Abu Bakar Et Al.. 2021. A Review of The Production Process of Bacteria-Based Polymeric Flocculants

Journal of Water Process Engineering 40 (2021) 101915
Contents lists available at ScienceDirect
Journal of Water Process Engineering

journal homepage: www.elsevier.com/locate/jwpe
A review of the production process of bacteria-based polymeric flocculants

Siti Nur Hatika Abu Bakar a, Hassimi Abu Hasan a, b, *, Siti Rozaimah Sheikh Abdullah a,
Nor Azman Kasan c, Mohd Hafizuddin Muhamad a, *, Setyo Budi Kurniawan a
a
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul
Ehsan, Malaysia
b
Research Centre for Sustainable Process Technology (CESPRO), Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi,
Selangor Darul Ehsan, Malaysia
c
Institute of Tropical Aquaculture and Fisheries Research (AKUATROP), Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia
A R T I C L E I N F O A B S T R A C T
Keywords: The utilisation of chemical flocculants in water and wastewater treatment has drawn several concerns especially
Bio-based polymeric flocculant in terms of further toxicity and its effect to the environment. The utilisation of chemical flocculants produces
Bioflocculant non-biodegradable and hazardous sludge as by-product of the treatment. Bioflocculants are observed as an
Bioflocculant extraction
alternative to chemical flocculants due to their comparable efficiencies. They are biodegradable, eco-friendly,
Bioflocculant purification
Bacteria-producing bioflocculant
and safe to handle. The identification and production of novel bioflocculants has gained much attention
worldwide and extensively reported. Amongst all, bacteria receive the most attention because of its availability
and ability to produce bioflocculants with many specialties. This review paper summarises the extraction and
purification processes in bioflocculant production as vital aspect that determine the production yield, which is
currently still scattered. Solvent extraction, ultrasound-assisted extraction, hydrothermal, microwave-assisted,
and enzyme-assisted are examples of extraction techniques, while lyophilisation, ion-exchange and dialysis
are the purification techniques often adopted in bioflocculant production. This review paper also provides brief
introduction on plant-, crustacean-, and microorganism-based bioflocculant as promising compounds with high
flocculating activity. The development of bioflocculants is representing important progress in sustainable envi
ronmental technology. The need to provide a selection of inexpensive, promising extraction, and purification
techniques for bioflocculant production are the challenges for bioflocculant production and future research di
rections on this subject should be emphasized to be applied further in water and wastewater treatment.
1. Introduction Coagulants and flocculants are applied to allow particles or pollutants

