in separate flasks were
adjusted using 1 N NaOH and 1 N HCl between
Thermogravimetric analysis (TGA) the ranges of 3 - 12 prior to the assessment of
To analyse the thermal stability of the purified flocculating activity. Flocculating activity was
bioflocculant, the thermogravimetric analyser measured as previously described using the
(Perkin-Elmer Thermal Analysis Pyris 6 TGA) was optimum bioflocculant concentration and cation.
used in a closed perforated aluminium pan under
nitrogen gas with a flow rate of 40 cc/min. 10 mg Thermal stability of a purified bioflocculant
of a bioflocculant was used over the temperature The effect of temperature on flocculating
range of 30 - 800 oC and heating rate of 10.00 oC/ activity of the purified bioflocculant was
min. determined following the procedure of Wang et al.
(2011). The optimum concentration of the
Flocculation properties of a bioflocculant bioflocculant obtained was used. The solution
containing 2 mL aliquots were transferred into
Effect of dosage concentration of a purified different Eppendorf tubes and were heated for 30
bioflocculant min at different temperatures (50 - 100 oC). The
To determine the dosage concentration of a residual flocculating activity was measured at
purified bioflocculant, the method described by Yu room temperature.
Bioscience Research, 2020 volume 18(1): 188-207 191Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
Bioflocculant solubility trypsinization, cells were cultivated into 48 well
To determine the bioflocculant solubility, plates and incubated at 37 oC for 18 - 24 h. The
solvents such as water, methanol, hexane, old medium was mixed with the freshly prepared
acetone, ethyl-acetate and benzene were used to medium (MEM + Glutamax + antibiotics). In three
dissolve the bioflocculant (Maliehe et al. 2016). folds, the microbial flocculant was introduced and
soaked for 4 h. The broth medium was then
Antimicrobial activity Assay eliminated and replaced by medium made up of
The selected bacteria (Bacillus cereus and MEM + Glutamax + antibiotics + 10% Fetal Bovine
Escherichia coli) were inoculated into nutrient Serum. After 2 days, the cells were treated with
broth and incubated at 37 oC overnight. 200 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
Afterwards, 1 mL from each of the bacterial tetrazolium bromide) (MTT) (5 mg/mL) in
species were pipetted into 9 mL of fresh prepared phosphate-buffered saline (PBS) and each well
nutrient broth in separate test tubes labelled with was filled with 200 µL medium and stored at 37 oC
corresponding microorganism. The test tubes for 4 h. Then, the broth was protracted from the
were incubated at 37 oC for overnight. After wells with resulted formazan crystals dissolved in
overnight incubation, absorbance of the selected 200 µL of dimethyl sulfoxide (DMSO). The
bacterial strains was read in the absorbance for each sample was measured using
spectrophotometer at 600 nm for determination of a microplate reader at 570 nm. The bioflocculant
their turbidity. The turbidity of the resulting impact on HEK 293 cell viability was determined
suspensions was diluted with nutrient broth to as the percentage of tetrazolium salts reduction
obtain an absorbance of 0.132. This absorbance by viable HEK 293 cells counter to the untreated
was compared with McFarland turbidity standard. cells. To estimate the cell viability, the following
The turbidity was estimated to be equivalent to equations was used:
1.5x108 CFU/mL (Qaralle et al. 2012). Cell viability (%) = ODtreated cells/ODuntreated cells X
100.
Minimum inhibitory concentration (MIC) The breast cancer (MCF 7) cells were
Sterile Mueller Hinton Broth (50 µL) was cultured in Dulbecco's Modified Eagle's Medium
added to all the wells of the bioflocculant (50 (DMEM) supplemented with 10% Fetal Bovine
mg/mL) and the bioflocculant solution (50 µL) was Serum (FBS) (100 U), 20 µg/mL penicillin, and
poured in the first rows and mixed well on 100 µg/mL streptomycin. Incubation was carried
separate plates. Mixture (5 µL) was removed from out at 37 °C with an atmosphere of 5% CO2.
all the wells in the row A to perform a 3-fold serial Normal breast (MCF 7) cells were cultured in 1:1
dilution down the columns, respectively. The last mixture of DMEM and Ham's F12 medium with 20
50 µL in the last column was discarded so that the mg/mL of epidermal growth factor (EGF), 100
total volume solution of each was 50 µL. The μg/mL cholera toxins, 0.01 mg/mL insulin and 500
selected bacterial strains (50 µL) were transferred μg/mL hydrocortisone, and 5% chelex treated
into the corresponding wells. Distilled water was horse serum. Purified berberine and tamoxifen
used as a negative control while Streptomycin (20 were dissolved in dimethyl sulfoxide (DMSO) and
µg/mL) was used as a positive control. The plates used for the bioassays. The anticancer activity of
were covered and incubated at 37 oC for samples on MCF 7 cells were determined by the
overnight. P-iodonitrotetrazodium violet (INT) MTT (3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl
solution (0.2 mg/mL) was used after incubation tetrazolium bromide) assay was used to assess
period and 40 µL of 0.2 mg/mL INT solution were the cytotoxicity (Horiuchi et al. 1988). Cells (1 ×
added to each well and incubated at 37 oC for 30 105/well) were plated in 0.2 mL of medium/well in
min. After the addition of INT solution, the 96-well plates. For MTT assay, the medium from
appearance of red colour indicated that there was the wells was removed carefully after incubation.
no antimicrobial activity while a clear colour Each well was washed with MEM (w/o) FCS for 2-
implies the presence of antimicrobial activity (Elof, 3 times and 200 µL of MTT (5 mg/mL) was added.
