Process Biochemistry 111 (2021) 181–190

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Dyestuffs from textile industry wastewaters: Trends and gaps in the use
of bioflocculants
Wagner Artifon *, Karina Cesca, Cristiano José de Andrade, Antônio Augusto Ulson de Souza,
Débora de Oliveira
Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina (UFSC), Florianópolis, Santa Catarina, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Regarding environmental impact, the textile industry is one of the most relevant sectors. Worldwide, it is esti­
Textile industry mated that 700,000 tons of dyes are generated annually, and 12 % of them are inappropriately disposal into
Bioflocculant aquatic resources. In this sense, flocculating systems can be a cost-effective treatment of dye-containing
Dyes
wastewater. One route that has attracted attention to this researching field is the flocculating effect of biolog­
Wastewater treatment
ical components. Known as bioflocculants, these extracellular molecules, mainly polysaccharides and proteins,
are produced by microorganisms and present relevant effects on precipitating suspended particles or diluted
substances from solutions. However, the flocculating phenomena related to bioflocculants and dyes are not fully
comprehended. The complex structure of extracellular substances and the variance in dye molecules make their
interactions hard to predict. This review aims to critically discuss the current state of the art and future trends
related to bioflocculants and dye-containing wastewaters from the textile industry. It includes biological systems
of bioflocculant-producing strains, chemical properties of bioflocculants and dyes, and physical parameters
employed on flocculation processes. This review would lead to a better understanding of challenges and cor­
responding strategies to open up new ways of bioflocculation in wastewater treatment.

1. Introduction
The increasing challenges related to water availability have
restricted environmental legislation, including industrial wastewater
disposal. Thus, it is essential to improve and develop cost-effective
wastewater treatments and encourage the reuse of water [1]. In this
sense, the textile industry is one of the most water consumers and pol­
luters. In addition, there are perspectives for textile industry expansion
in developing countries [2] and dye-containing wastewaters from the
textile industry are composed of recalcitrant molecules [3]. Hence, the
inappropriate disposal of textile wastewaters into aquatic bodies in­
terferes with the entire aquatic community since there are toxicological
effects that hamper light penetration. Consequently, the photosynthetic
aquatic community is primarily affected [4].
It is worth noting that inorganic and synthetic flocculating agents are
widely applied to dye-containing wastewaters. However, they are
related to environmental and human health concerns, for instance,
acrylamide has neurotoxic and carcinogenic properties [5], aluminum
salts may induce Alzheimer’s disease [6], among others. In this sense,
bioflocculation is a promising alternative. Flocculating agents can be
classified into three groups: (I) inorganic, such as aluminum sulfate and
poly-aluminum chlorides; (II) synthetic, such as polyacrylamide and
polyethyleneimine; and (III) natural or bioflocculants, such as chitosan,
alginate, and extracellular polymeric substances (EPS) [7,8]. On the
other hand, bioflocculants produced by microorganisms are considered
an eco-friendly alternative [9]. The dye-bioflocculant interactions are
induced by microbial flocculants’ chemical structure, which makes the
suspended material settle down by adsorption, bridging, and charge
neutralization [10,11].
Several review papers have reported the bio-aggregation dynamics,
bioflocculant producers, and bioflocculation modeling. The information
is segregated themselves, that is, they do not provide a correlation
among them. In this sense, this review aims to explore the current state
of the art of bioflocculation systems for dye-containing wastewaters,
including the available data for chemical interactions, bioflocculant
strains, and flocculation system parameters in order to deeply discuss

* Corresponding author at: EQA/CTC/ Universidade Federal de Santa Catarina (UFSC), Campus Trindade, Cx. Postal 476, 88010-970, Florianópolis, Santa Cat­
arina, Brazil.
E-mail address: wagner.artifon@posgrad.ufsc.br (W. Artifon).

https://doi.org/10.1016/j.procbio.2021.10.030
Received 1 July 2021; Received in revised form 22 September 2021; Accepted 23 October 2021
Available online 29 October 2021
1359-5113/© 2021 Elsevier Ltd. All rights reserved.
W. Artifon et al. Process Biochemistry 111 (2021) 181–190

