Accepted Manuscript

Recent advances in polysaccharide bio-based flocculants

Hossein Salehizadeh, Ning Yan, Ramin Farnood

PII: S0734-9750(17)30122-2
DOI: doi:10.1016/j.biotechadv.2017.10.002
Reference: JBA 7160
To appear in: Biotechnology Advances
Received date: 27 May 2017
Revised date: 9 September 2017
Accepted date: 5 October 2017

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 ACCEPTED MANUSCRIPT

Recent advances in polysaccharide bio-based flocculants

Hossein Salehizadeha, Ning Yana,b, Ramin Farnooda,*

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Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200

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College St., Toronto, Ontario, Canada M5S 3E5

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b
Faculty of Forestry, University of Toronto, 33 Willcocks St., Toronto, Ontario, Canada M5S
3B3

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Abstract

Natural polysaccharides, derived from biomass feedstocks, marine resources, and

microorganisms, have been attracting considerable attention as benign and environmentally

friendly substitutes for synthetic polymeric products. Besides many other applications, these

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biopolymers are rapidly emerging as viable alternatives to harmful synthetic flocculating agents

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for the removal of contaminants from water and wastewater. In recent years, a great deal of effort

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has been devoted to improve the production and performance of polysaccharide bio-based

flocculants. In this review, current trends in preparation and chemical modification of

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polysaccharide bio-based flocculants and their flocculation performance are discussed. Aspects
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including mechanisms of flocculation, biosynthesis, classification, purification and

characterization, chemical modification, the effect of physicochemical factors on flocculating

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activity, and recent applications of polysaccharide bio-based flocculants are summarized and
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presented.
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Keywords
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Bio-based, Bioflocculant, Biopolymer, Colloids, Flocculant, Flocculation, Grafting,

Polymerization, Polysaccharide, Wastewater treatment

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Corresponding authors: R. Farnood (E-mail: ramin.farnood@utoronto.ca, Tel: 416 946 7525);

N. Yan (E-mail: ning.yan@utoronto.ca, Tel: 416 946 8070); H. Salehizadeh (E-mail:

Hossein.salehizadeh@utoronto.ca)

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Abbreviations

ADAC: Anionic dialdehyde cellulose derivatives,

Alg: Alginate,

AM: Acrylamide,

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APTAC: 3-(acrylamidopropyl) trimethyl ammonium chloride,

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ATPPB: Allyl triphenyl phosphonium bromide binary,

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BOD: Biochemical oxygen demand,

BPC: Bamboo pulp cellulose,

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C: Conversion,

CAFS: Combinational aluminum, ferrous and starch,

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CAMFA: Chitosan-acrylamide-fulvic acid,

CEC: N-carboxyethylated chitosan,

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CHES: Cationized hydroxyethylated starch,

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CHPTAC: N-3-Chloro-2-hydroxypropyl trimethyl ammonium chloride,

CMC: Carboxymethyl chitosan,

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CMS: Carboxymethyl starch,

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CNC: Cellulose nanocrystals,

CNF: Cellulose nanofibirils,

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COD: Chemical oxygen demand,

CPC: Cetylpyridinium chloride;

CR: Color removal,

CTA: 3-chloro-2-hydroxypropyl trimethyl ammonium chloride,

CTAB: Cetyltrimethyl ammonium bromide,

DC: Degree of cationization,

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DCC: Dicarboxyl cellulose,

DD: Degree of deacetylation,

DOT: Dissolved oxygen tension;

Dp: Particle size,

DMA: N,N-dimethyl acrylamide,

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DP: Degree of polymerization,

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DS: Dry solids,

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EDTA: Ethylenediaminetetraacetic acid,

FA: Fraction of acetylated units,

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FA: Flocculating activity,

FC: Fecal coliforms,

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FCP: Filter cake permeability,
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G%: Precentage of grafting,

g: graft,
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G: (1,4) α-L-Guluronic acid,

GDPG: Guanidine diphosphoglucose,

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GE: Grafting efficiency,

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GR: Grafting ratio,

HE: Helminth eggs,

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HES: Hydroxyethyl starch,

HPTAC: Hydroxypropyltriammonium chloride,

Ku: Kundoor mucilage,

M: (1,4) β-D-Mannumaric acid,

METAC: 2-(methacryloyloxy) ethyl trimethyl ammonium chloride,

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MPA: Membrane-periplasmic,

MPN: Most probable number,

MW: Molecular weight,

NA: Not applicable,

NS: Not specified,

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NVP: N-vinyl-2-pyrrolidone,

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NTU: Nephelometric turbidity unit,

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OFC: Optimum flocculant concentration,

OMA: Outer membrane,

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P: Pullulan,

PAC: Polyaluminium chloride,

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PD: Polydispersity,
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PAF: Polyaluminium ferric chloride,

PAM: Polyacrylamide,
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PDMC: Poly(2-methacryloyloxyethyl) trimethyl ammonium chloride,

PMMA: Polymethyl methacrylate,

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PNVCL: poly(N-vinylcaprolactam),
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PSW: Potato starch wastewater,

QCMC: Quaternized carboxymethyl chitosan,

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QL: Trimethyl quaternary ammonium salt of lignin,

QS: Quorum sensing,

Quat 188: N-(3-chloro-2-hydroxypropyl) trimethyl ammonium chloride,

SAG: Sodium alginate,

SD: Substitute degree,

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S.G.: Specific gravity,

SRF: Specific resistance to filtration,

SS: Suspended solids,

STC: Starch,

T: Turbidity,

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TC: Total coliforms,

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TR: Turbidity removal,

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TDS: Total dissolved solids,

TOC: Total organic carbon,

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TS: Total solids,

TSS: Total suspended solids,

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TTF: Time to filter,
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UDPG: Uridine Diphosphoglucose,

Xyl: Xylan.
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1. Introduction

Flocculants are used as additives in a wide range of industrial applications, including water and

wastewater treatment, food and beverages, mining, dyes and textile, and fermentation and its

downstream processing. Flocculants bring colloidal and other particles suspended in a liquid

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together to form larger particles (or flocs) for promoting the settling of these particles from the

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stable suspension. Therefore, they have been extensively applied for removing turbidity,

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suspended and dissolved solids, colors and dyes, and chemical oxygen demand (COD) in

sedimentation and clarification processes (Lee et al., 2014a,b; Li et al., 2017; Liu et al., 2017;

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Pathak et al., 2017; Salehizadeh and Yan, 2014; Teh et al., 2016).
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In general, flocculants can be divided into three groups: i) inorganic flocculants, such as alum
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and polyaluminum chloride; ii) synthetic organic flocculants, such as polyacrylamide (PAM)

derivatives and polyethyleneimine (PEI), and iii) natural-based or naturally occurring polymeric
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flocculants, such as chitosan, sodium alginate, cellulose, lignin, tannin, and the microbial
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flocculants (bioflocculants).

Inorganic and synthetic organic flocculants are the most commonly used flocculants in industry
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today due to their effective flocculation activity and low cost. But, their usage poses some
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serious environmental and health challenges (Okaiyeto et al., 2016). For example, acrylamide

monomer is carcinogenic and neurotoxic to humans (Ruden, 2004) and aluminum salt

flocculants can induce Alzheimer's disease (Campbell, 2002). In addition, inorganic flocculants

have other limitations related to their relatively high dosage requirement, high pH sensitivity,

and poor efficiency for the coagulation of very fine particles (Sharma et al., 2006).

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Polysaccharide bio-based flocculants such as alginate, chitosan, cellulose and starch are

generally derived from seaweeds, arthropods, and plants. Figure 1 shows chemical structures of

major polysaccharide bio-based flocculants (Babu et al., 2013; Daza et al., 2016; Draget, 2009;

Prashanth and Tharanathan, 2007; Yang et al., 2011).

Among various kinds of flocculants, polysaccharide bio-based flocculants are particularly

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attractive because of their biodegradability and high capability (many) of removal of turbidity,

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COD, solids, colors and dyes. In recent years, polysaccharide bio-based flocculants have

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received considerable attentions for usage in water and wastewater treatment, textile, mining,

cosmetology, pharmacology, food and fermentation industries due to their many advantages,

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such as their large availability, environmental friendliness, biodegradability, and attractive
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molecular structural features (Grenda et al., 2017; Kolya et al., 2017; R. Yang et al., 2016; Saeed

et al., 2011). These flocculants are mainly derived from chitosan, alginate, cellulose, starch, and
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microbial-based raw materials. They are typically nontoxic, biodegradable, thermo-stable (to
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some extent), and shear-stable (R. Yang et al., 2016; Tao et al., 2017). Furthermore, they may be

chemically modified to improve their flocculation performance (Singh et al., 2014, Wu et al.,
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2015). Broad industrial application of polysaccharide bio-based flocculants depends on lowering

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the production cost by using cheaper biomass (substrate) sources and developing more efficient

fermentation and recovery processes, as well as applying cost-effective chemical modification of

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the extracted polysaccharides for improved performance (Babu et al., 2013; H. Li et al., 2015;

Nunez et al., 2013; Nurul-Adela et al., 2016; Oh et al., 2015, Salehizadeh and Yan, 2014; Zhao

et al., 2017).

This review focuses on the recent progress in polysaccharide bio-based flocculants. In particular,

the manuscript provides a summary of advances in production, purification, modification,

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characterization, and applications of these emerging chemicals. Challenges, limitations, and

knowledge gaps related to polysaccharide bio-based flocculants are presented and perspectives

for further studies are highlighted. The present paper provides an introduction to major bio-based

flocculants, such as alginate, chitosan, cellulose, starch, pullulan, xanthan, and pectin in section

1. Section 2 describes the marine, plant and microbial origins of polysaccharide bio-based

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flocculants. In section 3, biosynthesis and role of genetic engineering are explained and

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biosynthesis of various bio-based polysaccharides, such as chitosan, cellulose, xylose, alginate,

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and pullulan, are summarized. Production, purification and characterization of polysaccharide

bio-based flocculants are presented in section 4. In this section, production of polysaccharide

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bioflocculants by pure culture, preparation, extraction and purification, and their characteristics
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are presented. Section 5 describes mechanisms of polysaccharide bio-based flocculants,

especially focusing on bridging and charge neutralization mechanisms. In section 6, chemical

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modification of polysaccharide bio-based flocculants by grafting methods and the effects of

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reactants, reactants dosages and their ratios, reaction temperature, pH, and reaction time are

addressed. The effect of physic-chemical factors on flocculating activity of chemically modified

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bio-based polysaccharide flocculants and bioflocculants, such as flocculant dosage, flocculant

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molecular weight, initial pH, temperature, metal ions are discussed in section 7. Finally,

emerging application fields of polyssacharide flocculants including drinking water treatment,

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COD and turbidity removal, decolorization and dye removal, biomass harvesting and cell

recovery, mining and metal recovery of nanoparticles synthesis, and sludge dewatering for both

major polysaccharides flocculants and chemically modified polysaccharide flocculant are

summarized in section 8.

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1.1. Major types of polysaccharide bio-based flocculants

1.1.1. Alginate

Alginate is a naturally occurring linear, anionic polysaccharide consisting of (1→4)-linked β-D-

mannuronate (M-block) and α-L-guluronate (G-block) residues (Fig. 1a). Over 200 different

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alginates have been identified which vary in length and array of M and G monomer units

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(Draget, 2009; Lee and Mooney, 2012). Alginates were first extracted by Stanford from

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seaweeds in 1881, and their commercial production, mainly from the genera

Laminaria and Macrocystis (>40% dry matter of these seaweeds) started in the early twentieth

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century. Alginates are intracellular structural components in algae (brown seaweeds) and play a
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role similar to cellulose in plants (Remminghorst and Rehm, 2006), but they are also produced

extracellularly in bacteria. The production of alginates is estimated to be more than 30,000

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tonnes per year (Darget, 2009). Alginates are biodegradable, biocompatible, non-toxic, and non-
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immunogenic biopolymer polyelectrolytes (Yang et al., 2011). They have been applied widely in

biomedical, pharmaceutical and biotechnology fields due to their desirable characteristics,

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versatility and biocompatibility (Hay et al., 2013). The commercial sodium alginates have an
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average molecular weight in the range between 32,000 and 400,000 g/mol. Alginates with high

M-block content are more immunogenic (by 10 times) compared with those with high G-block
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contents. However, no considerable inflammatory response was reported using commercially

available, highly purified alginates (Lee and Mooney, 2012).

Over the past years, natural alginates have been extensively studied due to their flocculation and

decolourization capabilities (Diaz-Barrea et al., 2014; Rani et al., 2013; Sand et al., 2010; Yang

et al., 2011; Yuan et al., 2013; Zhao et al., 2012). The strong flocculation properties and

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adsorption ability of alginates stem from the free hydroxyl and carboxyl groups distributed along

their polymer chain (Yang et al., 2011).

1.1.2. Chitosan

Chitosan is one of the most important natural-based biopolymers, derived commercially by

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heterogeneous alkaline deacetylation of chitin. Chitin is a long-chain polymer and a derivative of

glucose that can be found naturally in many places including exoskeletons of crabs, lobsters, etc.

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Chitin is an abundant naturally occurring biopolymer (up to 1 × 10 13 kg) (Cauchie, 2002). It was

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first discovered in 1811 by Henri Braconnot, a French chemist. In 1843, Lassaigne reported the

presence of nitrogen in chitin structure and subsequently, in 1878, Ledderhose discovered that
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chitin was composed of glucosamine and acetic acid (Kavitha et al., 2011). Chitin is composed
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of a high amount of A-units which results in it being insoluble in water, while its high content of

D-units renders it soluble in acidic aqueous solutions (pH < 5.7). Chitosan is a linear, cationic
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polysaccharide composed of randomly repeating units of β (1→4)-linked 2-acetamido-2-deoxy-

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D-glucopyranose (A-unit; the neutral sugar unit GlcNAc) and 2-amino-2-deoxy-D-

glucopyranose (D-unit, the positively charged sugar unit GlcN) (Fig. 1b). The amount of D-units
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in chitosan is often more than 60%. It shows different degrees of deacetylation (DD) between
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40–90% and is available commercially in various molecular weights ranging from 50,000 to

2,000,000 Da. The contents of the A-units in chitosan can vary from 0.7 (70% acetylated) to 0

(0% acetylated, all units charged). Therefore, chitosan can be considered as ampholyte (enriched

in A-units) or polyelectrolyte (enriched in D-units) (Alves and Mano, 2008; Bhumkar et al.,

2006; Kaur and Dhillon, 2014; Nilsen-Nygaard et al., 2015).

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The annual commercial production of chitosan and glucosamine from chitin is estimated at 2000

and 4000 tons, respectively (Sandford, 2003). Chitosan is generally derived from arthropods,

crustaceans (shrimp and crabs), fungi, and yeast (Prashanth and Tharanathan, 2007). Crustacean

shells are a well-known source for industrial production of chitosan, however, this process: i)

requires large quantities of chemicals under higher temperatures and a long reaction time; ii) has

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negative environmental impact, seasonal supply limitation, and high cost and laborious process;

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iii) involves demineralization treatment to remove calcium carbonate; iv) results mainly in high

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molecular weight chitosan with protein contamination that limit applications in the biomedical

area.

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Chitosan is one of the most promising natural substitutes for commercial synthetic polymer
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flocculants. Chitosan exhibits unique flocculation properties among biopolymer flocculants,

especially due to the presence of primary amino groups (Guibal et al., 2006; Rojas-Reyna et al.,
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2010). The polycation of chitosan displays great properties such as biodegradability,

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biocompatibility, physical and biological activity, flocculability; also its monomers are non-

toxic, immunological, and antibacterial (Alves and Mano, 2008).

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1.1.3. Cellulose

Cellulose is a linear polysaccharide consisting of β (1→4) D-glucose units (Fig. 1c). Cellulose is
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the most abundant natural biopolymer resource on earth, with a global economic importance.

The annual production rate of cellulose is estimated at 10 11 –1012 t/y. Cellulose can be derived

from different origins, such as plants and wood, animals and microorganisms (Kim et al., 2006;

Roy et al., 2009).

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Cellulose was first discovered in 1838 by Anselme Payen by isolating from plant matter. The

polymeric structure of cellulose was first demonstrated by Staudinger in 1920. Five allomorphic

forms of cellulose have been known based on the location of hydrogen bonds between and

within strands. Natural cellulose, or cellulose I, has two different crystalline structures, namely

Iα and Iβ. Cellulose may be also found with other crystal structures including cellulose II, III and

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IV. Cellulose II is the most stable structure and can be obtained by alkali treatment of cellulose I

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(Roy et al., 2009). Cellulose is insoluble in water and most organic solvents. It becomes

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amorphous in water at 320 °C and 25 MPa. It can be converted chemically into its glucose units

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by reacting with concentrated acids at high temperatures (Deguchi et al., 2006).

Plant-based cellulose is usually found in a mixture with hemicellulose, lignin, pectin and other
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substances, while bacterial cellulose is completely pure, with a much higher water content and a
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higher tensile strength (Klemm et al., 2005). Hemicellulose materials include various monomers

such as xylan in their structure and can be converted chemically to polysaccharide bio-based
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flocculants (S. Wang et al., 2015).

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Bacterial cellulose is produced by certain bacteria, such as Acetobacter xylinum, was first

discovered by Brown in 1886 for protection against ultraviolet radiation, harsh chemical
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environment, and oxidation (Huang et al., 2014). Cellulose can be considered as an attractive
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alternative to produce environmentally friendly functional materials (Chauhan and Yan, 2016,

2017; Vollick et al., 2017) and chemicals such as biopolymer flocculants due to its physical

characteristics and chemical reactivity (Roy et al., 2009).

1.1.4. Starch

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Starch is a naturally occurring, linear, biodegradable, inexpensive polysaccharide biopolymer

originating from plants. Starch grains from the rhizomes of Typha have been found on grinding

stones in Europe dating back to about 30,000 years ago. Starch is a product of photosynthesis in

plants largely available in nature; it is derived from various botanical sources, including wheat,

rice, corn, barley, sorghum, millet, rye, legumes, banana, mango, potato, cassava and so on.

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Cassava and maize were known as the major sources of starch on a commercial scale for a long

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time (Ashogbon and Akintayo, 2014; Revedin et al., 2010).

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Starch is insoluble in cold water, alcohol, or other solvents but soluble in hot water. It has

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received much attention among natural biopolymers because of its properties including

biodegradability, large availability, low cost, and versatility in applications. Crude starch
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includes a mixture of amylase and amylopectin. Depending on the source of starch, amylase and

amylopectin are present at levels up to 25% and 95%, respectively. The Tg of starch varies in the
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range of -50 °C and 110 °C, and has a modulus similar to polyolefins. Amylose is a linear
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polymer of α (1→4) D-glucopyranosyl units (Fig. 1d), while amylopectin has a highly branched

polymer of D-glucopyranosyl residues linked together by α (1→4) and α (1→6) bonds. The
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molecular weight of starch varies in the range of 104 –107 g/mol (Athawale and Lele, 2000; Babu
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et al., 2013; Nair and Jyothi, 2014).

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1.1.5. Pullulan

Pullulan, α-1,6-linked maltotriose units (Fig. 1e), is a water-soluble linear exopolysaccharide

biopolymer. Pullulan was first discovered by Bauer in 1938. It can be produced by fermentation

of a yeast-like fungus, Aureobasidium pullulans. Pullulan has unique physicochemical properties

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due to a glycosidic linkage pattern such as high water solubility and low viscosity, adhesive

ability, the capacity to form thin and biodegradable film and so on. Pullulan has numerous

applications such as food and cosmetic additives, blood plasma substitutes, flocculant, resins,

thin-film and adhesive additives. It has been also used in pharmaceutical and biomedical

applications such as targeted drug, gene delivery, tissue engineering, wound healing, and even in

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diagnostic imaging medium (Babu et al., 2013; Cheng et al., 2011; Singh et al., 2008). The

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molecular weight of produced pullulan was reported in the range of 4.5 × 10 4 to 6 × 105 Da and

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greatly influenced by cultivation conditions (Cheng et al., 2011).