1998). The plates were incubated for 6 - 7 h in 5% CO2
incubator for cytotoxicity. After incubation, 1 mL of
Cytotoxicity assay of bioflocculant DMSO (solubilising chemical) was added to each
A method of Mosmann (1983) was used to well and mixed well by micropipette and left for 45
determine the cytotoxicity effect of a bioflocculant. sec. Presence of viable cells was visualized by
Sangamly human embryonic kidney 293 (HEK the development of purple colour due to formation
293) cells were grown in 25 mL flasks. After of formazan crystals. The mixture was dispensed
Bioscience Research, 2020 volume 18(1): 188-207 192Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
to the glass cuvette of a spectrophotometer and and 2 mL of the bioflocculant solution were added.
the OD (optical density) values were measured at The mixture was shaken at 200 rpm for 3 min and
595 nm by using DMSO as a blank. Standard the speed was reduced to 40 rpm for 5 min at
Graph was plotted by taking a concentration of the room temperature. The supernatant was taken
drug versus the relative cell viability. and used to measure chemical oxygen demand
Cell viability (%) = (ODtreated cells/ODuntreated) x (COD), biological oxygen demand (BOD), and
100% total nitrogen content utilizing test kits as
described in manufacture’s protocol. The
Dye removal efficiency of bioflocculant untreated wastewater sample was also assessed
Experiments for dye removal were done, for BOD, COD, and total nitrogen (T). The optical
where 2 mL of bioflocculant and 3 mL of 1% (w/v) density of both treated (top-layer) and untreated
Ba2+ were mixed with 100 mL of dye solution (4 samples were read using spectrophotometer
g/L) and shaken vigorously for 60 sec. The (Spectro-quant, Merck Pharo 100) at 680 nm. The
mixture was allowed to sediment for 10 min at removal efficiencies on T, BOD and COD were
room temperature. Dyes such as methylene blue, calculated using the following equation:
malachite green, safranin O and congo red were Removal efficiency (%)= (To -TA/To) X 100
used/ tested. The top-layer was collected and Where To and TA are the initial and final
assessed using UV-Vis spectrophotometer at 550 values attained prior to and after treatment with
nm. The optical densities attained for both treated microbial flocculant. Both Alum and ferric chloride
and untreated dye solutions were used to were used as standards and compared with
estimate the removal efficiency of a bioflocculant. bioflocculant for their removal efficiencies.
The dye removal efficiencies were calculated
using the formula below: Statistical analysis
Removal Efficiency (%) = (To-TA/To) X 100 All experiments were conducted in triplicates
Where To is the initial value and T A is the final with mean and standard deviation values
value after treatment with bioflocculant. determined where differences were considered
Therefore, it is essential to calculate residual significant at 0.05 at confidence level (p > 0.05) by
concentration of the dye in the samples after the use of Graph Pad Prism TM version 6.0.
treatment with the basis of the initial and final dye
contents (Dlamini et al. 2019). RESULTS AND DISCUSSION
Wastewater treatment using bioflocculant Characterization of a bioflocculant
Wastewater sample was freshly collected
aseptically from Tendele Coal Mine wastewater SEM Analysis of purified bioflocculant
plant situated at KwaSomkhele Area, KwaZulu- The surface morphological structure has a
Natal, RSA, for assessment of the potential significant impact on the bioflocculation process
removal effect of contaminants by the microbial as it is liable for flocculating efficiency of the
flocculant. The pH of wastewater was calibrated to bioflocculants (Zhang et al. 2007). Morphological
8 using 1.0 M NaOH and 1.0 M HCl. The jar test structure surface of the produced bioflocculant,
was performed following the method described by kaolin powder and flocculated kaolin powder were
Maliehe et al. (2019). To 100 mL of wastewater revealed using scanning electron microscope
sample adjusted to pH 8, 3 mL of 1% (w/v) Ba2+ (Figure 1).
Bioscience Research, 2020 volume 18(1): 188-207 193Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
A B C
Figure 1: SEM analysis of a bioflocculant (A), kaolin powder (B) and flocculated kaolin clay (C).
Element Wt%
C 50.22
O 38.07
Na 1.26
Mg 0.12
Al 0.11
P 0.86
S 3.06
Cl 0.97
K 2.11
Ca 3.22
Total 100.00
Figure 2: EDX analysis of a purified bioflocculant.