that are light, fine and subtle to coagulate and flocculate to form larger
Wastewater produced by industries through food and beverage particle sizes by neutralizing the charges on the particles [11,12]. This
production, mining, dyes and textiles, fermentation and its downstream condition allows the particles to separate from the water body and to
processes, palm oil mill effluent, pulp mill, sanitary landfill leachates sediment at the base of the tank due to the flocs higher density than that
and others contain very fine suspended solids, dissolved solids, inor of water [13]. The flocculation performance depends on the choice of
ganic particles, organic particles, metals, and other impurities [1–4]. flocculants used, with turbidity removal, residual turbidity, sludge vol
Solids particles in wastewater can affect the environment by decreasing ume, and settling rate become the parameters to define the overall
the light penetration in surface water and affecting the fish gill [5,6], performance. The settling rate of particles depends on the particle size
while organic materials can decrease oxygen availability which can formed through the coagulation and flocculation processes [11,14]. In
harm aquatic organisms [7,8]. However, because of its very fine and other words, larger particle sizes with higher densities will lead to
subtle characteristics, it is very hard to directly remove these contami higher settling rates and a better supernatant quality. As a consequence,
nants from wastewater. Thus, the task of bringing the particles closer the easy removal of particles is achieved since they are already physi
and making them heavier is necessary for settling purposes [9,10]. cally separated from the wastewater.
* Corresponding authors at: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor Darul Ehsan, Malaysia.
E-mail addresses: [email protected] (H. Abu Hasan), [email protected] (M.H. Muhamad).
https://doi.org/10.1016/j.jwpe.2021.101915
Received 17 November 2020; Received in revised form 2 January 2021; Accepted 5 January 2021
Available online 15 January 2021
2214-7144/© 2021 Elsevier Ltd. All rights reserved.
S.N.H. Abu Bakar et al. Journal of Water Process Engineering 40 (2021) 101915
Water and wastewater can be treated using various technologies such performance, but the excessive use of these compounds pose several
as physico-chemical treatments (coagulation/flocculation, precipita concerns including the trigger of Alzheimer’s disease, cancer and
tion, conventional chemical oxidation, and advanced oxidation), and neurotoxicity to human [22,23]. Inorganic and synthetic flocculants are
biological treatments (activated sludge process, integrated fixed-film not biodegradable [15,21,22], thus sludge produced as by product of
activated sludge, submerged aerobic attached growth, and emerging treatment using these compounds is also non-biodegradable. For this
biofilm process). Wastewater treatment using coagulants/flocculants is reason, bioflocculants derived from microorganisms have emerged as
relatively simple, having competitive cost with other treatment method the most suitable candidates to be explored. Bioflocculants has been
and able to separate various pollutants such as particles, dyes and heavy proven as nontoxic and eco-friendly behavior towards human and
metals [14,15]. According to Okaiyeto et al. [14] flocculation can be environment [16,24]. To date, many researchers have reported on bio
used as a substitute for filtration and centrifugation to separate micro flocculant production by microorganisms such as Aspergillus parasiticus
bial cells from broth in the food, beverage and pharmaceutical in [25], Ochrobacterium cicero W2 [26], Bacillus mojavensis [21], Pseudo
dustries. However, despite its high efficiency in wastewater treatment, monas koreensis, Pantoea sp. [27], Pseudomonas sp. HP2 [28], Entero
the flocculation process can produce small and fragile flocs that can coccus faecalis, Proteus mirabilis, Lysinibacillus sp. [29], Bacillus
easily be dishevelled when physical force is applied [14]. Therefore, it is velezensis [30] and many more. The methods of extraction and purifi
crucial to ensure that the flocs formed are sturdy and heavy enough for cation are different and specific in each study, while information related
the separation process to occur. to the production of bioflocculant compounds is currently still scattered,
Before choosing suitable flocculants, it is important to have infor thus causing complexity in full-scale production. The downstream pro
mation about flocculant classifications. The flocculants must have zero cesses in microorganism-based bioflocculant production are crucial to
or minimum hazardous impacts to human health and the environment obtain a bigger yield and high-quality bioflocculant. Databases analysis
[16]. Generally, flocculants can be divided into inorganic and polymeric including Elsevier (www.sciencedirect.com), Springer (www.springe
categories. Polymeric flocculants can be further divided into three rlink.com), Wiley (www.wiley.com), ResearchGate (www.researc
groups, namely, synthetic, grafted and bio-based polymers [15], as hgate.net) and Google Scholar was implemented using key words such
illustrated in Fig. 1. The use of inorganic or metal salts-derived floccu as ‘flocculation and coagulation’, ‘wastewater treatment technology’,
lants are common in industrial wastewater treatment [17]. Ferric sul ‘bioflocculant extraction’, ‘bioflocculant purification’, ‘bacteria bio
phate, ferric chloride, aluminium sulphate [18], aluminium chloride flocculant’ and ‘bioflocculant productions’. There were about 100 pa
and titanium chloride [19] are examples of inorganic flocculants that are pers selected, from the year 2000–2020 and > 80 % research papers
frequently used in the treatment of industrial wastewater. from 2015 to 2020 indicated that the bioflocculant production applied
The term of synthetic polymeric flocculants refers to chemical-based various unique methods which rarely summarized in a review paper.
polymeric flocculants that are frequently used as an alternative to Therefore, this paper aims to cover and deeply discuss the produc
inorganic flocculants. Polyethyleneimine (PEI), polyaluminium chloride tion, extraction, and purification methods for bioflocculant derived from
(PAC) and polyacrylamide (PAM) polymers are examples of synthetic potentially flocculant-producing organisms. Utilisation of wastewater as
polymeric flocculants. On the other hand, grafted flocculants are a class potential medium in the production of bioflocculant by microorganism
of polymeric flocculants that combine natural and synthetic polymers are highlighted in this review paper. Comparison of several extraction
and sometimes more with the aim of gaining the advantages of synthetic and purification methods are presented for the basic reference in
and natural polymers simultaneously [20]. Meanwhile, bio-based selecting appropriate method to obtain the optimum production yield.
polymeric flocculants or bioflocculants are those synthesized naturally The difficulties and challenges of bioflocculant production and appli
by either plants or microorganisms. Examples of bioflocculants from cation were also discussed to direct for future research and opportunities
plants are usually derived from beans, moringa, maize and cactus, while of applications in real wastewater treatment plants for various
examples of bioflocculants from microorganisms are derived from acti industries.
nomycetes, fungi, bacteria and algae [21].
Both inorganic and polymeric flocculants are excellent in terms of
Fig. 1. Classifications of flocculants.
2
2. Bio-based flocculant such as PAM to promote the flocculating activities of the respective
bioflocculants.
2.1. Plant- and crustacean-based flocculants
2.2. Microorganism-based flocculants
Bio-based flocculants are known as bioflocculants and are extracted
either from naturally occurring bioflocculants such as marine poly Another promising category of substitutes for chemical flocculants is
saccharide flocculants (sodium alginate and chitosan) and microbial microorganism-based flocculants. Microorganism-based flocculants are
flocculants or are produced from renewable sources such as plants (seeds known as biotechnological tools for use in wastewater treatment
and vegetable tannin) and eggshells [31]. Alginate is an anionic poly through the bioflocculation process [50], which are much safer and have
saccharide consisting of (1→4)-linked β-D mannuronate (M-block) and higher biodegradable ability [47] compared to synthetic chemical
α-L-guluronate (G-block) residues with biodegradable, biocompatible, flocculants. These bioflocculants are metabolites secreted by microor
non-toxic and non-immunogenic biopolymer polyelectrolyte character ganisms during their growth phase [51]. They can be produced by
istics that is often extracted from seaweed [13]. Chitosan is a poly bacteria, fungi, microalgae and actinomycetes. The first report of bio
saccharide made up of N-acetylglucosamine (GlcNAc) and/or flocculants was made by Louis Pasteur in 1876 using yeast as the bio
glucosamine (GlcN) linked through β-1,4-glycosidic linkages produced flocculant producer [52]. Bioflocculants derived from microorganisms
from the exoskeletons of crustaceans such as crabs, shrimps, prawns, are easily isolated, optimised, and characterised [52,53]. The selection
lobster and krill, and it has biocompatible, biodegradable and antimi of microorganisms for microbial bioflocculants is often based on their
crobial activity properties [32]. Similar to alginate, pectin is a complex morphology and ability to produce slimy extracellular polysaccharides
anionic polysaccharide composed of β-1,4-linked D-galacturonic acid (EPS) [21,54]. EPS, which is naturally adhesive and cohesive, helps in
residues found in plants that is able to form a gel with divalent cations to the aggregation of suspended solids wastewater [55]. According to
help in the flocculation and coagulation processes [33]. Shahadat et al. [16], there are two types of EPS, namely, loosely bound
In addition to crustaceans, plants also have potential as sources of EPS (LB-EPS) and tightly bound EPS (TB-EPS).
bioflocculants, including okra, cassava roots, pomelo peel, guar gum and According to Rebah et al. [21], in the polymer characterization of
bamboo [13,34]. Table 1 summarizes the types of bioflocculants, which bioflocculants, variations are observed, especially in the composition of
are divided into marine plant, crustacean and plant categories. Marine polysaccharides, proteins, DNA, cellulose, sugar, and polyamino acids
plants such as Ascophyllum nodosum, Durvillaea antartica, Laminaria during the growth phase of microorganisms. However, the concern only
digitata, Macrocyctis pyrifera and Sargassum sp. are types of brown sea focuses on the composition of polysaccharides and proteins, since these
weeds from which alginate is extracted. As shown in the table, there are substances make up the highest percentage of the bioflocculant polymer
many applications of alginate, especially in the remediation of waste [29] and indirectly affect the flocculating activity of the bioflocculant.
water from the pharmaceutical, cosmetic, food and biotechnology in To clearly observe the relationship between the composition of poly
dustries [13,35], as well as the removal of heavy metals [36]. saccharides and proteins and the flocculating activity, previous studies
Chitosan is extracted from crustaceans such as Metapenaeus mono on bioflocculant production from microorganisms, especially bacteria,
ceros, Penaeus longirostris and Chionoecetes opilio. The extracted chitosan are tabulated in Table 2. From Table 2, it can be observed that the
can be applied to remove heavy metals, particles, dissolved organic compositions (%) of polysaccharides (PS) and proteins (P) were different
matter [38] and in the removal of solid suspensions from food pro in each bioflocculant-producing strain. Bioflocculant polymers with a
cessing waste [39]. On the other hand, bioflocculants from plants such high composition of polysaccharides (>80 %) managed to achieve high
Cassia angustifolia have been used to remove colour from dye solutions flocculating activities (FA) (>90 %). Vimala et al. [29] found that the
[42,49]. Ummalyma et al. [45] reported that guar gum (guaran) can be structures of polysaccharide functional groups (carboxyl, hydroxyl,
used in microalgae biomass harvesting. Despite being biodegradable and amino and phosphate groups) can affect the flocculating activity of
safe for application, plant-based bioflocculants are still lacking in floc bioflocculants. A similar finding was also reported by Nwodo et al. [23],
culating activity. Therefore, they are often grafted onto other polymers in which hydroxyl, carboxyl and amino sugars, which are constituents of
Table 1
Naturally occurring bioflocculants.
Organisms Extraction sources Bioflocculant Application References
Marine
Ascophyllum nodosum Pharmaceuticals, cosmetic, food and biotechnology industries [13]
Durvillaea antarctica Raw materials for cell immobilization for wastewater treatment [13,36]
[13,36,
Laminaria digitata Brown seaweeds Alginate Removal of heavy metals such as cadmium, chromium and copper
37]
Macrocystis pyrifera Raw materials for cell immobilization for wastewater treatment [13,36]
Sargassum muticum Raw materials for cell immobilization for wastewater treatment [13,36]
Crustaceans
Metapenaeus monoceros Applied in removal of metals, particles and dissolved organic matter [13,38]
Shrimp shell
Penaeus longirostris Chitosan [13]
Chionoecetes opilio Crab shell Solid suspension from food processing waste [13,39]
Plants
Abelmoschus esculentus Okra seeds Anionic polysaccharide Water treatment and sludge dewatering application [13,40]
Cassava (Manihot esculenta) Cassava roots Starch Wastewater treatment [13,41]
Cassia tora; Cassia
Leguminous plant Cationic Cassia Decolourisation of dye solutions [13,42]
angustifolia
Citrus maxima Pomelo peel Pectin Pharmaceutical suspensions treatment [13,43]
Endosperm of Guar Polysaccharide [13,44,
Guar gum (Guaran) Microalgae harvesting
beans (Galactomannan) 45]
Phyllostachys heterocycla Bamboo forest Dicarboxyl cellulose Effluent treatment from surfactant manufacturer [13,46]
Tamarindus Indica Tamarind seeds Polysaccharide Textile wastewater treatment [13,47]
Water and wastewater treatment; Removal of dye from industrial
Opuntia ficus indica Pads of cactus Mucilage and pectin [33,48]
wastewater
Cactus latifaria Cactus Cactus extract Comparable studies with alum in reduction of kaolin [42]
3
Table 2
Production of bioflocculants from microorganisms.
Category of Carbon/Nitrogen Sources Strain for bioflocculant production Flocculating Composition of References
substrate used Activity (FA) polysaccharide (PS) and
protein (P)
Microbial consortium (Origins)