1.1.6. Xanthan
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Xanthan is a branched polysaccharide with high affinity for water and ability to increase the

solution viscosity at low concentrations. It can be derived from microbial sources. Xanthan was
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first discovered by Allene Rosalind Jeanes, and its commercial production was carried out by the
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Kelco Company under the trade name Kelzan in the early 1960s (Petri, 2015). Xanthan gum has

been produced mainly by the bacterium Xanthomonas campestris on a commercial scale

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(Palaniraj and Jayaraman, 2011; Rosalam and England, 2006). Xanthan has broad industrial
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applications including foods and fermentation, toiletries, oil recovery, cosmetics, wastewater

treatment, and so on, due to its superior rheological properties. The backbone of xanthan is the
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same as cellulose β (1→4) D-glucose and side-chains consisting of β (1→4) D-mannose, β

(1→2) D-glucuronic acid and D-mannose, which are linked to glucose residues in the backbone

by α (1→3) linkages (Fig. 1f) (Desplanques et al., 2012; Nwodo et al., 2012). Until now, there

have been only a few reports about the flocculation activity of xanthan and its applicability as a

flocculating agent (Ghorai et al., 2013; Zhu et al., 2009).

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1.1.7. Pectin

Pectin is a water-soluble heterogeneous polysaccharide with major components of α-D-

galacturonic acid and the carboxyl groups of α (1→4)-D-galacturonic acid, which are free or

esterified with methyl groups (Fig. 1g). Homogalacturonan is a major component of pectin and

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can be extracted using hot water or chelating agents. Pectin is derived mainly from plant cell

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walls and agricultural and food-industrial wastes of fruits, such as the peel of citrus fruits (e.g.,

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lemon, lime, orange and grapefruit), and apple pomace (Patova et al., 2014; Schols and Voragen,

1996). Pectin has flocculating activities and can be used as a flocculant in various suspensions.

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Flocculating activity of pectin in a kaolin suspension was considerably enhanced by the addition
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of Al3+ and Fe3+ to the suspension (Ho et al., 2009, 2010).

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2. Source of polysaccharide bio-based flocculants

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Polysaccharide bio-based flocculants can be classified into three main groups based on their

source: i) marine polysaccharide flocculants; ii) microbial polysaccharide flocculants, and iii)
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plant polysaccharide flocculants. Polysaccharides, as a sort of natural flocculant, can be

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generally derived from natural origins, such as crustacean shell wastes, agricultural/forestry

feedstocks, and microorganisms. Cellulose, starch, etc. have been derived from
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agricultural/forestry feedstocks, whereas alginates, chitosan and microbial polysaccharide are

commonly extracted from seaweeds, crustacean shells, and microorganisms. Microorganisms,

including bacteria, yeast, fungi, and algae, are the other important sources of polysaccharide

bioflocculants.

Table 1 lists some of the polysaccharide bio-based flocculant producers according to their origin.

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Plant-based polysaccharide flocculants derived mainly from Mellocactus sp. (Daza et al., 2016),

Plantago psyllium (Mishra et al., 2002, 2004), Malva sylvestris (Anastasakis et al., 2009),

Tamarindus indica (Mishra and Bajpai, 2006), Trigonella foenum-graecum (Srinivasan et al.,

2008), Plantago ovate (Al-Hamadani et al., 2011), and Abelmoschus esculentus (Lee et al.,

2014a, 2015).

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Very few genera of bacteria, such as Agrobacterium tumefaciens and Acetobacter xylenium, can

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produce cellulose extracellularly (Li et al., 2011; Keshk et al., 2014). A cellulose-based

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bioflocculant, produced by Agrobacterium tumefaciens F2 (former name, Rhizobium

radiobacter) isolated from soil, was able to flocculate various wastewaters (Li et al., 2011).

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Alginates are produced from microorganisms and brown seaweeds (Draget, 2009; Nedovic and
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Willaert, 2004). Those are commercially produced from three main algal species including

Macrocystis pyrifera (Cazon et al., 2014; Gomez et al., 2009), Laminaria hyperborean (Vea and
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Ask, 2011), and Ascophyllum nodosum (Ugarte and Sharp, 2012). Other brown seaweeds
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sources, such as Durvillaea Antarctica (Kelly and Brown, 2000), Laminaria japonica (Oh et al.,

2015), Laminaria digitara (Fertah et al., 2014), Sargassum siliquosum, Turbinaria conoides,
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Sargassum baccularia, Sargassum binderi (Chee et al., 2011), Eclonia maxima and Lesonia
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negrescens (Kim et al., 2011) are used less often to produce alginates. The properties of

polysaccharide bio-based flocculants vary with the origin from which they derive. For example,
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the alginate derived from L. hyperborea (G 45–70%) exhibited the highest content of L-

guluronic acid residues (G-block) than M. pyrifera (G 40%) and A. nodosum (G 35%) (Lee and

Mooney, 2012; Nedovic and Willaert, 2004).

Table 1 List of polysaccharide bio-based flocculants producers

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Microorganism/Plants/ Species Source Structure Reference

Marine polysaccharide flocculants

Ascophyllum nodosum Brown seaweeds Alginate (Ugarte and Sharp, 2012)

Durvillaea antarctica Brown seaweeds Alginate (Kelly and Brown, 2000)

Laminaria digitata Brown seaweeds Alginate (Fertah et al., 2014; Vauchel et

al., 2008)

Laminaria hyperborea Brown seaweeds Alginate (Lee and Mooney, 2012)

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Laminaria japonica Brown seaweeds Alginate (Oh et al., 2015)

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Macrocystis pyrifera Brown seaweeds Alginate (Gomez et al., 2009)

Sargassum sp. Brown seaweeds Alginate (Chee et al., 2011)

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(siliquosum, Turbinaria conoides,
Sargassum baccularia, Sargassum
binderi)
Metapenaeus monoceros Shrimp shell Chitosan (Younes et al., 2014)
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Penaeus longirostris Shrimp shell Chitosan (Sila et al., 2014)

Red queen crab Crab shell Chitosan (Setoguchi et al., 2012)

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Plant polysaccharide flocculants

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Abelmoschus esculentus Orka seeds Anionic Polysaccharide (Lee et al., 2015)

Cassava (Manihot esculenta) Cassava roots Starch (Jadhav and Mahajan, 2013)
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Cassia tora & Cassia obtusifolia Leguminous plant Cationic Cassia (Banerjee et al., 2014)

Citrus maxima Pomelo peel Pectin (Piriyaprasarth and Sriamornsak,

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2011)

Guar gum (Guaran) Endosperm of Guar Polysaccharide (Mukherjee et al., 2013)

beans (Galactomannan)
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Malva sylvestris Mallow seed pods and Anionic Polysaccharide (Anastasakis et al., 2009)
lobs

Phyllostachys heterocycla Bamboo forest Dicarboxyl cellulose (Zhu et al., 2015a)

Plantago ovata Isabgol seed husks Anionic Polysaccharide (Al-Hamadani et al., 2011)
(Xylan backbone)

Plantago psyllium Psyllium seed husks Anionic Polysaccharide (Agarwal et al., 2002)

Strychnos potatorum Nirmali seeds Anionic Polysaccharide (Jadhav and Mahajan, 2013)

Tamarindusindica T amarind seeds Polysaccharide (Mishra and Bajpai, 2006)

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Trigonella foenum -graecum Fenugreek seeds Polysaccharide (Srinivasan et al., 2008)

Microbial polysaccharide flocculants

Absidia butleri NS (NCIM 977) 1 Chitosan (Vaingankar and Juvekar, 2014)

Absidia coerulea NS (AT CC 14076)2 Chitosan (Nwe et al., 2011)

Agrobacterium tumefaciens Soil Cellulose (Li et al., 2011)

Aspergillus japonicus Culinary leaf Pullulan (Maghsoodi et al., 2009)

Aspergillus niger NS Chitosan (Maghsoodi et al., 2009;

(AT CC 9642) 2, (BBRC Pochanavanich and Suntornsuk,
20004)3 (TISTR3245)4 2002; Tayel et al., 2011 )

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Soil Chitosan (Cheng et al., 2014)
Aspergillus terreus

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Aureobasidium Soil Pullulan (Cheng et al, 2011)
pullulans

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Azotobacter chroococcum Soil Alginate (Khanafari et al., 2007)

Azotobacter indicus Soil Alginate (Patil et al., 2011)

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2
Azotobacter vinelandii Soil, NS (AT CC 9046) Alginate (Diaz-Barrea et al., 2014; Nunez et

al., 2013)
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Basidiomycetes (Agaricus sp., Pleurotus Forestry Plant Chitosan (Kannan et al., 2010)

sp. and Ganoderma sp.)

Botryodiplodia theobromae Medicinal Plant Chitosan (George et al., 2011)

M

4
Candida albicans NS (T IST R5239) Chitosan (Pochanavanich and Suntornsuk,

2002)
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Cladosporium cladosporioides Medicinal Plants Chitosan (George et al., 2011)

Cryphonectria parasitica Bark sample Pullulan (Forabosco et al., 2006)

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Cunninghamella bertholletiae NS (IFM ) 5 Chitosan (Amorim et al., 2006)

1
Cunninghamella blakesleeana NS (NCIM 687) Chitosan (Vaingankar and Juvekar, 2014)
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Cunninghamella echinulata NS (NCIM691)1 Chitosan (Vaingankar and Juvekar, 2014)

Fomitopsis pincola Soil Chitosan (Kaya et al., 2015)

Gluconabacter sp. Fruit Cellulose (Jahan et al., 2012)

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Gluconacetobacter hansenii Rotten apple Cellulose (Park et al., 2003)

Gluconacetobacter sacchari NS (AT CC10245) 2 Cellulose (El-saied et al., 2008; Gomes et al.,

2013)

Gluconacetobacter xylinus Nata (Coconut region) Cellulose (Keshk, 2014)

(Acetobacter xylinum)

Gongronella butleri NS (USDB 0201) 6 Chitosan (Maghsoodi et al., 2009; Streit et

al., 2009)

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Clover roots (Trifolium Cellulose (Robledo et al., 2012)

Rhizobium endoglucanase
repens)

Rhizomucor pusillus NS Chitosan (Driessel and Christov, 2002)

Rhizopus oryzae T IST R3189, NCIM Chitosan (Kleekayai and Suntornsuk, 2011)
1009, TISTR3189 4
Zygosaccharomyces rouxii NS (T IST R5058)4 Chitosan (Pochanavanich and Suntornsuk,
2002)
1
NCIM: National Collection of Industrial Microorganism (NCIM) Pune, India.
2
AT CC: American T ype Culture Collection, Manassas, USA.

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3
Biochemical and Bioenvironmental Research Centre, Sharif University of T echnology, T ehran, Iran.

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4
T IST R: T hailand Institute of Scientific and T echnological Research, Bangkok, T hailand.
5
IFM: Institute for Food Microbiology, Chiba University, Japan.

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6
USDB: Department of Biological Sciences, National University of Singapore, Singapore.

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3. Biosynthesis and role of genetic engineering

The biosynthesis pathway of exopolysaccharide bio-based flocculants can be generally classified

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into four main steps as follows: i) precursor synthesis; ii) cytoplasmic membrane transfer and
M

polymerization; iii) periplasmic modification/transit, and iv) export through the outer membrane

(Hay et al., 2013; Lee and Mooney, 2012). The biosynthesis of polysaccharide flocculant is a
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complex multi-stage process which involves many enzymes and proteins localized in the inner
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membrane and the outer membrane. Many precursors, nucleotide diphospho-sugars, are

contributed in transferring and synthesizing of polysaccharide-based flocculants attached to an

CE

acceptor. The enzyme specificity, primer substances, and the structure of the cytoplasmic
AC

membrane have influence on the structure, control and organization of specific polysaccharides

(Skorupska et al., 2006).

Though several studies have reported on the biosynthesis of polysaccharides flocculants (Chen et

al., 2017; Cheng et al., 2011; Hay et al., 2013; Jiang et al., 2013; O. Li et al., 2015; Pawar and

Edgar, 2012; Remminghorst and Rhem, 2006; Robledo and Rivera, 2012; Sandhu et al., 2009;

Skorupska et al., 2006; Stone et al., 2011; Yan et al., 2013), this process is not fully understood
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yet, and significant knowledge gaps remain regarding the role of genetic engineering and

biosynthesis pathways.

3.1. Chitosan biosynthesis

Chitosan are synthesized in different fungi, such as Aspergillus nidulans, Rhizopus oryzae,

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Colletotrichum lindemuthianium, and Mucor rouxii using two important enzymes, including

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chitin synthase (EC 2.4.1.16) and chitin deacetylase (CDA, EC 3.5.1.41). Firstly, chitin synthase

CR
forms a chain of chitin using the uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), and

monomers of GlcNAc are coupled in a reaction catalyzed by chitin synthase enzyme. The

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polymerization occur using UDP and GlcNAc in the presence of divalent cations as co-factors.
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Chitin biosynthesis includes three main steps: i) formation of chitin polymer chains using chitin

synthase; ii) the translocation of the synthesized polymer across the membrane and its secretion
M

into the medium, and iii) formation of crystalline microfibrils of chitosan. Then, chitin
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deacetylase enzyme hydrolyzes acetic groups from N-acetylglucosamine moieties of chitin,

transforming them into glucosamine moities and finally forming chitosan. The chitin synthase
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and chitin deacetylase affect directly the MW of the chitin and the degree of deacetylation, that is
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the content of GlcNAc groups in the chitosan molecule (Kaur and Dhillon, 2014).
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3.2. Cellulose biosynthesis

Despite several reviews and papers being published concerning cellulose biosynthesis (Esa et al.,

2014; Keshk, 2014), there is no ideal single system to study this process. Cellulose is synthesized

by a few bacteria belonging to the genera Gluconacetobacter xylinus, Agrobacterium

tumefaciens, Rhizobium, Aerobacter, and Sarcina. Plant cellulose (PC) and bacterial cellulose

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(BC) are chemically the same, i.e. β-1,4-glucans, but with different physical properties (Jahan et

al., 2012). Algal cellulose from different origins has proven difficult to investigate at the

molecular level. The mold Dictyostelium discoideum produces cellulose at different steps of its

life cycle, and the existence of cellulose synthase activity has been shown clearly in this

microorganism. The Saprolegnia mold includes 1,4-β-D-glucan and β-D-1,3-glucan in its cell

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walls, and separable enzymes have been detected that synthesize these glucans in vitro, but

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genetic studies are not advanced with this microorganism. Though there is no documentation that

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bacterial cellulose biosynthesis varies from those of green plants, Uridine Diphosphoglucose

(UDPG) is required in the bacterial biosynthesis of cellulose, whereas Guanidine

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Diphosphoglucose (GDPG) is used in green plant cellulose biosynthesis (Keshek, 2014).
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3.3. Xylose biosynthesis
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Xylose-containing polysaccharide can be synthesized by Paenibacillus elgii B69. The synthesis

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process starts with the nucleotide sugars, uridine diphosphate-xylose (UDP-Xyl) and UDP-

glucuronic acid (UDP-GlcA). UDP-Xyl is derived from UDP-GlcA by a UDP-GlcA

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decarboxylase, which has a key role as an enzyme in the synthesis of UDP-xylose to produce
CE

xylans during cell wall biosynthesis. These precursors are sequentially added to build the xylose-

containing polymers in fungi, bacteria, plants, and animals using various specific
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glycosyltransferases, followed by export and polymerization (O. Li et al., 2015; Nwodo et al.,

2012). The xyloglucan, xylan, xylogalacturonan, and capsular polysaccharides of some fungi are

best examples of these polymers including proteoglycans in the extracellular matrix, and on the

cell surface of plant polysaccharides (Bar-Peled et al., 2001; Du et al., 2013; Mohnen, 2008;

Pattathil et al., 2005).

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Undecaprenol diphosphate is well known as a sugar acceptor in most heteropolysaccharides

produced by Rhizobiaceae. The repeating unit is formed at the cytoplasmic membrane, while the

polymerization occurs at the periplasmic part of the inner membrane and is coupled to export

biopolymer to the cell surface using Wzy-like polymerase enzyme and Wzc-like inner

membrane-periplasmic protein (MPA). The latter protein is proposed to control the chain length

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of the growing heteropolysaccharide. The physical association of the proteins localized in both

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membranes plays an important role in effective translocation of extracellular polysaccharide. The

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translocation of polysaccharide relies on the outer membrane protein (OMA), which forms a

channel in the outer membrane to ease the growing polysaccharide in order for it to exit the cell

surface (Skorupska et al., 2006).

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3.4. Alginate biosynthesis
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Figure 2 indicates the proposed alginate biosynthesis by Pseudomonas sp. Alginate biosynthesis
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begins with the conversion of a carbon source to acetyl-CoA and then undergoes the Krebs cycle

to be converted to fructose-6-phosphate via gluconeogenesis. Fructose-6-phosphate goes through

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a series of biosynthetic transformations and finally is converted to GDP-mannuronic acid as a

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key precursor for alginate synthesis. GDP-mannuronic acid precursor synthesis, cytoplasmic

membrane transfer and polymerization to polymannuronic acid, periplasmic transfer and

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modification, and export through the outer membrane sequentially occur within alginate

biosynthesis. Then alginate can be modified by post-polymerization, where polymannuronic acid

is acetylated by several transacetylases. Epimerization is then performed by a family of

epimerase enzymes to convert some non-acetylated M residues to G residues, and eventually

alginate is secreted from the cell through transmembrane porins. Thirteen genes including alg A,

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algC, alg D, alg 8, alg44, alg K, alg X, alg I, alg J, alg F, alg G, alg L and alg E operate in

alginate biosynthesis (Fig. 2). Alg L contribitues to periplasmic translocation as well as may

control the length of the biopolymer. Genes control the biosynthesis of exopolysaccharides form

large clusters assigned on the chromosomes or megaplasmids. The genes contributing to alginate

biosynthesis are almost identical between Pseudomonas and Azotobacter, though they act

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slightly differently in regulation (Hay et al., 2013; Pawar and Edgar, 2012; Remminghorst and

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Rhem, 2006; Skorupska et al., 2006).

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3.5. Pullulan biosynthesis

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In pullulan biosynthesis by Aureobasidium pullulans, the glucose is converted to pullulan in the
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presence of key enzymes including α-phosphoglucose mutase, uridine diphosphoglucose

pyrophosphorylase (UDPG-pyrophosphorylase), and glucosyltransferase. Moreover, A. pullulans

M

requires hexokinase and isomerase when different carban sources such as sucrose, mannose,
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galactose, maltose, fructose, and even agricultural wastes are utilized. The pullulan precursor,

UDPG, begins the addition of a D-glucose residue to the lipid molecule. Then, a further transfer
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of the D-glucose residue from UPDG leads to lipid-linked isomaltose, and isomaltosyl reacts
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with lipid-linked glucose to form an isopanosyl residue as a precursor. Lastly, the pullulan chain

is synthesized intracellularly at the cell wall membrane by polymerization of isopanosyl residues

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and released to the cell surface to form a slimy layer (Cheng et al., 2011; Duan et al., 2008;

Leathers et al., 2003).