SEM analysis showed that the bioflocculant Elemental analysis
was whitish in colour and quadrilateral-like in According to Cosa et al. (2011), elemental
shape, which may be responsible for its high composition of the microbial flocculant has a
flocculating efficiency (Figure 1(A). significant role to play in the bioflocculant
On the other hand, kaolin powder particles structure as well as flocculation activity. Different
appeared fine and scattered before flocculation as elements give rise to the stability and flexibility of
shown in Figure 1(B). Flocculated kaolin particles the bioflocculants. Perceptible analysis of the
appeared to have formed flocs in clusters. These elemental make-up of the produced bioflocculant
appeared flocs are due to the absorption of kaolin was investigated and the results are depicted in
particles towards the binding sites of the Figure 2. The presence of elements such as C: O:
bioflocculant resulting in the formation of large Na: Mg: Al: P: S: Cl: K and Ca accountable for
flocs through their interaction (Figure 1(C). The 50.22: 38.07: 1.26: 0.12: 0.11: 0.86: 3.06: 0.97:
formation of large flocs is in line with rapid 2.11 and 3.22 (% Wt) respectively, shown in the
sedimentation due to weight (Xiong et al. 2010). elemental spectrum using EDX analysis. The
presence of C and O elements in the molecular
chain of the bioflocculant is an indication of the
Bioscience Research, 2020 volume 18(1): 188-207 194Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
glycoprotein-like polymer (Pathak et al. 2015). A effect of temperatures is shown in Figure 4. The
similar finding of the bioflocculant with carbon and highest weight loss of 60.35% bioflocculant was
oxygen as its main elements was reported by Sun reported at 400 oC and the bioflocculant was
et al. (2015). almost completely pyrolyzed at temperatures
above 400 oC, about approximately 11.0% of
Fourier transform infrared spectroscopy (FT- weight loss at 150 oC and 29.0% at 250 oC. This
IR) analysis bioflocculant seems to exhibit thermo-labile and
The functional groups of a purified thermostable molecular contents, as the mixture
bioflocculant from consortium of two marine of protein and carbohydrates materials are
bacteria were assessed and the results are shown presented as shown by examination. The first
in Figure 3. The FT-IR spectrum (Figure 3) degradation of the bioflocculant or weight loss
exhibited a broad stretching observed at around may be due to moisture content loss or just the
3456 cm-1 as an indication of the presence of -OH presence of C=O and -OH functional groups in the
group (Lian et al. 2008). The vibration band molecular chain of the bioflocculant linked with the
observed at 2929 cm -1 may be attributed by the glycoprotein-like particles (Tawila et al. 2019).
presence of C-H group towards the binding site of Both Wang et al. (2011) and Tawila et al. (2019)
the bioflocculant. The peak at 1856 cm -1 respectively reported similar observations with the
corresponds to the bond stretching due to the bioflocculant produced by consortium of multiple
availability of alkenes group. The strong stretching bacteria and a novel bioflocculant QZ-7 from
peaks between 1110 and 1000 cm -1 are for alkoxy Bacillus salmalaya 139SI.
group in the molecular chain of a bioflocculant.
The weak bands observed from 829.9 to 543.2 Optimisation of a purified bioflocculant
cm-1 represent or are characteristics of all sugar
derivatives. The results indicate clearly the Effect of bioflocculant dosage size on
presence of hydroxyl, alkoxy, and carboxyl groups flocculating activity
in the molecular chain of a bioflocculant which are Dosage concentration is still one of the
essential as they promote flocculation process. important parameters to take into consideration
Moreover, the functional groups, carboxyl group in when assessing the excellent conditions for the
particular, has an advantage of working as high performance of flocculants in the flocculation
functional moieties to generate novel and modified process. This is vital since inadequate dosage of
carbohydrate variants using various methods such bioflocculant leads to a poor bridging
as polymer engineering or new emergence phenomenon, thus resulting in low flocculating
designing, thereby linking the carbohydrate with activity, while excessive input induces re-
other produced polymer (Yim et al. 2007). The stabilization of kaolin particles (Hassan et al.
said functional groups have the advantage of 2009). Moreover, determination of the dosage
participating in the linkage of hydrogen ions and concentration becomes more important in terms of
hydroxyl ions present on the surface of the establishing the excellent microbial flocculating
particle resulting in the formation of the hydrogen dosage concentration. This could play a vital role
bonds when bioflocculant molecular chains in minimizing costs and resulting in the improved
approach surface of the particles (Cosa et al. flocculation rate for its application industrially. The
2013). Similar findings for functional groups optimum dosage size was determined using jar
present in the bioflocculant were reported by Feng test experiment and the highest flocculating
and Xu (2008). activity was obtained at 0.8 mg/mL (75%) of a
bioflocculant (Figure 5). All tested dosage
Thermogravimetric analysis (TGA) of a concentrations resulted in the flocculating activity
purified bioflocculant above 50% with 0.2 mg/mL dosage concentration
The bioflocculant from consortium of bacteria the lowest and this resulted in the low flocculating
was assessed to elucidate its thermogravimetric activity compared to other dosage concentrations.
behaviour at varied temperatures starting from 30 Below and above 0.8 mg/mL dosage
– 800 oC, and the results are depicted in Figure 4. concentrations, the flocculating activity decreased.
The thermogravimetric analysis is carried out to This may be due to the fact that low dosage
assist in recognizing the bioflocculant’s thermal concentrations cannot neutralize the functional
character after exposed to significantly elevated groups optimally and high dosage concentrations
heat (Yim et al. 2007). A weight loss of lead to poor colloids binding due to high viscosity.
bioflocculant expressed in percentage under the Therefore, for all subsequent tests 0.8 mg/mL was
Bioscience Research, 2020 volume 18(1): 188-207 195Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
used. Agunbiade et al. (2017) also reported 0.8 flocculating activity of 84% and was used in all
mg/mL as an optimum dosage for the subsequent tests. The bioflocculant produced in
bioflocculant produced by Arthrobacter humicola; this study seems to be cation-dependent as little
while Liu and Cheng (2010) reported that the activity (31%) was recorded when there was no
bioflocculant produced by C. daeguence where cation added. These findings are comparable to
more than 90% flocculating activity obtained using those of Zayed et al. (2019) and Zheng et al.
