Microbial Flocculants as an Alternative to Synthetic Polymers for Wastewater Treatment : A Review

Microorganisms such as bacteria, fungi, and microalgae have been used to produce bioflocculants with various structures. These polymers are active substances that are biodegradable, environmentally harmless, and have flocculation characteristics. Most of the developed microbial bioflocculants displayed significant flocculating activity (FA > 70–90%) depending on the strain used and on the operating parameters. These biopolymers have been investigated and successfully used for wastewater depollution in the laboratory. In various cases, selected efficient microbial flocculants could reduce significantly suspended solids (SS), turbidity, chemical oxygen demand (COD), total nitrogen (Nt), dye, and heavy metals, with removal percentages exceeding 90% depending on the bioflocculating materials and on the wastewater characteristics. Moreover, bioflocculants showed acceptable results for sludge conditioning (accepted levels of dry solids, specific resistance to filtration, moisture, etc.) compared to chemicals. This paper explores various bioflocculants produced by numerous microbial strains. Their production procedures and flocculating performance will be included. Furthermore, their efficiency in the depollution of wastewater will be discussed.


Introduction
The conventional coagulation-flocculation technique applied for wastewater treatment is widely used, showing significant treatment efficiency regarding the removal of organic material, suspended solids, and heavy metals [1].In addition, it provides benefits for the wastewater treatment system, such as higher resistance to toxic loadings and massive amounts of organics, conducting simplicity, energy savings, etc. [2].The chemical flocculants universally used in this process included inorganic (polyaluminum chloride, ferric chloride, etc.) and organic flocculants (such as polyacrylamide and its derivatives) [3].These chemicals stay in wastewater after treatment and sludge and may cause health and ecological complications [4].Consequently, the discarding of treated wastewater in the environment may cause serious disadvantages for human health, since the used chemicals are reported to be related to various health effects (Alzheimer's disease, neurotoxicity, carcinogenic, genotoxic properties, etc.) [5].Residual chemicals destroy aquatic life and make the water inappropriate for human consumption.Both synthetic organic and inorganic flocculants were reported to be responsible for neurotoxicity and carcinogenicity.It was also demonstrated that Alzheimer's disease is linked to aluminum remaining in treated water [6].A recent study showed the toxicity of both anionic polyacrylamide and cationic polymers for aquatic invertebrates and fish [7].In this context, it was reported that cationic polymers tend to accumulate in fish gills, interfering with gill function and ion regulation, causing fish death and consequently reducing the supply of healthy fish for human consumption [8].Moreover, monomers resulting from the degradation of polyacrylamide under specific environmental conditions are considered to be a likely human carcinogen and neurotoxin.Therefore, many authorities have restricted the use of chemical polymers in various industrial applications [9].In addition, these products are costly and may not be available locally.Hence, there is a need to consider other flocculants offering a new sustainable strategy.This sustainable approach is based on the use of bioflocculants in the coagulation-flocculation treatment for the removal of pollutants from wastewater [5].Natural biological origin materials, such as beans, moringa, maize, cactus, etc., were investigated [5].Recently, more attention has been diverted to microbial flocculants produced by various microorganisms (actinomycetes, fungi, bacteria, and algae) widely distributed in soil and water [10][11][12].Microbial flocculants that are produced during the microorganism growth varied in composition (polysaccharides, proteins, DNA, cellulose, sugar, protein, polyamino acids, etc.).They are active biocompounds, biodegradable, without degraded intermediate pollutants, environmentally harmless, and have flocculation properties.For these reasons, examinations were carried out to determine their efficiency for wastewater treatment from various origins.This review aims to explore the production of microbial flocculants and their applications in wastewater treatments.