4. Production, purification and characterization of polysaccharide bio-based flocculants

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Wider usage of polysaccharide bio-based flocculants is limited by their high production cost

compared to traditional synthetic flocculants. To reduce their production cost, a great deal of

effort has been devoted to developing better bacterial strains, cheaper biomass/carbon sources,

and more efficient production/fermentation and recovery processes (Ferreira et al., 2014; J. N.

Wang et al., 2013, Maghsoodi et al., 2009; Salehizadeh and Yan, 2014; Yan et al., 2013; Zhao et

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al., 2017). The production of polysaccharide bio-based flocculants is influenced by physical,

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chemical, and biological variables. In the case of microbial polysaccharide flocculants, the

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culture conditions including carbon and nitrogen sources, C/N ratio, initial pH, culture

temperature, metal ions, incubation conditions, agitation and aeration, ionic strength, trace

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elements, vitamins and other additives, such as microbial quorum sensing (QS) signaling
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molecules, play an important role in affecting yield and productivity of polysaccharide

flocculants (Diaz-Barrera et al., 2014; More et al., 2014; Patil et al., 2010; Salehizadeh and Yan,
M

2014, Wu et al., 2015; Yang et al., 2016). Recently, researchers found that the addition of
ED

microbial quorum sensing (QS) signaling molecules could improve the exopolysaccharide

bioflocculant production by microorganisms. The addition of N-hexanoyl-homoserine lactone

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was found to enhance the production of extracellular polysaccharide bioflocculant from

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Agrobacterium tumefaciens F2 by 1.6 fold and the flocculation activity of the bioflocculant was

increased by 10% (Yang et al., 2016). Similarly, the exopolysaccharide bioflocculant production
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by Agrobacterium tumefaciens and flocculation activity were improved by 1.55 times and

10.96%, respectively, in the presence of N-3-oxo-octanoyl-homoserine lactone, one of the N-

acyl-homoserine lactone classes of microbial quorum-sensing signaling molecules (Wu et al.,

2015).

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4.1. Production of polysaccharide bio-based flocculants by pure culture

Since the first discovery of bioflocculant secretion in yeast by Pasteur in 1876, more than 100

different polysaccharide-producing microorganisms have been identified. Until now, most of the

investigators have reported the production of polysaccharide bioflocculants by pure culture

(Diaz-Barrera et al., 2014; Ferreira et al., 2014; Gomes et al., 2013; Maghsoodi et al., 2009;

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Mishra and Suneetha, 2014; Vaingankar and Juvekar, 2014; Okaiyeto et al., 2015; Patil et al.,

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2010; Salehizadeh and Yan, 2014; Shen et al., 2015; Subudhi et al., 2014; Zhong et al., 2016).

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Table 2 provides an overview of the production of polysaccharide bio-based flocculants by pure

culture of microorganisms.

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Alginates are commonly produced from brown seaweeds (Phaeophyceae) including Laminaria
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digitata, Laminaria hyperborea, Laminaria japonica, Sargassum baccularia, Sargassum binderi,

Sargassum siliquosum, Turbinaria conoides, Ascophyllum nodosum and Macrocystis pyrifera

M

Bacterial alginates are generally produced by Pseudomonas aeruginosa, Azotobacter indicus,

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and Azotobacter Vinelandii (Chee et al., 2011; Darget, 2009; Diaz-Barrea et al., 2014; Hay et al.,

2009; Lee and Mooney, 2012; Patil et al., 2010).

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Chitosan is generally produced by crustaceans (shrimp and crabs), arthropods, fungi, and yeast
CE

(Prashanth and Tharanathan, 2007). Though the production of fungal chitosan is not usually

scaled up to an industrial process, the advantages of production of chitosan from fungi sources
AC

can be summarized as i) yield of medium-low molecular weight that is suitable for many

biomedical applications; ii) achievement of a higher degree of deacetylation; iii) absence of

allergenic shrimp protein; iv) controllable molecular weight and degree of deacetylation by

varying the fermentation conditions; v) unlimited supply of fungal biomass, largely from the

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biotechnological and pharmaceutical industries; and vi) use of low-cost wastes as economical

substrates for fungi culture (Aranaz et al., 2009; Dhillon et al. 2013).

Aureobasidium pullulans is the main microorganism for pullulan production (Cheng et al.,

2011). Many other microorganisms such as Rhodototula bacarum (Chi and Zhao, 2003),

Teloschistes flavicans (Reis et al., 2002), Cryphonectria parasitica (Forabosco et al., 2006), and

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Aspergillus japonicas (Mishra and Suneetha, 2014) were also known to secrete pullulan.

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Different strains of microorganisms produce polysaccharide flocculants at different growth

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stages. Thus, microbial polysaccharide flocculants can be divided into primary and secondary

bioproducts. For example, polysaccharide production by Aureobasidium pullulans was paralleled

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by the cell growth and reached its highest amount of pullulan (37.1 g/L) at 45 °C, pH 5.5 in 48 h
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the production of pullulan was stable after 48 h due to lack of degrading enzyme (namely

pullulanase) production (Singh et al., 2012). Recently, the hyper production of pullulan by a new
M

strain of Aspergillus japonicas was investigated and the results showed that the strain was able to
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produce a significant amount of pullulan (39 g/L) after 6 days using sucrose and yeast extract as

carbon and nitrogen sources, respectively. The presence of α (1→6) glycosidic linkage of α
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(1→4) linked maltotriose units were revealed by over 91% hydrolysis of pullulan by pullulanase
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enzyme (Mishra and Suneetha, 2014).

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So far, many strains including X. campestris, X. arboricola, X. axonopodis, X. citri, X. fragaria,

X. gummisudans, X. juglandis, X. phaseoli, and X. vasculorium, have been used for xanthan gum

production (Ferreira et al., 2014; Palaniraj and Jayaraman, 2011; Rosalam and England, 2006)

(Table 2).

Table 2 Culture conditions for polysaccharide bio-based flocculants production by pure culture

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Microorganism Carbon/Nitrog Culture Biopolymer Flocculant Bio- Reference

en source conditions flocculant properties flocculant

charcteristics Yield (g/L)

Azotobacter indicus Mahua extract pH 7.4, 48 h, 30 °C, Alginate - 6.10 (Patil et al., 2010)

5×108 CFU/ml

Azotobacter vinelandii Sucrose pH 7.3, 40h, 30 °C, Alginate 1250 kDa 3.8 (Diaz-Barrera et al., 2014)

600 rpm, 1 vvm

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Aspergillus japonicus Sucrose, Yeast pH 6.5, 6 days, 25 Pullulan, - 39 Mishra and Suneetha, 2014
6

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extract °C, 150 rpm, 5×10

Spores/ml

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Aureobasidium Sucrose, pH 5.5, 48 h, 42 °C, Pullulan T hermostable 37 Singh et al., 2012
7
pullulans (NH4 ) 2 SO4 120 rpm, 5×10

CFU/ml

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Mucor rouxii Molasses, pH 5, 3 days, 30 °C, Chitosan Crystalline 0.61 Chatterjee et al., 2005

NaNO3 150 rpm, 6.4×10 6

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Spores/ml

Absidia butleri Glucose, pH 5.5, 72 h,30 °C, Chitosan 8.7 × 10 4 g 1 Vaingankar and Juvekar,
M

T ryptone and 180 rpm, 1.8×10 8 mol-1 2014)

Yeast extract Spores/ml Glucosamine

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80.68%, DD

79.89%,

Viscosity
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73.22 ml/g
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Aspergillus niger Sabouro pH 5.5, 12 days, 30 Chitosan - 0.91 Maghsoodi et al., 2009

dextrose °C, 150 rpm, 3×10 6

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media Spores/ml

(Glucose 8%)

Gluconacetobacter Olive mill pH 5, 96 h, 30 °C Cellulose 297 × 10 3 g 0.85 Gomes et al.,2013

sacchari residue, -1
mol , 575

(NH4 ) 2 SO4 ml/g, M/G

1.17

Gluconabacter xylinus Molasses pH 5-6, 144h, 30 °C Cellulose Crystalline, 1.97(1.045), El-Saied et al., 2008

(Glucose), T hemaostable Yp/s : 0.2

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CSL g/g,

0.026g/L/h

Gluconacetobacter sp. Glucose, pH 6, 6 days, 30 °C, Cellulose - 4.5 g/L, Jahan et al., 2012

Yeast extract 100 rpm, Inoculum 0.075 g/L/h

size 4% v/v

Xanthomonas Soybean pH 7, 96 h, 28 °C, Xanthan Consistency 12.89 Ferreira et al., 2014

campestris biodiesel, 200 rpm, index (K) 937

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Urea mPa.s, Flow

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rate 0.61

Xanthomonas Sucrose, Yeast pH 6, 72 h, 30 °C, Xanthan Viscosity: 11.5, Yp/s: Carignatto et al., 2011
campestris 160 rpm, 10 7

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extract cells/ml 245 cP 0.404 g/g

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4.2. Extraction and purification of polysaccharide bio-based flocculants
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The extraction of polysaccharide bio-based flocculants is typically carried out using different

physical methods including centrifugation, filtration, heating, sonication, and chemical methods,
M

such as alkaline/acid treatment, solvent extraction, cation exchange resin, ethylene diamine tetra
ED

acetic acid (EDTA), and enzymatic extraction, depending on the type of polysaccharide focculant

and its origin. The loosely bound polysaccharide flocculants (LB-EPS) are usually extracted by
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mild methods, such as high-rate shear, heating at low temperatures, or high-speed centrifugation,
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while harsh methods such as heating at high temperatures, sonication or chemical extraction are

employed for tightly bound polysaccharide flocculants (Chee et al., 2011; Fertah et al., 2014;
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More et al., 2014; Okaiyeto et al., 2015; Sila et al., 2014; Subudhi et al., 2016; Younes et al.,

2014).

In addition to the above conventional methods, enzyme-assisted, and microwave-assisted

extraction methods have recently received increased attention because they enable

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polysaccharide extraction without any significant degradation (Michalak and Chojnacka, 2015;

Rodriguez-Jassoa et al., 2011).

Solvent extraction is perhaps the most common technique for bioflocculant separation and

purification. Figure 4 represents a schematic of extraction and purification processes for

microbial polysaccharide flocculants.

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Similarly, for other microbial-based polysaccharide flocculants, such as xanthan, pullulan, and so

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on, the purification process includes cell removal from the fermentation broth, and precipitation

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of bioflocculant using a suitable solvent, with further purification achieved using ultrafiltration

or chromatography (Cheng et al., 2011; Kachhawa et al., 2003). For xanthan produced by X.

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campestries, the broth was pasteurized to remove microorganisms, and then xanthan was
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precipitated by solvent extraction as a crude bioflocculant. Further purification of xanthan was

carried out by adsorption, enzymatic lysis, filtration, and repeated precipitation (Petri, 2015).
M

Other major polysaccharides such as alginate and chitosan are usually obtained through washing
ED

and removal of undesirable parts, extraction by solvents in aqueous solution, precipitation and

finally drying. For example, alginate was separated from seaweed by hot and cold extraction
PT

methods. CaCl2 solution was added to the air-dried seaweed sample. The soaked sample was left
CE

for 3 h at 50 °C in hot extraction, while it was incubated overnight at room temperature in the

cold method. To remove excess calcium chloride and HCl, sample was washed using water
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before and after the addition of acid. Subsequently, sodium carbonate was added to convert

alginic acid to sodium alginate and the mixture was centrifuged to recover sodium alginate.

Ethanol and water in a volume ratio of 50:50 were mixed and added to the supernatant to

precipitate out sodium alginate, which was then dried at 50 °C (Chee et al., 2011).

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Pre-treatment of seaweed by alkali, acid, formalin, and formaldehyde is suggested to fix phenolic

compounds and prevent discolouration. In a recent study, Fertah et al. (2014) dried seaweed

samples at 60 °C and soaked them for one night in a 2% formaldehyde solution to eliminate

pigments. The samples were washed with distilled water and added to a 0.2 M HCl solution

before being set aside for 24 h. After this period, the samples were washed once again with

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distilled water and extracted under agitation for 5 h with a 2% sodium carbonate solution.

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Finally, sodium alginate was purified twice with ethanol precipitation, then with methanol and

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acetone before being dried at room temperature.

Solvent extraction is widely used for the extraction and purification of grafted polysaccharide

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bioflocculants as well. Figure 5 indicates the purification steps of acrylamide grafted onto
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polysaccharide such as alginate. A mixture of acetic acid and formaldehyde with a volume ratio

of 1:1 was employed to extract bioflocculant in aqueous solution. Methanol was used to remove
M

solvents and then grafted polysaccharide flocculant was dried in a vacuum oven (Tripathy and
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Singh, 2001).

Figure 6A shows the extraction and purification of chitosan obtained from marine sources (e.g.;
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shrimps, crabs) and lignocellulosic sources such as fungi (Kaur and Dhillon, 2014). Though the
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extraction methodologies used for chitosan production from marine waste vary with the source

of chitosan, demineralization, deproteinization and finally deacetylation are common unit

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operations in all cases. In this process, chitin is first recovered through demineralization and

deproteinization and then chitosan is produced by deacetylation. Deproteinization of marine

wastes could be achieved using enzymatic processes. For instance, high levels of protein removal

(about 77 ± 3% and 78 ± 2%) were recorded using proteases obtained from Bacillus mojavensis

A21 and Balistes capriscus, respectively, by an enzyme/substrate ratio of 20 U/mg after 3 h of

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hydrolysis at 45 °C. These crude proteases have been reportedly used for chitin extraction (Fig.

6A) (Younes et al., 2014).

Figure 6B represents the extraction of chitosan from waste fungal mycelium feedstocks. The

dried mycelium was finely homogenized and pretreated with alkali to extract alkali-soluble

materials like glucan and protein from fungal biomass. Dry biomass was treated with NaOH (1

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N) solution (1:40, w/v) and autoclaved at 121 °C. The alkali-insoluble materials (AIM) were

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removed by centrifugation, washed to remove excess alkali and then reacted with acetic acid (2%

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1:40, w/v) at 95 ˚C for 6 h. The supernatant including the chitosan was separated from the acid-

insoluble parts. The chitosan was precipitated by adjusting the pH at 10 with sodium hydroxide.

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The precipitate was centrifuged and washed with distilled water to a neutral pH, and extracted
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using ethanol (95%, 1:20, w/v) and acetone (1:20, w/v), then dried at 60 ˚C (Vaingankar and

Juvekar, 2014).
M

Though microbial bioprocesses (Fig. 4) for bioflocculant production and extraction are
ED

environmental-friendly, controllable, efficient (some), but the production and recovery cost of

bioflocculants are high. Large quantities of solvents are consumed for the extraction of
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bioflocculants and can not be easily scaled up commercially (Salehizadeh and Yan, 2014).
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Despite the advantages of method (Fig. 5) employed for the purification of chemically grafted

polysaccharide bio-based flocculants which results in improving mechanical, physic-chemical

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properties of grafted flocculants, such as biodegradability of product to some extent, high

flocculating activity, high shear stability and controllability of molecular structure, this method is

still expensive and presents secondary pollution risks for environment and humans (R. Yang et

al., 2016).

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The chitosan derived by industrial processing of crustacean waste shells (Fig. 6A) through

chemical deacetylation either using hot (> 100 °C), concentrated base solution (40-50% w%) or

acids suffers from inconsistent level of deacetylation, protein contamination, and high molecular

weight that can cause variable chemical properties. Some environmental problems also exist

such as a large amount of generated alkaline and acidic wastes, demineralization treatment

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requirement, seasonal limitation of seafood supply, high production cost, and laborious process.

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Green synthesis method (Fig 6B) is effective, environmentally friendly, and inexpensive and

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offers a great potential for producing consistent products compared to alkaline or solvent based

extraction methods. In addition, a large amount of fungal waste biomass can be obtained by

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bioprocess industries without any limitation in geographical region or seasons. The chitosan
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flocculants produced by base extraction from crustacean shells are high molecular weight

polysaccharides which are not suitable for biomedical applications due to some allergic effects
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on body, while those derived by green synthesis method from fungal source are low or medium
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molecular weight, free of allergenic shrimp protein, and suitable for biomedical applications.

Green synthesis methods are yet to be commercialized (Kaur and Dhillon, 2014).
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4.3. Characteristics of polysaccharide bio-based flocculants

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The chemical composition, physico-chemical properties and flocculation activity of

polysaccharide bio-based flocculants depends on the source of flocculant and its production

method. For examples, the charge density and grafting ratio are important factors in determining

polysaccharide bio-based flocculant performance. Earlier studies indicate that flocculation

efficiency could be enhanced by optimizing the grafting ratio. A cellulose-based flocculants,

CMC-g-PDMC18 exhibited the highest color removal was found to be 97.3% for a lower
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grafting ratio of 181% and mass feed ratio of CMC:DMC 1:5 at a flocculant dosage of 90 mg/L.

The color removal efficiency decreased to 92.2% with increased grafting ratio to 337% at a

flocculant dosage of 60 mg/L (Cai et al., 2015).

Cellulose-g-PAM had been synthesized by using microwave irradiation with a grafting ratio of

57.4%. The result of elemental analysis for Cellulose-g-PAM exhibited the presence of C, H, N

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and O in mass ratio of 43.83:11.24:5.72:39.21, and the nitrogen content confirmed that PAM was

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successfully grafted onto cellulose (J.P. Wang et al., 2013).

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The bamboo pulp cellulose-g-ployacrylamide flocculant (BPC-g-PAM) had a grafting ratio of

43.8% and DS of 1.31. The BPC-g-PAM appeared an isoelectric point at pH 4 and showed a

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negative ZP over the isoelectric point with effective flocculating activity under acidic or neutral
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conditions (Szygul et al., 2009). Polyacrylic acid-grafted carboxymethyl cellulose (CMC-g-

PAA) was synthesized using a mass feed ratio of CMC to Acrylic acid of 1:10 w/w and
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microwave radiation for 5 min. The optimized CMC-g-PAA with grafting ratio and intrinsic
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viscosity of 15.5% and 13.38 (dl/g) respectively, showed the highest flocculation activity due to

its highest hydrodynamic volume, and a linear correlation was observed between the grafting
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ratio, intrinsic viscosity and flocculation efficacy (Song et al., 2011).

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Polymethyl methacrylate-grafted sodium alginate (SAG-g-PMMA) was prepared by the

microwave-assisted method. The grafting of the PMMA on the polysaccharide was proved by
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intrinsic viscosity study, FTIR spectroscopy, and elemental analysis. The results showed that the

intrinsic viscosity of all synthesized SAG-g-PMMA was higher than sodium alginate due to an

increase in hydrodynamic volume resulting from the grafting ratio. The elemental analysis of the

best grade of SAG-g-PMMA indicated an elemental composition that is intermediate between its

constituents (SAG and PMMA) (Roy et al., 2009).

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A strong cationic chitosan-based graft copolymer flocculant 3-chloro-2-hydroxypropyl trimethyl

ammonium chloride (CTA)-modified chitosan-g-polyacrylamide (chitosan-CTA-g-PAM) was

synthesized. The FT-IR showed a new peak at 1476 cm-1 , representing the presence of methyl of

the quaternary ammonium groups in grafted co-polymer, which confirms CTA was successfully

grafted in chitosan-g-PAM. H-NMR supported that a graft reaction also occurred because a new

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peak around 4.01 ppm was detected corresponding to the resonance of the methylene proton of

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CTA (H9 proton). The flocculating efficiency was enhanced with increase of the degree of

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substitution of CTA. The grafted polysaccharide flocculant with the highest degree of

substitution of CTA (44.7%), showed the maximum flocculating activity at the optimum dosage

of 1.3 mg/L (Dhillon et al., 2013).