1.2 mg/mL. (2008), where they reported various bivalent and
trivalent metal ions to optimally stimulate the
Effects of cations on the flocculating activity flocculating activities of the produced
Metal ions increase flocculating activity by bioflocculants. Contrary to the study findings, a
neutralizing and stabilizing the residual negative cation-independent bioflocculant ETH-2 produced
surface charge of both functional groups of kaolin by Enterobacter sp. ETH-2 was reported by Tang
clay particles and the bioflocculant (Wu and Ye, et al. (2014), where the bioflocculant produced an
2007). The results depicted in Figure 6 show that optimum flocculating activity without being
all tested metal ions stimulated the flocculating stimulated by metal ions.
activity of a bioflocculant produced against kaolin
clay suspension with great stimulation by Mg2+,
Ba2+ and Fe3+. Ba2+ which resulted in the optimum
15 0
10 0
T %
2936
50 3334 1629 1465
1098 597
0
4000 3000 2000 10 0 0 0
-1
W a v e le n g t h ( c m )
Figure 3: Fourier Transform Infrared Spectrum of purified bioflocculant.
Bioscience Research, 2020 volume 18(1): 188-207 196Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
150
W e ig h t lo s s ( % )
100
50
0
0 200 400 600 800
o
T e m p e ra tu re ( C )
Figure 4: Thermogravimetric analysis of a purified bioflocculant.
Figure 5: Effect of dosage size on the flocculating activity of a purified bioflocculant.
Figure 6: Effect of cations on the flocculating activity of a purified bioflocculant.
Bioscience Research, 2020 volume 18(1): 188-207 197Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
Figure 7: The effect of pH on flocculating activity of a purified bioflocculant.
Figure 8: The effect of temperature on flocculating activity of a purified bioflocculant.
The slight decrease in flocculation activity was
observed at acidic condition (3 - 6); which might
Effect of pH on the flocculating activity be associated with the protein content present in
The pH of the solution is a key parameter that the bioflocculant denaturation. According to
controls the flocculation /coagulation process Nwodo et al. (2014), this behaviour of the
(Maliehe et al. 2019). pH may cause some bioflocculant might be as a result of the absorption
modifications in microbial flocculant charge status of hydrogen ions at low pH tends to weaken
and the structural characteristics of the colloid bioflocculant-kaolin clay complex formation
particles in a solution and as a result affects process leading to poor flocculation and similar
flocculating efficiency (Li et al. 2007). The effect of effect at high pH due to hydroxyl ions. Therefore,
pH on flocculating activity of the purified pH 8.0 was used in all subsequent tests. Similar
bioflocculant was determined ranging from 3 - 12 results whereby the bioflocculant produced
and the results are depicted in Figure 7. The optimum flocculating activity at pH 8.0 was
produced bioflocculant was effective within a wide reported for bioflocculant produced by Bacillus
range of pH (3 -12) with flocculating activity equal tequilensis MMFG37 (Awady et al. 2018). Cosa et
to / greater than 60%. The optimum flocculating al. (2013) also reported a bioflocculant from
activity of 95% was obtained at pH 8.0 and lowest consortium of two bacterial species to be stable to
flocculating activity of 60% observed at pH 4.0. wide ranges of pH, however, but the slightly basic
Bioscience Research, 2020 volume 18(1): 188-207 198Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
conditions had the highest flocculating activity. and hydrophilic (Walker and Wilson, 2005;
Maliehe et al. 2016). Its protein and
Effect of thermal stability carbohydrates contents gave better explanation in
The correlation between heat and flocculating terms of its stability towards solvent tested. In
efficiency of the purified bioflocculant from general, bioflocculants have tendency to be
consortium of two bacterial strains was examined soluble in water as well as both acidic and alkaline
at a temperature range of 50 – 100 oC for 30 min solutions (Zaki et al. 2011). The presence of
and the results are depicted in Figure 8. This is hydroxyl groups in the molecular chain of a
done to assess its stability when exposed to bioflocculant (Figure 3) make- up strong attraction
higher temperatures. The microbial flocculant forces among bioflocculant molecules, leading to
retained over 70% flocculating activity when the formation of relatively hard crystalline solids;
exposed to heat at 100 oC for 30 min suggesting where the strong hydrogen bonding occurs. As
that the bioflocculant was stable. The flocculating other solvents were unable to dissociate these
activity of 80% was observed at 60 oC, when the forces, the bioflocculant became insoluble in all of
heat was increased above 60 oC, which resulted them (organic solvents). According to Okaiyeto et
in slight decrease in flocculation rate but al. (2015), for bioflocculant to dissolve in water or
interestingly, there was no significant difference in aqueous solution, it has to possess the hydroxyl
terms of statistical analysis. Therefore, it was group in its molecular chain that will form
deduced that the bioflocculant was thermostable hydrogen bonding with water molecules. Both
and its flocculating efficiency was not affected Zaki et al. (2011) and Maliehe et al. (2016)
when the temperature was elevated. In general, reported the bioflocculants that are insoluble in all
the availability of protein or peptide within the tested organic solvents but soluble in water.
structure of a bioflocculant is highly linked with its Table 1: Bioflocculant solubility in various
sensitivity towards heat and those with high sugar solvents.
content are more resistant to heat (Sun et al.