Glucose, urea, yeast extract, PS: n.m
Defined media Cobetia sp. OAUIFE, Bacillus sp. MAYA (sediment) 90.0 % [21]
ammonium sulphate P: 31 %
Halomonas sp. Okoh and Micrococcus sp. Leo
Glucose, yeast 86.0 % n.m [56]
(sediment)
Sodium carbonate, urea, yeast Halobacillus sp. Mvuyo and Oceanobacillus sp. Pinky
98.3 % n.m [57]
extract, ammonium sulphate (sediment)
Sucrose, peptone, magnesium Streptomyces sp. Gansen and Cellulomonas sp. Okoh PS: 34.4 %
98.9 % [23]
chloride (river) P: 18.56 %
Hydrolyzed wheat bran, rice Enterococcus faecalis MG996001, Proteus mirabilis
n.m n.m [29]
bran extract MG996004 and Lysini bacillus sp. MG996000(n.m)
Single strain (Origins)
Agrobacterium sp. M-503 (propylene epoxide PS: 85.0 %
Sucrose, yeast extract, urea 74.5 %, [21]
wastewater sludge) P: 3.0 %
PS: 69.7 %
Sucrose, peptone Aspergillus flavus S44-1 (readily isolated) > 90 % [58]
P: 28.5 %
PS: 65.4 %,
Glucose, yeast extract Bacillus agaradhaerens C9 (alkaline lake) 80.6 %,
P: 4.7 %
L-glutamic acid, ammonium 96.1 %, PS: 98.4 %,
Bacillus mojavensis 32A (salt production pond)
chloride P: 1.60.0 %
90.0 %, PS: 88.3 %,
Sucrose, peptone Bacillus subtilis F9 (wastewater sludge)
P: 10.1 %
98.0 %, PS: 91.5 %,
Beef extract, peptone Bacillus licheniformis X14 (soil) [21]
P: 8.4 %
> 98.0 %, PS: 98.0 %,
Glucose, yeast extract Bacillus velezensis 40B (brakish water)
P: 2.0 %
75.0 %, PS: 17.0 %
Starch, yeast Bacillus cereus (cultivated soil)
P: 3.0 %
76.3 %, PS: 15.2 %
Starch, yeast extract Bacillus thuringiensis (cultivated soil)
P: 84.7 %
Bacillus sp. TERI VB2 (Soil from India Habitat 99.0 % PS: 97.13 %
Glucose, yeast extract, urea [55]
Centre) P: 1.46 %
Cellulosimicrobium cellulans L804 (Corn farmland > 80 % PS: 68.6 %
Dry corn stover, yeast extract [59]
soil) P: 28.0 %
Chryseobacterium daeguense W6 (biological aerated 96.9 %, PS: 13.1 %
Glucose, tryptone
filter sludge) P: 32.4 %
Nutrient poor medium, (with 80.0 %, PS: 72.3 %
Klebsiella sp. PB12 (river water) [21]
glucose or lactose or mannose) P: 14.1 %
Sucrose, yeast extract, beef 86.9 %, PS: 84.6 %
Klebsiella sp. TG-1 (starch factory wastewater)
extract (using trona suspension) P: 11.1 %
Tryptone, yeast extract, sodium 93.9 % PS: 84.6 %
Klebsiella sp. ZZ-3 (Paper mill wastewater treatment) [60]
chloride P: 6.1 %
Klebsiella oxytoca GS-0-4-08 (China General PS: 46.3 %
Acetonitrile, [61]
Microbiological Culture Collection Centre) P: 20.6 %
Yeast extract, peptone, rice
Pseudomonas sp. HP2 (Forest soil) 92.5 % n.m [28]
straw biomass
Sucrose, yeast extract, sodium
Paenibacillus mucilaginosus (soil) >97 % PS: 97.8 % [62]
hydrogen phosphate
90.0 %, PS: 75.1 %
Sorbitol or starch, yeast extract Solibacillus silvestris W01 (activated sludge) [21]
P: 24.9 %
Glucose, yeast extract, malt Streptomyces platensis HBUM174787 (The 90 % PS: 83.0 %
[50]
extract Sterkfontein Dam) P: 4.6 %
99.2 % PS: 86.9 %
Streptomyces sp. (sediment) [63]
P: 12.8 %
Sago mill effluent 18.5 –
Bacillus velezensis [30]
Complex media Palm oil mill effluent 11.4 –
Potato starch wastewater Aspergillus niger A18 90.1 % [64]
76.8 % PS: 69.7 %
Palm oil mill effluent Aspergillus niger (readily isolated) [65]
P: 28.5 %
Palm oil mill effluent Bacillus marisflavi NA8 (Aerobic POME) 80.0 % n.m [66]
Bacillus licheniformis (China General Microbiological
Molasses > 90 % n.m [67]
Culture Collection Centre)
Dairy wastewater Klebsiella mobilis (soil and activated sludge) 95.4 % n.m [68]
Dairy wastewater Klebsiella mobilis 95.4 % n.m [67]
87.8 % PS: 89.4 %
Crude petroleum Psedomonas aeruginosa (activated sludge) [69]
P: 6.2 %
Fish meal wastewater Pseudomonas sp. 95.0 % n.m [67]
Potato starch wastewater Rhizopus sp. 95.5 % n.m [70]
Stenotrophomonas maltophilia ZZC-06 (recycled 95.2 % PS: 71.2 %
Phenol containing wastewater [71]
activated sludge) P: 27.9 %
Brewery wastewater 75.2 % n.m
Dairy wastewater 95.5 % n.m
Serratia ficaria (Soil) [72]
Soy sauce brewing wastewater 94.3 % n.m
Meat processing wastewater 92.3 % n.m
*n.m: not mentioned.