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Two kinds of starch-based flocculants, (2-hydroxypropyl)trimethylammonium chloride etherified

carboxymethyl starch (cationic CMS-CTA-P and anionic CMS-CTA-N, respectively), were

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successfully prepared with the different carboxymethyly groups and substitution degrees for
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CTA of 20.3% and 43.2% in CMS-CTA-P, and 62.3% and 27.4% in CMS-CTA-N, respectively.

The results of FTIR and H-NMR revealed that anionic and cationic moieties were successfully
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introduced onto the starch (H. Li et al., 2015).

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Quaternary phosphonium cationic starch (St-g-AM/ATPPB) flocculant was prepared at a

starch:AM:ATPPB wt. ratio of 1:1.4:0.93 (i.e., AM/ATPPB 1:0.124 mol) using corn starch and
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acrylamide (AM), allyl triphenyl phosphonium bromide binary (ATPPB) and by simultaneous

gamma-irradiation. The FTIR and 1 H NMR were used to characterize the St-g-AM/ATPPB. The

chemical shifts in 7.726, 7.637 and 7.574 ppm confirmed the presence of ATPPB moiety in St-g-

AM/ATPPB. The FT-IR spectrum of St-g-AM/ATPPB revealed the presence of all absorption

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peaks related to corn starch and the –CONH2 group in the acrylamide moiety (Kolya and

Tripathy, 2013).

A novel pullulan-based flocculant was synthesized by grafting (3-acrylamidopropyl)-

trimethylammonium chloride onto pullulan (Pullulan-g-pAPTAC) using potassium persulfate as

an initiator. The FT-IR spectrum showed the presence of the absorption peak at 1480 cm-1

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corresponding to the methyl groups of ammonium, confirming the grafting of the monomer on

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the pullulan. The highest flocculating activity was obtained at 52.69% using pullulan to APTAC

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in a molar feed ratio of 6.17 mmol to 4.83 mmol. The NMR spectroscopy represents the

absorption peaks relevant to the protons in the saccharide ring of pullulan at 3.4–5.8 ppm, and

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the representative peaks resulted from grafting APTAC units. The molar ratio of APTAC to
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pullulan was determined by measuring of methylene protons at 1.9–2 ppm, which was in

agreement with the APTAC and that of H-1 and H-6 protons of pullulan moieties detected at 5.6
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and 5.2 ppm (Revedin et al., 2010).

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The xanthan-based flocculant (Xan-g-PDMA) was synthesized by grafting N,N-

dimethylacrylamide (DMA) onto xanthan gum using ceric ion-induced graft co-polymerization.
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The FT-IR spectrum for Xan-g-PDMA confirmed the grafting of DMA successfully occurred on
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the xanthan gum; the grafting ratio and intrinsic viscosity of Xan-g-PDMA were obtained at

83.24% and 6.4 dl/g, when the feed ratio of xanthan to DMA was 1.5 g to 0.109 mol (Kolya et
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al., 2012).

The molecular weight of polysaccharide flocculants is an important factor that may influence

performance of polysaccharide flocculants and their properties, such as viscosity of solution

(Grenda et al., 2017; S. Wang et al., 2015). Polysacchride flocculants with higher molecular

weight and longer chain indicate higher viscosity than low molecular weight flocculants. The

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molecular weight of Xyl-g-METAC hemicellulose based flocculant was reported to be 102250

g/mol that was higher than xylan at 20480 g/mol, which led to copolymer with a higher viscosity

(S. Wang et al., 2015).

The molecular weights of bioflocculants are generally varied in the range of 10 4 –106 Da. For

example, Yang et al., (2014a) obtained the flocculating activity of 99% for chemically modified

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chitosan-based flocculant, Carboxymethyl chitosan-graft-poly(2-methacryloyloxyethyl)

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trimethylammonium chloride (CMC-g-PDMC) with a molecular weight of 8.34 × 105 g/mol. The

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flocculating activity of 97.8% was achieved by chemically modified xylan [2-(methacryloyloxy)

ethyl] trimethyl ammonium chloride (METAC) grafted on xylan (Xyl-g-METAC) with a

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molecular weight of 102250 g/mol for color removal for anionic-azo dye solutions (S. Wang et
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al., 2015). A ternary copolymer of chitosan with molecular weight of 2 × 10 5 Da grafted together

by acrylamide and fulvic acid has recently been synthesized as a novel polysaccharide based
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flocculant (CAMFA) and exhibited excellent abilities for dye removal in waste water treatment
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(Tao et al., 2017).

Pathak et al., (2017) characterized the glycoprotein biflocculant produced by P. aeruginosa

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consisting of 89.4% polysaccharide and 6.2% protein. The monomer constituents of

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bioflocculant were determined as D-xylose, L-Rhamnose, D-glucose, D-glucuronic acid and N-

acetyl D-glucosamine. Bacillus toyonensis produced a thermostable-glycoprotein bioflocculant

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consisting of polysaccharide (77.8%) and protein (11.5%), respectively (Okaiyeto et al., 2015).

Azotobacter vinelandii secreted an alginate polysaccharide bioflocculant with molecular weight

of 590 kDa in 14 L stirred fermenter and 1250 kDa in 3 L bioreactor (Diaz-Barrera et al., 2014).

Acinetobacter junii produced a polysaccharide bioflocculant with an average molecular weight

of 2 × 105 Da composed of neutral sugar (73.21%), uronic acid (10.12%), amino sugars (0.23%),

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α- amino acids (11.13%), and aromatic amino acids (1.23%) (Yadav et al., 2012). A novel

thermally stable bioflocculant M-C11 produced by Klebsiella sp. was consisted of 91.2% sugar,

4.6% protein and 3.9% nucleic acids (m/m) (J. Liu et al., 2014). The bioflocculant produced by

Klebsiella sp. ZZ-3 was polysaccharide in nature that was composed of 84.6% polysaccharides

and 6.1% protein with molecular weights of 603-1820 kDa. The main monomer components of

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the bioflocculant were mannose, glucose, and galactose (Yin et al., 2014). Klebsiella pneumonia

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ZCY-7 produced a novel bioflocculant including polysaccharide (82.4%) and protein (14.2%)

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(Zhong et al., 2014). A salt-tolerant, alkaliphilic Bacillus agaradhaerens C9 secreted a

polysaccharide bioflocculant which contained 65.42% polysaccharides, 4.70% proteins, and

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1.65% nucleic acids (Liu et al., 2015). Chemical analysis of the bioflocculant produced by
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Paenibacillus polymyxa showed that its polysaccharide content was up to 96.2%, with the molar

weight of 1.16 × 106 Da (Guo et al., 2015b).

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5. Mechanisms of polysaccharide bio-based flocculants

The flocculation mechanism of polysaccharide flocculants has been studied by many researchers
PT

in recent years (C. Zhang et al., 2016; Ghimici and Nichifor, 2009; More et al., 2014; Okaiyeto et
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al., 2016; Renault et al., 2009a; Szygul et al., 2009; Tao et al., 2017; Wu et al., 2015; Yang et al.,

2013, 2014b; Zemmouri et al., 2011). These studies show that parameters such as flocculant
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charge density and dosage, biopolymer molecular weight (MW), the nature of the colloids and

their concentration, pH, ionic strength, and temperature can mainly affect the flocculation

mechanism by polysaccharide flocculants (Cheng et al., 2005; Ghimichi and Nichifor, 2009;

More et al., 2014; Singh et al., 2003; Strand et al., 2003; Szygul et al., 2009; Tao et al., 2017; Wu

et al., 2015; Yang et al., 2013).

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The aggregation of colloidal particles and cells by polysaccharide flocculants is considered to be

due to the following main mechanisms: i) charge neutralization; ii) bridging (Fig. 3).

Charge neutralization occurs if the polysaccharide flocculant is oppositely charged compared to

the colloidal particles. In this case, the particle surface charge density is reduced by adsorption of

the polysaccharide flocculant, resulting in destabilization of particles by which repulsive

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electrostatic interactions are overcome by attractive forces. This mechanism has been found to be

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quite effective for low molecular weight polysaccharide flocculants that tend to adsorb and

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neutralize the opposite charges on the particles (R. Yang et al., 2016; Tao et al., 2017).

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Bridging is major mechanism of flocculation in like-charged or neutral polysaccharide

flocculants, especially when polysaccharide flocculant extends from the particle’s surface into
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the solution for a distance greater than the distance over which the interparticle repulsion occurs.
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In this case, segments of the polysaccharide flocculant are adsorbed onto the particle surface

resulting in loops and tails extending into solution with the possibility of attachment of dangling
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polysaccharide segments onto other adjacent particles to form flocs. Bridging could be due to
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van der Waals force, static, hydrogen bonds or even chemical reaction between some radical

groups of the polysaccharide molecule and the particle. This mechanism is known to be
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especially effective for high molecular weight like-charged polysaccharide flocculants.

The molecular weight (MW) of polysaccharide-based flocculant affected the bridging

mechanism. Higher molecular weight flocculants generally resulted in more adsorption (of

polysaccharide-based flocculant) onto particle surface, stronger bridging between the flocculants,

and higher flocculation efficiency than lower molecular weight flocculants (Cai et al., 2015;

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Ghimichi and Nichifor, 2009; More et al., 2014; Okaiyeto et al., 2016; Rani et al., 2013; Renault

et al., 2009a; Salehizadeh and Yan, 2014; Tao et al., 2017; Vandamme, 2013; Wu et al., 2015;

Zhang et al., 2015).

Polysaccharide flocculants usually include functional groups (such as carboxyl, hydroxyl, amino,

and phosphate groups) in their structures that play an important role in determining their

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flocculating activities, especially when bridging is the major mechanism for flocculation. For

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example, carboxyl groups in polysaccharide flocculants provided more sites for colloidal particle

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adsorption due to surface charge that facilitated a higher extend of bridging between the

bioflocculants and the particles (Farooq et al., 2010; Pathak et al., 2017, Tao et al., 2017; Zhang

et al., 2015).
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Table 3 summarizes the proposed flocculation mechanisms for various polysaccharide bio-based
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flocculants. According to literatures, flocculation process is mainly driven by bridging and

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charge neutralization mechanisms. However, electrostatic patch and sweeping mechanisms may

also contribute in a few cases.

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Electrostatic patch is caused by polymer flocculants of high charge density interacting with
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oppositely charged colloidal particles of low charge density. The net residual charge of the

polymer patch on one colloidal particle surface can adsorb onto the oppositely charged colloidal
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particle.

Sweeping is the other mechanism of flocculation in which flocculant forms a bulky precipitate

that enmeshed the colloidal particles. These particles are then either settled out or flocculate

together with the precipitate (Lin et al., 2012; Okaiyeto et al., 2016; Rahul et al., 2015; Xu et al.,

2013; Yang et al., 2013, 2014a).

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Table 3 Flocculation mechanism of some polysaccharide bio-based flocculants

T ype of flocculant Characteristics/Conditions Flocculation medium Flocculation Reference

mechanism

Alginate-based polysaccharide

QL-g-SAG QL:SAG 2:1 (w/w), pH 8 Dyeing wastewater Bridging (Zhang et al., 2013)
(Trimethyl quaternary ammonium salt (Methylene blue)

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of lignin-g-sodium alginate)
SAG&PAC SAG:PAC 0.5:1.5 g/g, pH Humic acid-kaolin Bridging & Charge (Zhao et al., 2012)

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(Combined Sodium alginate & 8.3-8.5 neutralization
P olyaluminium chloride) suspension

CR
SAG&PAF&PAM 2:1:1 (w%), pH 7.1 Wastewater samples Bridging & Charge (Zeng et al., 2011)
(Combined Sodium alginate and neutralization
polyaluminium ferric chloride and
cationic polyacrylamide)
SAG-g-NVP SAG:NVP 0.1:0.16, G% Coal fine suspension Bridging (Sand et al., 2010)

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(Sodium alginate-g-N-vinyl-2- 431.5%
pyrrolidone)
SAG-g-PAM AM:SAG 0.3:2.5 mol/g, Coal fine suspension Bridging (T ripathy et al.,
(Sodium alginate-g-polyacrylamide) MW 2.47×10 6 g/mol, G% (1%); Iron ore 2001)
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95.56%, PD 8.2 dl/g suspension (0.5%)

SAG-g-PMMA SAG:MMA1:7.5 g/g, G% Coal fine suspension Bridging (Rani et al., 2013)
(SAG-g-P olymethyl methacrylate) 87%, PD 8.8 dl/g (1% )
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Chitosan-based polysaccharide
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Chitosan DD87%, MW 125000 Anionic dye (Acid Charge neutralization (Szygul et al., 2009)
g/mol Blue 92/AB92 & Bridging
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Chitosan FA 0.49, MW 290000 E.coli (4×108 cells/ml) Bridging (Cai et al., 2015)
g/mol, pH 5- 6.5

Chitosan pH 3-5, DD 89.5%, MW Reactive Black 5 Charge neutralization (Guibal et al., 2006)
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308300 g/mol, PD 2.1 (anionic sulfonic dye)

Chitosan pH 6 Microalgae (Chlorella Charge neutralization (Xu et al., 2013)
sorokiniana) & Patch effect
Chitosan-acrylamide-fulvic acid Chitosan: AM:FA Acid blue113, Charge neutralization (T ao et al., 2017)
AC

(CAMFA) (1.5:3:3 g); 60 °C, 5 h Reactive black 5, & Bridging

Methyl orange
Chitosan-CT A-g-PAM DD 85.2%, G% 286%, Kaolin suspension, Charge neutralization (Lu et al., 2011)
(3-chloro-2-hydroxypropyl trimethyl MW 83.4×10 4 g/mol, Raw water & Bridging
ammonium chloride (CTA) modified Chitosan-g-PAM: CT A
chitosan-g-polyacrylamide) 2:4 (w/w)

CMC-g-PAM G% 74%, CMC:AM 1:1, Anionic dye (Methyl Charge neutralization, (Yang et al., 2013)
(Carboxymethyl chitosan-g- pH 4.0 & pH 11.0 Orange, MO) & Bridging & Sweeping
P olyacrylamide)
cationic dye (Basic effect
Bright Yellow, 7GL)

CMC-g-PDMC SD 48.3%, G% 251% E.coli Charge neutralization (Yang et al., 2014a)

(Carboxymethyl chitosan-g-poly(2- & Bridging
methacryloyloxyethyl) trimethyl

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ammonium chloride) (10 6 -10 8 CFU/mL)

CMC-g-PDMC DD 85.2%, MW 83.4×104 Cationic dyes (Acid Charge neutralization (Wu et al., 2015)
(Carboxymethyl chitosan-g-poly(2- g/mol, SD 48.3%, G% Green 25 (AG25) &
methacryloyloxyethyl) trimethyl Basic Bright
ammonium chloride) 150%
Yellow (7GL))

MAC DD 90%, 2-4×104 g/mol, Kaolin suspension Bridging & Charge (Zhang et al., 2015)
(Mercapto-acetyl chitosan) pH 6.3 neutralization

Cellulose-based polysaccharides

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ADAC & Alum ADAC 5 mg/L, Alum Kaolin suspension 5 Charge neutralization, (Liimatainen et al.,
(Combined anionic derivatives of 62.5 g/L g/L patch effect 2012)

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Dialdehyde cellulose & Alum)

BPC-g-PAM G% 43.8%, DS 1.33 Kaolin suspension Charge neutralization (H. Liu et al., 2014)

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(Bamboo pulp cellulose-g- & Bridging
Ployacrylamide)

CMC-g-PAA CMC:AA 1:10 g/g, G% River water Bridging (Jin et al., 2014)
(Carboxymethyl cellulose-g- 15.5%, C 41.29, PD 13.38

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Polyacrylic acid) dl/g

CMC-g-PDMC CMC:DMC 1:5, Anionic dye: Acid Charge neutralization (Sirvio et al., 2011)
(Carboxymethyl cellulose-graft- SDcarboxymethyl group 17.4 %, Green 25 (AG25), 100
poly(2-methacryloyloxyethyl) G% 181%
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(mg/L)
trimethyl ammonium chloride)

CMC-g-Quat 188 pH 6, MW CMC 10000 Ferric laurate Charge neutralization (Hebeish et al., 2010)
(Carboxymethyl cellulose-g-(N-3- suspension (Lauric
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chloro-2-hydroxypropyl trimethyl acid 10 g/L)

ammonium chloride)

CMCNa pH 8, DS 1.17, DP 480 Drinking water Bridging (Khiari et al., 2010)

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(Sodium carboxymethyl
cellulose)
DAC pH 4.5, Cationicity 4.27 Calcium carbonate Charge neutralization (Mishra et al., 2012)
(Cationizing dialdehyde cellulose) mmol/g, Aldehyde suspension (64 w%)
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content 11.77 mmol/g

DCCs DP 781, Carboxylate Kaolin suspension, Charge neutralization (Zhu et al., 2015b)
(Dicarboxyl cellulose) content>1 mmol/g paper mill effluents
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DEAE-HEC DS 0.85, MW 350,000– Bacillus Bridging (Mazeika et al.,

(Diethyl laminoethyl- hydroxyethyl 400,000 g/mol amyloliquefaciens 2015)
cellulose)
AC

Xyl-g-MET AC MET AC:Xylan 3 mol/mol Dye solution: Reactive Charge neutralization (S. Wang et al.,
(Xylan-g-2-(methacryloyloxy) Orange 16, Dye 2015)
ethyl trimethyl ammonium content ≥70%
chloride)

Starch-based polysaccarides

CAFS pH 5 Dye suspension: Charge neutralization; (J. P. Wang et al.,

(Combinational Aluminum ferrous & reactive brilliant red Bridging & Sweeping 2013)
Starch) X-3B (OD538 nm 3.55,
CODCr 107.4 mg/L)

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CHES pH 7.5; DS 0.57 Bacillus Bridging (H. Li et al., 2015,

(Cationized hydroxyethylated starch)
pH 4-7; SDCTA 3.2%,
SDcarboxymethyl 0.3%/(pH 7,
SDCTA 27.4%; Dcarboxymethyl
62.3%)
amyloliquefaciens Mazeika et al., 2015)
Kaolin suspension Charge neutralization
(1g/L)/(hematite & patching/(Bridging
suspension, 1 g/L) &Charge
neutralization)

DCS Starch: Epoxy: NaOH 1: Kaolin suspension (0.6 Patch effect (Klimaviciute et al.,
(Dispersible cationic starch) (0.4–0.7): 0.04, DS 0.48, μm) 2010)
Cationic group amount
0.64×10–5 to 0.68×10–5
eq./g DCS

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HES-g-PDMA pH 5.5, HES:DMA Silica suspension (1%, Bridging (Ghimici et al., 2010)
1.5:0.109 g/g, G% 726%, w)

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(Hydroxyethyl starch-g-poly-N,N- Intrinsic viscosity 7.4 dl/g
dimethyl acrylamide)

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Potato Starch pH 8, DS 0.6, MW 40 × Kaolin suspension Bridging & Patch (Bratskaya et al.,
(2-hydroxy-3-trimethylammonium- 10 6 Da (1%) effect 2005)
propyl starch chloride)

Starch (Green Floc 120) pH 5-10, DS 0.15 Freshwater Bridging & patch (Lin et al. , 2012)
microalgaes: effect

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Parachlorella kessleri,
Scenedesmus Obliquus
and Parachlorella
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ST C-g-MAPT AC DS 0.82, pH 8 Microalgae suspension Charge neutralization (Kolya et al., 2013)
(Starch-g-3-methacryloyl amino propyl (S. obliquus, 200–
trimethyl ammonium chloride) 250 mg/L. pH 7)