2012); hence it can be deduced that the Solvent Solubility
bioflocculant produced by the consortium of Distilled water +
Bacillus safensis and Bacillus sp. consisted of Methanol -
polysaccharide as its main component. The heat- Acetone -
resistance of this product suggests the presence Hexane -
of a hydroxyl group in its chain responsible for Benzene -
emergence of hydrogen bonds in its structure Ethyl acetate -
(Ntombela et al. 2019). Cosa and Okoh (2013) Chloroform -
reported a flocculating activity of more than 80%
for a purified bioflocculant produced by the Antimicrobial activity
bacterial consortium of Halobacillus sp. Mvuyo The antimicrobial activity assay was
and Oceanobacillus sp. Pinky retained after conducted to assess the effect of bioflocculant on
heating at 100 oC for 30 min, thus indicating its Gram- positive and Gram-negative bacterial
thermo stability character. strains. The test strains were Escherichia coli
which is Gram- negative and the Gram-positive
Bioflocculant solubility in different solvents Bacillus subtilis. Results attained show that both
Microbial flocculant biomolecules are microbial strains were not affected after incubation
dissimilar in the balance of charged, polar and (Table 2). The reddish colour was observed after
hydrophobic components they have on their the addition of INT indicator, which is the
surface. The bioflocculant in this study was only indication of INT being reduced by the metabolic
soluble in water and insoluble in all other tested activity of microorganisms to form formazan, a
solvents (Table 1). Bioflocculants mainly positive indicator for microbial activity. This means
composed of carbohydrates are more of that the bioflocculant doesn’t have antimicrobial
hydrophilic fraction as opposed to hydrophobicity activity to inhibit the growth of microbes rather; it
nature of protein-dominated bioflocculants (Jorand is thought that bioflocculant can only form flocs
et al. 1998; More et al. 2014). The produced with microorganisms being incorporated into
bioflocculant in this study is mainly a them, settle down with them and removed them
carbohydrate, thus carrying charged and polar together with flocs. The 20 µL Streptomycin
groups easily solvated by water molecules and (control) used showed inhibitory effect all test
therefore, making the microbial flocculant soluble microorganisms. A bioflocculant passivated
Bioscience Research, 2020 volume 18(1): 188-207 199Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
copper nanoparticles was reported to have shown highest bioflocculant concentration (100 µg/µL)
good properties on inhibiting and killing both showed less than 25% of cytotoxicity effect
Gram-negative and Gram-positive against the tested cells. Increasing the
microorganisms tested (Dlamini et al. 2019). bioflocculant concentration causes a slightly
Table 2: Antimicrobial assay of the decrease in the MFC 7 cells number (Figure 10).
bioflocculant. Sharma et al. (2017) also reported the toxic- free
bioflocculant produced by Acinetobacter
Bacterial strains
MIC Streptomycin haemolyticus against sheep blood cells and in the
Test (MIC Assay) in-vivo study done on rats; no toxicity effects were
Bacillus cereus - 3.125 mg/mL reported. Mahmoud et al. (2015) also reported no
Escherichia coli clinical symptoms on rats tested against the
- 6.25 mg/mL
(ATCC 25922) produced bioflocculant.
In- vitro cytotoxicity effect of bioflocculant on Dye removal potential of a bioflocculant
HEK 293 and MFC7 Cell-line The effect of bioflocculant on staining dye
Bioflocculants have been declared as non- removal was investigated and the results are
toxic compounds by number of researchers shown in Figure 11. The produced bioflocculant
(Sponza, 2002; Sun et al. 2015; Czemierska et al. showed good and high affinity for all tested dyes
2017), but there is a need to be evaluated for their (Congo red, malachite green, methylene blue and
toxicity before use for biosafety reasons (Pathak safranin) with the removal efficiency above 85%.
et al. 2015). This is done as some bioflocculants This microbial flocculant is efficacious in
may have toxic effect (Abdullah et al. 2017). eliminating stains in wastewater from various
Cytotoxicity effect of a bioflocculant for this study industries including clothing industry. The
was also evaluated and the results are depicted in bioflocculant was able to eliminate all various
Figure 9 and 10. The bioflocculant revealed a dyes assessed, possibly, by causing aggregation
margin of safety, as no significant cytotoxicity of particles and cells due to charge neutralization
effect on the assessed cell line. Above 90% and bridging (Salehizadeh and Shojaosadati,
viability of HEK 293 cell-line was exhibited in all 2001). Bridging is taking place when the
tested bioflocculant concentrations and the bioflocculant extends from the surface into the
highest concentration of bioflocculant (100 µg/µL) medium for a distance greater than the distance
showed about 96% viability of HEK 293 cell-line over which the inter-particle repulsion acts. Here,
exhibition (Figure 9). Therefore, the bioflocculant the bioflocculant can attach to other particles to
produced in study has demonstrated a good form flocs (Buthelezi et al. 2012).
safety property which could be probably safe for
implementation in various industrial processes.
The cytotoxicity test for bioflocculant on MFC 7
cell line revealed above 75% cell survival in all
tested bioflocculant concentrations with the
Figure 9: In-vitro cytotoxicity of various amounts of bioflocculant on HEK 293 cell line.
Bioscience Research, 2020 volume 18(1): 188-207 200Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
150
C e ll s u rv iv a l (% )
a a ,b
100 a ,b
a
90 83
85 78
50
0
0
5
0
5
10
2
5
7
C o n c e n t r a t i o n ( g / l)
Figure 10: In-vitro cytotoxicity of the bioflocculant on MFC 7 cells.
