The most important physical aspects of natural coagulant that could be studied are surface morphology and surface charges. Surface morphology refers to the imaging of an exposed surface of any object under the microscope, which cannot be seen by the naked eye. By analysing the surface morphology, the active groups attributed to flocculation function can be identified, for example, the citral. According to the Essential Oil-Bearing Grasses, the genus Cymbopogon by Akhila, the oil in citral would help in the blood coagulation–fibrinolysis system [111]. Besides, citral is an antimicrobial element that will protect coagulant such as chitosan from microbial damage [112]. Moreover, the presence of pores (micro-, macro- and mesopores) on natural coagulant could be clearly identified via surface morphology analysis, and they are favourable for the attachment of suspended particles through adsorption, intraparticle bridging or electrostatic contacts during coagulation and flocculation. In addition, the previous study by Obiora-Okafo and Onukwuli [107] proved that a compact net structure coagulant showed higher flocculating activity as compared with a branched structure. Furthermore, changes to the surface morphology of coagulants after coagulation and flocculation show proof of interaction between the coagulants and suspended particles. In view of surface morphology as a strategy to enhance the flocculating activity, modification on physical structures such as grafting could be done to create a high density of pores and ultimately more favourable to coagulation. With these, the mass harvesting of microalgae in the industrial scale is applicable.

 

On the other hand, surface charge, or zeta potential, is one of the factors that will affect the flocculating activity. Theoretically, zeta potential is the measure of the electrical charge of particles that are suspended in liquid [113]. Practically, the higher the negative surface charge of natural coagulant, the greater it’s flocculating activity against positive suspended particles and vice versa for the positive surface charge of natural coagulant against negatively suspended particles. Thus, the study of surface charge shows a preliminary estimation of flocculating activity of natural coagulant. Besides, the nature of surface charge (positive or negative) indicates the potential treated group of suspended particles, to illustrate, a negatively charged coagulant is used to remove cation heavy metals or the other way round. Chemically and structurally modified of natural coagulant such as quaternary agent 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) grafted on cellulose nanocrystals (CNC) could be applied to enhance the zeta potential to extreme positive or negative [114]. Above all, natural coagulant with positive zeta potential is favourable in microalgae harvesting due to the anionic nature of microalgae.

 

Moreover, different molecules of the same compound could have different molecular masses because they contain different isotopes with different mass number. The physical aspect of coagulants, molecular weight, could reflect their flocculating mechanism and activity. Yin [2] noted that high molecular weight of natural coagulant played a role in improving aggregation. The higher the molecular weight of natural coagulant, the stronger the bridge formed onto the particle surface than natural coagulant with a lower molecular weight. Thus, the formed flocs were stronger, larger and denser for a larger molecular weight natural coagulant and permitted better settling, also improving the harvesting efficiency [115]. Additionally, the high molecular weight allows natural coagulant’s chains to stretch sufficiently far from the particle surfaces; thus, favourable for bridging to form [81]. Another study by Muylaert et al. [116] also showed that the high molecular weight polyelectrolytes (i.e., lignosulfonate) were a better bridging agent. On the other hand, the molecular mass of natural coagulant often reveals its undergoing mechanism in flocculation, for example, the lower molecular weight of natural coagulants, such as polyethyleneamine are usually undergoing flocculation via the charge patch mechanism [116]. It had also been well reported that the high molecular weight of natural coagulant would usually predominant in bridging mechanisms. Yin [2] also suggested that the dimeric cationic proteins with the molecular mass of 12–14 kDa and isoelectric point (pI) between 10 and 11 were predominant in adsorption and charge neutralisation mechanisms. Therefore, by studying the molecular weights of natural coagulants in advance prior to the application, the underlying coagulation mechanism of natural coagulant could be defined and modification could be made based on their respective mechanism. All in all, by knowing the molecular weight, the same compounds can be operated as dispersants (e.g., dextrin, low molecular weight) or coagulants (e.g., starch, high molecular weight). Generally, a dispersant is used to prevent fine particles from aggregating and normally being utilised in a selective flocculation process, in which gangue minerals are dispersed while flocculating valuable or desired minerals [117]. Such approach is suitable to be used in microalgae harvesting.