this promising research and technological field.
2. Dye-containing wastewaters from the textile industry
Dyes are compounds responsible for giving color to a material [12].
The chemical structure of dyes is composed of two main parts, (I) the
auxochrome moiety, which is related to their water solubility and
binding the textile fibers, and (II) the chromophore group that confers
the color to them [13]. Dyes have a wide range of chemical structures
[14]. Nevertheless, the chemical characteristic that corresponds to each
color is unique [15]. In this sense, Table 1 presents the classification of
textile dyes according to their chemical structures and the color index
system [16]. Most dyes present chemical functional groups such as
carboxylic, amine, azo groups, sometimes conjugated with aromatic
structures [17]. The increase of aromatic nucleus in the assemblage of
the dye matrix augments double bonds and molecule complexity [18].
Due to these complex structures, synthetic dyes usually present very low
degradability-recalcitrance [19].
Benkhaya et al. [20] classified the textile dyes based on their appli­
cation and fiber ionic attraction, as acid, azoic, basic, direct, disperse,
reactive, sulfur, and others. The Color Index list comprises> 8,000
synthetic dyes and ≈ 40,000 trade names that indicate the class, color,
and order of dyes [21]. According to Subramani and Thinakaran [2], ≈
700,000 tons of dyes are produced annually, in which the textile in­
dustry consumes ≈ 466 tons of dyes/year, that is, two-thirds out of total.
Table 1
Chemical structure of textile dye groups.
Group of Dye Chemical Structure Reference
Azo -N¼N- [91]
Phthalocyanines [92]
Furthermore, ≈ 12.5 % out of total (87.500 tones) are disposal inade­
quately into the environment [2,22].
The textile industry is a massive water-consuming sector [2,23].
Furthermore, the wastewater generated in this industrial sector is
considered a relevant source of pollution due to characteristics such as
oxidative substances, persistent color, low biodegradability, and alka­
linity [24]. These liquids are a complex mixture of substances such as
acids (e.g., polycyclic acids), alkalis (e.g., NaOH), auxiliaries (e.g., en­
zymes), surfactants (e.g., alcohol sulfates), heavy metals (e.g., copper),
dyes and pigments based on organic chlorine [25]. In addition, the
liquid discharged is a descendent runoff from a series of chemical baths,
which produces high volumes [26].
The textile dyes show high molecular stability to chemical oxidation
and light degradation (recalcitrant molecules). In addition, they may
present biomagnification, toxicity, and even carcinogenic properties
which affect humans health [27–29]. Hence, textile wastewaters are a
concern that should be carefully treated to mitigate their environmental
impact.
The conventional wastewater treatment for the textile industry in­
cludes flocculation/coagulation operations to induce the precipitation
of suspended and soluble substances. An inevitable byproduct inherent
to textile industry plants is the textile dyeing sludge [30]. According to
Huang et al. [31], ≈ 25 m3 of activated sludge are generated per million
tons of textile wastewater. In 2016, the China Environment Statistical
Yearbook revealed that > 4.5 million tons of textile dyeing sludge were
produced (80 % moisture content) [32]. The activated sludge is
composed of toxic organic matter and heavy metals, among others [33].
Some procedures have been considered to minimize the deleterious
environmental effects of textile dyeing sludge, such as incineration [31],
oxidation [32], gasification [34], pyrolysis/carbonization [35], use of
landfills, dewatering [36]. Although the textile dyeing sludge treatment
from conventional wastewater systems is reported in the literature, the
activated sludge from bioflocculation of dyes is a non-covered area.
After bio-aggregation, the bioflocculant and dye complex deserves
attention and appropriate disposal.

3. Bioflocculants

Xanthene [93] Bioflocculants are specific microbial extracellular molecules. Their

biosynthesis is related to the cellular attachment on a surface that assists
biofilm formation, so, the components are environmentally resistant
Nitro [93]
with high adsorption properties [37]. The produced bioflocculants can
be used in a wide range of wastewater treatments [38–40], such as
decolorization [41], metal removal [42], sludge dewatering [43],
Quinoline [94] microalgae harvesting [44], and among others.

3.1. Chemical composition

Indigo [95]
The chemical composition of extracellular polymeric substances
varies according to microbial strain and growth conditions, among
others. Nevertheless, the composition of bioflocculants is roughly con­
Acridine [93] stant (mainly carbohydrates and minority protein, nucleic, lipids, and
non-secreted fraction represented by humic substances) [45,46].
The polysaccharides produced by microorganisms are classified as
homo and heteropolysaccharides [47,48]. Homopolysaccharides, such
as cellulose, dextran, and curdlan, are typically neutrally charged with
Azine ¼N-N¼ [96]
Anthraquinone [97]
different branching degrees and can be classified according to their most
present linkage: α-D-glucans, β-D-glucans, and fructans [49]. Hetero­
polysaccharides, such as alginate, xanthan, and gellan gum, present
distinct physical properties according to their monosaccharides and
branching [50]. The uronic acid content and their derivates, such as
Triarylmethane [93] acetate ester, pyruvate ketals, succinates, phosphates, and sulfates,
define the molecular charge of bioflocculant [50]. The bioflocculant
polysaccharide content and properties depend on several factors related
to microbial growth [37]. The extracellular products released during the
microbial consortium growth also present a wide range of compositions