100
F loccu latin g activity (% )
80
60
94 92 93
87
40
20
0
re
d
ee
n ue ne
o gr bl ni
ng te ne fra
Co i le Sa
ch hy
a la et
M M
D ye
Figure 11: Decolourization of solution by a bioflocculant.
Table 3: Treatment of coal mine wastewater using the bioflocculant.
BOD5 COD Nitrogen Flocculating
Flocculants
(mg/L) (mg/L) (mg/L) activity at OD550 nm
Before treatment 62 98 7.2 2.037
After treatment 08 14 4.4 0.100
Microbial
Removal rate /Flocculation
87 86 39 95
efficiency (%)
Before treatment 62 98 7.2 2.037
After treatment 15 19 5.1 0.298
Alum Removal rate /
76 81 29 85
Flocculation efficiency (%)
Before treatment 62 98 7.2 2.037
After treatment 11 22 5.2 0.150
FeCl3 Removal rate /
82 78 28 93
Flocculation efficiency (%)
Note: Values are means of triplicate measurements.
The bioflocculant concentration of 0.8 mg/mL demonstrated to be more effective as more than
was optimum for the removal of all dyes used and 85% of dye removal efficiency was observed in
Bioscience Research, 2020 volume 18(1): 188-207 201Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
the presence of Ba2+ as a stimulating agent. stimulating agent. The heat-stable bioflocculant
Buthelezi et al. (2012) and Elkady et al. (2017) with functional groups such as hydroxyl, amino,
also reported different microbial biopolymers with carboxyl and carbonyl exhibited no antimicrobial
potential of removing dyes from wastewater. activity when tested. The bioflocculant was
Functional groups in the molecular chain of a revealed to be non-toxic and possess great
bioflocculant should be capable to interrelate with properties for flocculation, pollutant removal in
sites on the surface of the suspended particle for coal mine wastewater as well as the
it to be effectual (Aljuboori et al. 2015). decolourization of different dye solutions. Over
85% of COD and BOD removal efficiency, 95%
Pollutants removal efficiency from wastewater flocculating rate and 39% of total nitrogen was
The availability of excessive concentrations of observed for coal mine wastewater, compared to
COD and BOD in water is often associated with predominant traditional flocculants. In all tested
the anaerobic conditions, unacceptable odours dye solutions, the bioflocculant showed great
and stagnant waters that have a negative effect potential with removal efficiency above 86% for all
towards aquatic life (Dlamini et al. 2019). The tested dye solutions. The revealed characteristics
presence of nutrients and metals in water, such as suggested its potential industrial applications.
sulfur, nitrogen, phosphate, aluminium and nitrate, Presently, the team is focussing on the
in excess result in eutrophication (Sigee, 2005). biosynthesis of nanoparticles using this
The ability of the produced bioflocculant to bioflocculant as a capping and stabilizing agent.
eliminate suspended particles in Tendele coal
mine wastewater is shown in Table 3, in CONFLICT OF INTEREST
comparison with conventional chemical The authors declared that present study was
flocculants (Alum and iron (III) chloride). performed in absence of any conflict of interest.
Bioflocculant showed better removal efficiency on
all tested parameters. ACKNOWLEGEMENT
In Table 3, bioflocculant showed the ability of Zuzingcebo Ntombela likes to acknowledge
removing about 39% of total nitrogen from coal the Research and Innovation Committee of the
mine wastewater which is better than alum and University of Zululand and the staff and
iron (III) chloride (both below 30%). These results postgraduate students (Biofloc Group) in the
suggest that the bioflocculant can be a suitable Biochemistry and Microbiology Department for
alternative to replace chemical flocculants. It also their support. The authors would like also to
produced a better flocculating activity (95%) than acknowledge the Electron Microscope Unit at the
both conventional chemical flocculants. The University of KwaZulu-Natal, Westville campus,
bioflocculant had the best removal efficiency for for providing support by letting us use the SEM-
COD (86%) and BOD (87%) with alum and FeCl3 EDX facility for characterization of a bioflocculant.
removed up to 81% (COD) and 82% (BOD). The Rajasekhar Pullabhotla would like to acknowledge
removal efficiency of the bioflocculant generally the National Research Foundation (NRF, South
could be attributed to the structure of the Africa) for the financial support in the form of the
bioflocculant, chemical composition and the Incentive Fund Grant (Grant No: 103691).
functional groups. These findings suggest that the
bioflocculant possesses high potential for AUTHOR CONTRIBUTIONS
industrial application as an alternative means to Conceptualization: AKB, VSRP and ME;
the currently predominant traditional flocculants. Formal analysis: ZGN, AKB and VSRP;
In addition, the effectiveness of bioflocculant investigation: ZGN; supervision: AKB, VSRP and
recommends that they also have the potential to ME; writing (original draft): ZGN; writing (review
eliminate the adverse effects of synthetic chemical and editing): AKB, VSRP and ME; cytotoxicity
flocculants being used (Lin and Haricund, 2011). experiments: MS.