ST C-g-PDMC pH 10 (-28 Mv, pH 10.0) Kaolin suspension Charge neutralization (Anthony and Sims,
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( Starch-graft-poly (2- (0.25 w%, 2 µm) 2013)

methacryloyloxyethyl) trimethyl
ammonium chloride)
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Plant-based & other

Polysaccharides
CHPT AC-g-Cassia Cationic polysaccharide Microalgae Bridging & Patch (Rahul et al., 2015)
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(N-3-Chloro-2-hydroxypropyl trimethyl (1,4-b-D-mannopyranose (Chlamydomonas sp. effect

ammonium chloride (CHP TAC)-g- units with 1,6 linked a-D- CRP7 and Chlorella
Cassia) galactopyranose), DC sp. CB4)
0.1779
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Guar gum Non-ionic Polysaccharide Rubber industry Bridging (Subramanian et al.,

(Galactomannan) effluent(pH 7.33, 2010)
T urbidity 87.8 NT U,
Color 200 T CU,COD
AC

867 mg/L)

Inulin-g-CHPT AC Cationic Inulin composed Microalgae Bridging (Agarwal et al.,

(Inulin-g-3-chloro-2-hydroxypropyl) of: D-(2→1)- (Botryococcus sp.) 2003)
trimethylammonium chloride) fructofuranosyl units+D-
(1→2)-glucopyranosyl

Ku-g-PAM Anionic polysaccharide; T extile wastewater Bridging (Mishra et al., 2004)

(Kundoor from Coccinia indica samples
-g-Polyacrylamide) G% 68.6%

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Okra Gum (Hibiscus esculentus) Anionic polysaccharide T annery wastewater Bridging (Mishra et al., 2006)
(D-galactose, L- sample (pH8.27,
rhamnose conductivity 7.87 mS,
and L-galacturonic acid) T 45.6 NTU, COD
1835 mg/L, TS 9936
mg/L, SS 2213 mg/L)

Okra seed (Hibiscus esculentus) Anionic Polysaccharide Brewery effluent (pH Bridging (Mukherjee et
7.68, T urbidity 316.63 al.,2013)
NT U, Conductivity
5290 μ/cm, BOD 640
mg/L, T DS 3438
mg/L, T SS 30.41

T
mg/L)

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Psy-g-PAM Anionic polysaccharide T extile wastewater Bridging (Okolo et al., 2014)
(Psyllium from Plantago psyllium samples (pH 4-4.2,
mucilage-g-Polyacrylamide) conductivity 5.6-6 mS,

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COD 815-910 mg/L,
T S 5590-5625 mg/L,
SS 95-120 mg/L)

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Cationic Dextran 1.10 mol Amino Clay suspension Charge neutralization (Ghimici and
(Dextran-g-N-ethyl-N, N-dimethyl-2- group/100 (Kaolin 47%, Nichifor, 2009)
hydroxypropylene ammonium Montmorillonite 22%,
Chloride)
glucopyranosidic units)
Quartz 31%)
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Pullulan-g-APT AC P:APT AC 6.17:4.84, Clay suspension Charge neutralization (Guo et al. 2015)
Pullulan-graft-(3- MW: 200,000 g/mol, G% (kaolin 47%, & Bridging
acrylamidopropyl)- 52.69% montmorillonite 22%,
quartz 31%)
trimethylammonium chloride)
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Xanthan-g-PAM/SiO2 MW: 4.09 × 10 7 g/mol Kaolin and iron-ore Bridging (pH > 4), (Ghorai et al., 2013)
(Polyacrylamide-g-Xanthan / (0.25%) Charge neutralization
Silica) suspensions (pH < 4)
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6. Chemical modification of polysaccharide bio-based flocculants by grafting methods

Grafted biopolymer flocculants offer great potential due to their unique characteristics and strong
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flocculating activity compared to the conventional floccuants (Lee et al., 2014b; Lee and

Mooney, 2012). Although polysaccharides are biodegradable and often more shear-stable than
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synthetic organic flocculants, they usually show lower flocculating activity and require higher

flocculant dosage. Therefore, chemical modification of polysaccharide flocculants is often used

to enhance their flocculation efficiency (Lee et al., 2014b; Mishra et al., 2012).

There are many ways for achieving novel flocculants through introducing functional groups onto

the polysaccharide chains. The modification of polysaccharides is generally occurred in two

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major forms: i) physical reactions, such as ionic interaction, polysaccharide-protein interaction,

and ii) chemical reactions, such as graft co-polymerization, crosslinking with aldehyde,

esterification, etherification, amination, carboxyalkylation, hydroxyalkylation and other addition

reactions, and condensation (Ahmad et al., 2015; Lewicka et al., 2015; Laurichesse and Avérous,

2014; Roy et al., 2009).

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So far, many graft copolymers have been synthesized by chemical modification of natural

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polysaccharides, such as poly-acrylamide grafted xanthan gum/silica-based nanocomposite

CR
flocculant (XG-g-PAM/SiO 2 ) (Ghorari et al., 2013), (3-Chloro-2-

hydroxypropyl)trimethylammonium chloride chitosan (HTCC) (Qin et al., 2015), poly(2-

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methacryloyloxyethyl)trimethyl ammonium chloride grafted starch (STC-g-PDMC) (J. P. Wang
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et al., 2013), quaternized carboxymethyl chitosan (QCMC) (C. Dong et al., 2014), polymethyl

methacrylate grafted sodium alginate (SAG-g-PMMA) (Rani et al., 2013), [2-(methacryloyloxy)

M

ethyl] trimethylammonium chloride (METAC) grafted xylan (S. Wang et al., 2015), (3-Chloro-2-
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hydroxypropyl)trimethylammonium chloride etherified carboxymethyl starch (CMS-CTA) (H.

Li et al., 2015), Poly-N,N-dimethylacrylamide-co-acrylic acid grafted hydroxyethyl starch (HES-

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g-Poly-(DMA-co-AA)) (Kolya and Tripathy, 2013), 3-(acrylamidopropyl)trimethylammonium

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chloride grafted pullulan (Pullulan-g-pAPTAC) (Ghimici et al., 2010), Xanthan-g-N,N-

dimethylacrylamide, (Xanthan-g-PDMA) (Kolya et al., 2012), poly-acrylamide grafted starch

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(St-g-PAM) (Mishra et al., 2011), and mercapto-acetyl grafted chitosan (MAC) (Zhang et al.,

2015), cellulose-g-PAM (Razak et al., 2014), P4VN-g-CNC (Kan et al., 2013), CMC-g-PAA

(Mishra et al., 2012), alginate-g- N-vinyl-2-pyrrolidone (Sand et al., 2010), sodium alginate-g-

PMMA (Rani et al., 2013), CMC-g-PDMC (Yang et al., 2014a,b), CMC-g-PAM (Yang et al.,

2013), chitosan-CTA-g-PAM (Lu et al., 2011), 2-hydroxypropyl)trimethylammonium chloride

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etherified carboxymethyl starch (CMS-CTA-P) (H. Li et al., 2015), Quaternary phosphonium

cationic starch (St-g-AM/ATPPB) (Song et al., 2013), pullan-g-pAPTAC (Ghimici et al., 2010),

and Xan-g-PDMA (Kolya et al., 2012), to name a few (Table 4).

Among different physical/chemical modification methods, grafting co-polymerization is the most

effective approach to add new properties to natural polysaccharides. The main methods for

T
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polysaccharide modification in aqueous solution could be classified into three groups (Kumar et

al., 2012): i) conventional method, ii) microwave initiated grafting method, and iii) microwave

CR
assisted grafting method.

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In the conventional method, a chemical-free radical initiator such as ceric ammonium nitrate or
AN
CAN is employed under an inert atmosphere to produce free radical sites on the polymer in order

to allow the addition of monomer to form the graft chain. This method has low reproducibility
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and is not suitable for commercial scale synthesis of polysaccharides. A better grafting method
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for polysaccharide modification is the use of microwave irradiation. In the microwave assisted

grafting method, the ions are produced by addition of external redox initiators to the reduction
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mixture, and generation of free radical initiators facilitates the grafting reaction. In microwave-
CE

initiated grafting reactions, however, no initiators are added and a small amount of hydroquinone

as a radical inhibitor is used to inhibit the grafting reactions (Kumar et al., 2012; Lewicka et al.,
AC

2015; Rani et al., 2013; Sen et al., 2009; Singh et al., 2012).

Many synthesis factors influence flocculation performance of modified polysaccharide bio-based

flocculants when using grafting methods. These include reactants/initiator/crosslinker dosage and

their dose ratios, reaction temperature, pH, reaction time and so on, which are reviewed in this

section.
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6.1. Effect of reactants, reactants dosages and their ratios

The reactants, reactant concentration and their ratios can affect structural factors such as grafting

ratio (GR), degree of polymerization, functional group fraction and hence the flocculation

performance of grafted polysaccharide flocculants.

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For example, a novel chemically modified hemicellulose flocculant, METAC-g-Xylan, was

IP
synthesized with a charge density of 2.6 meq/g and a grafting ratio 110.61%. The optimum

CR
values of METAC/xylan obtained was 3 mol:1 mol (S. Wang et al., 2015). Dicarboxylic acid

nanocellulose flocculant was produced using periodate oxidation and followed by chlorite

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oxidation and was employed effectively to flocculate municipal wastewater (Suopajarvi et al.,
AN
2013). Song et al. (2011) synthesized acrylamide-modified cellulose flocculants with the ability

to flocculate ferric hydroxide colloids, using the Michael addition reaction and by saponification
M

hydrolysis of acylamino groups in NaOH-Urea aqueous solution. Similarly, dicarboxyl cellulose

ED

flocculant was synthesized via the Schiff base route and using bamboo pulp cellulose extracted

from Phyllostachys heterocycla. The bamboo cellulose was dissolved in NaOH-Urea solution
PT

and oxidized by NaOI4 to form dicarboxyl cellulose flocculant (Zhu et al., 2015a,b). Cellulose-g-
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PAM and BPC-g-PAM flocculants have been synthesized by grafting ployacrylamide onto the

cellulose and bamboo pulp using free-radical graft copolymerization in aqueous solution (H. Liu
AC

et al., 2014; Razak et al., 2014). In a later study, a novel cellulose-based flocculant was produced

by the TEMPO/NaBr/NaClO oxidation of kraft pulp and successive ultrasonic treatment, and

applied for destabilization of kaolin suspension (Jin et al., 2014). Pang et al. (2013) grafted

cationic etherifying (3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) onto

the cellulose chains of corncob powder using hydroxyl radical made from Fenton reagent (H 2 O2 -

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FeSO 4 ) and used it as a polysaccharide bio-based flocculant. A pH responsive polymer-grafted

cellulose nanocrystal flocculant (P4VP-g-CNCs) was synthesized by surface-initiated graft

polymerization of 4-vinylpyridine with the initiator ceric (IV) ammonium nitrate. The elemental

analysis of P4VN-g-CNC showed the presence of C, H, N, and O in mass ratio of

44.62:6.03:0.95:0.61 and the nitrogen content revealed that the grafting reaction effectively

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occurred (Kan et al., 2013).

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In the case of [2-(methacryloyloxy) ethyl] trimethyl ammonium chloride (METAC) grafted on

CR
xylan (Xyl-g-METAC), both grafting ratio and charge density were increased by increasing the

concentration of METAC to xylan in the range of 0.5–3 mol/mol. More monomer dosages

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resulted in higher free concentration for reacting with the xylan macro radicals and yielded more
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xylan-g-METAC copolymer. The availability of free METAC radicals was enhanced with the

increase of xylan concentration and reached a maximum at 25 g/L. However, the viscosity of the
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reaction solution was also increased with higher xylan concentration, leading to mass transfer
ED

limitation (Zou et al., 2011). Similarly, the flocculation performance of carboxymethyl cellulose-

graft-poly[(2-methacryloyloxyethyl) trimethyl ammonium chloride] (CMC-g-PDMC) bio-based

PT

flocculant enhanced by increasing the mass feed ratio of carboxymethyl cellulose to (2-
CE

methacryloyloxyethyl) trimethyl ammonium chloride (CMC:DMC) during the grafting reaction

(Moad, 2011). A similar finding was reported for chitosan-grafted starch where the flocculating
AC

efficiency improved by increasing the starch-to-chitosan ratio (You et al., 2009).

Chemical modification of chitosan has been explored to improve its flocculating properties. For

example, the flocculation effectiveness of the highly soluble modified chitosan using Glycidyl-

trimethyl-ammonium chloride with the degree of substitution of 0.77 and a DA of 85% was

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shown to be significantly better than unmodified commercially available chitosan (Rojas-Reyna

et al., 2010).

In chitosan-grafted polysaccharide flocculants, the degree of deacetylation (DD) of chitosan can

influences the flocculation activity. Results showed that at the same dose of flocculant, a higher

DD improves the flocculation performance. This correlation can be attributed to the higher

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grafting efficiency (GE) and better flocculation activity at higher DD. A higher GE causes a

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stronger bridging adsorption, leading more particles to be adsorbed on grafted biopolymer, and

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hence flocculation efficiency was improved (Y. Zhang et al., 2012).

Until now, many types of modified starch-based flocculants have been reported in the literature

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with improved solubility, charge density, and molecular weight for effective flocculation. For
AN
example, cationic corn starch and cationic potato starch flocculants with the degree of

substitution of 1.34 and 0.82 were synthesized using 3-methacryloyl amino propyl trimethyl
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ammonium chloride by ceric ammonium nitrate for municipal wastewater treatment. In another
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study, cassava starch-graft-polymethacrylamide (PMAM) copolymers were synthesized by a

free-radical-initiated polymerization reaction, and used as efficient flocculants and textile sizing
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agents. Amylopectin grafted with PAM by a microwave (MW) assisted technique exhibited a
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maximum flocculation efficiency in kaolin suspension (Adhikary and Krishnamoorthi, 2013;

Anthony and Sim, 2013; Lewicka et al., 2015; Nair and Jyothi, 2014).
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Optimizing the reactant concentration could improve the water content of the sludge formed in

the settling process. In the case of inorganic–organic composite bio-based flocculant, CSSAD,

synthesized by the reaction of corn starch, acrylamide (AM), and (2-methacryloyloxyethyl)

trimethyl ammonium chloride (DMC) with a SiO 2 sol using the aqueous solution polymerization

process, results showed that the weight removal increased from 92.56 to 97.56 and the water

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content decreased from 33.51 to 24.10 with the increase in the mass ratio of AM to starch from 1

to 3. However, the flocculating activity of CSSAD decreased at the dosage ratio above 3 because

the solubility of CSSAD in water was reduced due to limited movement of polysaccharide

flocculant molecules with increasing molecular weight of CSSAD. Similarly, by increasing the

mass ratio of initiator (K 2 S2 O8 ) to AM from 0.1% to 0.5%, the weight removal increased from

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89.15% to 98.35%, and the water content decreased from 35.72% to 23.67%. At higher K 2 S2 O8

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to AM mass ratios, the concentration of free radicals and the grafting ratio as well as the

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molecular weight of CSSAD both increased. However, at high initiator to AM mass ratios

(>0.5%) the flocculating activity of CSSAD decreased due to the decrease in the polysaccharide

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flocculant molecular weight caused by the termination of free radicals (Zou et al., 2011).
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6.2. Effect of reaction temperature

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Higher temperatures cause the breakage of molecular chains and induce structural deformation in

biopolymer configuration and length, preventing formation of effective bridges between particles
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in the suspension. The grafting ratio and charge density of Xyl-g-METAC flocculant enhanced

by increasing temperature from 50 °C to 80 °C. This was attributed to the increased generation of
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free radicals with increasing temperature and greater access of METAC to xylan. However, no
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more improvement in charge density and grafting ratio was observed when temperature

increased from 80 to 90 °C, likely due to chain termination and side reactions (S. Wang et al.,

2015). Similarly, the flocculating activity of a corn starch and chitosan-based polysaccharide

flocculant (CATCS) increased as crosslinking temperature rose from 40 to 70 °C, then decreased

for temperatures above 70 °C. In fact, both the degree of crosslinking and the molecular weight

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of CATCS increase at higher reaction temperatures, so flocculation activity of CATCS was

enhanced (You et al., 2009). Tao et al. (2017) reported that color removal efficiency of chitosan-

acrylamid-fulvic acid copolymer (CAMFA) prepared at 60 °C was higher than those synthesized

at 50 °C and 70 °C.

The flocculating ability of CSSAD was explored for the polymerization reaction temperature in

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the range of 50 to 80 °C when the mass ratio of DMC to starch, mass ratio of starch to AM, dose

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ratio of initiator to AM, and reaction time were 1.5:1, 1:3, 0.5%, and 4 h, respectively. The

CR
flocculating activity was enhanced by increasing the reaction temperature from 50 to 70 °C, and

the maximum value was observed at 70 °C. The degree of substitution of starch and the

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molecular weight of flocculant were first increased by raising the reaction temperature, resulting
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in improvement of the flocculating activity of CSSAD. The reduction of flocculating activity for

temperatures above 70 °C can be attributed to the breakage of molecular chains of CSSAD at

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higher temperatures (Zou et al., 2011).

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6.3. Effect of pH
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The effect of pH of the reaction medium is another key factor affecting the grafting reaction and
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hence the flocculation activity of the chemically modified polysaccharide flocculant. In the case

of Xyl-g-METAC, charge density and grafting ratio were reported to reach their maximum
AC

values at pH 7 and then decrease by increasing pH to 11. The charge reduction at alkaline

conditions was suggested to be due to hydroxyl ions attacking the hydrogen linked at β-carbon to

the quaternary ammonium group, resulting in instability and decomposition of quaternary

ammonium groups into tertiary ammonium groups (S. Wang et al., 2015).

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6.4. Effect of reaction time

Reaction time is another important factor in chemical modification of polysaccharide bio-based

flocculants. In co-polymerization reaction for synthesis of Xyl-g-METAC flocculant, more

radicals were produced and the reaction rate was accelerated by extending reaction time. The

charge density and grafting ratio improved when increasing the reaction time to 3 h. More

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mobility of METAC monomers occurred when increasing the reaction time, which caused more

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collisions of the METAC monomers with xylan. However, both charge density and grafting ratio

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were reduced by further increasing the time above 3 h due to synthesis of PMETAC (S. Wang et

al., 2015). In the case of corn starch and chitosan-based flocculant (CATCS), crosslinking

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between the reactants increased with an increase of reaction time to 1.5 h, then reached its limit
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by reducing of reactants with the greater molecular weight (You et al., 2009). For CSSAD bio-

based flocculant, the flocculating activity was enhanced by increasing reaction time from 1 h to 4
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h and then decreased after 4 h when the mass ratio of DMC to starch, mass ratio of starch to AM,
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dose ratio of initiator to AM, and reaction temperature were 1.5:1, 1:3, 0.5%, and 65 °C,

respectively. The reaction was terminated by hydrolysis of starch and DMC, with increase in the
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reaction time above 4 h (Zou et al., 2011).