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W. Artifon et al.
[51]. Different carbon and nutrient sources [52] and extraction methods
[53] also interfere with flocculant efficiency.
Proteins and enzymes are relevant components of flocculation sys­
tems. Non-enzymatic proteins such as lectins, responsible for producing
carbohydrates matrix network [54], and glycoproteins, accountable for
form-giving and shape-maintaining function of the bacteria [55], are,
usually, present in the bioflocculant matrix. Liu et al. [56] reported a
consistent amount of proteins when strains were fed with different
carbon sources. Nevertheless, the EPS presented differences in protein
content when varying nitrogen sources and availability [57].
The bioflocculant matrix also includes minor components such as
nucleic acid and humic substances. The former is an intracellular sub­
stance released after cell lysis and found in large amounts in wastewa­
ters after microbial growth [54]. Whereas the humic substances,
generated by the hydrolysis of organic substances, are associated with
several substances such as amino acids, pectin, lignin, and carbohy­
drates. Distinct groups represent the humic substances such as the fulvic
and humic acids that correspond to the soluble fragments and the
insoluble fraction named humin [58].
Several functional groups are inherent to bioflocculant configura­
tion. Hydroxyl and aliphatic structures, typical from carbohydrates, and
carboxyl and amide groups, classic from proteins, integrate the main
functional groups responsible for floc formation. This chemical
composition of bioflocculants and their molecular functional groups is
strictly related to microbial strain, carbon source, nitrogen source,
cultivation parameters, and extraction techniques. These properties are
essential to determine their effects on flocculation aptitude and appli­
cation suitability [37].
3.2. Flocculation mechanisms
The mechanisms related to the chemical-flocculating agents are well-
known. However, the bioflocculating agents have not been fully
explored. The bioflocculating agents act, very likely, by bridging and
charge neutralization mechanisms [7,59]. Fig. 1 illustrates the contri­
bution of both phenomena on the formation of a floc between dyes and
bioflocculants.
The presence of biopolymers in the flocculation systems creates
threads or fibers. The bridging phenomenon occurs when the bio­
flocculant acts as a neutralizing and stabilizing agent to the charged
particles suspended in the medium by forming bridges in between [7].
This process could initiate due to chemical reaction, van der Waals force,
static or simple hydrogen bonds. Even neutral bioflocculant could link
particles by extending itself into the solution from a distance greater
than the interparticle repulsion edge. Bioflocculants with high molecu­
lar weight may promote larger flocs by binding particles during floc­
culation reaction, which induces an increase in floc density, size, and
resistance to shear [60]. The functional groups on the surface of the
Fig. 1. Flocculation mechanisms scheme of charged dyes promoted by bioflocculants.
183
Process Biochemistry 111 (2021) 181–190
bioflocculant as well as their configuration induced by pH are key fac­
tors for the bridging effect of attractive adsorption sites [61].
The presence of negatively charged particles provides electrostatic
repulsion forces in a solution, which are stronger than the van der Waals
forces and prevent settling and floc establishment. In the charge
neutralization process, the surface charge density of the particles is
reduced by the adsorption sites of the bioflocculant [7]. The repulsive
electrostatic interactions among particles give place to attractive forces,
which destabilize particles and start the flocculation. It has been found
that low molecular biomolecules are quite effective for charge neutral­
ization [62]. Several studies reported the enhancement of bio­
flocculation efficacy by adding metallic cations such as K+, Mg2+, Ca2+.
These ions can assist the neutralization of negative charges (functional
groups) and contribute to bridge formation [46,63]. The flocculation
process starts with the formation of small flocs; the charge of the par­
ticles decreases negativity augmenting attraction forces and making the
flocs larger by the time contact with the suspension [64].
4. Bioflocculant-based treatment for dye-containing textile
wastewaters
The flocculating effect of microbial substances was reported for the
first time in the 18th century [65]. Since then, bioflocculants have been
applied in several fields for coagulation systems [38]. In order to iden­
tify the most recent trend on bioflocculant-based treatment for
dye-containing textile wastewaters, research on the Scopus database
was carried out considering the terms (I) "Bioflocculant" and (II) "Bio­
flocculant" AND "Dye" that are presented in the title, abstract, and
keywords of published papers and patents. The analysis of data revealed
an increasing trend in the last 20 years. Fig. 2 shows publications related
to the terms I (entire bar) and II (red part) from 2001 until 2020. During
this period, 649 articles were found for "Bioflocculant" and 50 for
"Bioflocculant" AND "Dye", which represent an average mean of ≈ 8%.
Furthermore, the number of patents during the same period was 57
and 12 for I and II, respectively. Regarding the countries, China was the
first one with 359 published articles, followed by India with 63. Addi­
tionally, the National Natural Science Foundation in China is reported to
be the most prominent funding sponsor.
Bioflocculants from several microorganism strains have been used on
textile wastewaters. It presents coagulation effects on a variety of dyes.
In this sense, Tables 2 and 3 summarize essential data for bioflocculant-
producing strains and specific experimental conditions applied on the
removal of commercial dyes from textile wastewater, respectively.
4.1. Bioflocculant-producing strains
New strategies for color removal by applying bioflocculants pro­
duced by low-cost and widely available microorganisms have been
W. Artifon et al. Process Biochemistry 111 (2021) 181–190

Table 2
Bioflocculant producer strains and their chemical composition.
Microorganism Strain Bioflocculant Reference
Origin Chemical
Composition

Laboratory strain Aspergillus parasiticus PS 76 %; PR 22 % [76]