Bioflocculant-Producing Microorganisms
Bacteria, fungi, and microalgae were showed to produce bioflocculants.Microorganisms are selected based on various factors (morphology, the presence of slimy extracellular polysaccharides, etc.) using different methods and reagents (Congo red, crystal violet and CuSO 4 solution, chelating agents, colorimetric methods, etc.).The flocculating activity (FA) was commonly evaluated using a kaolin suspension.The produced bioflocculant was also subject to qualitative analyses.Fourier transfer infrared radiation was used to analyze the structure of the bioflocculant.Interestingly, many sources (sludge, soil, sediments, river, seawater, etc.) were investigated to isolate microorganisms that yield flocculating substances (e.g., polysaccharides, proteins, and glycoproteins).

Bacteria
Several bacterial strains belonging to various classes (Actinobacteria, Alphaproteobacteria, Bacilli, Deltaproteobacteria, Gammaproteobacteria, Proteobacteria, etc.) have been reported to produce flocculants (Table 1) .For example, salt production pond Bacillus mojavensis strain 32A was found to produce proteoglycan flocculant (98.4% polysaccharide and 1.6% protein) with an interesting FA of 96.11% recorded at pH 10 [14].In the presence of specific growth conditions (L-glutamic acid and NH 4 Cl as nutrient sources), this strain yields 5.2 g/L of the extracted biopolymer.Another Bacillus strain isolated from freshwater (Bacillus pumilus ZAP 028) produced a thermostable and wide pH range flocculating agent (FA = 69.8%)[18].In this case, results were obtained in the presence of maltose and several nitrogen sources (e.g., yeast extract, urea and ammonium sulfate) with 4% (v/v) of inoculum and pH 7. The bioflocculant content was 75.4% polysaccharide, 5.3% protein, and 15.4% uronic acid [18].More recently, two isolates from Egyptian soil (Bacillus cereus and Bacillus thuringiensis) were reported to produce a significant amount of bioflocculants [21].The total carbohydrate bioflocculant contents were 16.99% and 15.27%, while the total protein content were 83.01%and 84.73%, respectively, for Bacillus cereus and Bacillus thuringiensis.The maximum FA (75% to 76.3%, respectively, for Bacillus cereus and Bacillus thuringiensis) were obtained at pH 7-8 and temperature 30-40 • C during the growth period from 72 to 96 h and in the presence of starch and yeast extract [21].Different carbon sources were used for the growth of sludge-isolated strain (Bacillus sp.XF-56).However, glucose was most favorable for bioflocculant production (FA = 93.5%).Interestingly, at the initial pH (5-10), this strain yields hydrogen (optimum yield at pH = 7) and bioflocculants (optimum yield at pH = 8).This strain resists higher salt concentration, offering broad application potential for fresh and marine wastewater [13].
Bacterial strains belonging to the Actinobacteria class (Arthrobacter, Rhodococcus sp.Streptomyces, etc.) were investigated by many researchers.Rhodococcus erythropolis isolated from activated sludge was cultivated in swine wastewater as a carbon and nitrogen source.The produced flocculant consist mainly of protein (99.2%) with higher FA of 99.2% [39].However, another strain (Rhodococcus rhodochrous) growing in the presence of glucose and NH 4 Cl produced proteoglycan flocculant (62.86% polysaccharide and 10.3% protein) with a lower FA (22.5%)) [40].