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Table 4 Properties of chemically modified polysaccharide Bio-based flocculants

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Biopolymer Reactants T pH Reaction Other properties Flocculation Reference

time efficiency

PyMeBnOO-g-CNC Cotton wool cellulose, 80 °C 4-11 - C:H:N:S 49.58: 100% Vandamme

(Cellulose nanocrystals) Pyridinium salts, 4-(1- 5.77:1.84:0.99, Br et al., 2015
bromoethyl)benzoic 4.95, DS 0.38 harvesting)
acid)
CAMFA Chitosan: AM:FA 60 °C 5h Reaction yield 55.8%, 97% T ao et al.,
(Chitosan-acrylamide-fulvic 1.5:3:3 g/g, Chitosan (85%), Mw 2017
acid) 2× 10 5 Da

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QL-g-SAG Lignin salt:Alginate 70°C 8-10 4h N content 3.29%, 97% Zhang et

(Trimethyl quaternary 2:1 (w/w) Carboxyl content 2.66- al., 2013
ammonium salt of lignin-g- 6.47%
sodium alginate)
SAG-g-PMMA SAG:MMA1:7.5 g/g 65 °C 50 s GR 87%, Viscosity 8.8 97% Rani et al.,
( SAG-g-Polymethyl methacrylate) dl/g, C:H:N:O 2013
34.53:5.962:0.00:54.88
CMC-g-PAM CMC:AM 1:1, pH 4.0 - - 3h GR 74% Chitosan (DD 94.97% Yang et al.,
(Carboxymethyl chitosan-g- & pH 11.0 85.2%, 8.34 ×10 5 g 2013
Polyacrylamide) mol-1 )

HES-g-(PDMA-co-AA) HES 2 g, DMA 0.078 35 °C 7.2 5h MW 12 × 10 6 Da >90% Kolya et

(Hydroxyethyl starch-g- mol, AA 0.117 mol al., 2017
poly(N,N dimethylacryamide-

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co-acrylic acid))

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Xyl-g-MET AC MET AC/Xylose 3 80 °C 7 3h GR 110.61%, MW 97.8% S. Wang et
(Xylan-g-[(2- mol/mol, Xylan 25 g/L 102250 g/mol, al., 2015
(methacryloyloxy) ethyl] C:N:H:O

CR
trimethyl ammonium 43.7:3.54:8.5:40,
chloride) Charge density 2.6
meq/g

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CMC-g-PDMC CMC:DMC 1:5 80 °C 7 3h GR 181%, Degree of 97.2% Cai et al.,
(Cellulose-g-poly[(2- carboxymethyl groups 2015
methacryloyloxyethyl) 17.4%
trimethyl ammonium
chloride])
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Xylan-g-MET AC MET AC:Xylose 80 °C 7 3h C:H;N:O 97.8% S. Wang et
Hemicellulose flocculant 3mol:mol 43.7:8.5,3.54,40, MW. al., 2015
(Xylan-g-[2- 102250 g/mol , Charge
(methacryloyloxy) ethyl] density 2.6 meq/g
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trimethylammonium chloride)
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7. Effect of physic-chemical factors on flocculating activity

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The role of physical-chemical parameters such as flocculant dosage, molecular weight, pH,
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temperature, and metal ions on flocculating activity of polysaccharide bio-based flocculant in a

AC

colloid system are explained in this section. Polysaccharide bioflocculants have shown

advantages over other types of flocculants. They are generally heat-stable, cation-dependent, and

show flocculating activity over a wide range of pH and temperatures. They may be either

produced at high rates or recovered from culture broths.

7.1. Effect of flocculant dosage on flocculating activity

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The polysaccharide flocculant dosage plays a key role in determining flocculating activity of a

colloid system. When the polysaccharide flocculant dosage is lower than the optimum, the

degree of flocculation is insufficient in a colloid system that results in restabilization and charge

reversal of the colloidal particles. In this case, there are not enough polysaccharide flocculant

molecules to adsorb onto the colloidal particle surfaces to bridge between these particles. An

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overloaded dosage of the polysaccharide flocculants increases the electrostatic repulsion forces

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between the colloidal particles and consequently increases the distance between the particles to

CR
inhibit floc formation and precipitation.

For example, the maximum flocculation efficiency of polymethyl methacrylate grafted sodium

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alginate (SAG-g-PMMA) was observed at the optimum biopolymer flocculant dosage of 0.375
AN
mg/L in a coal suspension of 1% w, and the results of the jar test in a practical scale exhibited

that the usage of 1 g of SAG-g-PMMA effectively treated 2667 L of coal washery effluent (Rani
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et al., 2013). Modified starches, both cationic and anionic, were proven to be effective in treating
ED

wastewater for improved water quality. The flocculation behavior of anionic and cationic (3-

Chloro-2-hydroxypropyl)trimethyl ammonium chloride etherified carboxymethyl starch(CMS-

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CTA-N and CMS-CTA-P) was studied in hematite and kaolin synthetic wastewater samples, and
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the results showed a flocculation efficiency of 98.6 and 97.2%, respectively, at optimum

flocculant dosages of 3 and 0.6 mg/L, and pH 7 (H. Li et al., 2015). Similarly, the optimum
AC

dosage for chemically modified chitosan based flocculant, i.e. carboxymethyl chitosan-graft-

poly(2-methacryloyloxyethyl) trimethylammonium chloride (CMC-g-PDMC), was obtained at

50 mg/L (Yang et al., 2014a). The highest flocculating activity of 97.3 was reported for

cellulose-graft-poly (2-methacryloyloxyethyl) trimethyl ammonium chloride (CMC-g-PDMC),

at an optimum dosage of 90 mg/L (Cai et al., 2015).

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The optimal dosage of the carboxymethyl cellulose-graft-poly[(2-methacryloyloxyethyl)

trimethyl ammonium chloride] (CMC-g-PDMC) was decreased from 210 to 60 mg/L by

increasing the grafting ratio from 44.5% to 337% at each pH level. The dye (Acid Green 25)

removal efficiency and floc sizes as well as flocculation performance were also improved by

increasing the graft ratio of CMC-g-PDMC. CMC-g-PDMC with higher grafting ratio exhibited

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higher molecular weight, resulting in an enhanced bridging effect and enhanced flocculation

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efficiency. Therefore, the flocculation performance of CMC-g-PDMC (GR 181%) proved pH-

CR
independent and always remained at a high level (> 90%) (Cai et al., 2015). Zhu et al., (2015b)

used dicarboxyl cellulose (DCC) flocculant for treatment of paper mill effluents and the

optimum concentration was determined as 40 mg/L.

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The highest dye removal efficiency of 97.8% was achieved for cationic Xyl-g-METAC

hemicellulose based flocculant at an optimum dosage of 160 mg/L. Similarly the same dye
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removal ability for homopolymer of 2-(methacryloyloxy) ethyl trimethyl ammonium chloride

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(PMETAC) was reported at 100 mg/L. A higher efficiency of PMETAC floccculant compared to

Xyl-g-METAC flocculant can be attributed to the effect of charge density because PMETAC has
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a higher charge density (4.8 meq/g) than Xyl-g-METAC (2.6 meq/g). The results showed that for
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dosages higher than the optimum concentration (160 mg/L), the flocculation efficiency of

cationic Xyl-g-METAC flocculant decreased with increasing repulsive forces due to positively
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over-charged surface of dye/copolymer, causing the restabilization of the colloid suspensions (S.

Wang et al., 2015). In fact, overdosing will result in over-charging if the flocculants are charged.

Most of natural flocculants do not have any charge and their main interaction is based on

hydrogen boding and bridging is the main flocculation mechanism. Thus overcharging may not

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be a significant problem for these flocculants (B. Zhang et al., 2013; Salehizadeh and Yan, 2014,

S. Wang et al., 2015; Zhu et al., 2015b).

The optimum dosage for bioflocculants produced by Bacillus toyonensis was reported to be 0.1

mg/mL (Okaiyeto et al., 2015). Bioflocculants produced by Klebsiella ZZ-3 and K. pneumonia

MBF5 were cation-independent, and the highest flocculating activity in kaolin suspension was

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determined to be over 80% and 92% for optimum bioflocculant dosages of 54.38 mg/L and 5

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mg/L, respectively (Yin et al., 2014). In the case of A. flavus, optimum dosage of bioflocculant

CR
was 5 mg/L for activated carbon and soil solids removal. The highest flocculating activity

observed was 95% at 30 mg/L of bioflocculant concentration in yeast cell suspension, and the

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flocculation activity was dramatically stimulated from 20.8 to 82.3%, when bioflocculant
AN
increased from 10 to 15 mg/L (Rajab Aljuboori et al., 2015).
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7.2. Effect of flocculant molecular weight on flocculating activity

ED

The molecular weight of polysaccharide bio-based flocculant affects not only the properties of

flocculant, but also its flocculation efficiency (Grenda et al., 2017). The effectiveness of bridging
PT

mechanism depends on the molecular weight of polysaccharide flocculant. The molecular weight
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of polysaccharide flocculants varies in the range of 10 4 –107 g/mol (Koyla et al., 2017;

Salehizadeh and Yan, 2014; S. Wang et al., 2015, Tao et al., 2017). For example, the molecular
AC

weight of chemically modified polysaccharide flocculants such as CAMFA (Tao et al., 2017),

CMC-g-PAM (Yang et al., 2013), and HES-g-(PDMA-co-AA) (Koyla et al., 2017) were 2× 105

Da, 8.34 ×105 g/mol, and 12 × 106 Da, respectively. The bioflocculant produced by K.

pneumonia was a polysaccharide with a molecular weight of 2 × 10 6 Da and its flocculating

activity was 96.5% at bioflocculant dosage of 50 mg/L at neutral pH (Luo et al., 2014). The

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molecular weight of polysaccharide biofloccualnt from Paenibacillus polymyxa was reported at

1.16×106 Da and the achieved flocculating activity was 81.7% (Guo et al., 2015b). The

molecular weight of bioflocculant produced by Acinetobacter junii was estimated at 2 × 105 Da

and its flocculating activity was determined as 97% (Yadav et al., 2012). The smallest molecular

weight has been recorded for polysaccharide bioflocculant from Aspergillus flavus with a

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molecular weight of 25470 Da, and the reported flocculating activity was 91.6% (Rajab

IP
Aljuboori et al., 2015).

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7.3. Effect of initial pH on flocculating activity

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The initial pH of the medium is one of the main environmental parameters influencing
AN
flocculating activity. The flocculation performance of polysaccharide bio-based flocculants

depends directly on pH of colloid suspension, and the optimum pH of the colloid systems for
M

flocculants varies with the change of chemical structure and composition of flocculant, the nature
ED

of colloid particles, metal ions additives, etc. (More et al., 2014).

The color removal ability of a chemically modified polysaccharide flocculant, CAMFA, was
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evaluated in the range of pH 5-9 for acid blue 113 (Ab-113), reactive black (RB-5), and methyl
CE

orange (MO). The results showed that the removal efficiencies of Ab-113 and RB-5 were

enhanced with stronger acidity and alkalinity conditions, while a higher color removal ratio
AC

achieved for lower pH because of strengthened protonation of NH2 under acidic solution and the

increased effect of charge neutralization (Tao et al., 2017). The color removal efficiency of 95%

for methylene blue was obtained using a mixture of a chemically cationized cellulose-based

flocculant (DAC) and bentonite at an acidic condition (pH=4) after 10 min. Decolourization

capacity was lower than 80% even with a higher dosage of DAC for higher pH’s after 24 h

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(Grenda et al., 2017). The settling behaviour of quartz using chitosan was greatly affected by pH,

with the largest volume of sediments achieved at pH 9. Chitosan desorption from the quartz

surface occurred by changing pH from 9 to 3 (Feng et al., 2017).

A cation-independent bioflocculant produced by Acinetobacter junii showed flocculating activity

above 94% in the pH range of 4–5 with bioflocculant dosage of 30 mg/L, while flocculation

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dropped down by increasing pH from 6 to 10. This can be attributed to decreasing negative

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charges of the clay particles at pH 4–5, causing the reduction of distance between kaolin

CR
particles, and improving the bridging mechanism. Above pH 6, the negative charge of

bioflocculant increased due to deprotonating of the functional amide groups, and repulsive forces

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preventing approach of clay particles, resulting in reduction of flocculating activity (Yadav et al.,
AN
2012). The effects of the reaction mixture pH on flocculating activity of bioflocculant (MBF-6)

produced by K. pneumoniae YZ-6 were investigated over the pH range of 2–12 in kaolin
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suspension. As a result, the flocculation activity reached over 80% in the pH range of 3–11, and
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the maximum flocculation activity of 87% was observed at pH 7 (Luo et al., 2014). Bioflocculant

(MBF-7) produced from K. pneumonia ZCY-7 using H-acid wastewater had the highest
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flocculating activity at optimum pH 5 that was not influenced by the pH range of 3–6 and
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decreased gradually at higher pH in the range of 7–12. Similarly, the biofloccculant of

Gyrodinium impudicum KG03 was active in pH range of 3–6, and a maximum flocculating
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activity was obtained at pH 4 (Zhong et al., 2014). For Micrococcus sp. Leo, the flocculating

activity was more than 50% at the pH values ranging from 2 to 9, and a maximum flocculating

activity of 80.7% was achieved at optimum pH 4. The lowest flocculating activity of 1.16% was

observed at pH 12 (Okaiyeto et al., 2014). The highest flocculating activity of alkaliphilic

Bacillus agaradhaerens was obtained with a value of 93.5% at pH 6.5, with bioflocculant dosage

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of 1.5 mg/L without addition of Ca2+; and flocculation efficiency was over 80% in the pH range

from 2.38 to 9.72 (Liu et al., 2015). Bioflocculant produced by Bacillus toyonensis was pH

tolerant and showed strong flocculating activity either under acidic (pH 5) or alkaline condition

(pH 10) (Okaiyeto et al., 2015).

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7.4. Effect of temperature on flocculating activity

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Temperature is an important physical parameter affecting flocculating activity. The bio-based

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flocculants with polysaccharide-based structure are generally thermal-stable, but those with

protein are sensitive to temperature (Salehizadeh and Yan, 2014). The effect of temperature on

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flocculation activity of alginate was investigated and the results showed that the polysaccharide

biofloculant was thermally stable in the range of 20-100 °C and over 50% of flocculating activity
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was retained after heating at 100 °C (Auhim and Odaa, 2013). The bioflocculant produced by B.
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Agaradhaerens was thermally stable and showed good flocculating activity over a board range of
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temperatures. More than 80% of the flocculating activity remained at all evaluated temperatures.

The maximum flocculating activity of 91.5% was obtained at 29 °C, with a high flocculating
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activity of 91.25% achieved at 63 °C. This can be attributed to the presence of polysaccharide as
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the main component in the bioflocculant structure (Liu et al., 2015). The flocculating activity of

bioflocculant from Enterobacter sp. was decreased from 91.5% to 80% by increasing the
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temperature from 40 °C to 100 °C, indicating that this bioflocculant was relatively thermo-stable

due to heat resistance of the polysaccharide component (Tang et al., 2014). For Paenibacillus

polymyxa, the flocculating activity reduced around 0.3–10% after being heated for 30 min at

different temperatures in the range of 30–110 °C (Guo et al., 2015b).

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The highest flocculating activity for bioflocculant from Acinetobacter junii was achieved at 20

°C in the temperature range of 10–100 °C. The flocculating activity decreased 23% when

temperature dropped to 10 °C or rose to 40 °C. A reduction of 15% and 10% was observed by

increasing temperature from 50 to 80 °C, and 80 to 100 °C, respectively. It may be explained by

the denaturation of the protein component in bioflocculant and a simultaneous increase in kinetic

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energy of kaolin particles due to a rise in temperature (Yadav et al., 2012). In the case of K.

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pneumonia, the highest flocculating activity achieved at 30 °C was 96.5%, and the flocculating

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activity was above 80% in the range of 50–60 °C (Luo et al., 2014).

7.5. Effect of metal ions on flocculating activity

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Cations increase the adsorption of polysaccharide bio-based flocculant onto the surface of

particles by neutralizing the negative charges of both flocculant and colloid particles. Multivalent
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cations can bridge with negative functional groups on the biopolymer chain. Polysaccharide bio-
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based flocculants with higher negative net charge on biopolymers surface cause higher

hydrophilic parts, resulting more interaction with metal ions (Salehizadeh and Yan, 2014).
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The macromolecule flocculant, mercaptoacetyl-g-chitosan (MAC) was prepared by amidization

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reaction of chitosan and L-cysteine. The results indicated that the removal rate of Cu2+ increased

by raising turbidity and the highest removal efficiency obtained was 96.73%. It was reported that
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the Cu2+ removal efficiency was stimulated in the presence of co-existing cations in order of

Ca2+~Mg2+>K +~Na+, and anions hindered the removal rate of Cu2+ (Zhang et al., 2015). For

example, divalent and monovalent cations stimulated the flocculating activity of bioflocculant

produced by Bacillus toyonensis by more than 80%. The maximum flocculating activity was

observed with Ca2+ (87%), K + (85%), Na+ (81.3%), Mg2+ (82.2%) and Al3+ (71.3%). The

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flocculating activity of bioflocculant was completely inhibited by Fe 3+ due to its immoderate

adsorption onto the suspension particles (Okaiyeto et al., 2015). The bioflocculant produced by

Klebsiella sp. ZZ-3 was cation-dependent, and the flocculating activity was dramatically

stimulated by Fe3+ and slightly increased by Al3+. Monovalents (Na+, K +) and divalents did not

influence flocculating activity (Yin et al., 2014). The cations including K +, Ca2+, Mn2+, Ba2+,

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Fe3+, Al3+ stimulated the flocculating activity of bioflocculant secreted by Micrococcus sp. Leo,

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and the highest flocculating activity of 85.2% was observed with Al3+, while Na+, Li+ and Fe3+

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had an inhibitory effect leading to flocculating activity less than 50% (Okaiyeto et al., 2014). In

the case of mixed culture of Rhizopus sp. M9&M17, addition of divalent cations, especially

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Mg2+ and Ca2+, enhanced the flocculating activity, whereas the monovalent cations (Na+, K +) and
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trivalent cations (Al3+, Fe3+) indicated a negative effect and flocculating activity decreased below

70% (Pu et al., 2014).

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8. Practical applications of biopolymer flocculants

Polysaccharide bio-based flocculants are widely used for drinking water and wastewater
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treatment, food production, fermentation process, mining operation, etc.. Several important
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practical applications of polysaccharide bio-based flocculants are summarized as follows:

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8.1. Drinking water treatment

Several studies have been reported on the application of polysaccharide bio-based flocculants as

an alternative to the synthetic polymer flocculants in water treatment. Chitosan has been widely

examined for the water treatment applications for its good turbidity removal efficiency over a

wide range of pH (R. Yang et al., 2016, Zhang et al., 2015). Zemmouri et al. (2011) reported a
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turbidity reduction of 87% for a surface water sample with the initial turbidity of 20 NTU at

chitosan dosage 0.15 mg/L (DD 85%) over a wide range of pH. The combination of chitosan and

alum exhibited more efficiency in removing turbidity, showing maximum reduction of 97% with

a chitosan dosage of 0.2 mg/L obtained after 30 min settling time. Chitosan has also been used to

remove silt from river water (Divakaran and Pillai, 2002). As discussed earlier, grafting could

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further enhance flocculation efficiency of chitosan and hence its performance in treating water.

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The strong cationic flocculant formed by grafting (3-chloro-2-hydroxypropyl trimethyl

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ammonium chloride (CTA) on chitosan-g-polyacrylamide has been used for the treatment of the

Zhenjiang water Supply. The flocculant decreased the turbidity of the water sample from 35.6

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NTU to 0.34 NTU at the dosage of 1.3 mg/L, mixing rate 150 rpm, and 28.5 °C after 20 min (Lu
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et al., 2011). The mercaptoacetyl-chitosan flocculant showed excellent ability to treat water

samples containing heavy metals and turbidity and the highest turbidity removal rate of 100%
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was achieved (Zhang et al., 2015).

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Alginate-based flocculants are other groups of polysaccharide flocculants that have been adopted
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for water treatment applications. In particular, calcium alginate has been reported to be an

effective flocculant for high turbidity (150 NTU) and medium turbidity (80 NTU) water (Coruh,
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2005). At optimum alginate dosage of 0.4 mg/L in the presence of Ca2+ the final turbidity levels
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of treated surface water samples met the requirements of drinking water standards. Combining

sodium alginate (SAG) and other polymers PAC could have synergistic effects and enhance the

overall flocculation efficiency. For example, PAC plus SAG had a synergistic effect on the

removal of dissolved organic carbon, and turbidity. In this case, the addition of SAG to PAC had

a positive effect on both floc growth rate and floc size, and it highly improved floc recovery after

floc breakage compared with PAC (Zhao et al., 2012).

