The biodegradability and safety of the bioflocculants make them a potential alternative to non-biodegradable chemical flocculants for wastewater treatment. However, low yield and production cost has been reported to be the limiting factor for large scale bioflocculant production. Although the utilization of cheap nutrient sources is generally appealing for large scale bioproduct production, exploration to meet the demand for them is still low. Although much progress has been achieved at laboratory scale, Industrial production and application of bioflocculant is yet to be viable due to cost of the production medium and low yield. Thus, the prospects of bioflocculant application as an alternative to chemical flocculants is linked to evaluation and utilization of cheap alternative and renewable nutrient sources. This review evaluates the latest literature on the utilization of waste/wastewater as an alternative substitute for conventional expensive nutrient sources. It focuses on the mechanisms and metabolic pathways involved in microbial flocculant synthesis, culture conditions and nutrient requirements for bioflocculant production, pre-treatment, and also optimization of waste substrate for bioflocculant synthesis and bioflocculant production from waste and their efficiencies. Utilization of wastes as a microbial nutrient source drastically reduces the cost of bioflocculant production and increases the appeal of bioflocculant as a cost-effective alternative to chemical flocculants.

The continuous scientific advance is a key factor for improvement of living standards and is principally driven by economic feasibilities. When seeking alternatives for existing basic products, sustainability, compatibility and cost-effectiveness should be considered. One of the goals for evolving countries is practical inventions that can bring the elementary facilities or products within the reach of all. Such products ought to be of good quality and relatively inexpensive. This is possible when they are synthesized using abundant renewable sources with positive impact on the environment. Biomass, being a renewable source of microbial nutrient, can meet these criteria as an alternative nutrient source for bioproduct production (Patil et al. 2010; Liu et al. 2017).

In response to resultant secondary pollutant accumulation associated with chemical flocculants such as aluminium salts and poly-acrylamide (PAA) derivatives for wastewater treatment and industrial processes, several efforts have been devoted to safer substitutes including bioflocculants. Bioflocculant refers to the extracellular polymeric substances secreted by bacteria, fungi and algae as a product of their cellular proliferation. They are composed of composite multi-chain, polymeric substances with repeated pieces of sugar units, glycoprotein, uronic acids and nucleic acids. They have defined molecular chain length and composition and can flocculate even tiny particles (Mohammed & Dagang 2019a). These exclusive characteristics have enhanced the appeal of bioflocculants for application in treatment of water and wastewater (Aljuboori et al. 2014), heavy metal and colour removal, biosynthesis of nanoparticles (Sathiyanarayanan et al. 2013), cell removal and biomass recovery (Drakou et al. 2017). The massive scientific and biotechnological attention accorded bioflocculants are due to their biodegradability, the non-toxic nature of their end products, and application prospects (Dlangamandla et al. 2018). Although much progress has been recorded at the laboratory scale, large scale production of bioflocculants is faced with economic drawbacks owing to the cost of production and low yield.

The major component required for a microbial medium for bioflocculant production includes carbon, nitrogen, sulphur and phosphorus, and these generally contribute major production costs. Nitrogen sources such as yeast extract, peptone and carbon sources such as glucose, fructose and sucrose and other conventional media sources are exorbitant components as they are commercially produced from valuable and comparatively expensive products (Mohammed & Dagang 2019b). This contributes to an extra cost of media formulation for microbial bioflocculant synthesis and limits the market potentials of this product. In seeking substitutes for expensive commercial nutrient sources, cheap renewable sources from wastewater, animal waste constituents, and industrial wastes received tremendous scientific investigations for their bioflocculant yielding potential especially when fermented with suitable microorganisms under appropriate culture conditions (Bukhari et al. 2018).

Most of the wastes contain high biological oxygen demand (BOD) and chemical oxygen demand (COD) concentrations. They are made up of biodegradable carbohydrates, proteins and lipids that can serve the same purpose as the exorbitant conventional media (Wang et al. 2007). In addition to harnessing these wastes for bioflocculant production, the environmental challenges in the form of pollution, diseases and loss of waste of important bioresources such as proteins, enzymes and lipids caused by their inappropriate disposal have also been combatted (Lasekan et al. 2013).

However, utilization of these wastes for bioflocculant production depends largely on isolation or use of already isolated bioflocculant producing microbial strains with competency in fermenting the cost-effective substrates, optimization of the substrate concentration and culture conditions. The present study is an up-to-date evaluation of the recent studies on utilization of waste and wastewater as a cost-effective alternative nutrient source for bioflocculant production.

Microorganisms are capable of synthesizing metabolites including polymeric substances that have diverse biological roles and substantial characteristics for industrial and medicinal applications. A good consideration of primary processes involved in their biological synthesis and control of such processes will help to improve their production rate and efficiency (Rehm 2010). This can also unlock novel avenues for engineering of microorganisms toward synthesis of tailor-made polymers with improved characteristics.

Generally, the synthesis of both extracellular and intracellular biopolymers occurs by the action of highly conserved and organized gene clusters (Okaiyeto et al. 2016) that are subjected to extended transcriptional regulation, which involves two-component signal transduction pathways, quorum sensing (Sakuragi & Kolter 2007), alternative RNA polymerase σ-factors and anti-σ-factors, and incorporation host factor (IHF) (Ionescu & Belkin 2009). These genes or their enzyme products accumulate polysaccharides by consecutively adding sugars to membrane attached reoccurring units which are later exported outside the membrane as extracellular polymer (Bai et al. 2008). Bacterial heteropolysaccharide polymers are typically synthesized intracellularly and exported over to the extracellular environment. The homopolysaccharides like levan, dextran, and mutan are generated extracellularly exported with the aid of secreted enzymes located in different portions of the cell (Ates 2015).

The metabolic pathway involves substrate uptake by the microorganisms via active and passive transportation systems. The substrate is subsequently catabolized intracellularly via the phosphorylative pathway with the aid of intracellular enzymes like hexokinase that can phosphorylate glucose to glucose-6-phosphate and participate in other cellular metabolisms (Ates 2015). The substrate can also be oxidized via the direct periplasmic pathway found only in some microbial strains. Both systems have been described in most bioflocculant-producing strains and are able to function concurrently (Okaiyeto et al. 2016). The cytoplasmic substrate is catabolized via glycolysis to form the initial precursors for formation of biomolecules such as amino acids and monosaccharides. The polysaccharide accumulation entails synthesis of active precursors such as nucleoside phosphate sugars, which originate from phosphorylated sugars.

The direct precursors for bacterial polymer production have been extensively reviewed in the work of Rehm (2010) and include adenosine diphosphate-glucose (nucleoside sugars), guanosine diphosphate-mannuronic acid (nucleoside sugar acids) and uridine diphosphate – N-acetyl glucosamine (nucleoside sugar derivatives). These precursors require the presence of polymer–specific synthesis enzymes such as pyrophosphorylases and dehydrogenases for their synthesis and activation. The synthesis of the activated precursor is the first step in polymer biosynthesis and is the target of metabolic engineering to boost biopolymer production and biosynthesis of tailor-made polymers (Ruffing & Chen 2006). The glycosyltransferases (GTFs) are found within the cell periplasmic membrane and are responsible for transferring the sugar nucleotides to a repeating unit attached to glycosyl carrier lipid.