Laboratory strain Staphylococcus sp. – [72]
and Pseudomonas
Soil Klebsiella mobilis PS 100 % [83]
Conglutination mud Rothia sp. PS 100 % [9]
Cassava-processing – PS 42 %; PR 27 % [81]
wastewater sludge
Conglutination mud ZHT3-9 and ZHT4- – [82]
13
Bacillus subtilis
Exiguobacterium
acetylicum
Klebsiella terrigena
Staphylococcus
Activated sludge – [63]
aureus
Fig. 2. Trends in bioflocculant and dye researching field publications in the last Pseudomonas
pseudoalcaligenes
20 years.
Pseudomonas
plecoglossicida
studied [66,67]. The properties of produced bioflocculants depend Soil Paenibacillus elgii PS 100 % [52]
dramatically on the parameters employed on the microorganism culti­ Acinetobacter
Laboratory strain PS 14 %; PR 3 % [90]
baumannii
vation. Fig. 3 correlates the bioflocculant-producing strains to the
Rhodococcus PS 91 %; PR 8 %; 1
number of times that they were cited as bioflocculant-producing for dye Laboratory strain [77]
erythropolis % DNA
removal. Bacillus sp. and Pseudomonas sp. are the genus most reported on Bacillus licheniformis PS 167 ug/mL; PR
these and many other applications [66]. Laboratory strain
(Bl) 48 ug/mL
[51]
PS 90 ug/mL; PR 30
Isolated Bacillus sp. from soil was employed to produce an efficient Bacillus firmus (Bf)
ug/mL
bioflocculant for wastewater decolorization containing indigo dye [68]. Activated sludge Haloplanus vescus PS 79 %; PR 21 % [71]
The reduction in dye color, chemical oxygen demand (COD), total sus­ Bacillus velezensis
pended solids (TSS), and chloride ions were 83, 92, 74, and 82 %. (Bv) detailed amino acids
Regarding the composition of bioflocculating agents from the same Egyptian Ecosystem Bacillus mojavensis and sugar [47]
(Bm) composition
microbial genus, Karthiga Devi and Natarajan [51] cultivated Bacillus
Pseudomonas (Ps)
licheniformis and Bacillus firmus. They compared them according to their Seawater Alteromonas sp. PS 70 %; PR 22 % [75]
capacity for producing bioflocculant agents. B. licheniformis produced 80 Hydrocarbon
PS 72 %; PR 2.8 %;
% more bioflocculant, which resulted in a better dye removal efficacy, contaminated Kocuria rosea
UA 16 %
[46]
sediments
and 77 % of it corresponds to polysaccharides. whereas B. firmus pro­
Pseudomonas
duced fewer flocculant agents 75 % of them were polysaccharides. It is Papermill sludge
boreopolis
– [89]
also possible to highlight that dye decolorization with bioflocculant Laboratory strain Periphytic biofilms PS 53 %; PR 20 % [70]
from the same genus strains, at the same experimental conditions, is Soil Bacillus sp. PS 97 %; PR 1.4 % [68]
alike. Elkady et al. [47] also compared two Bacillus strains and obtained PS 67 %; PR 0.54 %;
Laboratory strain Serratia sp. [78]
LI 10.5 %
a very close decolorization rate for basic yellow dye.
Ruditapes
Similarly, Pseudomonas montelli and Pseudomonas putida were philippinarum Vibrio and Bacillus PS ~100 % [87]
compared among four different microorganisms by Saha et al. [69], and conglutination mud
significant differences between polysaccharide content, specific growth Ramie degumming
Alcaligenes faecalis PS 75 %; PR 21 % [74]
wastewater
rate, and bioflocculant yield were found. The decolorization results also
Pseudomonas PR 84 %; PS 3.6 %;
presented variances. P. montelli presented a higher dye removal for the monteilii (Pm) LI 0.8 %
anionic dye congo red, and P. putida was more efficient in removing the Paenibacillus PR 85.5 %; PS 8.2 %;
cationic dye rhodamine-B. These results are attributed to the differences Water treatment xylanilyticus (Px) LI 1.6 %
[69]
in chemical composition and functional groups inherent to each strain. plant PR 83.5 %; PS 4.3 %;
Bacillus pumilus (Bp)
LI 1.25 %
The bioflocculant-producing strains seem to be a cost-effective
Pseudomonas putida PR 82 %; PS 7.4 %;
strategy for textile wastewater treatment, particularly Bacillus sp. and (Pp) LI 1.5 %
Pseudomonas sp. Wastewaters in general present several microorganisms
PS (Polysaccharide); PR (Protein); UA (Uronic Acid); LI (Lipid).
that are responsible for the precipitation of substances. The screening of
new bioflocculant-producing strains, their specific components, and the
optimization of their growth conditions represent a promising research physical properties and functional groups exposed on both components,
area. is still a non-fully comprehended phenomenon.
Considering the chemical composition of bioflocculant presented in
Table 2, it is possible to conclude that polysaccharides generally
4.2. Bioflocculant chemical composition and flocculation mechanism
constitute the most fraction of bioflocculants. The characterization of
the bioflocculant produced by Paenibacillus elgii indicated glucose, glu­
Polysaccharides and proteins present long molecular chains with
curonic acid, xylose, mannose, and homogeneous carbohydrates [52].
many active radicals known as the main components of bioflocculants.
On the other hand, EPS extracted from periphytic biofilms presented
However, these biomolecules comprehend a wide range of components
most heteropolysaccharides [70], which are cited to increase floccula­
that, along with their intermediates from extraction and purification
tion yield [71]. The detailed fractions of amino acids and reducing sugar
steps, demand in-depth research to detail their chemical composition
compounds that constitute the bioflocculant agents are reported in the
entirely. The interaction between bioflocculants and dyes, which covers