4
polysaccharides, were found to be able to enhance the flocculating ac must be followed. As shown in Fig. 2, the steps involve the isolation of
tivity of the bioflocculant. Therefore, in the search for bioflocculants bioflocculant-producing bacteria (BPB), the identification of BPB, the
with high flocculating activities, the presence of functional groups on growth of the BPB seed, fermentation for bioflocculant production, the
the polysaccharides needs to be highly considered. Vimala et al. [29] determination of the flocculating activity, and last, the purification of
also added that bioflocculants with high compositions of poly the bioflocculant itself.
saccharides were thermally stable, while bioflocculants with high
compositions of proteins were thermally sensitive. This knowledge is 3.1. Isolation and identification
especially helpful in the application of bioflocculants in wastewater
treatment. The isolation of BPB can be done from various sources using common
Based on Table 2, the types of carbon sources used were also different medium of nutrient broth for the isolation. Usually, the isolated strain of
and were categorized into defined and complex media. Media with BPB can be identified at the genus level by performing polymerase chain
known ingredients were called defined media, whereas media with reaction (PCR) and sequencing (16S RNA) analyses to identify the
undetermined chemical compositions were called complex media. The taxonomic locus of the strain. The sequencing data are then used to
complex media tabulated in Table 2 normally comes from various types construct a phylogenetic tree to recognize the isolated bacteria lineage
of industrial wastewater and were considered low-cost carbon sources [63]. According to Rebah et al. [21], sludge from aerobic activated
since the wastewater was available in abundance. The search for low- sludge systems is often selected as origins for bacteria isolation for
cost carbon sources has been widely covered by many researchers bioflocculation production purposes. The reason for this process is that
because the production of bioflocculants using defined media contrib bioflocculation often reflects a dynamic process that occurs inside aer
utes to the high production cost. Palm oil mill effluent [30,65,66], po obic activated sludge systems. Thus, the sludge from these systems
tato starch wastewater [64,70] and dairy wastewater [67,68,72] are contains significant numbers of bacteria that are able to produce bio
example of wastewater with potential as carbon sources for bio flocculants. For example, an activated sludge sample from paper mill
flocculant production from bacteria. Among all low-cost carbon sources, wastewater contained Klebsiella sp. ZZ-3, which has the ability to pro
dairy wastewater shows the ability to produce bioflocculants with high duce a bioflocculant [60].
flocculating activity (>95 %). This is because dairy wastewater contains In addition, Zhang et al. [76] solely used activated sludge obtained
easily degradable carbohydrates (lactose), proteins and lipids [68] that from a sewage plant to produce bioflocculant. Both processes show that
are crucial for microbial growth. activated sludge can be a good source either for the isolation of bacteria
The derivation of bioflocculants from bacteria can be performed involved in bioflocculant production or for use as the sole source in
using either single or mixed cultures [53]. The cultivation of bacteria production. A later study by Qi et al. [28] used Pseudomonas sp. HP2
can be performed using carbon and nitrogen sources such as glucose and isolated from forest soil for bioflocculant production. The bioflocculant
yeast extract, respectively [21]. In addition, according to Abdullah et al. produced managed to achieve a 92.5 % FA. Streptomyces sp. isolated
[53] and Rebah et al. [21] bioflocculants derived from cultivation via from sediment showed the most promising performance, with a 99.2 %
mixed culture are able to produce higher flocculating activity (FA) FA [63], which was the highest FA amongst all isolated strains. There
compared to those from single-culture cultivation techniques. Bio fore, the isolation sources for BPB can be varied but are mostly
flocculants produced from a mixture of Streptomyces sp. Gansen and concentrated in areas believed to be niches for BPB such as soil, acti
Cellulomonas sp. Okoh managed to achieve a 98.9 % FA [55], and a vated sludge and sediment.
mixture of Halobacillus sp. Mvuyo and Oceanobacillus sp. Pinky achieved After the source for isolation is chosen, the microorganisms, espe
a 98.3 % FA [57]. However, the FA of bioflocculants from single strains cially bacteria, are then selected based on physical and chemical char
are also on a par with those of bioflocculants produced from mixed acteristics and chemo-taxonomical characteristics. Physical
cultures. Bacillus sp. TERI VB2 and Pseudomonas sp. HP2 managed to characteristics that should be considered are the morphology of the
achieve 99 % and 99.2 % FA [55,63], respectively. Pseudomas sp. HP2 bacteria and the existence of slimy EPS upon cultivation on selected
managed to record the highest FA among all strains. In addition to Ba media. This slimy EPS contributes to flocculating activity in addition to
cillus velezensis 40B (>98 % FA), Bacillus licheniformis X14 (98 % FA) assisting in the adherence of cells to a surface and acts as a source of
[21] and Paenibacillus mucilaginosus (>97 % FA) [62] also showed energy for microbes during starvation [69]. The chemical characteristics
promising performances. of BPB can be evaluated using Congo red, crystal violet and copper
Bacillus sp. is the most studied bacterial species for bioflocculant sulphate solution, as well as chelating agents and colorimetric methods
production. This finding is in agreement with Lachhwani [11], who [21].
labelled Bacillus sp. 101 as capable of producing flocculating substances
with good flocculating activity, despite different carbon and nitrogen 3.2. Growth and fermentation of BPB
sources. Microorganisms from the class Bacilli also capable of producing
higher quantities of extracellular polymeric substances (EPS) with pri The next step in bioflocculant production is the growth and
mary constituents of bioflocculants comparable to those of lactic acid fermentation of BPB in the cultivation medium that is optimal for the
bacteria (LAB) and are the most widely reported [54,73,74]. Klebsiella bioflocculant yield. There are two types of conditions in which a bio
sp. are also capable of producing bioflocculants with flocculating ac flocculant can exist. First, bioflocculant materials secreted into the
tivities of more than 80 %. According to Yin et al. [60], there are seven production medium which can be easily obtained by filtration of the
Klebsiella sp. capable of producing bioflocculants, namely, Klebsiella sp. production medium. Second, bioflocculant materials adhered to the cell
strain S11, Klebsiella pneumonia H12, Klebsiella sp. MYC, Klebsiella or adsorbed to the cell surface require an extraction method to take place
mobilis, Klebsiella terrigena, Klebsiella pneumonia strain NY1 and Klebsiella [77].
pneumonia strain MBF-5. Aspergillus sp. is also often reported by re As can be observed in Table 2, the cultivation of BPB using defined
searchers due to the ability of the microorganism to produce high-FA media usually consists of carbon (glucose, sucrose) and nitrogen (urea,
bioflocculants. The filamentous characteristic of Aspergillus sp. allows yeast extract) sources to support the growth of the bacteria. The carbon
them to entrap solid particles to form aggregates and strengthens the source is very crucial for microbial growth; however, different carbon
flocculation structure [75]. sources often lead to different efficiencies in bioflocculant production.
This could be because of different metabolic pathways involved during
3. Process of microorganism-based flocculants production the utilisation of the carbon source by BPB, which thus interfere with the
EPS production that is often related to the bioflocculant [73]. However,
Generally, to produce a bioflocculant, there are several steps that the cost to purchase such carbon and nitrogen sources can be
5
Fig. 2. Process of bioflocculant production.
problematic, especially in the production of bioflocculants at a large particles during the flocculating activity test, while doses that are too