Meanwhile, polymerization and exudation of both extracellular and capsular polymers are habitually rate restrictive and can considerably affect the flow of carbon towards the progressive synthesis of extracellular polymers with high-molecular weight. The synthases, or their catalytic units, are often found in association with the translocation proteins situated in the cytoplasmic membrane. On demand, they help to translocate the synthesized polymer across the cytoplasmic membrane and the outer membrane. The metabolic pathways associated with synthesis of common polymers is depicted in Figure 1 below. Regulation of the metabolic pathways that affects gene expression and enzymatic functions and use of suitable substrate and microbial strain are important considerations in bioflocculant production.

Figure 1

Metabolic pathways for microbial conversion of substrate to polymers.

Figure 1

Metabolic pathways for microbial conversion of substrate to polymers.

Close modal

Bioflocculant as a product of microbial fermentation is affected by several factors including nutrient composition culture conditions and physicochemical parameters (Ren et al. 2013; Okaiyeto et al. 2016; Shahadat et al. 2017). The influence of the nutrient composition was extensively demonstrated in the works of Abdel-Aziz et al. (2011) and Aljuboori et al. (2013). Other factors of importance include carbon and nitrogen source, initial ionic concentration of the production broth, incubation period, presence and type of metal ion, agitation speed, culture temperature and inoculum volume (Okaiyeto et al. 2016). To properly appraise their role in bioflocculant production, the factors are regrouped and extensively discussed under the following headings.

Nutrient requirement for bioflocculant production

Generally, a good production medium for bioflocculant production should be composed of appropriate carbon and nitrogen sources, sulphur and phosphorus, vitamins and trace elements (Shahadat et al. 2017). While carbon is the most required element for cell building in microorganisms, nitrogen play a significant role in the synthesis of enzymes, proteins and nucleic acids. Using optimum carbon and nitrogen sources and C/N proportion can help in maximizing bioflocculant yield within a short incubation period (Xiong et al. 2010). Good numbers of studies (Gong et al. 2008; Gao et al. 2009; Cosa et al. 2011; Mabinya et al. 2011; Aljuboori et al. 2013; Rao et al. 2013; Wan et al. 2013; Liu et al. 2015a; Liu et al. 2016b; Luo et al. 2016; Subudhi et al. 2016) have produced bioflocculant from simple carbon sources such as glucose, sucrose, fructose, starch, maltose and nitrogen sources such as yeast extract, peptone (NH4)2SO4, tryptone and urea. Ethanol, propionic and acetic acids have also been reported as carbon sources for bioflocculant production (Fujita et al. 2000; Fujita et al. 2001).

Further, sulphur and phosphorus are important elements for synthesizing proteins and co-enzymes in microbial cells and can be sourced from inorganic salts such as magnesium sulphate, calcium sulphate or from sulphur-containing amino acids (Shahadat et al. 2017). Trace elements, for example, iron, copper and cobalt, are needed in small amounts for cellular activities of flocculant-producing microbes. They are required as co-factors in activation and functioning of the enzymes in the microbial cells (Mehta et al. 2010). Vitamins also serve the role of coenzymes in microbes and can be derived from vitamin-containing nitrogen sources such as peptone and yeast extract (Shahadat et al. 2017).

However, exploring non-fossil sources to formulate media for bioproduct formation in general is a key consideration in reducing the consequences of global warming. Exploration of sustainable and renewable nutrients from non-edible sources would save us from competition with food and associated conflicts. Thus, use of agro-waste in an eco-friendly manner is a promising strategy in this regard. While plant-based agro-wastes such as straws, husks, brans and molasses could serve as the main carbon source for bioflocculant production, especially when pre-treated, animal-based bioproducts such as poultry viscera and fish viscera are good sources of proteins, amino acids and nitrogen.

Although scientists all over the world are using their expertise to manage wastes generated from different sources including those generated from animals, utilization of these wastes in raw or pre-treated form has not been totally effective. Their utilization at molecular level, whereby the important organic compounds are recovered and used to solve other environmental problems, is another better means of managing these wastes (Lasekan et al. 2013). In nutrition, patients who are unable to digest complex or simple proteins are placed on hydrolysates from food sources, while in microbiology the hydrolysates are used as carbon and nitrogen sources in growth media.

Generally, biomolecules including protein hydrolysates, amino acids, sugars, proteases and polyunsaturated fatty acids have all been recovered from different animal by-products. Recovery of these biomolecules involves their initial extraction from the organic material with water, alkali or acid (Soerensen et al. 2016). The extraction is also dependent on the pH at which the molecules are soluble. Partial enzymatic, chemical and chemical-enzymatic hydrolysis can also produce a hydrolysate containing a combination of peptides of different length and free amino acids (Lasekan et al. 2013).

Culture conditions

Microbial growth is generally influenced by the cultivating conditions including temperature, agitation, and initial pH of the culture medium, as well as the inoculum size. Bioflocculant, being a product of fermentation, has a direct relationship with microbial growth and is therefore affected by these conditions. Culture temperature has a great influence on microbial flocculant yield since the genes and enzymes required for bioflocculant synthesis are activated at specific optimum temperatures for various microbial strains (Zhang et al. 2007).

The optimum temperature for better yield has been reported to be between 25 °C and 40 °C (Nie et al. 2011; Aljuboori et al. 2013; Cosa & Okoh 2014; Liu et al. 2016b). Temperature below or above the optimum requirement for a given organism could lead to lower bioflocculant yield (Shahadat et al. 2017). For instance, the molecular chain length of the enzymes can be affected at high temperature, thereby retarding the bioflocculant enzyme activities (Salehizadeh & Shojaosadati 2003). Bioflocculant yield increased rapidly as the cultivation temperature was raised from 25 to 30 °C; a further slight increase in yield was recorded as the temperature increased to 35 °C (Salehizadeh & Shojaosadati 2001; Wu & Ye 2007).

Agitation speed, conversely, regulates the amount of oxygen within the culture system and has a direct relationship with nutrient absorption and enzyme activities (Okaiyeto et al. 2016). While aerobic microbes need oxygen for metabolism and catabolism, facultative anaerobes are endowed with certain characteristics that enable them to fine-tune from aerobic to anaerobic or vice versa depending on the environment in which they find themselves.