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W. Artifon et al. Process Biochemistry 111 (2021) 181–190

Table 3
Dye removal efficacy of bioflocculants and experimental conditions.
Dye/Charge nature pH Temperature (◦ C) Bioflocculant dosage Cation added Dye removal Reference

Direct blue 1/Anionic 6 58 %

Reactive orange 16/Anionic 6 68 %
Reactive blue 4/Anionic 6 92 %
Basic blue B/Cationic 6 Ambient 50 mL broth/L – 2% [76]
Acid yellow 25/Anionic 6 93 %
Acid orange 8/Anionic 6 62 %
Acid Blue 45/Anionic 6 43 %
Indigo/Neutral 9 Ambient 17 mL broth/L Ca2+ 86 % [72]
Disperse yellow/Non-ionic 85 %
Disperse violet/Non-ionic 91 %
7 Ambient 4 mL broth/L Ca2+ [83]
Reactive light-yellow/Anionic 60 %
Reactive turquoise blue/Anionic 65 %
Methylene blue/Cationic 86 %
Crystal violet/Cationic Natural Ambient 200 mg/L – 98 % [9]
Malaquite green/Cationic 99 %
Methylene blue/Cationic 83 %
7 Ambient 5 mg/L Ca2+ [81]
Fast blue/Anionic 78 %
Methylene blue/Cationic 3− 11 10− 50 200 mg/L 96 %
Crystal violet/Cationic 3− 11 10− 50 200 mg/L 96 %
Ca2+ [82]
Malaquite green/Cationic 3− 11 10− 50 600 mg/L 95 %
Ink Blue/Anionic 9− 11 10− 20 400 mg/L 82 %
Whale dye/Anionic 7 97 %
Mediblue dye/Anionic 7 81 %
35− 45 10 mg/L Ca2+ Mn2+ Mg2+ CTAB [63]
Fawn/Anionic 10 95 %
Mixed dyes 10 82 %
Methylene blue/Cationic 65 %
Red X-GRL/Cationic Natural Ambient 15 mg/L – 72 % [52]
Anionic and Neutral <50 %
Congo red/Anionic 7 Ambient 0.009 mg/gDS Ca2+ 79 % [90]
Reactive brilliant red/Anionic 23 %
2+
Direct sky blue/Anionic 7 Ambient 30− 65 mg/L Cu 87 % [77]
Dispersive yellow/Non-ionic 74 %
Orange G/Anionic Bl; Bf*
Methylene blue/Cationic 67 %; 58 %
Crystal violet/Cationic Natural Ambient 800 mg/L – 89 %; 90 % [51]
90 %; 84 %
Malachite green/Cationic
83 %; 72 %
Acid brilliant scarlet GR/Anionic 2 Ambient 150 mg/L Ca2+ 95 % [71]
Bv; Bm; Ps*
Basic yellow/Cationic 7 Ambient – – [47]
91 %; 89 %; 88 %
Congo Red/Anionic 11 100 mg/L 98 %
Direct Black/Anionic 11 Ambient 200 mg/L – 98 % [75]
Methylene Blue/Cationic 11 180 mg/L 72 %
Reactive blue/Anionic 76 %
Acid yellow/Anionic 73 %
Natural Ambient 15 mg/L Ca2+ [46]
Basic red/Cationic 23 %
Basic blue/Cationic 11 %
CBBR-250/Anionic Natural Ambient 300 mg/L Ca2+ 89 % [89]
Aniline blue/Anionic – Ambient 1.2 mg/L Ca2+ 56 % [70]
Indigo/Non-ionic 7.8 Ambient 2% (w/v) – 83 % [68]
Trypan blue/Anionic 40 %
Acridine orange/Cationic 80 %
Methyl orange/Anionic 25 %
7 Ambient – Ca2+ [78]
Bromothymol blue/Anionic 75 %
Aniline blue/Anionic 60 %
Crystal violet/Cationic 95 %
Methylene blue/Cationic 26000 mg/L 99 %
Crystal violet/Cationic 7.5 Ambient 10000 mg/L – 89 % [87]
Malaquite green/Cationic 14000 mg/L 99 %
Dispersive Blue/Non-ionic 8 Ambient 1000 mg/L – 86 % [74]
Congo red/Anionic 6− 8 25− 40 10− 14 mg/L Na+ k+ Ca2+ Mn2+ Mg2+ Al3+ Fe3+ Pm; Px; Bp; Pp*
79%; 96%; 90%; 69% [69]
Rhodamine-B/Cationic CTAB
54%; 98%; 77%; 88%
*
Refers to the producer strain (see Table 2).