scale. Therefore, the quest for a cultivation substrate that is economical, high can cause a repulsive force to build up between the kaolin clay
easily available and can support the optimal growth of bacteria is of particles and bioflocculant [13,60]. Furthermore, from the flocculating
huge concern [64]. In addition, Rebah et al. [21] also mentioned that activity test, a flocculating activity curve can be constructed based on
wastewater and sludge contain sufficient macronutrients besides car the flocculating activity value obtained and dose amount used [69]. This
bon, nitrogen and phosphorus that can support the growth of microor will eventually allow the optimization of parameters to be carried out
ganisms, leading to bioflocculant production. To achieve the according to the flocculating activity of the targeted bacteria species or
aforementioned statement, Table 2 lists cost-effective substrates that are consortium.
capable of producing bioflocculants with promising flocculating Metal ions are also believed to affect the flocculating activity of
activities. bioflocculants. This is because the addition of cations decreases the
Wastewater, which is abundantly available, can be the best candi negative ions in kaolin particles and biopolymer flocculants. Mono
date as a substitute for the substrate frequently used in the production of valent and divalent cations are reported to help increase the flocculating
bioflocculants. Dairy [67,68,72] and potato starch [64,70] wastewaters activity of the bioflocculants produced, while trivalent cations cause
are the most effective cultivation substrates in the production of bio decreases of flocculating activity in terms of both floc formation and the
flocculants, which each yield flocculating activities higher than 90 %. optical density percentage [78]. Sodium chloride (NaCl) is an example
However, the use of wastewater such as palm oil mill effluent (POME) of a monovalent cation that can be added during the flocculating activity
may face some difficulties since the colour of the POME itself can affect assay. Calcium chloride (CaCl2), magnesium chloride (MgCl2) and
the absorbance reading during testing of the flocculating activity of the aluminium chloride (AlCl3) are examples of divalent and trivalent cat
bioflocculant produced [66]. Hence, cultivation using cultivation a ions, respectively. A study by Zulkeflee et al. [78] on bioflocculant
substrate made from wastewater with a high turbidity and colour re production from Bacillus sp. reported that the addition of AlCl3 during
quires pre-treatment or dilution in order to omit absorbance distur the flocculating activity assay only resulted in a 34.8 % FA compared to
bances and to obtain the optimal cultivation medium for bioflocculant the 70.5 % FA in the control set. This shows that the trivalent cation
production. failed to neutralize the zeta potential in the kaolin suspension; thus,
In addition, dilution of the wastewater is also required to control the there was no attraction between the kaolin particles and the bio
substrate concentration that exists in wastewater. Aljuboori et al. [65] flocculant polymer. Despite this finding, Okaiyeto et al. [56] reported
used a series of POME concentrations to produce PM-5 bioflocculant that no addition of cations was needed in the flocculating activity assay
from Aspergillus niger. They found that the substrate concentration does for a bioflocculant produced from Chryserbacterium daeguense and Ba
have a significant impact on the bioflocculant production beyond those cillus sp. F19.
of the culture operating parameters. Parameters such as the C/N ratio, Although flocculating activity can still be observed within wide
inoculum size, metal ions, cultivation period, dissolved oxygen (DO), pH range of pH values and temperatures, these factors can still affect the
and temperature are culture operating parameters that need to be taken overall flocculating activity of the bioflocculant produced. For example,
into serious consideration when dealing with real wastewater as the the pH value of the solution determines the flocculating activity and
cultivation medium [21,58,65]. It can also be said that each microor affects the stability of the suspended particles and floc formation [56]. In
ganism has its own optimal conditions for producing a high yield bio addition, variations in the pH value cause differences in the electrostatic
flocculant with high flocculating activity. charge of the bioflocculant and suspended particles, thus affecting the
bridging efficiency for kaolin clay particles [56]. Highly acidic condi
3.3. Flocculating activity tions (pH values < 5) and highly alkaline conditions (pH values > 9)
decreased flocculation activity (< 70 %) of bioflocculant produced by
Once the selection of bacteria and cultivation in media is done, the Bacillus salmalaya, with optimum pH occurred at 7 ± 0.2 [52].
determination of the flocculating activity will take place by the standard
Kaolin suspension method, applying Eq. 1 [69]. 3.4. Extraction and purification
Flocculating activity, FA (%) = (B − A)/B × 100 % (1)
The final step in the production of a bioflocculant is the purification
Here, A is defined as the absorbance of the sample at 550 nm, and B is of the bioflocculant after the extraction of the bioflocculant is carried
defined as the absorbance of the control at the same wavelength. out. The extraction methods for bioflocculants are usually unique and
The flocculating activity of the bioflocculant could be influenced by diverse in application, depending on the type of bioflocculant sources
the dose used, molecular weight, pH, temperature and metal ions [13, and the expected yield. The extraction methods often applied in bio
60]. Among all factors, the bioflocculant dosage can greatly influence flocculant production include the water extraction method, ethanol
flocculating activity. This is because polysaccharide-based bio extraction, hydrothermal and microwave extraction and centrifugation.
flocculants are often stable under heating and are able to show floccu Afterwards, the purification of the bioflocculant is conducted. The bio
lating activity within a wide range of pH values and temperatures [13]. flocculant produced is often lyophilized and vacuum dried [23,62] prior
In discussing the effects of the dosage used, for example, lower doses of to further processing to obtain the crude bioflocculant to avoid deteri
bioflocculants may be insufficient for the adsorption of kaolin clay oration of the organic polymeric content of the bioflocculant.
6
4. Extraction of bioflocculants into the matrix of substances, (2) the solvent dissolves the solute, (3) the
solute diffuses from the solid matrix, and (4) the extracted solute is
The extraction methods for bioflocculants often vary according to the collected [82].
type of bioflocculant producers and yield. Due to these variations, there Zhang et al. [82] reported that a solvent extraction is often influ
are many possible methods to be utilized in bioflocculant extraction. enced by the temperature, extraction time and solvent-to-solid ratio.
There are two distinct extraction methods that are frequently applied, High temperatures during the extraction process will promote the sol
which are physical and chemical methods, as shown in Fig. 3. Centri ubility and diffusion rate. However, a temperature that is too high can
fugation, filtration, sonication, and heating are classified as physical decompose thermo-sensitive materials and case solvent loss. Increases in
extraction methods. Meanwhile, alkaline/acid treatment, cation ex the extraction time will eventually increase the bioflocculant yield until
change resin, solvent extraction, ethylene diamine tetra acetic acid extraction equilibrium is reached. Therefore, it is important to deter
(EDTA) treatment, and enzymatic treatment are examples of chemical mine the optimum extraction time to avoid lost time. High bioflocculant
extraction methods. The selection of extraction methods plays a vital yields can be achieved at high solvent-to-solid ratios; however, using too
role in bioflocculant production because the extraction methods not much solvent can be wasteful and not cost-effective in the extraction of
only determine the yield of the produced bioflocculants but also define bioflocculants. Table 3 lists the extraction techniques applied in bio
the cost of bioflocculants production [79]. flocculants production. From the list, it can be observed that solvent