Nevertheless, many strains belonging to the Proteobacteria class possess the ability to synthesize bioflocculants.For example, a strain identified as Agrobacterium sp.M-503 (from propylene epoxide wastewater sludge) produced a biopolymer (85%p polysaccharide and 3% protein) with an acceptable level of FA (75%) [43].Amjres et al. (2015) [41] isolated Halomonas stenophila HK30 from saline wetland that is able to produce sulphated heteropolysaccharide with an efficient FA (72.06%).
Based on the collected information, the majority of research activities has mainly been oriented to the isolation of new strains and the production of bioflocculant using pure culture.However, the possibility was reported recently of combining microbial strains to produce bioflocculants with better FA than pure strains.Table 2 [50][51][52][53][54][55][56][57][58] presents several consortia evaluated for their bioflocculant production.A culture mixture of Cobetia sp.OAUIFE and Bacillus sp.MAYA produces a bioflocculant containing 66% uronic acid and 31% protein.At a flocculant dose of 0.8 mg/mL, at pH 8 and in the presence of Ca 2+ , optimum FA (90%) was obtained [50].Similarly, a produced bioflocculant by growing both Halomonas sp.Okoh and Micrococcus sp.Leo was shown to be controlled by Ca 2+ , Mn 2+ and Al 3+ , thermostable and active at pH (2-10), with an optimum FA of 86% at pH 8. Consequently, the bioflocculant may be used to replace the synthetic flocculants widely used in wastewater treatment [54].An interesting bioflocculant (FA = 98.9%) was also produced by a mixture of Streptomyces sp.Gansen and Cellulomonas sp.Okoh [56] grown in an optimized medium (containing sucrose, peptone, and magnesium chloride).It has been reported that the extracted bioflocculant contains polysaccharides (neutral sugar, amino sugar, and uronic acids) and proteins [56].Because bioflocculation represents a dynamic process occurring in an aerobic activated sludge system, the sludge may contain large numbers of bioflocculant-producing microbial strains.Microorganism aggregate of the biological sludge secretes mainly flocculating materials (e.g., polysaccharides, proteins, glycoproteins, etc.) at different concentrations.Biological sludge from municipal wastewater was used for bioflocculant production by Zhang et al. (2013) [57] and Sun et al. (2012) [58].In these studies, hydrochloric acid treatments were used to extract flocculating active ingredients.The highest flocculating rate fraction can be purified from the crude bioflocculant.The conducted experiment allowed the purification of an amino-polysaccharide bioflocculant with an optimum FA of 98.5% at pH 10.5 and 3.0% (v/v) as a dose [57].Likewise, FA of the purified polysaccharide reached 99.5% at the same conditions (3.0% v/v and pH 10.5) [58].
Regarding the flocculating capability of microalgae, various species have been reported to produce flocculants during the stationary phase in batch cultures.However, fewer studies on microalgal flocculant properties have been recorded.For the first time, Guo et al. (2013) studied an extracellular biopolymer from Scenedesmus obliquus AS-6-1.The produced bioflocculant is a 127.9 kDa polysaccharide that flocculates freely-suspended microalgal cells.However, another strain, Scenedesmus quadricauda, was shown to produce significant amounts of bioflocculant composed of sugar (56.7%) and protein (41%) [68].The self-flocculation efficiency reached 96.8% at pH 7 with biomass concentration 0.21-39 g/L [67].