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Cellulose-derived flocculants are another group of polysaccharide flocculants with good

potential for water treatment applications. Sodium carboxymethylcellulose (CMCNa) removed

95% of the turbidity of a surface water sample at a dosage of 100 mg/L in the pH range of 7–8

(Khiari et al., 2010), while polyacrylic acid grafted carboxymethyl cellulose, CMC-g-PAA,

reduce more than 86% of the turbidity of a river water sample at a dosage of only 0.75 mg/L.

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Specially, the chemically grafted CMC-g-PAA was highly effective in removing Cr6+, total iron,

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Ni2+, and Mn2+from the river water, organic load (COD) and TSS (Mishra et al., 2012).

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Similarly, the bio-based flocculant (DXSL-1) formed by grafting cationic (3-chloro-2-

hydroxypropyl) trimethyl ammonium chloride (CHPTAC) onto the corncobs removed more than

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95% of the turbidity from an aqueous solution after 2 min (Pang et al., 2013).
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8.2. COD and turbidity removal
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The application of polysaccharide bio-based flocculant for removing suspended solids and
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improving effluent quality of municipal and industrial wastewaters has been explored by many
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researchers in recent years (Table 5) (Grenda et al., 2017; H. Li et al., 2015; Kolya et al., 2017;

Lee et al., 2014b; Qin et al., 2015; Renault et al., 2009b; Yuan et al., 2013).
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Once again, chitosan-based flocculants have been widely reported for the wastewater treatment
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applications (Lee et al., 2014b; Wu et al., 2015). In one study, chitosan (MW 1.8×105 g/mol) was

used to treat cardboard-mill wastewater (Renault et al., 2009b). This polysaccharide flocculant

achieved higher treatment efficiency than polyaluminium chloride under optimum conditions,

decreasing COD and turbidity by more than 80 and 85%, respectively, compared to 40–45% and

55–60% for PAC. Chitosan was also able to remove a considerable amount of color and resulted

in a significant reduction in the heavy metal concentration (Renault et al., 2009b). In another
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study, the flocculating activity of chitosan was improved from 2.73 to 20 by grafting on corn

starch (CATCS) at the optimum flocculant dosage of 0.75 mg/L at pH 7.6 (You et al., 2009).

Alginate has been also used in combination with other common flocculants for both industrial

and municipal effluent treatment. The flocculating effectiveness of a composite flocculant,

including sodium alginate, polyaluminium chloride, and ferric chloride and cationic

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polyacrylamide (mass ratio of 2:1:1) was investigated for wastewater treatment, and the

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maximum turbidity removal and COD reduction of 99.2% and 89.6%, respectively, were

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achieved at the optimum flocculant dosage of 20 mg/L, pH 7.1 and 30 °C (Zeng et al., 2011).

In a separate study, the turbidity/colour removal ability and COD reduction from sewage using a

combination of alginate/chitosan biopolymer

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flocculants achieved 100% and 90.01%,
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respectively, at pH 5.5 and 70 °C after 2.5 h (Yuan et al., 2013). The alginate-like polysaccharide

flocculant produced by Azotobacter indicus decreased the BOD (38–80%), COD (37–79%), and
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SS (41–68%) of wastewater samples (Patil et al., 2011).

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Mukherjee et al. (2014) evaluated the flocculation effect of different biopolymers and alum on

the recovery of pulp fibers from paper mill effluent and reported that guar gum and xanthan
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recovered up to 3.86 mg/L and 3.82 mg/L of pulp fiber. The highest turbidity removal for guar
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gum, xanthan, locust bean gum, and alum obtained were 94.68%, 92.39%, 92.46% and 97.46%,

respectively (Mukherjee et al., 2014).

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Various types of cellulose-derived floccunalts have been reported in the literature for effluent

treatment. The dicarboxyl cellulose (DCC) bio-based flocculants showed excellent flocculation

performance in the paper mill effluent in the presence of CaCl2 and the results revealed that the

DCC was able to decrease turbidity by 87.6%, CODcr by 67.2%, and BODcr by 64.7%,

respectively (Zhu et al., 2015a). The bio-based flocculant sodium carboxymethyl cellulose (Na-

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CMC) decreased the turbidity of a pond ash water sample to 19.9 NTU, and 17.7 NTU after 5

and 30 min, respectively, with a flocculant dosage of 5 × 10 -6 (Prasad and Kumar, 2013).

Suopajärvi et al. (2013) studied the effect of the different dicarboxylic acid nanocellulose (DCC)

bio-based flocculants. The results showed that the best flocculation efficiency was obtained with

high charge density and high nanofibril content of the DCC. A combined FeSO 4 and DCC bio-

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based flocculant was used to flocculate wastewater samples, achieving COD reduction of 40–

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80% and turbidity removal by 40–60%, respectively, using bio-based flocculant dosage of 2.5–5

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mg/L in the presence of coagulant concentration of 25 mg/L (Suopajarvi et al., 2013). Guar gum

is another bio-based flocculant that has found its application in wastewater treatment.

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Hydroxypropyltriammonium chloride guar gum (HPTAC-guar) was employed to decrease
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biological contaminants of wastewater containing total coliforms (TC), fecal coliforms (FC) and

helminth eggs (HE) with initial values of 2.8 × 10 7 MPN/100 ml, 8.48 × 106 MPN/100 ml and
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470 HE/L, respectively. The removal efficiency observed for biological contaminants was 82%
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for TC, 94% for FC and 99% for HE, along with COD reduction of 46%, and turbidity removal

of 39%. The use of combined HPTAC-guar flocculant and Ca(OH)2 coagulant resulted in the
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reduction of 52% for TC, 100% for HE, 47% for COD and 30% for turbidity (Zamudio-Perez et
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al., 2013).

The cationic glycogen polysaccharide flocculant decreased the turbidity, total solids (TS), total
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dissolved solids (TDS), total suspended solids (TSS), and chemical oxygen demands (COD) of

municipal wastewater from 64 to 4 NTU, 630 to150 mg/L, 280 to 125 mg/L, 350 to25 mg/L, and

540 to 175 mg/L, respectively, using an optimum flocculant dosage of 9 mg/L (Singh et al.,

2014). Kolya et al. (2017) reported that the best flocculation efficiency for polysaccharide-g-

(PDMA-co-AA) in textile effluent and paper mill effluent, whereas polysaccharide-g-PDMA

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showed the best result in sewage wastewater. Starch, amylopectin and hydroxyethyl starch were

used as the model polysaccharides. The graft copolymers based on HES exhibited greatest

flocculation efficiency compared to some commercially available flocculants such as telfloc

2230, magnafloc 1011, and percol 181 in all colloidal suspensions, industrial effluents and

municipal wastewaters. HES-g-PDMA (Hydroxyethyl Starch-g- poly (N,N-dimethylacrylamide)

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acid efficiently reduced the turbidity, COD, TSS, TDS, TS of municipal wastewater from 90

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NTU, 650, 325 mg/L, 530 mg/L, and 855 mg/L to 8.5 NTU, 138, 30 mg/L, 65 mg/L, and 95

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mg/L, respectively (Kolya et al., 2017).

Similarly, polyacrylamide grafted xanthan gum/silica-based nanocomposite bio-based flocculant

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(XG-g-PAM/SiO 2 ) was able to decrease turbidity, TS, TDS, TSS, and COD of Fe-ore and kaolin
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containing wastewater (Ghorai er al., 2013). In another work, a compound flocculant was

synthesized by grafting acrylic acid/acrylamide (AA/AM) onto xanthan gum (XG) with sepiolite
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fiber to treat oil wastewater (Zhu et al., 2009).

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Table 5 Flocculation properties of bio-based polysaccharide flocculants in wastewater treatment

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Bio-based flocculant Conditions Optimum Flocculating Remarks Ref.

dosage activity/Removal
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(mg/L) efficiency
Alginate pH 7 , CaCl2 10 500 SS 63%, COD T reat sugar, wool, (Patil et al., 2011)
(Azotobacter indicus) mg/L, SS 450 mg/L , 76%, BOD 80% starch, and dairy
COD 630 mg/L, wastewaters
BOD 350
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CAT CS pH 7.6, COD 551 0.75 86% (FA 2.73 T reat wastewater (You et al., 2009)
(starch and chitosan- based mg/L, SS 666 mg/L reach to 20)
flocculant)
Cationic Glycogen 64 NT U, T S 630 9 4 NT U, T S150 T reat municipal (Singh et al., 2014)
mg/L,T DS 280 mg/L,T DS 125 sewage wastewater
mg/L, T SS 350 mg/L,T SS 25
mg/L, COD 540 mg/L, COD 175
mg/L), 75 rpm, mg/L
settling time 15 min,
Chitosan pH 6.3-7.7 7ml/L (3% T R 85%, COD T reat wastewater, (Renault et al., 2009b)
w/v) 80% used either heavy
metals removal and
decolourization

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Chitosan pH 8, Settling time 500-1500 SS 89%, BOD T reat unhairing (Sila et al., 2014)
30 min 33.3%, COD tanning effluent
58.7%

Chitosan pH 5, Settling time 5 150 T R 93%, T SS T reat dairy (Devi et al., 2012)
min 73%, COD 79% wastewater

Chitosan + Sodium Alginate pH 5.5, 70 °C, 15mg/L+ T R/CR 100%, T reat sewage water (Yuan et al., 2013)
Settling time 2.5 h 7.5 mg/L COD 90.01%

HES-g-PDMA pH 6.8, 90 NT U 12 mg/L T R 90.5% T reat municipal (Kolya et al., 2017)

(Hydroxyethyl Starch-g- COD 78.8%, wastewater
poly (N,N- T SS 90.7%,
T DS 87.7%

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dimethylacrylamide) T S 88.9%
acid

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CMS-CT A-N & CMS-CT A- Hematite suspension: 3& (0.6 98.6% & (97.2%) T reat synthetic (H. Li et al., 2015)
P (2- pH 7,1 g/L& mg/L) wastewater
hydroxypropyl)trimethylam (Kaolin suspension: suspensions

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monium chloride etherified pH 7, 1 g/L)
carboxymethyl anionic &
cationic starch

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DCC Paper mill: 381 40 FA 88.5%, 47.1 T reat paper mill (Zhu et al., 2015b)
(Dicarboxyl cellulose) NT U, COD 5568, NT U, COD 67.2% effluent
BOD 918 mg/L, or 1824 mg/L,
Added Ca2+ 300 BOD 64.7% or
mg/L 324 mg/L
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DCC pH 7, T SS 368 2.5-5 T R 40-80%, T reat wastewater (Suopajarvi et al.,
(anionic nanofibrillated mg/L,COD 435 COD 40-60% 2013)
dicarboxylated cellulose) mg/L, 158 NT U,
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FeSO4 25 mg/L
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HT CC T annic acid 10 g/L, 2 350 63.4% T reat tannic acid (Qin et al., 2015)
(N-hydroxypropyl trimethyl h, pH 4.5 extract
ammonium
chloride chitosan)
Na-CMC Ash suspension 5× 10 -6 17.7 NT U, Water T reat pond ash (Prasad and Kumar,
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(Sodium Carboxymethyl (SiO2 81.85%, Al2O3 drainage 88.28% wastewater 2013)

Cellulose) 30.48%), Settling in 45 min
time 30 min
Pullulan and PAC pH 7-7.3, 24-96 0.6&15 T R 95%, CODcr T reat municipal (Yang et al., 2007)
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NT U, SS 60-100 mg/L 58%, T otal sewage wastewater

mg/L, COD 6-140 phosphorus 91%,
mg/L) NH3-N 15%
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SAG-g-PMMA Coal fine suspension 0.375 O.D< 0.1 1 g of SAG-g- (Rani et al., 2013)
(Polymethyl methacrylate (1% w), Settling PMMA is sufficient
grafted sodium alginate) time 5 min for the
treatment of 2667 L
of coal washery
effluent.
Sodium alginate+ pH 7.1, COD 1208 20 T R 99.2%, COD T reat paper making (Zeng et al., 2011)
polyaluminium ferric chlo- mg/L, 415 NT U, 89.6% wastewater
ride+cationic SAG:FeCl3:PAM
polyacrylamide (2:1:1)
Xanthan 1-2 mg/L 94.68%; Fiber T reat paper mill (Mukherjee et al.,
recovery 3.82 effluent 2014)
mg/L, settling
velocity 0.1
cm/min
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8.3. Decolourization and dye removal

Some polysaccharide bio-based flocculants are known to be effective biosorbents for the removal

of dyes and colors. Biosorption capacity of these bio-based flocculants depends on the bio-based

flocculant concentration and composition, pH, temperature, and type of dye (Cai et al., 2015;

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C.H. Yang et al., 2014; Kono, 2017; Kolya and Tripathy, 2013; Kolya et al., 2017; S Wang et al.,

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2015). Several water soluble cellulose flocculants with different substitution degrees were

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prepared from cellulose dissolved in the urea/NaOH. Cellulose-based flocculants showed great

flocculation abilities against anionic dyes. Their flocculating ability depended on the degree of

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substitution, but not influenced by temperature and pH of the dye solution (Kono, 2017). Grenda
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et al. (2017) reported the use of a dual system containing bentonite and cationic celullose fiber

with the highest degree of substitution as flocculant for dye removal, and better results were
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achieved compared with a commercial polyacrylamide flocculant.

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Other types of polysaccharide bio-based flocculants are also able to remove contaminants via

biosorption, including [2-(methacryloyloxy) ethyl] trimethylammonium chloride (METAC)

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grafted xylan for removing Reactive Orange 16 (S. Wang et al., 2015), cellulose-g-poly[(2-
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methacryloyloxyethyl)trimethylammonium chloride], and CMC-g-PDMC, for removing acid

green 25 (Cai et al., 2015), cationic glycogen for removing reactive black dye (Singh et al.,
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2014), starch-g-poly-N,N-dimethylacrylamide-co-acrylic acid and HES-g-Poly-(DMA-co-AA)

for malachite green removal (Kolya and Tripathy, 2013), and trimethyl quaternary ammonium

salt of lignin-g-sodium alginate for removing methylene blue and acid black ATT (Zhang et al.,

2013) (Table 6). Xyaln-METAC copolymer, as a chemically modified hemicellulose flocculant,

was employed for effective removal of azo-dyes (reactive orange 16) from textile wastewater. A

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decolourization efficiency of 97.8% was obtained using the modified cationic hemicellulose at

an optimum dosage of 160 mg/L of flocculant and dye solution containing 100 mg/L of dye.

8.4. Biomass harvesting and cell recovery

Although microalgaes have been attracting much attention for biofuel production, microalgae

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biodiesel production is limited by high cost in biomass harvest, and includes 20–30% of the total

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production cost. Bio-based flocculants can be considered as a low-cost alternative for harvesting

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the microalgae biomass (C. Dong et al., 2014; Lee et al., 2010; Liu et al., 2015, 2017; Rakesh et

al., 2013; Rashid et al., 2013; Wan et al., 2013). Lee et al. (2010) studied the energy requirement

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and economic analysis of a full-scale bioflocculant system for microalgae removal. The overall
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cost of the process, and the required mixing energy for flocculation of microalgae were predicted

to be 0.893 kWh per 103 kg of dry mass flocculated and 0.13 US$/m3 culture medium. The area
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needed for algal harvesting was estimated at 0.7% of the proposed 1 km2 high rate algal pond.
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For example, the maximum removal efficiency of 99% was obtained for C. vulgaris microalgae

using 120 mg/L chitosan at pH 6 after 3 min (Rashid et al., 2013). In comparison, the removal
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algal harvesting using quaternized carboxymethyl chitosan (QCMC) generated larger, stringier
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and denser flocs than those formed using traditional chemical flocculants. Furthermore, the

turbidity removal efficiencies of QCMC were higher than those obtained using chitosan, PAM,
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alum and FeCl3 (C. Dong et al., 2014).

The carboxymethyl chitosan-g-poly[(2-methacryloyloxyethyl)trimethylammonium chloride],

(CMC-g-PDMC), flocculant was employed to remove E.coli (2.5×107 CFU/mL) from synthetic

wastewater samples. The results showed 90–95% bacterial removal efficiency at optimum

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flocculant dosage of 50 mg/L, and pH of 6–9. While for 99% removal efficiency, the required

bio-based flocculant concentration had to increase as pH increased (Yang et al., 2014a).

The flocculation ability of cationized hydroxyethylated starch (CHES) and aminated

hydroxyethylcellulose (DEAE-HEC) to separate cell disintegrate of Bacillus amyloliquefaciens

H (BamHI) was explored, and the best yield for BamHI recovery were 82.7% and 86.0% using

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CHES and aminated hydroxyethylcellulose(DEAE-HEC) bio-based flocculants, respectively.

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DEAE-HEC was also showed good flocculation properties for protein purification (Mazeika et

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al., 2015). The harvesting efficiency of S. oblique using cationic corn starch and cationic potato

starch were 99% and 85%, respectively. The removal efficiency of cationic corn starch, cationic

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potato starch, and alum for total phosphorus were determined to be 33%, 29%, and 42% at the
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coagulant/algae ratio of 1.4:1 (Anthony and Sim, 2013). Liberatore et al. (2016) suggested that

99% of Chlamydomonas reinhardtii cells were removed using 0.1-0.6 g/L of cationically
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modified cellulose at less than 60 minutes. The use of cellulose nanofibirils (CNF) has recently
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explored as a flocculant for harvesting microalgaes (Chlamydomonas reinhardtii) and results

showed that CNF required no surface modification (such as cationic cellulose nanocrystals
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(CNC) and its flocculation behavior derived from the network geometry of nano size cellulose
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fibrils (Yu et al., 2016).

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8.5. Mining and metal recovery

The use of polysaccharide bio-based flocculants is expected to increase in mineral processing

and mine wastewater treatment due to their effective adsorption efficiency for heavy metals at

low flocculant dosages as well as their biodegradability.

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Chitosan and its derivatives are known to be effective for removing heavy metals and mineral

particulates from wastewater. Examples include chitosan grafted acrylamide (CTS-g-AM) bio-

based flocculant for Cu (II) and Cr (VI) (Zheng et al., 2012), N-carboxyethylated chitosans

(CEC) for Cu2+ , Zn2+ , and Ni2+ (Bratskaya et al., 2009), chitosan and poly(N-vinylcaprolactam)

(PNVCL) for silica (Aerosil OX50) suspension (Licea-Claverie et al., 2013), and acid soluble

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chitosan alone and in combination with alum for bentonite removal (Chen and Chung, 2011).