The type of EPS (LB-EPS or TB-EPS) eventually affect the selection of extraction often uses water, ethanol, and salt solutions—especially
extraction methods to be used. For LB-EPS, centrifugation at a high cetylpyridinium chloride solution (CPC) and sodium chloride (NaCl)—
speed, low-temperature heating and high- shear rate processes are more as well as acid and alkali solvents such as hydrochloric acid (HCl) and
suitable since the EPS is easily extracted from the respective mixture. sodium hydroxide (NaOH).
Meanwhile, the extraction of TB-EPS needs more extreme techniques
such as heating at high temperatures, sonication and chemical methods 4.1.1. Water extraction method
[13]. These extraction methods are called conventional extraction The water extraction method is one of the most commonly applied
methods and are applicable not only to microorganism bioflocculants methods, wherein water is used as the extraction medium or solvent.
but also to plant-based bioflocculants. Therefore, more advanced Water extraction methods can be carried out either with or without heat.
methods such as enzyme treatment and microwave-assisted techniques However, the involvement of heat was observed to be able to assist in the
are now receiving attention due to their ability to preserve the poly extraction procedure of bioflocculant, as increases in temperature are
saccharides during the extraction process. The next section of this paper able to reduce the viscosity of the solvent and enhance the penetration
emphasise the methods used for bioflocculants extraction. between the solvent and cell, thus leading to rapid mass transfer of
water-soluble active compounds into the extraction medium [83,92]. In
addition, the application of heat decreases the van der Waals forces and
4.1. Solvent extraction
hydrogen bonding [93], thus enhancing the solubility between the sol
vent and cells. However, the value of the temperature applied in bio
Solvent extraction methods are frequently used in the extraction of
flocculant extraction may differ according to the source of the
either plant-based or microorganism-based bioflocculants. Solvent
bioflocculant. The extraction of bioflocculants from microorganisms, for
extraction methods are known as the main conventional extraction
example, normally uses temperatures less than 70 ◦ C to avoid cell lysis.
techniques and use one or combination of two different solvents. They
Zhou et al. [93] found that as the temperature exceeded 70 ◦ C, signifi
are often called liquid-liquid extractions and use mainly water as the
cant evidence of cell lysis and the release of intracellular soluble organic
primary solvent with an organic solution such as hexane, dichloro
materials was detected.
methane of ethanol as the secondary solvent [80,81]. Additionally,
Lee et al. [83] studied the extraction of bioflocculants form okra and
solid-liquid separation can be used to extract dissolved or suspended
Chinese yams and applied a water extraction method with an additional
extractants in the respective mixture by applying special treatments,
incubation period (90 ◦ C; 30 min), while Tsuge et al. [77] carried out a
especially heat and high-speed mixing [81]. However, in the extraction
water extraction method without an additional incubation period. In the
of bioflocculants, not all extraction techniques can be applied due to
study by Tsuge et al. [77] on bioflocculant production from activated
unsuitability of the solvents and external forces applied. Basically, sol
sludge, they initially centrifuged the activated sludge at 5,000 rpm for
vent extraction involves the following steps: (1) the solvent penetrates
Fig. 3. Extraction methods for bioflocculants.
7
Table 3
Solvent extraction methods applied in bioflocculant extraction.
Bioflocculant producer Extraction method Solvent Bioflocculant Yield Flocculating activity References
Okra and chinese yam Water extraction Deionized water 74.2 % – [83]
Activated sludge Water extraction Distilled water 24.6 % 20.8 % [77]
Bacillus agaradhaerens C9 Ethanol extraction Ethanol 2.5 g L− 1 93.1 % [84]
Bacillus agaradhaerens C9 Ethanol extraction Ethanol 6.92 g L− 1 96.3 % [85]
Pantoea sp. 2.98 g L− 1 71.0 %
Ethanol extraction Ethanol [27]
P. koreensis 3.26 g L− 1 51.0 %
Bacillus thuringiensis Ethanol extraction Ethanol – 93.8 % [86]
Stenotrophomonas maltophilia ZZC-06 Ethanol extraction Ethanol 4.99 g L− 1 >90.0 % [71]
Moringa oleifera Ethanol extraction Ethanol – – [87]
Bacillus licheniformis 16.55 g L− 1 96.0 %
Ethanol extraction Ethanol [88]
Bacillus firmus 10 g L− 1 89.0 %
Aspergillus flavus Salt solution extraction CPC and NaCl 0.4 g L− 1 87.2 % [58]
Bacillus mojavensis strain 3A Salt solution extraction CPC and NaCl 5.2 g L− 1 96.1 % [89]
Rhodococcus erythropolis Acid and alkali extraction NaOH – 94.5 % [90]
Biological sludge Acid and alkali extraction HCl and NaOH – 96.0 % [76]
Sludge Acid and alkali extraction HCl – 99.5 % [91]
*Notes: CPC: cetylpyridinium chloride solution.
20 min before suspending the concentrated activated sludge in 300 mL Bacillus licheniformis and Bacillus firmus compared to acetone and
distilled water and mechanically stirring at 4 ◦ C for 30 min. Centrifu methanol, respectively. This shows that ethanol is more compatible for
gation at 6000 rpm for 30 min was carried out afterwards to obtain the use in the extraction of bioflocculants from microorganisms compared to
supernatant before lyophilization and storage at 4 ◦ C. The flocculating other organic solvents. Furthermore, it was noticeable that the ethanol
activity obtained was 24.6 %, with an extraction yield of 20.83 %. In extraction method usually required two centrifugation processes (1) for
contrast, in the study carried out by Lee et al. [83], plant materials were solid-liquid separation and (2) for the collection of the precipitate
ground to break the cell walls and enhance the solubility and were formed (crude bioflocculant), in contrast to the water extraction
mixed with 100 mL of deionized water before placement in a shaker bath method. This was because ethanol extraction methods are normally
at 300 rpm for 3 h. The extract was then incubated at 90 ◦ C for 30 min applied when retrieving bioflocculants from both LB-EPS and TB-EPS,
and left at room temperature for another hour to complete the extraction which require extra solvent solubility [92] in the absence of heat.
process. Then, the bioflocculant was obtained by filtration and centri
fugation at 7,000 rpm for 20 min before oven- or freeze-drying the 4.1.3. Salt solution extraction
product for storage at 4 ◦ C. The yield gained was 74.2 %. Thus, the Salt solutions such as sodium chloride (NaCl), potassium chloride
extraction processes of bioflocculants using water extraction methods (KCl) and cetylpyridinium chloride (CPC) solutions have also been
may be different, depending on the sample condition. applied in the extraction of bioflocculants. The extraction of bio
flocculants using water is said to be the most accepted technique, yet
4.1.2. Ethanol extraction method extractions using salt solutions are viewed as more efficient compared to
Ethanol extraction methods have the ability to separate water and water extractions [94]. A study by Madrona et al. [95] found that there
lipo-soluble components [91]. Retrieving bioflocculants via ethanol were significant effects from the salt solution concentration on the
extraction methods can extend the lifetime of the extraction solution performance of the extracted bioflocculant. In their study, the extraction
compared to water extraction methods. Wu et al. [86], for instance, of a bioflocculant from Moringa oleifera revealed that extraction using 1
utilized an ethanol extraction method to extract a bioflocculant from M KCl was far more effective (99.7 % turbidity removal) compared to
Bacillus thuringiensis. First, the solution containing the bioflocculant was 0.1 M and 0.01 M KCl in real applications in producing drinking water.
centrifuged at 8000 rpm for 15 min. A volume of ethanol was then added They claimed that a high concentration of the salt solution was more
to the supernatant collected and mixed thoroughly before centrifuging effective in extracting the active compound in the bioflocculant, which
again at 5000 rpm for 15 min at room temperature for solid-liquid was a protein that led to a higher flocculating activity.
separation purposes. Afterwards, the flocculant precipitate was Elkady et al. [89] used CPC to extract a bioflocculant from Bacillus