Microorganism Growth Conditions for Bioflocculant Production
Microbial growth bioflocculant production has been reported to be influenced by various factors such as carbon sources, nitrogen sources, oligoelements, and operating parameters (temperature, pH, inoculum size, aeration rate, etc.).In order to increase the bioflocculant production, growth factors were optimized using statistical analyses.As reported before, a wide variety of microorganisms isolated from various sources, utilizing various nutrient sources and growing under various conditions, are able to produce bioflocculants with different characteristics.
At the beginning of the microbial bioflocculant investigation, media containing simple carbon and nitrogen were utilized for culturing new isolates.Carbon sources included sugar alcohols and organic acid.However, nitrogen sources included peptone, urea, yeast extract, NH 4 Cl, etc. Tables 1-3 show the favorable nutrient sources allowing the obtention of flocculanting polymers with significant FA.Glucose and yeast extract were efficient for several microbial strains such as Bacillus velezensis 40B [20] and Rhodococcus opacus [40], allowing the production of bioflocculants having different rates of FA (98% for Bacillus velezensis 40B and 22.5% for Rhodococcus opacus).L-glutamic acid has been reported to have an effective role in the culture of Bacillus mojavensis 32A and flocculant productivity [14].Interestingly, using glucose for Penicillium sp.HHE-P7 allowed the production of bioflocculant having a FA of 95% [59].For Solibacillus silvestris W01, an optimal flocculant amount was reached with maltose [24].Similarly, Zhang et al. (2010) [29] concluded that glucose and peptone were favorable for Proteus mirabilis to synthesize bioflocculant [29].Compared with various nitrogen sources, peptone was highly appropriate for Paenibacillus elgii B69 to produce bioflocculant [25].In the case of Aspergillus flavus, sucrose allowed the obtention of the maximum flocculant amount.However, this amount was negatively affected by fructose and glycerol.Interestingly, growth media supplemented by yeast extract and urea significantly enhanced the bioflocculant production [3].Similarly, nitrogen sources such as NaNO 3 , NH 4 Cl, and urea stimulate the growth of Klebsiella sp.ZZ-3, allowing the production of glycoprotein with FA ranging from 90.4% to 94.5% [31].In addition, glycerol and ammonium enhance significantly the growth of Bacillus.However, the produced flocculant showed a lower FA of 23% [23].
Byproducts and wastes generated by the agroindustrial sector contain a considerable amount of nutrients (carbon, nitrogen, oligoelements, etc.) useful for microbial growth.In this context, multiple studies demonstrated that agroindustrial residues (sugarcane, starch molasses, corn-steep liquor, soybean juice, etc.), which are mainly composed of polysaccharides, could be used as substrates for microbial growth and bioflocculant production.Similarly, wastewater and sludge, which are abundant raw materials containing enough carbon, nitrogen, phosphorus, and micronutrients, could sustain microbial growth for bioflocculant production.According to Zhao et al. (2017), Rhizobium radiobacter and Bacillus sphaericus were able to synthesize flocculating materials while growing in wastewater supernatant of anaerobic co-digestion (corn straw and molasses wastewater) [22].Methanol wastewater was used as a growth medium to produce bioflocculant useful for arsenite removal [34].Potato starch wastewater was used for the culture of two strains of Rhizopus, allowing the production of an efficient bioflocculant MBF917 for wastewater treatment [66].Moreover, Guo et al. (2014) reported the use of wastewater sludge to prepare biopolymers with flocculating activity exceeding 92% [39].
The pH is also an important factor in the microbial culture.It was reported to affect the growth, bioflocculant production, and FA.Each microbial strain has an optimum pH for growth and bioflocculant production.The impact of pH can be illustrated by some examples reported for various microbial strains.For example, the A. flavus bioflocculant was produced at pH values ranging between 5 and 9 and the highest FA was obtained at neutral pH [3].In the case of S. silvestris, the pH of growth was in the range pH 7-9 with an optimum FA at pH 8 [24].For the C. daeguense strain, the maximum amount of flocculating material was reached at pH 6 [29].For Penicillium purpurogenum, pH 5.5 allowed the highest bioflocculant production [59].
Based on this reported information, it is vital to point out the role of pH during the flocculation procedure.In the presence of protein as a bioflocculant, an alkaline pH is required to ensure the flocculation [68].However, polysaccharidic bioflocculants tolerate a pH ranging from slightly acidic to slightly alkaline conditions.
Similar to pH, the temperature plays an essential role in microbial culture.Depending on the microbial stain, the growth temperature significantly affects the growth, the bioflocculant production, and FA.As reported by many authors, a high temperature may change the protein or peptide structure of the bioflocculant, causing polymer damage and reducing the FA.In addition, the inoculum size and the time course of bioflocculant production are considered two critical factors.Both the bioflocculant production characteristics and the FA varied during the growth depending on the microbial strain and the inoculum size.For many strains such as S. silvestris [24] and Streptomyces sp.[56], the bioflocculant was produced during the logarithmic growth phase.For others strains such as A. flavus, the bioflocculant was produced at the same time as the cell growth with a maximum at the stationary phase [3].
Therefore, it is apparent that every microorganism has its specific operating parameters to maximize bioflocculant yield and FA.In order to determine the precise time at which the microbial culture should be stopped, the growth medium composition (carbon, nitrogen, and growth factors) and culture operating parameters (pH, temperature, aeration, inoculum size, etc.) should be optimized for each microbial strain.