Microorganisms differ in their agitation speed requirement; such disparity is attributed to the variation of oxygen requirements with the growth phases. At the initial growth phase, the cell mass and the produced bioflocculant are both low and can only make the culture broth slightly viscous and thus little oxygen is needed for continuous growth and bioflocculant synthesis. However, at the log and stationary phases, more biomass and bioflocculant accumulate within the system, which makes the broth very viscous, thereby increasing the oxygen demand (Li et al. 2009a). Ruperez & Leal (1981) demonstrated that agitation of Aspergillus parasiticus culture yielded more EPS than a static culture. Okaiyeto et al. (2014) and Zhang et al. (2007) reported 160 and 180 rpm as the optimum speed for Micrococcus sp. and multiple microbial consortia respectively. In another report, Klebsiella pneumoniae strain NY1 yielded maximum bioflocculant of about 14 g/L at the optimum speed of 200 rpm (Nie et al. 2011).

The initial pH of the medium can influence the electric charge of the microorganisms and their oxidation–reduction potential. Similarly, it can affect nutrient absorption and the enzymatic activities within the microbial cells (Salehizadeh & Shojaosadati 2001). Based on pH requirement, microorganisms are grouped into acidophilic, neutrophilic and alkaliphilic organisms. The acidophiles grow in pH range of 0.1–5.4, neutrophiles exhibit favourable growth in pH range 5.4–8.0 and alkalophiles do well in pH range 7.0–11.5 (Deng et al. 2005). While most optimum bioflocculant production occurs at or close to neutral pH (Deng et al. 2005; Gong et al. 2008; Xia et al. 2008; Liu et al. 2010b; Xiong et al. 2010; Elkady et al. 2011; Gomaa 2012; Aljuboori et al. 2013; Nwodo & Okoh 2013; Wang et al. 2013; Zaki et al. 2013), some studies reported optimum production at both acidic and basic pH. For example, production of bioflocculant by Bacillus megaterium was maximized at pH 9.0 and completely inhibited at acidic pH (Zheng et al. 2008). Similarly, bioflocculant production by Klebsiella pneumoniae (Zhao et al. 2013) and Agrobacterium sp. (Li et al. 2010) were respectively favoured at pH 3.3 and pH 11.

The volume of the microbial inoculum inoculated into the culture medium has a significant role in bioflocculant production by any given microorganisms (Makapela et al. 2016). While lower inoculum size can lead to an extended lag phase for a bioflocculant-producing microorganism, greater size could initiate excessive overlapping of the microbial niche and halt the production. Similarly, higher inoculum size could easily exhaust the nutrient in the medium within a short period because of the increase in microbial population, which in turn can generate higher turbidity at the early stage of growth. This is not favorable for bioflocculant production. Xiong et al. (2010) and Makapela et al. (2016) both reported optimum bioflocculant yield at 4% inoculum size. Others such as Gong et al. (2008), Li et al. (2009b) and Luo et al. (2014) recorded optimum bioflocculant production at 1% (v/v) inoculum using various microorganisms. In general, identification of a suitable organism and substrate, and determination and optimization of culture conditions remain key requirements for maximization of bioflocculant yield.

Growth phase and incubation time

The type of substrate, along with other variables, play a vital role in all microbial growth phases (lag, log/exponential, stationary, and death phase) in relation to incubation time. Bioflocculant production may be growth independent, growth synonymous or growth dependent. Basically, microbial growth tends to be low at the lag phase due to acclimatization. The duration of acclimatization also depends on whether the organism is a fast grower or slow grower (Okaiyeto et al. 2016).

Most studies have demonstrated no or low bioflocculant production and efficiency at the lag phase and higher production and efficiency at the log and early stationary phase of microbial growth. This is because at the lag phase, the rate of microbial proliferation is quite low due to the time required to adapt to the new environment; however, as the incubation time increases, the cells grow at an exponential rate due to profuse nutrient in the system (Nwodo & Okoh 2013). More organisms have been reported to produce bioflocculant during the stationary phase than other growth phases. For example, the bioflocculant yield by Aspergillus flavus has been demonstrated to be growth dependent, with optimum flocculating efficiency of 78.2% at 60 h in the late stationary phase (Aljuboori et al. 2013). Similarly bioflocculant production by Pseudomonas fluorescens WR-1 (Raza et al. 2012) was related to the cell growth with the highest flocculation rate and production at 60 h and 72 h respectively (late stationary phase).

The flocculation efficiency of bioflocculants produced by Pseudomonas aeruginosa also peaked at the stationary phase at 72 h (Gomaa 2012). Other studies that reported production at the stationary phase include those of Xia et al. (2008) and Raza et al. (2012). However, declined bioflocculant yield beyond the stationary phase can be attributed to degradation of the flocculant serving as nutrient and energy source for microbial growth in the absence of the primary nutrient in the system (More et al. 2014), and synthesis of bioflocculant-degrading enzymes within the system (Elkady et al. 2011; Okaiyeto et al. 2015).

As the nutrient become exhausted in the system, the rate of cellular death surpasses cellular multiplication and thus reduces the microbial load. The lysed cells release their intracellular component in the system (Okaiyeto et al. 2016). Intracellular bioflocculants are thus produced at the death phase of microbial growth and are growth independent. Liu et al. (2010b) found that flocculation efficiency of the bioflocculant produced by Corynebacterium daeguense peaked at over 90% during the death phase. The authors in their explanation associated the bioflocculant production with cell autolysis and described the bioflocculant as an intracellular product. Overall, identification of the growth phase at which microorganisms synthesize bioflocculant and determination of their extracellular or intracellular nature can help to maximize bioflocculant harvest from the culture broth.

The performance of the bioflocculant is affected by several factors. The effect of concentration of crude or purified bioflocculant is linked to its chemical composition and molecular weight. The effect of pH of the wastewater to be treated depends on the ionic content of the wastewater and ionic nature of the bioflocculant itself (Mohammed & Dagang 2019a). The effect of temperature on performance of the bioflocculant depends on whether the bioflocculant is made up of polysaccharide or proteins. Those with high polysaccharide content can perform well even at high temperature while the proteinous bioflocculants are sensitive to high temperature due to their denaturing property at high temperature. Other factors that influence bioflocculant performance are the salinity of the wastewater, mixing speed and time, and suspended solids concentration of the target wastewater (Aljuboori et al. 2015).

Generally, the industrial and agricultural wastes, most especially the solid and semi- solid forms, require pre-treatment to make their nutrient available for microbial fermentation. Depending on the nature of the solid and semi solid wastes, shrouding and homogenization is the first step of pre-treatment. The shrouded and homogenised solid/semi-solid wastes can be directly fermented using lignocellulose-degrading microbial strains. This is reported in the case of an alkaliphilic bacteria, Bacillus agaradhaerens C9, which effectively fermented untreated rice bran to produced bioflocculant (Liu et al. 2017). Its ability to utilize the untreated bran directly was due to secretion of xylanase and cellulase, two important enzymes needed for fermentation of lignocellulose materials. Utilization of such organisms has the advantage of circumventing the cost of microbial bioflocculant production. However, a vast number of studies hydrolysed the homogenized solids to release the available nutrient for microbial fermentation. This is especially important if the microbial strain of interest is not a lignocellulose or cellulose-degrading one or cannot secrete enzymes required for direct break-down of solid wastes. The basic types of hydrolysis include acid hydrolysis, enzymatic hydrolysis (Bukhari et al. 2018) and thermal hydrolysis. It is, however, worth mentioning that combination of two or more hydrolysis procedures may be require for some complex solid wastes. Also hydrolysates produced through dilute acid hydrolysis may contain inhibitory elements that should be detoxified prior to microbial inoculation. Guo & Ma (2015), however, applied alkaline-thermal pre-treatment to wastewater sludge for bioflocculant production and demonstrated no presence of inhibitory elements in the sludge. Optimization of hydrolysis conditions and procedures could also facilitate the release of available nutrients in the solid wastes, as demonstrated by Bukhari et al. (2018).