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In addition to chitosan, other polysaccharide bio-based flocculants such as alginate, cellulose,

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starch, xanthan, and pectin have also been successfully developed for fine and heavy metal

removal. For example, Rani et al. (2013) described the synthesis of a PMMA-g-ALG by the

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microwave-assisted method that reduced the turbidity of a 1% coal fine suspension sample to 0.1
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(OD < 0.1) at a dosage of 0.375 mg/L. N-vinyl-2-pyrrolidone grafted onto alginate (SAG-g-

NVP) was reported to effectively remove Pb 2+, Ni2+ and Zn2+ from wastewater (Sand et al.,
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2010). Similarly, polymethacrylic acid-g-cellulose (PMAAC) was reported to remove divalent

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cations including Cu2+, Co2+, and Ni2+ (Abdel-Halim and Al-Deyab, 2012). In another study,

starch derivatives, hydroxyethyl starch-g-polyacrylamide (HSE-g-PAM) and hydroxyethyl

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starch-g-N, N-dimethyl acrylamide (HSE-g-PDMA) were found to be effective in removing

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Hg2+, Cu2+, Zn2+, Ni2+, Pb2+ (Kolya and Tripathy, 2013). Pullulan grafted 3-(acrylamidopropyl)

trimethyl ammonium chloride (pullulan-g-pAPTAC) could fully remove suspended particles

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from kaolin, montmorillonite, and quartz (Ghimici et al., 2010). The cationic glycogen

flocculated various ore suspensions including silica-ore suspension, Fe–ore suspension, and Mn-

ore suspension (Singh et al., 2014). The polysaccharide flocculant xanthan-g-N,N-

dimethylacrylamide (Xanthan-g-PDMA) was able to flocculate kaolin suspension, Fe-ore slime

suspension and silica suspension (Kolya et al., 2012). Ghorai et al. (2013) reported the synthesis

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of a novel xanthan-based hybrid nanocomposite flocculant, (XG-g-PAM/SiO2), which showed

excellent efficiency as an adsorbent for removal of Pb (II) ions from aqueous solution. Pectin

alone or in combination with alum (Ho et al., 2009) or polyacrylamide (PAM, MW 6.00×107 Da)

(Ho et al., 2010) has been reported to be effective in flocculating kaolin suspension.

Flocculation performance of nine chemically modified polysaccharide flocculants including

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polysaccharide-g-PAM, polysaccharide-g-PDMA and polysaccharide-g-(PDMA-co-AA), where

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starch, amylopectin and hydroxyethyl starch were employed as the polysaccharide backbone

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were investigated using four solid suspensions, including 0.25 wt% iron ore slime, 1.0 wt%

kaolin clay, 1.0 wt% coal and 1.0 wt% silica. Polysaccharide-g-PDMA flocculants had a higher

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flocculation activity in coal and iron ore slime suspensions, while polysaccharide-g-(PDMA-co-
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AA) achieved the best result in kaolin suspension (Kolya et al., 2017). The alginate produced by

Azotobacter vinelandii was able to remove 131 mg Cu2+/g Alginate at pH 4.5 and 30 °C
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(Moral and Yildiz, 2016). In the other case, chemically modified cellulose composed of
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hydroxypropyl cellulose and polyacrylamide (HPC/PAAm) was employed for metal removal

with removal performance for Pb at 53%, Ni at 92%, and Cu at 51%, respectively at 40 °C

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(Zamarripa-Ceron et al., 2016).

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8.6. Nanoparticles synthesis

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Nanoparticles could be formed in combination with polysaccharide bio-based flocculants.

Polysaccharide bio-based flocculants have been reported to be effective as stabilizing and

reducing agent in nanoparticle synthesis (Liu et al., 2012; Kang et al., 2016; Kanmani and Lim,

2013; Khan et al., 2013; Krishna et al., 2012; Salehizadeh et al., 2012; You et al., 2013; Y. Y.

Dong et al., 2014; Yumei et al., 2017). For instance, Silver nanoparticles with an average size
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ranging from 9 nm to 72 nm were synthesized using polysaccharide produced by Arthobacter sp.

B4, under the optimum conditions: AgNO 3 5 g/L, pH 7-8 at 80 °C (Yumei et al., 2017). Rasulov

et al. (2017) reported the synthesis of Ag/AgCl nanoparticles with different sizes (6-50 nm)

using extracellular polysaccharide secreted by Azotobacter chroococcum and AgNO 3 (10 mM).

Kanmani and Lim (2013) used pullulan for the synthesis of silver nanoparticles with average

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diameter in the range of 2–30 nm at 121°C for 15 min. The pullulan-mediated silver

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nanoparticles were stable for 3 months. Salehizadeh et al. (2012) reported the role of chitosan as

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both stabilizer and reducing agent of gold and Fe 3 O4 nanoparticles at room temperature, and

proposed a green, simple, effective strategy using chitosan-mediated synthesis of gold

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nanoparticles with average size of 15 nm. An eco-friendly method for synthesis of the silver
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nanoparticles was employed by the cellulose produced from a non-pathogenic

Gluconacetobacter xylinum and the synthesized nanoparticles showed antibacterial activity

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against E.coli and S. aureus (Liu et al., 2012). Other examples of the use of polysaccharide bio-
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based flocculants for the synthesis of silver nanoparticles, include chitosan oligomers (Kang et

al., 2016), chitosan (Krishna et al., 2012), cellulose (You et al., 2013; Y. Y. Dong et al., 2014),
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and starch (Khan et al., 2013).

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8.7. Sludge dewatering

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Large amounts of synthetic flocculants are used in the sludge dewatering process, causing this

operation to be an expensive process in wastewater treatment. Polysaccharide bio-based

flocculants could play a significant role in improving the efficiency of sludge dewatering process

(Guo et al., 2015a; J. P. Wang et al., 2013; Khachan et al., 2014; More et al., 2012; Yang et al.,

2012).

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The starch-g-poly[(2-methacryloyloxyethyl)trimethylammonium chloride] (STC-g-PDMC)

showed great flocculation and dewatering properties on anaerobic sludge. The flocculant

decreased SRF of sludge to 0.04 × 10 13 m/kg at the flocculant dosage of 1.3%, while at the same

flocculant dosage of PAM the SRF was 1.506 × 10 13 m/kg. The sludge conditioned by STC-g-

PDMC was easily filtered with the flocculant dosage below 0.696% of the dry weight of sludge

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(J. P. Wang et al, 2013). A novel inorganic-organic starch-based flocculant (CSSAD) composed

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of corn starch, acrylamide (AM), and 2-(methacryloyloxyethyl) trimethyl ammonium chloride

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(DMC) and a SiO 2 sol, was used for dewatering the waste drilling fluid; the lowest water content

of 21.34% and weight removal of 98.15% were obtained with the optimum flocculant dosage of

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0.3 g per 100 g waste drilling fluid (Zou et al., 2011).
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The flocculation characteristics and dewatering ability of pullulan as a polysaccharide bio-based

flocculant were studied by several researchers (Ghimici and Constantin, 2011; Yang et al.,
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2007). For example, the primary treatment of low-concentration municipal wastewater was
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efficiently enhanced using a composite flocculant combining pullulan and poly-aluminum-

chloride (PAC). This process resulted in better disposal efficiency than the normal two-stage
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wastewater treatment process due to low concentration of municipal wastewater. It also had
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advantages of low treatment costs and good ability to resist the shock of loading rate. The

maximum efficiency of a composite flocculant for removal of turbidity, CODcr, total phosphate,
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and NH3 -N was obtained at 95%, 58%, 91%, and 15%, respectively, at optimum dosage of

pullulan 0.6 mg/L and PAC 15 mg/L. Moreover, the combined bio-based flocculant increased the

sludge settling and dewatering properties (Yang et al., 2007) (Table 6).

Table 6 Applications of polysaccharide bio-based flocculants

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Applicaions Bio-based Flocculants Remarks Refernces

Biomass harvesting and cells recovery
Cationic modified cellulose Remove Chlamydomonas reinhardtii (Liberatore et al., 2016)
microalgae

Cationic cassia gum Remove Chlorella vulgaris microalgae (Banerjee et al., 2014)

Cationic corn starch Remove Scenedesmus obliquus microalgae (Anthony and Sim, 2013)
Cationic starch Remove Chlorella vulgaris microalgae (Vandamme et al., 2012)
Greenfloc 120
CHES Remove Bacillus amyloliquefaciens (Mazeika et al., 2015)
(cationized hydroxyethylated starch)
Chitosan Remove Chlorella vulgaris microalgae (Vandamme et al., 2012)

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Chitosan Remove Chlorella vulgaris microalgae (Rashid et al., 2013)

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Chitosan Remove Chlorella sorokiniana microalgae (Xu et al., 2013)
Chitosan Remove Chlorella sp. microalgae (Ahmad et al., 2011)
Chitosan Remove E. coli (Cai et al., 2015)

CMC-g-PDMC
(Carboxymethyl chitosan-graft-poly(2-
methacryloyloxyethyl)
trimethylammonium chloride)
DEAE-HEC
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Remove E. coli and improve bacterial (Yang et al., 2014a)
disinfection
Remove Bacillus amyloliquefaciens (Mazeika et al., 2015)

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(aminated hydroxyethylcellulose)
QCMC (quaternized carboxymethyl Remove M. aeruginosa (C. Dong et al., 2014)
chitosan)
Rice starch Remove Botryococcus sp.microalgae (Rakesh et al., 2014)
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Dye re moval and decolorization
CAMFA Decolourize reactive black, acid blue and T ao et al., 2017
(Chitosan-Acrylamide-fulvic Acid methyl orange dye solutions
Copolymer)
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CMC-g-PDMC Decolorize acid green 25 (AG25) dye solution (Cai et al., 2015)
(Cellulose-graft- poly[(2-
methacryloyloxyethyl) trimethyl
ammonium chloride])
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HES-g-Poly-(DMA-co-AA) Decolourize malachite green dye solution (Kolya and T ripathy,

(Hydroxyethyl Starch-g-Poly-N,N- 2013)
dimethylacrylamide-co-acrylic acid)
QL-g-SAG Remove acid black and methylene blue (Zhang et al., 2013)
(T rimethyl quaternary ammonium salt of
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lignin-g-sodium alginate)
Xylan-g-MET AC Hemicellulose based T reat anionic azo-dye (S. Wang et al., 2015)
flocculant
(Xylan-g-[2-(methacryloyloxy) ethyl]
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trimethylammonium chloride)
Mining and metal removal
Alginate Remove Cu2+ Moral and Yildiz, 2016
(Azotobacter
vinelandii)
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Cationic glycogen Flocculation of silica, Fe-ore, Mn-ore (Singh et al., 2014)

suspensions
CEC Remove of Cu2+, Zn 2+ and Ni2+ (Cu2+ >Zn2+ (Bratskaya et al., 2009)
(N-carboxyethylated chitosans) >Ni2+)
Chitosan Floccualtion of bentonite suspension (Chen and Chung, 2011)
Chitosan (Aspergillus terreus) Remove Sn 2+ (Cheng et al., 2014)
HES-g-PAM (Hydroxyethyl starch-g- Remove Hg2+, Cu2+, Zn2+, Ni2+, Pb2+ (Kolya et al., 2013)
PAM) &
HSE-g-PDMA
(Hydroxyethyl starch-g-N,N-dimethyl
acrylamide)

MAC Remove of Cu2+ (Zhang et al., 2015)

(Mercapto-acetyl chitosan)
Pectin Flocculation of kaolin suspension (Ho et al., 2009)
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HPC/PAAm Remove Pb, Ni, Cu Zamarripa-Ceron et al.,

(Hydroxypropyl cellulose/ and 2016
polyacryamide)
PMAAC Remove Cu2+, Co 2+, Ni2+ (Abdel-Halim and Al-
(polymethacrylic acid-g-cellulose) Deyab, 2012)
PNVCL+Chitosan Floccualtion of Silica suspension (Licea-Claverie et al.,
(poly(N-vinylcaprolactam) 2013)
+Chitosan)
MAC Heavy metals removal Zhang et al., 2015
(Mercapto-acetyl-g-chitosan)
Pullulan-g- pAPT AC Floccualtion of clay suspension (Ghimici et al., 2010)
(Pullulan-g-3-acrylamidopropyl)-
trimethylammonium chloride)

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SAG-g-NVP Flocculation of coal fines and metals removal (Sand et al., 2010)
(Sodium alginate-g-N-vinyl-2-

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pyrrolidone)
Xanthan-g-PAM/SiO2 T reatment of mine slurry (Ghorai et al., 2013)
(Xanthan gum-g- polyacrylamide grafted
/silica)

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Xanthan-g-PDMA Flocculation of kaolin, Iron-ore, and silica (Kolya et al., 2012)
(Xanthan-g-N,N-dimethylacrylamide) suspensions

Nanoparticles synthesis
Chitosan oligomer Synthesis of silver nanoparticles (42 nm) Kang et al., 2016

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Chitosan Synthesis of gold nanoparticles (15 nm) (Salehizadeh et al., 2012)
Pullulan Synthesis of Ag nanoparticles (2-30 nm), (Kanmani and Lim,
Stablility period 3 months 2013)
Starch Synthesis of Ag nanoparticles (Khan et al., 2013)
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Polysaccharide Synthesis of Ag nanoparticles (9-72 nm) (Yumei et al., 2017)
(Arthobacter sp.)
Polysaccharide Synthesis of Ag/AgCl nanoparticles (6-50 nm) Rasulov et al., 2017
Azotobacter chroococcum
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Sludge dewatering
Cationic Starches Enhancing dewaterability of the Tully sand, (Khachan et al., 2014)
T ully fines, and Elliot soil slurries as
conditioner
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CSSAD Enhancing dewaterability of the waste drilling (Zou et al., 2011)

(Corn starch-SiO2- AM- DMC) slurry as conditioner

MBFGA1(Paenibacillus polymyxa) & Enhancing dewaterability of the secondary (Guo et al., 2015a)
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P(AM-DMC) settling tank sludge as conditioner

(MBFGA1&poly-acrylamide[2-
(methacryloyloxy)ethyl]-
trimethylammonium chloride)
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ST C-g-PDMC Enhancing dewaterability of the anaerobic (J. P. Wang et al., 2013)

(starch-graft-poly (2- sludge as conditioner
methacryloyloxyethyl) trimethyl
ammonium chloride)
CO D and turbidity removal
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Alginate (Azotobacter indicus) T reat sugar, wool, starch, and dairy (Patil et al., 2011)
wastewaters
CAT CS T reat wastewater (You et al., 2009)
(starch and chitosan- based flocculant)
Cationic Glycogen T reat municipal sewage wastewater (Singh et al., 2014)
Chitosan T reat wastewater, used either heavy metals (Renault et al., 2009b)
removal and decolourization
Chitosan T reat unhairing tanning effluent (Sila et al., 2014)
Chitosan T reat dairy wastewater (Devi et al., 2012)
Chitosan + Sodium Alginate T reat sewage water (Yuan et al., 2013)
HES-g-PDMA T reat municipal wastewater (Kolya et al., 2017)
(Hydroxyethyl Starch-g- poly (N,N-
dimethylacrylamide) acid
CMS-CT A-N & CMS-CT A-P (2- T reat synthetic wastewater suspensions (H. Li et al., 2015)
(Hydroxypropyl)trimethylammonium
chloride etherified carboxymethyl
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anionic & cationic starch

DCC T reat paper mill effluent (Zhu et al., 2015b)
(Dicarboxyl cellulose)
DCC T reat wastewater (Suopajarvi et al., 2013)
(anionic nanofibrillated dicarboxylated
cellulose)
HT CC T reat tannic acid extract (Qin et al., 2015)
(N-hydroxypropyl trimethyl ammonium
chloride chitosan)
Na-CMC T reat pond ash wastewater (Prasad and Kumar,
(Sodium Carboxymethyl Cellulose) 2013)

Pullulan and PAC T reat municipal sewage wastewater (Yang et al., 2007)
SAG-g-PMMA T reat coal washery effluent (1 g of SAG-g- (Rani et al., 2013)

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(Polymethyl methacrylate grafted sodium PMMA is sufficient for the
alginate) treatment of 2667 L of coal washery effluent)

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Sodium alginate+ polyaluminium ferric T reat paper making wastewater (Zeng et al., 2011)
chlo-ride+cationic polyacrylamide
Xanthan T reat paper mill effluent (Mukherjee et al., 2014)

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Drinking water treatment
Chitosan T reat river silt water (Divakaran and Pillai,
2002)
Chitosan and Alum T reat surface water (Zemmouri et al., 2011)

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Chitosan-CT A-g-PAM T reat raw water (Lu et al., 2011)
CMC-g-PAA T reat river water (Mishra et al.,2012)
(polyacrylic acid grafted carboxymethyl
cellulose)
CMCNa T reat surface water (Khiari et al., 2010)
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(Sodium carboxymethylcellulose)
MAC T reat tap water Zhang et al., 2015
(Mercapto-acetyl chitosan)
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9. Conclusions and future outlook

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Polysaccharide bio-based flocculants are non-toxic, benign to the environment, fairly shear

stable, and generally display effective removal ability for turbidity, suspended solids, colors and
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dyes in larger dosage compared to organic synthetic flocculants. Their use has not caused
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environmental and health issues, whereas inorganic flocculants such as aluminum salts may

present significant health hazards. The application of polysaccharide bio-based flocculants

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indicates a vital development in sustainable environmental technology and improving the quality

of life.

The grafted polysaccharide bio-based flocculants are generally superior in terms of flocculation

efficiency and shear-stability compared to unmodified polysaccharide and currently used

conventional chemical polymers. They show synergistic flocculation characteristics and

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controllable biodegradability. The results showed that the molecular weight and charge density

are two important structural parameters on the flocculating activity of polysaccharide flocculants.

The current challenges with chemical modification of polysaccharide bio-based flocculants,

especially by grafting methods, include the complexity of the synthesis process, the extent of

biodegradability, health and safety concerns of the presence of some chemical monomers within

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the product, and time and energy consumption in terms of bio-based flocculants production and

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scale up. So far, very limited work has been carried out on the industrial scale production of

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chemically modified polysaccharide bio-based flocculants and bioflocculants, with most focused

only on lab scale research. Extensive research is required to improve feasibility of bio-based

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flocculants production and bioflocculants in a large scale.
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Applications of polysaccharide bioflocculants in various industrial operations reveal great
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potential of these flocculants for improving product quality and productivities in many industries

such as bioprocessing and water and wastewater treatment. Further investigation is required for
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media optimization and the study of kinetic of bioflocculant production, especially by mixed
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culture and using wastewater and agricultural wastes as cheap substrates. The use of some

additives in medium such as microbial quorum sensing (QS) signaling molecules can enhance
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the extracellular production of bioflocculants. Knowledge of biosynthetic pathway of

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bioflocculants production can be an asset and leads to better yield and productivity of

bioflocculants. Extraction methods can affect the bioflocculant performance. Therefore, suitable

and mild extraction methods should be developed to enhance purity and efficiency of

bioflocculants. More research is still needed to understand the mechanisms of flocculation by

bioflocculants in order to control flocs density and removal ability of impurities in wastewater.

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Most of the reported bioflocculants are cation-dependent. Further research should be directed to

producing cation-independent bioflocculants to decrease flocculation cost.

Finally, to eliminate the traditional bottlenecks in cost and performance issues related to

bioflocculants; the use of low-cost biomass sources and development of new

fermentation/recovery strategies for large scale productions of polysaccharide-based flocculants

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and bioflocculants are recommended as key focus in future research.

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Acknowledgments

Financial support from Natural Sciences and Engineering Research Council of Canada (NSERC)

Discovery Grant program is gratefully acknowledged.

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Figure Captions

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Fig. 1. Chemical structures of major polysaccharide bio-based flocculants.

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Fig. 2. Alginate biosynthesis pathway (Hay et al., 2013; Remminghorst and Rhem, 2006).

Fig. 3. Schematic of flocculation mechanisms; a) charge neutralization, b) bridging, c) electrostatic patch,

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d) sweeping (Vandamme, 2013).
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Fig. 4. Extraction and purification processes for microbial polysaccharide flocculants.

Fig. 5. Purification of grafted polysaccharide bio-based flocculants (Polyacrylamid-g-sodium

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alginate) (Salim et al., 2011).

Fig. 6. Extraction and purification of chitosan from different sources: A) marine source, B)
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fungal source (Kaur and Dhillon, 2014).

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