Similar results were presented by number of
authors including Luo et al. (2014) and Maliehe et Copyrights: © 2020@ author (s).
al. (2019). This is an open access article distributed under the
terms of the Creative Commons Attribution License
CONCLUSION
(CC BY 4.0), which permits unrestricted use,
The bioflocculant showed an excellent
flocculating property at 0.8 mg/mL dosage. It distribution, and reproduction in any medium,
performed very well in the presence of Ba2+ as a provided the original author(s) and source are
Bioscience Research, 2020 volume 18(1): 188-207 202Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
credited and that the original publication in this 15(4) :3581-3592.
journal is cited, in accordance with accepted Bisht V, Lal B (2019). Exploration of performance
academic practice. No use, distribution or kinetics and mechanism of action of a
reproduction is permitted which does not comply potential novel bioflocculant BF-VB2 on clay
with these terms. and dye wastewater flocculation. Front.
Microbiol.
https://doi.org/10.3389/fmicb.2019.01288.
REFERENCES
Buthelezi SP, Olaniran AO, Pillay B (2012).
Ab Kadir NN, Shahadat M, Ismail S (2017).
Textile dye removal from wastewater
Formulation study for softening of hard water
effluents using bioflocculants produced by
using surfactant modified bentonite
indigenous bacterial isolates. Molecu. 17:
adsorbent coating. Appl. Clay Sci. 137: 168-
14260-14274.
175.
Chen X, Oh SW, Zheng Z, Chen HW, Shin H, Hou
Abdullah M, Roslan A, Kamarulzaman M, Erat M
SX (2003). Cyclin d-cdk4 and cyclin e-cdk2
(2017). Colloids removal from water
regulate the Jak/STAT signal transduction
resources using natural coagulant: Acacia
pathway in Drosophila. Dev. Cell. 4(2): 179 -
auriculiformis. In AIP conference
190.
proceedings; AIP publish. Melville, NY, USA.
Chen X, Stewart PS (2002). Role of electrostatic
Agunbiade OM, Van Heerden E, Pohl CH, Ashafa
interactions in cohesion of bacterial biofilms.
AT (2017). Flocculating performance of a
Appl. Microbial. Biotechnol. 59: 718-720.
bioflocculant produced by Arthrobacter
Cosa S, Mabinya LV, Olarian AO, Okoh OO,
humicola in sewage waste water treatment.
Bernard K, Deyzel S, Okoh AL (2011).
BMC Biotechnol. 17(51).
Bioflocculant production by Virgibacillus sp
Ahmad HRA, Azni I, Hamid HRA, Yoshimitsu U,
Rob isolated from the bottom sediment of
Abubakar BSUI (2015). Flocculation behavior
Algoa Bay in the Eastern Cape, South Africa.
and mechanism of bioflocculant produced by
Pol. J. Environ. 16:2431-24442.
Aspergillus flavus. J. Environ. Manage. 150:
Cosa S, Okoh A (2013). Bioflocculant production
466-471.
by a consortium of two bacterial species and
Akapo CSO, Ntombela ZG, Pullabhotla VSR,
its potential application in industrial
Basson AK (2019). Isolation, optimization,
wastewater and river water treatment.
characterization and application of
Applied and environmental microbiology
bioflocculant BA-CGB produced by novel
study. 23(3): 689-696.
Bacillus atrophaeus isolated from Richards
Cosa S, Ugbenyen MA, Mabinya LV, Okoh AI
Bay Harbour, South Africa. Biosci. Res.
(2013). Characterization of a thermostable
16(4): 3873-3902.
polysaccharide bioflocculant produced by
Aljeboree AM, Alshirifi AN, Alkaim AF (2017).
Virgibacillus species isolated from Algoa
Kinetics and equilibrium study for the
Bay. Afr. J. Microbiol. Res. 7: 2925-2938.
adsorption of textile dyes on coconut shell
Czemierska M, Szcześ A, Holysez L, Wiater A,
activated carbon. Ara. J. Chem. 10: 3381-
Jarosz-Wilkolazka A (2017). Characterization
3393.
of exopolymer R-202 isolated from
Aljuboori AHR, Idris A, Al-joubory HHR, Uemura
Rhodococcusrhodochrous and its
Y, Abubakar BI (2015). Flocculation
flocculating properties. Eur. Polym. J. 88: 21-
behaviour and mechanism of bioflocculant
33.
produced by Aspergillus flavus. J. Environ.
Dilling L, Lemos MC (2011). Creating usable
Manag. 150: 466-471.
science: opportunities and constraints for
Arafa RA, El-Rouby MN, Abass HA, El-khier ZA
climate knowledge use and their implications
(2014). Bioflocculant produced by bacterial
for science policy. Glob. Environ. Chang. 21:
Isolates from Egyptian soil 1-characterization
680-689.
and application of extracellular bioflocculants
Dlamini NG, Basson AK, Pullabhotla VSRR
and nanoparticles for treatment of River Nile
(2019). Optimization and application of
water. J. Pharm. Biol. Sci. 9(5): 103-114.
bioflocculant passivated copper
Awady E EL, Ghada Ibrahim S, Ayaz AO, Mouafi
nanoparticles in the wastewater treatment.
FE, Mahmou MG (2018). Production and
Int. J. Environ. Res. Pub. Healt. 16: 2185.
characterization of bioflocculant from isolated
Dlangamandla C, Ntwampe SKO, Basitere M
Bacillus tequilensis MMFG37. Biosc. Reas.