In order to optimize bioflocculant production from an agro-industrial waste (sugar beet molasses), Sam et al. (2011) used different pre-treatment methods including clarification and pH adjustment, sulphuric acid and activated carbon pre-treatment and tricalcium phosphate, sulphuric acid and activated carbon pre-treatment. In clarification and pH adjustment, the pH of the supernatant collected after centrifugation was adjusted to 7.0 prior to fermentation. For the sulphuric acid and carbon pre-treatment, the pH of the molasses was first adjusted to 3.0 using sulphuric acid and allowed to mix for 24 h before activated carbon was added and insolubles removed via centrifugation. The tricalcium phosphate, sulphuric acid and activated carbon pre-treatment involved treating the molasses with tricalcium phosphate, autoclaving at 105 °C for 5 min and treating it with activated carbon as stated above. Although the bioflocculant recovered from fermentation of all the pre-treated molasses had good flocculation efficiency on synthetic wastewater, the flocculants from sulphuric acid and carbon pre-treated molasses stand out in terms of flocculation efficiency.

Similarly, Bukhari et al. (2018) optimized enzymatic hydrolysis of palm oil mill effluent using Plackett–Burman and central composite design of Design Expert to facilitate the release of bioconvertible sugars for bioflocculant production. The authors studied five variables, namely shaking speed, enzyme dose, incubation period, POME concentration and Triton X-100 concentration and reported a highest fermentable sugar concentration of 38.44 g/L. POME concentration, shaking speed and enzyme dose were found to exert greater impact on the hydrolysis.

Some waste/wastewater requires little or no pre-treatment, considering their liquid nature and how they are generated. After characterization of wastewater, a bioflocculant producing strain is inoculated into sterilized wastewater for fermentation. The unsterilized wastewater can be cultivated with the indigenous microbial strains to yield bioflocculant (Zulkeflee & Sánchez 2014) just as in the case of isolating bioflocculant directly from activated sludge. However, it is important to isolate the indigenous strains and test them individually on the sterilized wastewater to identify those with bioflocculant yielding potential.

In another study, Peng et al. (2014) compared the efficiency of bioflocculant produced through Rhodococcus erythropolis fermentation of excess sludge pre-treated by various methods; namely, alkaline pre-treatment, thermal pre-treatment, acid pre-treatment, ultrasonic pre-treatment, microwave pre-treatment and combinations of these pre-treatment methods. These authors reported the highest flocculation efficiency from the sludge pre-treated with 120 °C in combination with alkaline treatment and 70 °C in combination with alkaline treatment. Thermal pre-treatment at low temperature could comparatively reduce cost (Skiadas et al. 2005) while thermal pre-treatment at high temperature could form complex organic substances that are rarely biodegradable and add to the production cost (Peng et al. 2014). Conclusively, the nutrient-rich solid wastes can release much fermentable nutrient upon adequate pretreatment and could yield more efficient bioflocculant. Nutrient-rich wastewater that requires little or no pretreatment is a better alternative to further reduce the cost of bioflocculant production, since it does not require the cost of pretreatment. Identification of new microbial strains or engineered strains that are capable of degrading solid wastes without prior pretreatment is crucial to further reduce the cost of bioflocculant production.

The effects of culture conditions on bioflocculant synthesis has been highlighted in the previous section above. To maximize bioflocculant yield from waste substrates, utilization of bioflocculant yielding strains that have competency in degrading low-cost substrates (Shahadat et al. 2017) and optimization of culture conditions and substrate concentration are basic requirements (Okaiyeto et al. 2016). Various microorganisms have optimum culture conditions at which metabolites production can be maximized. As such, for every waste and microorganism, it is important to optimize the culture conditions as well as the substrate concentration.

Although optimizations can be achieved through conventional optimization of multiple factors by studying a factor or component at a time. This approach involves varying one independent factor while keeping other variables fixed. This method is laborious, time consuming, expensive and does not revealed the alternate impact between the variables (Zhang et al. 2007; Guo et al. 2015) An effective alternative method is response surface methodology; a combination of arithmetical and statistical approach used for modelling and optimization of several variables to determine best process conditions through combination of experimental designs and using first or second order polynomial equations to test the procedure. It shows the connections among the process parameters in classified order with few experimental runs and are preferable to the conventional method. This approach prevails on the feebleness of traditional methods of optimization and has been demonstrated to work for optimization of microbial flocculant yield in several studies (Li et al. 2013; Nwodo & Okoh 2013; Peng et al. 2014). Adequate identification of multifactors that affect bioflocculant production and their optimization are generally key requirements for yield maximization.

As stated earlier, in selecting a nutrient source for bioflocculant production, it is important to consider the cost of production and industrial application coupled with the used of cheap nutrient sources with high production capacity. In line with this, recent research in bioflocculant production focused on evaluation of complex and cheap nutrient sources such as wastewaters, agricultural and industrial wastes as well as harvesting EPS with flocculation capacity from activated sludges.

In fact, our literature search on bioflocculant production generally revealed that the highest reported bioflocculant yield come from studies that utilize waste/wastewater as the nutrient sources (Table 1). The yield from the waste sources can be attributed to their rich nutrient composition, such as the carbon and nitrogen. For example, cultivation of Cloacibacterium normanense in sterilized wastewater sludge fortified with glycerol yielded 25 g/L of extracellular polymeric substance that successfully flocculated Kaolin suspension (95.3%) and heavy metal; Ni (85.0%) (Nouha et al. 2016b). The high production recorded was due to the presence of much carbon sources such as glucose, proteins, volatile fatty acids in the wastewater as the EPS synthesise almost stopped at the late stationary phase despite the presence of about 5.5 g/L of residual glycerol in the system. The highest EPS extraction achieved with EDTA, as compared to other extraction methods used in the above study, and its efficiency is a remarkable one as it further highlights the importance of different extraction methods.