Applications of Microbial Bioflocculants for Wastewater Treatment
Microbial bioflocculants are eco-friendly materials, harmless and biodegradable.They are composed of polysaccharides, proteins, and glycoproteins.Their degraded intermediates are safe for humans and the environment.Moreover, microbial enzymes responsible for bioflocculant degradation are present in the environment (wastewater, sludge, soil, sea, etc.).Because of the increasing requirement for environmental quality, bioflocculant performance has been investigated for wastewater treatment to remove solids, organic pollutants, and heavy metals.Sludge conditioning was also studied using microbial bioflocculants.
Wastewater from various origins such as swine, municipal use, breweries, aquaculture, potato starch, landfill leachate, and tannery wastewater was subject to microbial flocculation.Studies were related to the optimization of culture conditions (pH, temperature, inoculum size, bioflocculant dosage, etc.) in order to maximize bioflocculant yields, FA, and pollutant removal.Obtained results varied depending on the used strain for bioflocculant production and on the wastewater characteristics (pH, COD, SS, P t , N t , etc.).For example, Tang et al. ( 2014) reported a flocculant from Paenibacillus mucilaginosus with removal efficiencies of 70-75.2% and 81.5-88%, respectively, for COD and SS from paper mill wastewater.The same bioflocculant allowed the removal of 88.8-92% of SS from biological factory wastewater, with 83.2-86.9%reduction of COD [73].Similar work was conducted with garbage incineration plant wastewater (pH 6.08), giving a COD ranging from 59.7% to 60.7% and SS removal in the range of 68-69.8%.However, it is important to point out that the used polymer doses were between 0.5 and 4 mg/L [72].
An interesting opportunity based on the direct addition of microbial strains to wastewater was performed by other authors.In this context, it was demonstrated that adding Bacillus megaterium SP1 (inoculation: 1 × 10 4 CFU/mL, 30 • C, pH 7) to aquaculture wastewater could efficiently reduce the COD and SS levels and accelerate the bioflocculation process [70].Therefore, a microbial polymer could substitute chemicals (e.g., Fe 2 (SO 4 ) 3 , AlCl 3 , etc.), during the treatment of industrial wastewater.A heterogeneous biopolymer prepared by a consortium (Rhizopus sp.M9 and M17) allowed many advantages (working at lower dose, without pH control, cheap cost of preparation, and significant elimination rates of turbidity and COD) while treating potato starch wastewater [66].
More recently, it was demonstrated that Rhizobium radiobacter and Bacillus sphaericus were effective for the elimination of Zn 2+ , Cu 2+ , Cr 6+ , and Ni 2+ from simulated electroplating wastewater.A bioflocculant dose of 374 mg/L (acting at pH 6 for 40 min) allowed 90% removal for both Zn 2+ and Cu 2+ , 65% for Ni 2+ , and only 30% for Cr 6+ [22].In addition, Yao et al. (2013) [77] reported significant removal of Fe 3+ and Pb 2+ from wastewater using B. mucilaginosus bioflocculant.Interestingly, heavy metals were removed by adsorption and by the formation of carbonate minerals in the presence of CO 2 , which can cause waste disposal problems [77].Another microbial bioflocculant showed maximum removal efficiency of arsenic (84.6%) and arsenite (98.9%) from synthetic wastewater [78].Also, a maximum Pb (II) ion removal efficiency of 99.85% was reported by Feng et al. (2013) [79].This removal level could be related to the charge neutralization and adsorption bridging.
Experiments with real wastewater were also conducted using a microbial culture broth or extracted bioflocculant.Among these experiments, a culture broth of microbial consortium (Bacillus cereus and Pichia membranifaciens) was used to treat printing and dyeing wastewater.A culture broth of 2% v/v, pH 7 and in the presence of 1% (v/v) of CaCl 2 allowed the removal of 57% of COD, 63% of SS, and 78% of the turbidity [49].In the same operating conditions, the use of each strain alone showed different performance.Bacillus cereus allowed the removal of 49% of COD, 73% of SS, and 70% of the turbidity; however, Pichia membranifaciens removed 45% of COD, 58% of SS, and 49% of the turbidity [49].The variability in performance could be explained by the variability of functional groups (hydroxyl, amino, phosphate, and carboxyl groups) present in the polymer molecules produced by each strain.Interestingly, the combination of the two strains may allow most substances present in wastewater to bond and, consequently, enhanced the removal efficiency.
In another study, tannery wastewater (initial COD: 1082.2 mg/L) treatment was assessed using the bioflocculant that resulted from the growth of three bacterial strains (Bacillus cereus CZ1001, B. subtilis CZ1002, and B. fusiformis CZ1003).The study showed the variability of the optimum bioflocculant doses for the removal of COD, chrominance, and total nitrogen.It showed that 0.2 g/L of the flocculant allowed COD removal percentages in the range 22.71-25.97%,while a 0.11 g/L dose allowed nitrogen removal percentages in the range 22.71-38.43%.At the same concentration, the chrominance removal reached 12.74-70.97%[81].Thus, microbial bioflocculant can be considered as a potential agent to treat industrial wastewater containing dyes in high concentrations.However, various parameters (pH, temperature, flocculant doses, etc.) should be optimized depending on the characteristics of the wastewater to be treated.