literature. Their contribution to the stabilization and precipitation of
dye molecules is not deeply reported/investigated.
The hydroxyl groups, classical for carbohydrates, are responsible for
inducing the bridging effect on dyes [68]. It is indicated that the floc
formation between dye and bioflocculant occurs by hydrophilic and
hydrophobic interactions; amine and hydroxyl groups interact with
negative charge surfaces and may be responsible for hydrogen-bridging.
Macromolecules as proteins and carbohydrates with long molecular
chains and many active radicles give the bioflocculant the ability to
neutralize negative charges and absorb colloids. It leads to the formation
of heavy three-dimensional flocs that later suffer settlement [72].
Elkady et al. [47] screened three bioflocculant-producing strains to
compare decolorizing synthetic and real wastewater with basic yellow, a
cationic dye. The much lower flocculating activity was reported for real
wastewater when compared to synthetic dye solution due to the higher
hydrogen ions concentration in the real wastewater and its competition

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W. Artifon et al.
Fig. 3. Bioflocculant strain producers applied for dye removal.
with cationic ions of dye. Low dye concentrations exhibited rapid
sorption, and equilibrium was attained quickly, indicating a monolayer
coverage formation by the dye molecules onto the surface of the bio­
flocculant. The kinetic study carried out presented a correlation with the
Langmuir model and predicted a single-stage equilibrium operation in
the batch biosorption process. The negative value of free energy
confirmed the affinity between bioflocculant and dye, and it indicated
the dye sorption is a spontaneous reaction.
The presence of uronic acid content on bioflocculant provides hy­
droxyl groups to the molecular chain and is directly related to adsorp­
tion and flocculating phenomena [73]. Chen et al. [74] cultivated the
strain Alcaligenes faecalis in ramie degumming wastewater medium for
bioflocculant releasing. The liberation of uronic acid from cells and a
high level of proteins favored the number of active sites for selective
adsorption due to the amino and carboxyl groups. They also presented a
dye removal efficacy of 86 % dispersive blue solution. The authors re­
ported that under acidic pH, the carboxylate and amine groups might
accept hydrogen ions. It results in an electrostatic repulsion force be­
tween bioflocculant and dye, conducting to lower flocculation activity.
Another study reported that Alteromonas sp., isolated from seawater,
was used to produce a bioflocculant for decolorizing congo red, meth­
ylene blue, and direct blue wastewaters; the results were 98, 98, and 72
%, respectively [75]. The dye removal results suggest that the bio­
flocculant showed a better affinity with anionic dyes, probably due to
the availability and strength of positive charges in the solution.
The cultivation of the strain Kocuria rosea was optimized to produce
extracellular polymeric compounds for flocculation [46]. The flocculant
was employed on reactive blue, acid yellow, basic red, and basic blue,
and the decolorization reached 76, 73, 23, and 11 %, respectively. The
results indicated a higher affinity between bioflocculant and anionic
dyes, mainly with reactive blue, attributed to the anthraquinone struc­
ture that contains hydroxyl groups responsible for binding the dye to
EPS molecules. The size of dye molecules and its inherent number of
sulfonic groups act as a barrier inhibiting other molecules’ adsorption
and may be another reason for this attraction [76].
The chemical functional groups present on the surface of bio­
flocculant play an essential role in bridging and adsorption processes
[75]. Further analysis conducted by several authors revealed the most
common chemical functional groups schematically represented in Fig. 4.
The most groups reported are hydroxyl [9,68,77], carboxyl [46,47,70],
methoxyl [47,51,69], and amine groups [47,71,76] all related to floc­
culation activity [46]. Other chemical groups are acetyl and amide [46],
methyl [78], and carbonyl [70].
The produced compounds reported by Karthiga Devi and Natarajan
[51] presented hydroxyl, carboxyl, and methoxyl functional groups; the
authors described that the second could stretch out by electrostatic
186
Process Biochemistry 111 (2021) 181–190
Fig. 4. Schematic representation of bioflocculants and their most reported
chemical functional groups.
repulsion and it may outcome in more effective sites for adsorption. On
the opposite side, the EPS compounds produced by both strains pre­
sented strong affinity with methylene blue, crystal violet, and malachite
green (cationic dyes), nevertheless lower attraction for orange G
(anionic dye). Buthelezi et al. [63] cited that acid dyes are the most
difficult to remove due to their inert chemical structure. Li et al. [52]
also reported a bioflocculant obtained from Paenibacillus elgii that pre­
sented better decolorization ability for cationic dyes and indicated two
mechanisms for adsorption. The first is related to the electrostatic
attraction of two oppositely charged ions due to the large number of
adsorption sites the long molecules of EPS offers [79]. The second is
based on the high amount of mannose present in the EPS, which may
promote van der Waals interactions and hydrogen bonding [80]. Table 3
presents more outcomes from studies that corroborate this higher af­
finity between bioflocculants and cationic dyes [78,81,82]. The possible
explanation may lie in the relation between composition and functional
groups of both dye and flocculant agents [9,63].
Similarly, Klebsiella mobilis was cultivated in diluted wastewater for
bioflocculant extraction. The bioflocculant produced was more active on
removing dispersive dyes, yellow 85 %, and violet 91 %, than reactive