Table 1

Bioflocculant yield from different waste/wastewaters

MicroorganismWaste/nitrogen sourceConditionsYield g/LReference
Staphylococcus sp. Pseudomonas sp. Brewery waste/yeast extract, urea, (NH4)2SO4 Conc. 1.0 L, 30 °C, 72 h, 160 rpm pH 6.0 15.00 Zhang et al. (2007)  
Bacillus subtilis AB110598.1 Waste fermenting liquor pH 4–5, 30 °C mix ratio 41:0.1, 24 h 2.10 You et al. (2008)  
B. agaradhaerens C9 Rice bran and yeast extract Conc. 20 g/L, 37 °C, 24 h. 12.94 Liu et al. (2017)  
Azotobacter indicus ATCC 9540 Madhuca latifolia L
flower extract/yeast extract 
Conc. 20 g/L, 30 °C, 144 h, 180 rpm 6.1 Patil et al. (2010)  
Ochrobactium cicceri W2 Corn stover hydrolysates,
Urea, and yeast extract 
Conc. 230 mL/L, pH 7.5 30 °C, 16 h 3.80 Wang et al. (2013)  
Rhodococcus erythropolis Livestock wastewater Sludge/wastewater 7:1 (v/v), 130 rpm 30 °C, pH 7.0 1.60 Peng et al. (2014)  
Paenibacillus polymyxa Potato starch wastewater/urea Conc. 1.0 L, 150 rpm 30 °C, 24 h 0.81 Guo et al. (2015)  
Rhodococcus erythropolis Rice stover/urea and yeast extract Conc. 200 mL/L, 150 rpm, 35 °C, pH 7.0, 24 h 2.37 Guo et al. (2017)  
Pseudomonas veronii L918 Peanut hull hydrolysate/yeast extract Conc. 300 mL/L, 180 rpm, 28 °C, pH 7.0 3.39 Liu et al. (2016a)  
Stenotrophomonas maltophilia ZZC-06 Phenol wastewater Conc. 800 mg/L, pH 6.0 150 rpm 30 °C, 120 h 4.99 Chen et al. (2016)  
Cloacibacterium normanense Sterilized sludge/glycerol
sterilized sludge 
Conc. 150 mL, 180 rpm, 30 °C, 96 h 25.5
13.3 
Nouha et al. (2015)  
Turicibacter sanguinis Methanol wastewater/NH4(SO4)2, yeast extract Conc. 100.8 mg/L, pH 7.7 120 rpm 30 °C, 144 h 4.61 Cao et al. (2015)  
Bacillus megaterium Swine wastewater Conc. 200 mL, 150 rpm 30 °C,60 h 3.11 Guo & Chen (2017)  
Rhizobium radiobacter
Bacillus sphaericus 
Supernatant of co-digestion of corn straw and molasses wastewater treatment 140 rpm 30 °C, pH 7.0, 24 h 2.32 Zhao et al. (2017)  
Cloacibacterium normanense Activated sludge/glycerol C/N 25, pH 7.0, 72 h. 22.50 Nouha et al. (2017)  
Klebsiella variicola B16 Corn ethanol wastewater Conc. 100 mL/L, 30 °C, pH 8.1 3.08 Xia et al. (2018)  
Bacillus marisflavi NA8 Enzymatic hydrolysate of POME/yeast extract Conc. 200 mL/L, 37 °C, pH 7.0 9.72 Bukhari et al. (2018)  
MicroorganismWaste/nitrogen sourceConditionsYield g/LReference
Staphylococcus sp. Pseudomonas sp. Brewery waste/yeast extract, urea, (NH4)2SO4 Conc. 1.0 L, 30 °C, 72 h, 160 rpm pH 6.0 15.00 Zhang et al. (2007)  
Bacillus subtilis AB110598.1 Waste fermenting liquor pH 4–5, 30 °C mix ratio 41:0.1, 24 h 2.10 You et al. (2008)  
B. agaradhaerens C9 Rice bran and yeast extract Conc. 20 g/L, 37 °C, 24 h. 12.94 Liu et al. (2017)  
Azotobacter indicus ATCC 9540 Madhuca latifolia L
flower extract/yeast extract 
Conc. 20 g/L, 30 °C, 144 h, 180 rpm 6.1 Patil et al. (2010)  
Ochrobactium cicceri W2 Corn stover hydrolysates,
Urea, and yeast extract 
Conc. 230 mL/L, pH 7.5 30 °C, 16 h 3.80 Wang et al. (2013)  
Rhodococcus erythropolis Livestock wastewater Sludge/wastewater 7:1 (v/v), 130 rpm 30 °C, pH 7.0 1.60 Peng et al. (2014)  
Paenibacillus polymyxa Potato starch wastewater/urea Conc. 1.0 L, 150 rpm 30 °C, 24 h 0.81 Guo et al. (2015)  
Rhodococcus erythropolis Rice stover/urea and yeast extract Conc. 200 mL/L, 150 rpm, 35 °C, pH 7.0, 24 h 2.37 Guo et al. (2017)  
Pseudomonas veronii L918 Peanut hull hydrolysate/yeast extract Conc. 300 mL/L, 180 rpm, 28 °C, pH 7.0 3.39 Liu et al. (2016a)  
Stenotrophomonas maltophilia ZZC-06 Phenol wastewater Conc. 800 mg/L, pH 6.0 150 rpm 30 °C, 120 h 4.99 Chen et al. (2016)  
Cloacibacterium normanense Sterilized sludge/glycerol
sterilized sludge 
Conc. 150 mL, 180 rpm, 30 °C, 96 h 25.5
13.3 
Nouha et al. (2015)  
Turicibacter sanguinis Methanol wastewater/NH4(SO4)2, yeast extract Conc. 100.8 mg/L, pH 7.7 120 rpm 30 °C, 144 h 4.61 Cao et al. (2015)  
Bacillus megaterium Swine wastewater Conc. 200 mL, 150 rpm 30 °C,60 h 3.11 Guo & Chen (2017)  
Rhizobium radiobacter
Bacillus sphaericus 
Supernatant of co-digestion of corn straw and molasses wastewater treatment 140 rpm 30 °C, pH 7.0, 24 h 2.32 Zhao et al. (2017)  
Cloacibacterium normanense Activated sludge/glycerol C/N 25, pH 7.0, 72 h. 22.50 Nouha et al. (2017)  
Klebsiella variicola B16 Corn ethanol wastewater Conc. 100 mL/L, 30 °C, pH 8.1 3.08 Xia et al. (2018)  
Bacillus marisflavi NA8 Enzymatic hydrolysate of POME/yeast extract Conc. 200 mL/L, 37 °C, pH 7.0 9.72 Bukhari et al. (2018)  

Meanwhile, extracellular polymer producing microorganisms synthesized much EPS at a certain C/N ratio. In the presence of optimum nitrogen in the system, the organisms used the nitrogen to effectively accumulate the required enzymes and concurrently used the available carbon for EPS synthesis (Liu et al. 2010a). This was further confirmed in a separate study by Nouha et al. (2017) in which fermentation of activated sludge reinvigorated with crude glycerol using Cloacibacterium normanense yielded 22.5 g/L bioflocculant at a specific C/N ratio.