The flocculating activity of natural coagulants also depends on the specific chemical properties of the polymer. One of the key polymer characteristics includes various functional groups. The particular functional groups to be evaluated are COO⁻ and OH⁻ as their existence usually contributes to the flocculating activity of natural coagulant. Besides, the increase in positively charged functional groups allows more interactions with the negatively charged suspended particles, and thus improve the binding capabilities of natural coagulants [116]. Modification on functional groups of natural coagulants is also proposed and evinced by researchers in the past studies to increase the flocculating activity. For example, functionalising of cationic starch and TANFLOC, in which, the starch and tannins added with quaternary ammonium groups to increase the flocculating activity and serve as the low-cost as well as more effective alternatives for flocculation process [116]. Additionally, natural coagulants often perform poorly in harvesting marine microalgae [118]. The underlying reason is the high ionic strength of seawater will cause coiling, and this will decrease the effective size of natural coagulants. Therefore, an alternative had been proposed to modify the structure to a more rigid molecule such as tannin-based natural coagulants or functionalised nanoparticles, namely, nanocellulose [116]. Furthermore, in microalgae harvesting, the functional group of natural coagulants can be furthered enhanced with magnetoresponsive Fe₃O₄ nanoparticle to separate the flocculated microalgae from the medium using a magnetic field [119]. To summarise, the modification of functional group begins with characterisation of natural coagulant, which is an important factor that influences the effectiveness of natural coagulant in microalgae harvesting.

Elemental property of natural coagulant affects the flocculating activity. The trivalent cation is the most efficient in flocculating the negatively charged suspended particles. However, trivalent cation is commonly found only in inorganic coagulant such as alum. In plant-based coagulants, divalent cation is predominant instead. Besides, numerous studies have shown that when there are more phenolic groups available in a tannin structure, the coagulation capability could be enhanced [2]. Correspond to this statement, it was reported that the legume-based coagulant was rich in phenolic compounds, and it had also proven to exhibit antibacterial property [3]. These could aid in removing pathogenic bacteria such as Salmonella parathyphi that is presented in wastewater due to the leaching of sewage effluents. Thus, phenolic groups provide the –OH group not only for bridging, but to indirectly inactivate the pathogenic bacteria in the wastewater [3]. Therefore, the phenolic group deserves attention in wastewater treatment as well as microalgae harvesting, especially in extraction of DHA microalgae oil. Moreover, there is also one characteristic that has been ubiquitously used as a preselection criterion for new plant-based coagulants, namely, mucilage. Mucilage is a thick, gluey and adhesive substance produced by nearly all plants and some microorganisms. Evidently, the high bridging–coagulation capability of Opuntia with the presence of mucilage will promote the bounding action of particulates to mucilage without directly contact of particulate and has been widely used in water treatment in North America [2,120]. Besides, the recent study on biopolymer coagulant showed that 73% (from 320.0 to 88.0 mg·L⁻¹) of Fe³⁺ reduction and ~36% of COD removal with an addition of 3.20 mg·L⁻¹ of okra mucilage during the harvesting process [120]. The presence of galacturonic acid in mucilage will act as an active coagulating agent and provide a bridge for particles adsorption. Further, the partial deprotonation of carboxylic functional group of mucilage in aqueous solution has given rise to the chemisorption between charged particle with COO⁻ and OH⁻ [2]. Therefore, it will aid in flocculating activity. To conclude, the selection of natural coagulant for microalgae harvesting should be focused on mucilage as its primary concern.

The thermal stability of natural coagulants is also a crucial parameter to be studied in enhancing the flocculating activity. Indeed, an optimum temperature will increase the flocculating activity. However, the temperature higher than 80 °C will usually destroy the chemical composition of natural coagulants [121]. Moreover, the temperature has direct effects on floc formation, breakage and reformation. To illustrate, floc formation is slower at a lower temperature, whereas breakage of floc is greater at higher temperatures. On the other hand, thermogravimetry analysis determines the minimum temperature causing decomposition of organic components in natural coagulant and differential scanning calorimetry allows study relating to the heat flow required to decompose the natural coagulant. In general, the thermal characteristics reveal the thermal stability of natural coagulant and it has no direct impact on microalgae harvesting because coagulation will not occur in extreme temperature.