ones, light-yellow 60 %, and turquoise blue 65 %. The higher molecular
volumes and hydrophobicity inherent to reactive dyes are pointed out to
justify the outcomes [83].
Several studies describe the decolorization yields due to the inter­
action between chemical groups present in both dye and flocculant
agent structures. However, the identification of chemical groups from
dyes and extracellular substances is often superficial, which makes the
comparison among data not conclusive. Further evaluation of dye-
bioflocculant pairs should be conducted to infer their chemical in­
teractions and make it possible to correlate groups of dyes with bio­
flocculant components.
4.3. Effect of temperature on bioflocculant
Regarding the textile industry that discharges dyeing-bath at high
temperatures, wastewater treatment may be conducted at high tem­
peratures. Thus, thermally stable bioflocculants are desirable during the
flocculation step. Although most bioflocculant wastewater treatments
studies are carried out at room temperature, a few reports correlated the
bioflocculation with temperature variation on dye solutions [69,82].
Wei et al. [82] applied the bioflocculant obtained from two isolated
strains from R. philippinarum conglutination mud on Methylene blue,
Crystal violet, Malaquite green, and Ink blue dyes. The temperature
range (10− 50 ◦ C) interfered critically with the decolorization of Ink blue
solution. Nevertheless, no significant decolorization-temperature effect
was observed for Methylene blue, Crystal violet, and Malaquite green.
W. Artifon et al.
The decolorization of Ink blue dye decreased according to an increase in
temperature. It indicates that proteins are the main responsible for the
flocculation of this dye due to its low thermal stability at high
temperatures.
Similarly, Saha et al. [69] evaluated the temperature range 25− 45 ◦ C
and the highest bioflocculation activity on dyes occurred at 40 ◦ C. This
increase in decolorization directly correlated to higher temperature in­
dicates an endothermic interaction between agents. Buthelezi et al. [63]
found the best temperature for removal of whale, mediblue, and fawn
dyes around 40 ◦ C. The analysis of these results indicates that the
optimal operation condition is near room temperatures, independently
of bioflocculant sources. It is worth noting that it is desirable to operate
at mild temperatures due to lower environmental impact and energy
demand.
4.4. Effect of pH on bioflocculant
The pH of a solution is a relevant factor due to interference in the
adsorptive process by dissociation of functional groups on active sites
[47]. For instance, the increase in pH decreases the electrostatic
attraction, resulting in negative properties for bioflocculant molecules
due to the complete deprotonation of functional groups. It may generate
electrostatic repulsion and inhibit the approximation between flocculant
and dye [76]. However, the color removal by bioflocculants is often
evaluated at not regulated pHs - usually neutral or slightly alkaline
(Table 3) [63]. Elkady et al. [47] described that the flocculant efficacy
increased linearly from pH 1 up to 7, and tended to deplete as it becomes
more alkaline. It is inferred that at pH 7 bioflocculants molecules
became negatively charged due to the ionization of hydroxyl and
carboxyl groups. It enhances the sorption of the positive dye cations by
electrostatic forces of attraction [84].
Saha et al. [69] studied the pH range 6–10 and the best pH for congo
red and rhodamine removal was 8 and 7, respectively. The surface
charge, structural features, and bridging ability are all related to the
solution pH [85]. The positively charged sites at the surface of the
bioflocculant are present at lower pH, and it probably favors the anionic
dye removal due to its negative charge. The repulsion between
Rhodamine-B monomeric cationic form and positively charged bio­
flocculant surface may interfere in decolorization [86]. On the other
hand, Zhong et al. [71] evaluated the pH range 2− 10. They reported the
decolorization of wastewater containing acid brilliant scarlet GR
increased with the decreasing pH to nearly 2, achieving a maximum
removal of 82 and 95 % on COD and color removal. The authors sug­
gested that dye can present variant speciation forms at different pH
levels. This parameter can change the redox potential of dye and the
bioflocculant, making the dye removal intensely dependent on pH.
The pH can significantly affect the surfaces of bioflocculants and
dyes, that is, on the bioflocculation systems. The optimal dye removal at
neutral or slightly basic pH is aligned to the eco-friendly profile of
bioflocculants.
4.5. Effect of bioflocculant concentration
There is a wide range of bioflocculant dosage often used (Table 2) in
terms of concentration (mg/L) or volume of growth broth (mL broth/L)
from the cultivation medium. Karthiga Devi and Natarajan [51] re­
ported that dye removal activity decreased due to excess flocculant
agent dosage. Very likely, the saturation of polysaccharides on the sur­
face of particles hamper the interaction with dye. On the other hand, Mu
et al. [87] described the decolorization (>99 %) of methylene blue and
malachite green when a high concentration of bioflocculants (more than
10 g/L) was applied.
The synergistic effects between bioflocculants and commercial floc­
culants are already reported. In this sense, Huang et al. [41] and Huang
et al. [88] obtained an increment of 7% on decolorization of dispersive
yellow dye by applying bioflocculant simultaneously with
187
Process Biochemistry 111 (2021) 181–190
polyaluminum chloride and aluminum sulfate. The adsorption and
bridging effect due to bioflocculant can positively result in flocs size
aluminum-based coagulant systems. The authors highlighted that the
removal mechanism is not dominated by charge neutralization since
there are effects of bioflocculant increments on zeta potential. The