Similarly, Zhang et al. (2007) used microbial consortia of Staphylococcus sp. and Pseudomonas sp. to ferment brewery wastewater along with yeast extract, urea, (NH4)2SO4 and reported 15 g/L bioflocculant, which had a flocculating efficiency of 96.8%. Apart from the high nutrient composition (COD 5,000 mg/L) of the wastewater used in their study, protocooperation between the two strains benefits both organism and promote their bioflocculant yielding potential.

While researchers (Wang et al. 2013; Bukhari et al. 2018) promoted pre-treatment of agricultural and industrial wastes for downstream fermentation to yield bioflocculant, Liu et al. (2017) in a recent study argued that the poisonous residues accumulated during the pre-treatment step, especially the dilute acid hydrolysis step, could reduce the effective fermentation of the hydrolysate generated. Thus, the microbial strains that can utilize lignocellulose directly to produce bioflocculants can evade the pre-treatment and simultaneously reduce the production cost. In support of this, Liu et al. (2017) reported 12.94 g/L bioflocculant from B. agaradhaerens C9 grown on untreated rice bran supplemented with yeast extract. This bioflocculant showed 91.05% efficiency in flocculating microalga, Chlorella minutissima. The ability of this organism to convert lignocellulose substrate directly is attributed to secretion of xylanase and cellulase (Liu et al. 2015b).

Similarly, Ochrobactium cicceri W2 fermentation of corn stover hydrolysates along with urea and yeast extract yielded 3.8 g/L bioflocculant (Wang et al. 2013), potato starch wastewater and urea (Guo et al. 2015), peanut hull hydrolysate and yeast extract (Liu et al. 2016a) and methanol wastewater and NH4(SO4)2, yeast extract (Cao et al. 2015) yielded 3.8, 0.81, 3.39 and 4.61 g/L of bioflocculant respectively. Use of piggery wastewater with COD 3,000 mg/L and TN 170 mg/L as a cost-effective substrate yielded 1.5 g/L bioflocculant, thereby mitigating the cost of substrate by above 90% (Pei et al. 2013).

Although the majority of studies supplemented the wastes and wastewater with additives, mainly nitrogen sources, owing to the intricacy of the components that can support microbial growth (Wang et al. 2007) and likely due to the scarcity of single wastes that can be used as a sole substrate, utilization of rich waste substrates that contain the major nutrients for fermentation to yield bioflocculant will no doubt further reduce the cost of production that may be incurred from the additives, and should be accorded much attention. Combination of two waste substrates; for example, a sugar-containing waste substrate can be supplemented with another waste known to contain proteins in the form of nitrogen.

A study by Joshi et al. (2017) cultivated Klebsiella pneumoniae strain NJ7 on starch wastewater that contained both sugar and proteins as a sole nutrient source to produce bioflocculant. The authors recovered about 0.111 mg/100 mL purified bioflocculant that was efficient (85.79%) in flocculating the same starch wastewater at optimized conditions. Another study reported 2.1 g/L of bioflocculant with flocculation efficiency of about 99% from Bacillus subtilis grown on waste fermenting liquor from hydrogen fermentation process as the sole nutrient (You et al. 2008). Also, cultivation of a phenol-degrading strain, Stenotrophomonas maltophilia ZZC-06, on phenol (800 mg/L) containing wastewater as a sole substrate yielded 4.99 g/L bioflocculant with a flocculation efficiency of over 90% (Chen et al. 2016).

The efficiency of the bioflocculant depends on the nutrient source, because it determines the sugar residue variation in the bioflocculants. In the study by Nouha et al. (2016b), after the addition of glycerol, the organism changed its carbon source from wastewater to glycerol and a slight decline in flocculation activity was observed and attributed to variation in the sugar composition of the EPS. The efficiency of the bioflocculants also depends on the functional groups of the bioflocculants, which serve as the binding sites for the suspended colloids in the target water to be treated. The functional groups are in turn determined by the residual composition of the bioflocculant. A separate study by Liu et al. (2017) also supported this argument. The authors showed that the fermentation broth of B. agaradhaerens C9 cultured on different lignocellulosic materials (rice bran, corn cob, corn storver and peanut hull) had varying flocculation activities. Table 2 highlights the residual composition, functional groups and efficiencies of the bioflocculant produced with some waste.

Table 2

Efficiency, composition and functional groups of bioflocculants produced from waste/wastewater