Microbial Flocculants for Sludge Dewatering
In order to prepare sludge for dewatering processes, a conditioning process using chemical polymers should be applied.Sludge dewatering allowed the obtention of a product that was dry enough, thereby reducing the storage volume and limiting the energy used during the process of sludge incineration.As indicated above, the use of chemical polymers presents various disadvantages.Interestingly, using biomaterials for wastewater sludge conditioning represents a new sustainable technology.To the best of our knowledge, very few works have described the possibility of using microbial bioflocculants for sludge conditioning.Sludge dewatering was evaluated regarding dry solids (DS) and specific resistance to filtration (SRF).As indicated in Table 7 [29,[84][85][86][87][88], bioflocculants showed significant results similar to those obtained with chemical polymers such as polyacrylamide (PAM), polyaluminum chloride (PAC), FeCl 3 , and Al 2 (SO 4 ) 3 .For example, Acidithiobacillus ferrooxidans flocculant improved the dewaterability of anaerobically digested sludge compared to PAM.The microbial polymer significantly reduced capillary suction time (CST) and the SRF of sludge by 74% and 89%, respectively, and these values are higher than with PAM.Interestingly, the Acidithiobacillus ferrooxidans biopolymer reduces the moisture content of sludge to 70% and improves the clarity of the filtrate in terms of removal of total suspended solids and total dissolved solids [84].Moreover, microbial bioflocculants offered the lowest optimum dosage, various conditions, are able to produce bioflocculants with different characteristics.Microbial growth conditions (strain, inoculum, nutrient sources, operating parameters, etc.) are studied in order to determine a typical procedure to maximize both bioflocculant production and flocculating activity.Bioflocculant chemical characteristics such as polymer content are related to the microbial strain and substrates used.Hence, data related to the conditions of microbial flocculant production are required to establish a strategy for scientific research and the commercial application of biopolymers in wastewater treatment.As discussed above, the potential use of microbial flocculants for wastewater treatment has been verified.They have shown significant results in removing pollutants from wastewater such as suspended solids, turbidity, COD, total nitrogen, dye, and heavy metals.At a laboratory scale, many examples of bioflocculants displayed significant flocculating activity, where the efficiency of pollutant removal exceeded 90% depending on the microbial strain used to produce the flocculant and on the wastewater characteristics.Therefore, extensive research is required to determine the optimal bioflocculation procedures for each type of wastewater.Also, in order to understand the bioflocculation mechanism, more experiments needed to be conducted taking into account the modifications in different treatment systems.Finally, the efficacy of the bioflocculation should be examined at a large scale, in real conditions and for a variety of wastewater systems, followed by a techno-economic assessment.

Table 1 .
Examples of bacteria investigated for flocculant production.

Table 2 .
Examples of microbial consortia investigated for bioflocculant production.

Table 3 .
Examples of fungi and algae investigated for bioflocculant production.

Table 4 .
Effluent treated by microbial flocculants for turbidity and organic pollutant removal.

Table 5 .
Heavy metal removal by microbial flocculants.