washed with a suitable volume of deionized sterile water and soaked mojavensis strain 3A with a flocculating activity of 96.1 % and a yield of
again with ethanol two more times. The extracted flocculants were then 5.2 g L− 1. The cell-free supernatant of the respective strain was
freeze-dried, weighed, sealed and stored at a temperature below -20 ◦ C. concentrated to 0.2 vol using a rotary evaporator and left to dialyze
The flocculating activity obtained by Wu et al. [86] was 93.8 %. overnight at 4 ◦ C in deionized water. Afterward, three volumes of cold
Ayangbenro et al. [27] also utilized an ethanol extraction in their anhydrous ethanol at 4 ◦ C were mixed into the mixture for precipitation.
study to retrieve bioflocculants from Pantoea sp. and P. koreensis. They The precipitate formed was then dissolved again in deionized water,
centrifuged the fermentation broth at 3000 rpm for 30 min at 15 ◦ C followed by stirring with 10 % CPC in the solution. Centrifugation at
before mixing the supernatant with cold ethanol (1:4 v/v) for bio 5000 rpm for 15 min was applied to collect the precipitate prior to
flocculant precipitation and kept the mixture at 4 ◦ C for 16 h. After dissolving in 0.5 M NaCl. Later, three volumes of cold anhydrous ethanol
wards, the mixture was again centrifuged (1000 rpm, 30 min, 15 ◦ C) for (4 ◦ C) were added for precipitation before washing with 75 % ethanol
precipitate collection prior to dissolving the precipitate in distilled water three times, lyophilization and storage for further use. Additionally,
(1:4 v/v) and lyophilization. The maximum yields gained for Pantoea sp. Aljuboori et al. [58] also used CPC (50 mL of 2% CPC) in their study to
and P. koreensis were 2.98 g L− 1 (FA: 71 %) and 3.26 g L− 1 (FA: 51 %), extract a bioflocculant from Aspergillus flavus (yield: 0.4 g L− 1, FA: 87.2
respectively. %), with the precipitate collected by dissolving it in 100 mL of 0.5 M
In the study by Karthiga and Natarajan [88], ethanol, acetone and NaCl and washing with two volumes of cold ethanol.
methanol were used to extract bioflocculants from Bacillus licheniformis
and Bacillus firmus. By using double the volume of cold solvent ethanol, 4.1.4. Acid and alkali extraction
acetone and methanol (1:2 v/v), extraction using ethanol as the Acid and alkali solutions are also frequently used for the extraction of
extraction solvent yielded the highest amounts of bioflocculants from bioflocculants. In the study by Zhang et al. [76] on producing
8
bioflocculants from biological sludge, a combination of acid and alkali heat then perform external evaporation (of solvent) and internal evap
solutions was applied. First, 60 mL of 3 % (w/w) of HCl was used on oration (inside compound’s matrix) to produce solid compounds [79].
sludge that was formerly treated with 100 mL of 4 % (w/w) NaOH, and This method is currently widely applied, while energy consumption
the mixture was mixed with a magnetic stirrer at 400 rpm for 15 min. from high temperature requirement and the long period of extraction
Afterward, the mixture was centrifuged at 8000 rpm for 15 min and become several considerations to seek for an optional method.
filtered with 3 μm filter paper. The supernatant was then diluted with The MAE uses discrete heating mechanisms that result in lower
150 mL of deionized water, and the pH was adjusted to 1 before treat extraction times compared to conventional hydrothermal extraction
ment with 4 % (w/w) NaOH to increase the pH of the mixture, followed methods using water and ethanol [79]. Additionally, MAE techniques
by centrifuging at 8000 rpm for 15 min. The best flocculating FA also require smaller equipment sizes and are much simpler compared to
recorded was 96 %. conventional extraction methods [101]. Commercially, there are two
In another study by Guo and Ma [90], they combined an alkali so types of MAE systems, namely, closed extraction vessels and focused
lution and thermal treatment (ALT) to extract a bioflocculant from microwave ovens [102]. In microwave extraction methods, the selection
Rhodococcus erythropolis (FA: 94.5 %). In the treatment, the pH value of of the solvent is crucial since microwaves work best in solvents that
the sample was adjusted to pH 10 using 1.0 mol L− 1 NaOH at room possess high dielectric or polar properties such as water and organic
temperature prior to autoclaving at 121 ◦ C for 30 min. HCl was also used solvents [79,82]. Zhang et al. [82] reported that MAE can be carried out
by Sun et al. [91] to extract a bioflocculant (FA: 99.5 %) from sludge due in two ways, which are solvent-free extraction (often applied for volatile
to the ability of HCl to extract flocculation-active ingredients effectively. compounds) and solvent extraction (often applied for non-volatile
They suspended the sludge in HCl (18 %, w/w) and stirred the mixture at compounds). The existence of heat in the extraction process does in
500 rpm for 15 min. Afterwards, the suspension was centrifuged at 8000 crease the yield of the extraction product; however, in MAE processes,
rpm for 15 min to remove the sediment. In addition to a high extraction the temperature must be controlled to not surpass the optimum tem
efficiency, it is also important to note that extraction using acid and perature (water solvent = 50–60 ◦ C, alcohol-based solvent = 79–90 ◦ C)
alkali solutions at high concentrations may cause denaturalization of the because high microwave power and extraction temperatures can cause
bioflocculant itself [96]. thermal degradation of the active constituents, thus leading to a low
extraction yield [79].
4.2. Ultrasound-assisted extraction In the implementation of MAE in the extraction of a bioflocculant
from okra by Lee et al. [101], they used sliced okra and deionized water,
Ultrasound-assisted extraction (UAE) is often called sonication and is which were exposed to a microwave heating system operated at 2450
applied in extraction methods to create cavitation that allow bubbles to MHz since no heating occurred when the frequency was lower or greater
form. During the sonication process, the cavitation that holds the bub than 2450 MHz [102]. After treatment with microwaves, the mixture
bles will become pressurized, and implosion will occur, thus creating a was left for 1 h for cooling and the complete release of the extractants
high-temperature and -pressure microenvironment and accelerating the into water. The supernatant was collected via centrifugation (4000 rpm,
extraction process [81]. Annegowda et al. [97] claimed that sonication 30 min) before drying at 105 ◦ C until a constant mass was achieved. The
was able to reduce the processing period while consuming less energy yield obtained was 53 % under 90 ◦ C heating.
and being environmentally friendly in addition to cost-effective, simple
and reliable. Moreover, sonication can also be used in the extraction of 4.4. Enzyme-assisted extraction
inorganic or organic substances in either the solid or liquid state [81].
However, it is best if the solvents used in UAE are acidic or organic Enzyme-assisted extraction (EAE) is considered an advanced
solvents with low viscosities and surface tensions to avoid the hindrance extraction method with advantages such as efficiency, eco-friendliness,
of cavitation [98]. Moreover, ultrasonic baths and probe units are two sustainability, selectivity and specificity according to the desired
devices that are usually used in the application of ultrasound in the extractant [103]. According to Nadar et al. [103], extraction via EAE can
extraction process [99]. eliminate the excess usage of hazardous solvents and is suitable for
A study by Li et al. [100] used ultrasonic treatment for cell disrup thermo-sensitive products since the application of EAE is carried out
tion. Prior to the sonication treatment, the fermented broth was filtered under controlled temperatures with higher production yields. In the
using a 0.22 μm filter, collected and resuspended in deionized water for extraction of a polysaccharide bioflocculant from Astragalus mem
the ultrasonication process. Ultrasonication was carried out at 200, 250 branaceus, using seven types of enzymes—amyloglucosidade, hemicel
and 300 W and 20 kHz for 20 min. The temperature was set below 20 ◦ C lulose, glucose oxidase, bacterial amylase, fungal amylase, pectinase and
to avoid the deterioration of thermo-sensitive components of the bio cellulose—the amounts of enzymes used were in the range of 0.5 %–5.5