MicroorganismProduction substrateFlocculant compositionFunctional groupsFlocculant doseWastewater treatedEfficiency (%)Reference
Staphylococcus sp. Pseudomonas sp. Brewery waster, yeast extract, urea, (NH4)2SO4 Polysaccharides, proteins NA 1.7 mL Indigotin waste,
Kaolin suspension 
79.20
96.8 
Zhang et al. (2007)  
Bacillus subtilis
AB110598.1 
Waste fermenting liquor Carbohydrates and proteins NA 10 mL Kaolin suspension >99.00 You et al. (2008)  
B. agaradhaerens C9 Rice bran and yeast extract Polysaccharides, proteins C–H, C–O 60 mg/L Chlorella minutissima 91.10 Liu et al. (2017)  
Azotobacter indicus
ATCC 9540 
Madhuca latifolia L
flower extract/yeast extract 
O-acetyl, Orcinol
Uronic acid 
–COOH, –OH, C–O 500 mg/L Kaolin suspension 72.00 Patil et al. (2010)  
Halomonas sp. AAD6 Sugar beet molasses NA NA 20 mg/L Synthetic sea water,
natural sea water 
60.00
85.00 
Sam et al. (2011)  
Rhizobium radiobacter
Bacillus sphaericus 
Rice straw fermentation liquor NA NA 10 mL Kaolin suspension 92.45 Zhao et al. (2012)  
Ochrobactium cicceri W2 Corn stover hydrolysates/urea, and yeast extract Polysaccharides and proteins C–H, amide I, amide II 10 mL/L Kaolin suspension 92.00 Wang et al. (2013)  
Aspergillus niger POME/glutamic acid Carbohydrate and protein –OH, C–H,
–COO − , C–O 
1 mL/100 mL
35 mg/L 
Kaolin suspension,
Real river water 
76.80
63.00 
Aljuboori et al. (2014)  
Rhodococcus erythropolis Livestock wastewater Polysaccharides protein and DNA –OH, –NH2, –CONH2 40 mg/L Dye wastewater 93.9 Peng et al. (2014)  
Paenibacillus polymyxa Potato starch wastewater/urea polysaccharide –OH, N–H, C = O, C–H, C–O 20 mg/L 30 mg/L Kaolin suspension,
Potato starch water 
95.4
81.7 
Guo et al. (2015)  
Pseudomonas veronii L918 Peanut hull hydrolysate/yeast extract Polysaccharides, proteins –COO-, –OH, C–H 2.83 mg/L Ash-flushing wastewater 92.5 Liu et al. (2016a)  
Turicibacter sanguinis Methanol wastewater/NH4(SO4)2, yeast extract Polysaccharide, protein, uronic acid OH, N–H, COH, CN,
C–O, S–S 
1 ML 500 mg/L Kaolin suspensions,
arsenite solution 
95.7
86.1 
Cao et al. (2015)  
Cloacibacterium normanense Sterilized wastewater sludge/glycerol Carbohydrate,
protein, nucleic acid 
CH2, CH, NH,
–CO, NH, C–N,
C–O, C–O–C 
23.1 mg
35 mg/L 
Kaolin suspension, Heavy metal (Ni) 95.3
85.0 
Nouha et al. (2016b),
Nouha et al. (2016a)  
Rhodococcus erythropolis Rice stover/urea and yeast extract Polysaccharide and protein OH, N–H, C = O 12 mg/L Domestic wastewater 89.7 Guo et al. (2017)  
Rhizobium radiobacter
Bacillus sphaericus 
Supernatant of co-digestion of corn straw and molasses wastewater treatment NA NA 374 mg/L
10 mL 
Electroplating wastewater,
kaolin suspension, 
>90.0
91.3 
Zhao et al. (2017)  
Bacillus megaterium Swine wastewater Polysaccharides, proteins, uronic OH, –NH2.
–CO, –CONH2 
1 mL/100 mL
5 × 10−2% w/w 
 Kaolin suspension,
arsenite solution 
90.2
99.2 
Guo & Chen (2017)  
Klebsiella variicola B16 Corn ethanol wastewater Neutral and amino sugars, uronic acid C-C, C = O,
C-N-(H), C = N 
0.333 g/L
5.88 mg/L 
Dye wastewater,
kaolin suspension 
96.18
>90.0 
Xia et al. (2018)  
Bacillus marisflavi NA8 Enzymatic hydrolysate of POME/yeast extract Polysaccharides, protein, DNA –OH, –NH, C = O, C–H 100 mg/L Chlorella vulgaris 90.0 Bukhari et al. (2018)  
Aspergillus niger Potato starch wastewater/glucose/urea Polysaccharide –OH, C–H,
C = O, C–O 
1.89 g/L PSW,
Kaolin suspension 
91.15
90.06 
Pu et al. (2018)  
Aspergillus flavus Chicken viscera Polysaccharide, uronic acids, amine groups –NH2,
C–C, C = O 
4 mL/100 mL
9 mL/100 mL
5 mL/100 mL 
Kaolin suspension,
activated carbon
soil solution 
83.1%
92%
94.8% 
Mohammed & Dagang (2019b)  
Indigenous microbes Soybean residues – OH, OCH3, C = O 0.5 mL/50 mL Kaolin suspension 70% Zulkeflee & Sánchez (2014)  
MicroorganismProduction substrateFlocculant compositionFunctional groupsFlocculant doseWastewater treatedEfficiency (%)Reference
Staphylococcus sp. Pseudomonas sp. Brewery waster, yeast extract, urea, (NH4)2SO4 Polysaccharides, proteins NA 1.7 mL Indigotin waste,
Kaolin suspension 
79.20
96.8 
Zhang et al. (2007)  
Bacillus subtilis
AB110598.1 
Waste fermenting liquor Carbohydrates and proteins NA 10 mL Kaolin suspension >99.00 You et al. (2008)  
B. agaradhaerens C9 Rice bran and yeast extract Polysaccharides, proteins C–H, C–O 60 mg/L Chlorella minutissima 91.10 Liu et al. (2017)  
Azotobacter indicus
ATCC 9540 
Madhuca latifolia L
flower extract/yeast extract 
O-acetyl, Orcinol
Uronic acid 
–COOH, –OH, C–O 500 mg/L Kaolin suspension 72.00 Patil et al. (2010)  
Halomonas sp. AAD6 Sugar beet molasses NA NA 20 mg/L Synthetic sea water,
natural sea water 
60.00
85.00 
Sam et al. (2011)  
Rhizobium radiobacter
Bacillus sphaericus 
Rice straw fermentation liquor NA NA 10 mL Kaolin suspension 92.45 Zhao et al. (2012)  
Ochrobactium cicceri W2 Corn stover hydrolysates/urea, and yeast extract Polysaccharides and proteins C–H, amide I, amide II 10 mL/L Kaolin suspension 92.00 Wang et al. (2013)  
Aspergillus niger POME/glutamic acid Carbohydrate and protein –OH, C–H,
–COO − , C–O 
1 mL/100 mL
35 mg/L 
Kaolin suspension,
Real river water 
76.80
63.00 
Aljuboori et al. (2014)  
Rhodococcus erythropolis Livestock wastewater Polysaccharides protein and DNA –OH, –NH2, –CONH2 40 mg/L Dye wastewater 93.9 Peng et al. (2014)  
Paenibacillus polymyxa Potato starch wastewater/urea polysaccharide –OH, N–H, C = O, C–H, C–O 20 mg/L 30 mg/L Kaolin suspension,
Potato starch water 
95.4
81.7 
Guo et al. (2015)  
Pseudomonas veronii L918 Peanut hull hydrolysate/yeast extract Polysaccharides, proteins –COO-, –OH, C–H 2.83 mg/L Ash-flushing wastewater 92.5 Liu et al. (2016a)  
Turicibacter sanguinis Methanol wastewater/NH4(SO4)2, yeast extract Polysaccharide, protein, uronic acid OH, N–H, COH, CN,
C–O, S–S 
1 ML 500 mg/L Kaolin suspensions,
arsenite solution 
95.7
86.1 
Cao et al. (2015)  
Cloacibacterium normanense Sterilized wastewater sludge/glycerol Carbohydrate,
protein, nucleic acid 
CH2, CH, NH,
–CO, NH, C–N,
C–O, C–O–C 
23.1 mg
35 mg/L 
Kaolin suspension, Heavy metal (Ni) 95.3
85.0 
Nouha et al. (2016b),
Nouha et al. (2016a)  
Rhodococcus erythropolis Rice stover/urea and yeast extract Polysaccharide and protein OH, N–H, C = O 12 mg/L Domestic wastewater 89.7 Guo et al. (2017)  
Rhizobium radiobacter
Bacillus sphaericus 
Supernatant of co-digestion of corn straw and molasses wastewater treatment NA NA 374 mg/L
10 mL 
Electroplating wastewater,
kaolin suspension, 
>90.0
91.3 
Zhao et al. (2017)  
Bacillus megaterium Swine wastewater Polysaccharides, proteins, uronic OH, –NH2.
–CO, –CONH2 
1 mL/100 mL
5 × 10−2% w/w 
 Kaolin suspension,
arsenite solution 
90.2
99.2 
Guo & Chen (2017)  
Klebsiella variicola B16 Corn ethanol wastewater Neutral and amino sugars, uronic acid C-C, C = O,
C-N-(H), C = N 
0.333 g/L
5.88 mg/L 
Dye wastewater,
kaolin suspension 
96.18
>90.0 
Xia et al. (2018)  
Bacillus marisflavi NA8 Enzymatic hydrolysate of POME/yeast extract Polysaccharides, protein, DNA –OH, –NH, C = O, C–H 100 mg/L Chlorella vulgaris 90.0 Bukhari et al. (2018)  
Aspergillus niger Potato starch wastewater/glucose/urea Polysaccharide –OH, C–H,
C = O, C–O 
1.89 g/L PSW,
Kaolin suspension 
91.15
90.06 
Pu et al. (2018)  
Aspergillus flavus Chicken viscera Polysaccharide, uronic acids, amine groups –NH2,
C–C, C = O 
4 mL/100 mL
9 mL/100 mL
5 mL/100 mL 
Kaolin suspension,
activated carbon
soil solution 
83.1%
92%
94.8% 
Mohammed & Dagang (2019b)  
Indigenous microbes Soybean residues – OH, OCH3, C = O 0.5 mL/50 mL Kaolin suspension 70% Zulkeflee & Sánchez (2014)  