(2018). A bioflocculant-supported dissolved
Bioscience Research, 2020 volume 18(1): 188-207 203Ntombela et al. Removal efficiency of a bioflocculant produced by bacterial consortium
air flotation system for suspended solids, Liu B, Zheng H, Wang Y, Chen X, Zhao C, An Y
lipids, and protein matter from poultry (2018). A novel carboxyl-rich chitosan-based
slaughterhouse wastewater. Wat. Sci. polymer and its application for clay
Technol. 78(2): 452-458. flocculation and cationic dye removal. Sci.
Elkady M, Zaki S, Farag S, Abd-Haleem D (2017). Tot. Environ. 107-115, 640-641.
Bioflocculation of basic dye onto isolated Liu L, Cheng W (2010). Characteristics and
microbial biopolymers. Chem. Biochem. Eng. culture conditions of a bioflocculant produced
Q. 31(3): 209-224. by Penicillium sp. Biomed. Enviro. Sci. 23:
Elof JN (1998). A sensitive and quickly microplate 213-218.
method to determine the minimal inhibitory Liu W, Yuan H, Yang, J, Li B (2009).
concentration of plant extracts for bacteria. Characterization of bioflocculants from
Planta. Med. 64 (8): 711-13. biologically aerated filter backwashed sludge
Feng DL, Xu SH (2008). Characterization of and its application in dying wastewater
bioflocculant MBF3‐3 produced by an treatment. Bioresour. Technol. 100: 2629-
isolated Bacillus sp. World J. Microbiol. 2632.
Biotechnol. 24:1627-1632. Luo Z, Li C, Changhong C, Wei Z, Ming L, Ye H,
Hassan MAA, Li TP, Noor ZZ (2009). Coagulation Jiangang Z (2014). Production and
and flocculation treatment of wastewater in charecteristics of bioflocculant by Klebsiella
textile industry using chitosan. J. Chem. Nat. pneumonia YZ-6 isolated from human saliva.
Res. Eng. 4: 43-53. Appl. Biochem. Biotechnol. 172: 1282-1292.
Horiuchi N, Nakagava K, Sasaki Y, Minato K, Mahmoud N, Hend A, Salma M, Rawhia A, Abd E,
Fujiwara Y, Nezi K (1988). In vitro antitumor Zakia A (2015). Bioflocculants produced by
activity of mitomycin C derivative (RM-49) isolated bacteria from Egyptian soil II-
and a new anticancer antibiotic (FK973) cytophathological effect of extracellular and
against lung cancer cell lines determined by intracellular bacterial extracts. Glob. J.
tetrazolium dye (MTT) assay. Canc. Biotechnol. Biochem. 10: 47-61.
Chemother. Pharmacol. 22: 246-50. Maliehe TS, Basson AK, Dlamini NG (2019).
Jorand F, Boue-Bigne F, Block JC, Urbain V Removal of pollutants in mine wastewater by
(1998). Hydrophobicity/hydrophilicity a non-cytotoxic polymeric bioflocculant from
properties of activated sludge exopolymeric Alcaligenes faecalis HCB2. Int. J. Environ.
substances. Water. Sci. Technol. 37: 307- Res. Pub. Health. 16(20): 4001.
315. Maliehe TS, Selepe NT, Ntombela GZ, Simonis
Kurane R, Hatamochi K, Kakuno T, Kiyohara M, JJ, Basson AK, Ngema S, Xaba PS, Mpanza
Hirano M, Taniguchi Y (1994). Production of F (2016). Production and characteristics of
a bioflocculant by Rhodococcus erythropolis bioflocculant TPT-1 from a consortium of
S-1 grown on alcohols. Biosci. Biotechnol. Bacillus pumilus JX860616 and Alcaligenes
Biochem. 58(2): 428-429. faecalis HCB2. Afric. J. Microbiol. Res.
Lee SR, John F, Mei C. Chong B (2014. A review 10(37): 1561-1575.
on application of flocculants in wastewater More TT, Yadav JSS, Yan S, Tyagi RD,
treatment. Proc. Safety Environ. Protect. 92: Surampalli RY (2014). Extracellular
489-508. polymeric substances of bacteria and their
Li XM, Yang Q, Huang K, Zheng GM, Xiao DX, potential environmental applications. J.
Liu JJ, Long WF (2007). Screening and Environ. Manage. 144: 1-25.
characterization of a bioflocculant produced Mosmann T (1983). Rapid colorimetric assay for
by Aeromonas sp. Biomed. Environ. Sci. 20: cellular growth and survival: Application to
274-278. proliteration and cytotoxicity assays. J.
Lian B, Chen Y, Zhao J, Teng HH, Zhu LJ, Yuan Immunol. Meth. 65: 55-63.
S (2008). Microbial flocculation by Bacillus Nie M, Yin X, Jia T, Liu S, Shen Q, Lia P, Wang Z
mucilaginosus: applications and (2011). Production of a novel bioflocculant
mechanisms. Bioresour. Technol. 99: 4825– MNXY1 by Klebsiella pneumonia strain NY1
4831. and application in precipitation of
Lin J, Harichund C (2012). Production and cyanobacteria and municipal wastewater
characterization of heavy metal removing treatment. Applied microbiol.111: 547-558.
bacterial bioflocculants. Afr. J. Biotech. Nouha K, Kumar RS, Balasubramanian S, Tyagi
11(40): 9619-9629. RD (2017). Critical review of EPS production,
Bioscience Research, 2020 volume 18(1): 188-207 204You can also read