flocculant. However, it was observed that for cell disruption in plants, % at 30–70 ◦ C temperatures for 1–7 days of reaction time and pH values
ultrasonication often uses frequencies greater than 20 kHz. Ramandi ranging from 3.5–13.0 [104]. In the study by Chen et al. [104], the se
et al. [99] used 40 kHz in an ultrasonic water bath with hexane as the lection of enzyme was conducted using response surface methodology
extracting solvent at 48 ◦ C for 30 min. The temperature can be optimised with the highest extraction yield as the indicator of the optimal enzyme.
in the UAE where it should not exceed the boiling point of the extracting Optimising the extraction conditions (enzyme ratio, temperature, pH,
solvent. The effectiveness of UAE in increasing the extraction efficiency and reaction time) is interesting and useful for the application of this
is also supported by Liu et al. [96], who used low-frequency ultrasound technique in future [105,106]. The extraction was further continued by
(80 W, 10 min) in an ice bath to pre-treating sludge before applying a ultrasonic treatment for 30 min. The precipitation of the desired product
solvent extraction technique. was achieved by adding 95 % ethanol for 36 h at 4 ◦ C, and the product
collected via centrifugation at 6000 rpm for 15 min before lyophilization
4.3. Hydrothermal and microwave-assisted extraction to obtain the crude product.
Based on the study by Chen et al. [104], the types of enzymes used
Hydrothermal and microwave-assisted extraction methods (MAE) during extraction process play a vital role in determining the yield of the
are considered advanced and modern extraction techniques. Hydro desired product in addition to the amounts of enzymes applied. This is
thermal extraction utilizes water bath accompanied with rigorous because using too little enzymes causes incomplete enzyme treatment
agitation and heating processes. This technique requires high tempera and using too much is not cost effective [103]. Moreover, the effect of
ture (above 70 ◦ C) with a long extraction time (minimum of 30 min. with the pH and temperature in performing EAE cannot be neglected since
average of 2 h). Extraction was occurred via heat transfer from the both factors play specific roles for every enzyme used. Most enzymes
heating medium (in water bath) to the extraction vessel. Transferred have an optimum pH value within the range of the isoelectric pH of
9
proteins [103] and work best at temperatures ranging from 30 to 55 ◦ C drying and dialysis to purify a bioflocculant. The bioflocculant was
[104]. The application of EAE beyond these temperatures will eventu hydrated for 2 h in order to eliminate all water and solvent using a rotary
ally result in a lower yield of the desired product due to the loss of evaporator prior to dialysis against distilled water. The mixture was then
enzyme activities [103,104]. kept overnight at 4 ◦ C until further use. Vacuum drying was also utilized
in the study by Sun et al. [91]. However, they used 40 mL of cold ethanol
5. Purification of bioflocculants and adjusted the mixture to pH 8 using 1 % (w/w) NaOH with contin
uous stirring, with the mixture kept at 4 ◦ C for precipitation to occur.
The final step in the production of a bioflocculant is purification. The The precipitate was then collected, centrifuged at rpm for 15 minutes15
purification step is necessary to extract the active coagulating agents and min, washed with distilled water, evaporated for 2 h in a rotary evap
eliminate the undesired substances from the extracted bioflocculant orator and vacuum-dried for 12 h.
[94]. The purification of bioflocculants can be carried out thorough
lyophilisation, chromatography, and dialysis as summarised in Fig. 4. 6. Scaling-up challenges of bioflocculant production
Agunbiade et al. [50] adopted lyophilisation to obtain the purified
bioflocculant. However, before the lyophilisation steps were carried out, Although most studies have reported the effectiveness of bio
they dissolved the crude bioflocculant in water before a volume of flocculants in water and wastewater treatment, most of this research was
chloroform and n-butyl alcohol (5:2 v/v) mixture was added. After carried out at the laboratory scale and is not widely applicable in real
wards, the mixture was stirred thoroughly, poured into a separating wastewater treatment plants (WWTPs). The development and applica
funnel and left for 12 h at room temperature. After the supernatant was tion of bioflocculants at the industrial scale is observed to be restricted
removed, two volumes of ethanol were added for bioflocculant precip due to the sensitivity of the products to the preparation process, mod
itation before lyophilisation took place. erate flocculating efficiencies [62], easy degradability over time and
Fujita et al. [107] used chromatography in an aqueous solution to higher production cost (Bukahri et al., 2020)—especially downstream
obtain a purified bioflocculant. Approximately 10 mM phosphate buffer costs (extraction and purification)—compared to commercial floccu
was used to adjust the pH of the crude bioflocculant to pH 5 before lants [9]. Salehizadeh et al. [13] reported that the production of bio
application to a Gigapite amphoteric column (Seikagaku Kogyo Co., flocculants through microbial bioprocesses is high cost in terms of
Tokyo; 3.2 cm × 18 cm) with the same buffer used (flowrate of 28 mL carbon sources [85], and the huge amount of solvents required during
h− 1). The bioflocculant was further purified through subjection to a the extraction and purification processes make them consequently hard
Sephadex 200HR column (Pharmacia Co., Sweden; 3.2 cm × 120 cm) to scale up commercially.
before elution by 10 mM phosphate buffer at flowrate of 8.2 mL h− 1. Mu The production of bioflocculants from mixtures of microorganisms,
et al. [108] also used chromatography to purify bioflocculants with on the other hand, is quite difficult to understand since the bio
some modifications. For double purification using chromatography, flocculants produced are also complex in form, and their functional
first, they resuspended the bioflocculant in 3 mL deionized water and molecules have not yet been thoroughly studied [108]. Additionally, the
loaded it onto an anion-exchange chromatography column of DEAE-52 production of bioflocculant from natural resources might also yield a
Cellulose (1.5 × 50 cm). Sodium chloride (NaCl) was used to remove proportion of ineffective ingredients for flocculation, thus affecting the
the absorbed substance at flow rate of 1 mL min− 1, and the eluent was overall production cost due to the requirement of eliminating the inef
collected and loaded onto a Sephadex G-100 for gel permeation chro fective ingredients [108]. Moreover, conflict in deciding the most suit
matography (1.5 × 50 cm), with deionized water used to elute the able bioflocculant with which to treat wastewater may arise since the
mixture before the lyophilisation step to yield a pure fraction of characteristics of the produced bioflocculants often vary according to
bioflocculant. the bacterial species and carbon and nutrient sources. In addition to
On the other hand, Karthiga and Natarajan [88] utilized vacuum challenges from the production of bioflocculant itself, the application of
Fig. 4. Purification method for bioflocculant.
10
bioflocculants in wastewater treatment may be inappropriate due to the [8] T. Rashid, F. Sher, A. Hazafa, R.Q. Hashmi, A. Zafar, T. Rasheed, S. Hussain,
Design and feasibility study of novel paraboloid graphite based microbial fuel cell
extreme pH values and concentrations of wastewater [94]. Optimal
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Abu Bakar Et Al.. 2021. A Review of The Production Process of Bacteria-Based Polymeric Flocculants

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Journal of Water Process Engineering 40 (2021) 101915

Contents lists available at ScienceDirect

Journal of Water Process Engineering

A review of the production process of bacteria-based polymeric flocculants

1. Introduction Coagulants and flocculants are applied to allow particles or pollutants

Fig. 1. Classifications of flocculants.

Microbial consortium (Origins)

*n.m: not mentioned.

Fig. 2. Process of bioflocculant production.

Fig. 3. Extraction methods for bioflocculants.

*Notes: CPC: cetylpyridinium chloride solution.

Fig. 4. Purification method for bioflocculant.

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