The functional groups of the bioflocculant determine their reactivity with the suspended particles, which are in most cases negatively charged. Generally, these functional groups have been determined using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). Dlangamandla et al. (2016), using the FTIR techniques, revealed the functional groups of a bioflocculant produced by a biofilm-forming strain, Comamonas sp. BF-3 to be similar to those found in proteins including hydroxyl, carboxyl, alkane and amine functional groups. Similarly, a wide deep peak of 3,340 cm−1, absorption of 2,933 cm−1 and 1,147–1,380 cm−1 signifying hydroxyl and amine groups, stretch of –CH2 groups, and carboxyl group were respectively reported for bioflocculant generated by Bacillus amyloliquefaciens (Rao et al. 2013). A bioflocculant produced by Achromobacter xylosoxidans showed absorption peaks corresponding to carboxyl, hydroxyl and amino groups, typical of glycoprotein, as confirmed by the nuclear magnetic resonance (Subudhi et al. 2016).

The nature of functional groups found in a bioflocculant and the ionic composition of the wastewater to be flocculated has great influence on the flocculation efficiency of the bioflocculant. Cationization of the bioflocculant with more negatively charged functional sites is a good strategy used to stimulate the efficiency of such bioflocculant on wastewater with anionic compositions. Details on cationization of the bioflocculant can be found in our previous work (Mohammed & Dagang 2019a).

The micrographic structure of the purified bioflocculant can be seen with the aid of scanning electron microscopy. Using this technique, Subudhi et al. (2016) showed a purified exopolysaccharide bioflocculant produced by metal-tolerant bacteria as an entrenched membrane structure in closed pack resembling a maze, sometimes interstice with spaces amid the large heap of sheets. The authors opined that the closely packed and clumped structure of the bioflocculant might contribute to its flocculation capacity. Further, the SEM images of bioflocculant IH-7 produced by A. flavus and the flocculated particles were used to explain the flocculation mechanism of the bioflocculant (Aljuboori et al. 2015). The structure of bioflocculants produced from pure strains of Halobacillus sp. and Oceanobacillus sp. were reported to be an amorphous and a crystal-linear-like structure respectively. Similarily, SEM examination of the surface of the freeze-dried mixed-cultured bioflocculant produced by both species revealed a white, amorphous-crystal-like structure (Cosa & Okoh 2014). A characterization study of a lipid bioflocculant produced by Rhodococcus erythropolis using gel permeation column chromatography showed it to be made up of trehalose monomycolate, glucose monomycolate, and trehalose dimycolate (Kurane et al. 1994).

Generally, the disparity in flocculation efficiency of bioflocculants harvested with different techniques and solvents and different dosage requirements can be attributed to changes in their residual component, molecular weight and target wastewater to be flocculated, which are major determinants of their efficiency. To buttress this, Lee et al. (2007) showed that the molecular mass of the EPS produced by Ganoderma applanatum grown on glucose or maltose was above 2,000 kDa, while it fell below 2,000 kDa when the same organism was cultured on lactose, fructose or sucrose. Further, as highlighted above, the residual composition of bioflocculant depends on their extraction method and can thus change their molecular weight and functional groups, which significantly affects their bio-flocculation capacities.

To effectively harness the flocculation efficiency of bioflocculants, there is a need to determine their optimum dose. For instance, the flocculation efficiency of S-EPS produced through fermentation of sterilized activated sludge was about 72% in 20 minutes' settling time at an EPS dose of 13 mg/g sludge, beyond which no improved flocculation was recorded, while about 10 mg Zetag (a chemical flocculant)/g sludge showed about 66% efficiency in 42 minutes (Nouha et al. 2017).

Conventional media are effective nutrient sources for microbial fermentation to yield bioflocculants, and have been widely used at the laboratory scale. Due to the exorbitant production cost associated with bioflocculant production from conventional media and attempts to make bioflocculant appealing for industrial application, cheap nutrient sources such as waste/wastewater are receiving huge research attention as substrates for microbial fermentation to yield bioflocculant. The waste/wastewater has been shown to reduce production cost to about 60%. Agricultural and industrial wastes that contains fermentable sugars, nitrogen and other nutrients required for microbial fermentation to yield bioflocculant are still underexplored and remain a significant resource for large scale production and application of bioflocculant. Apart from investigation of unexplored waste for bioflocculant production, optimization of pre-treatment procedures, fermentation conditions and substrate concentrations for both solid and liquid wastes remain good strategies for successful utilization of waste for bioflocculant production. Furthermore, the exploitation of xylanase, cellulase and bioflocculant producing microbial strains in consortia will reduce the cost of pre-treatment most especially for solids and will further reduce the entire production cost. Generally, there is need to look into molecular, genetic and biochemical investigation of bioflocculant synthesis by microorganisms to engineer the genes and modify the metabolic pathways for higher production. Extensive studies are needed to ascertain the satisfactory quality and property of bioflocculant produced from waste, as more than 90% of the studies only assume the bioflocculant to be safe without a prior toxicity study.

Although synthesis of bioflocculants from cheap and renewable nutrient sources will help to realize the goal of commercialization, the low availability of data on applications of bioflocculants compared to the chemical flocculants calls for further research into application of bioflocculants. Similarly, about 85% of bioflocculant production from waste and bioflocculant-aided wastewater treatment and other applications are based on test tube trials, mostly with kaolin suspension as the model wastewater, which does not represent their real performance. Complex production methods and use of co-reagents are also major traditional issues associated with bioflocculant production that require further research. There is also inadequate reporting on the effect of extraction methods on the structure and chemical composition of bioflocculants and the introduction of contaminants during extraction. These can have adverse consequences on the flocculation efficiency and need further and systematic investigations to boost the future prospect of bioflocculants as a sustainable alternative to chemical flocculants. Further, genetics and biochemistry studies of bioflocculant synthesis and the number of microorganisms with diversity for agricultural wastes utilization are quite low. Further research into these aspects will no doubt boost the future prospect of bioflocculant production from waste.

The authors appreciate Universiti Teknologi Malaysia, GUP Tier1 (Q. J130000.2545.13H22) and Demand-Driven Innovation grant (R. J130000.7845.4L190) for their financial support.

The authors declare no financial or commercial conflict of interest.

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