sweep of colloids inside coagulant precipitates and adsorption may
control the flocculation process. Floc breakage factors were also evalu­
ated and suffered augment by insertion of bioflocculant, which indicates
that the dispersive yellow flocs are more robust against shear stress.
Bioflocculant dosage is intimately related to the decolorization of
dye-containing wastewaters, that is, the biopolymer efficiency in pro­
moting complexation with dyes. In this sense, production and purifica­
tion steps must be optimized to generate high-efficient bioflocculant
compounds.
4.6. Effect of cations on bioflocculant
Low concentrations of cations as K+, Mn2+, Mg2+, Ca2+, Fe3+ were
applied with bioflocculant for dye removal-synergistic effects [52,68,69,
81]. Buthelezi et al. [63] reported that flocculation results were strongly
impacted by pH, temperature, bioflocculant concentration, and divalent
cations (Mn2+, Mg2+, Ca2+), which resulted in the enhancement of dye
removal. The presence of metal cation was essential, for it caused
sweeping and bridging of the dye [46]. Metal cations are conductive and
induce an electrostatic repulsion between dyes and flocculants due to
neutralization and stabilization of negative charges of their functional
groups [63]. Similar results related to cation addition on enhancing dye
precipitation are reported [70,89,90] and the most efficient and
commonly used one is the divalent Ca2+.
The simultaneous application of cations with bioflocculants for
decolorization treatment systems is an interesting alternative since it
reduces the bioflocculant requirements and leads to more economically
feasible processes.
5. Prospection on bioflocculant-based treatment for dye-
containing textile wastewaters
Biopolymers have been identified as an emerging solution to pro­
mote the flocculation of substances in several wastewater systems.
However, some researching areas should be further explored to glean
insights into the bioflocculation field. Most studies related to bio­
flocculant and dyes report laboratory-scale experiments with synthetic
dye solutions. The lack of pilot-scale implementation with real textile
wastewater limits the evaluation of this promising technique.
Moreover, the usage of laboratory culture media as carbon and ni­
trogen sources, necessary for microorganism growth and bioflocculant
releasing, is a drawback of the process due to the associated high cost.
Industrial and agriculture residues or wastewaters may be considered as
alternative culture media. It reduces the demand for treatment and
serves as low-cost carbon and nutrient source for microbial growth. It is
also important to highlight the wide quantity of biomass, such as algae
or brewing and food industry subproducts, that may present potential as
flocculant agents by themselves after conversion. Bioflocculant pro­
duction might valorize these subproducts, that is, a promising research
area.
Furthermore, the flocculation mechanisms associated with
bioflocculant-dye systems should be carefully evaluated. The wide range
of dye molecule structures and the not identified sub-fraction compo­
nents of bioflocculants may generate controversial data and mask rele­
vant results. In this sense, the detailed composition and identification of
both bioflocculants and dyes are essential to reveal the unique influence
of each component and, then elucidates the bio-aggregation mecha­
nisms. Moreover, different groups of dyes may behave unpredictably in
the presence of bioflocculants with different chemical compositions.
Therefore, any treatment system should be aligned to each class of dyes
applied by the textile industry.
W. Artifon et al.
In addition, the inappropriate disposal of textile dyeing sludge
biomass with toxic and recalcitrant properties, very likely, leads to
deleterious effects on the environment. The development of textile
dyeing sludge purification methods, valorization techniques, as well as
their chemical identification are remarkable researching areas. Thus,
new treatment systems and traditional approaches should be
investigated.
6. Conclusion
The elucidation of the bioflocculant process is a key factor for
decolorizing and detoxifying dye-containing wastewaters. In this sense,
this review comprises researching studies on the effects of biological,
chemical, and physical parameters related to bioflocculants and dyes
available in the literature. The flocculation and settlement of dyes are
attributed to biomolecules, such as polysaccharides and proteins, which
induce bio-aggregation by bridging and charge neutralization. The most
prominent bioflocculant-producing strains for color removal applica­
tions were Bacillus sp. and Pseudomonas sp. Bioflocculant dosage, tem­
perature, and pH parameters considerably influence flocculation
efficacy. Moreover, the best color removal results for both anionic and
cationic dyes were attained mostly around neutral pH and at room
temperature. In some studies, low cation ions addition improved dye
precipitation for stabilization of functional group negative charges.
Bioflocculants are cost-effective, eco-friendly, and have great potential
to be employed to remove textile industrial dyes from wastewaters.
Authors contributions
Wagner Artifon: Conceptualization, Methodology, Writing original
draft, Review, and Editing. Karina Cesca: Conceptualization, Method­
ology, Review, and Editing. Cristiano José de Andrade: Methodology,
Review, and Supervision. Antônio Augusto Ulson de Souza: Review and
Supervision. Débora de Oliveira: Conceptualization, Review, Editing,
and Supervision.
Funding
This work was funded by the National Council for Scientific and
Technological Development (CNPq) and Coordination of Superior Level
Staff Improvement (CAPES-PRINT), Project number 88887.310560/
2018-00.
Declaration of Competing Interest
The authors report no declarations of interest.
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