# Flocculation Harvesting Techniques for Microalgae: A Review
## 2 Auto-Flocculation  
In auto-flocculation, suspended algal cells spontaneously aggregate, forming large flocs, which induce their simple gravitational sedimentation (Figure 2). This phenomenon has been observed in various algal species particularly under non-ideal culture conditions such as change in pH and cultural aging, as summarized in Table 1. Both alkaline and acidic conditions have been reported to reduce the intensities of the negative surface charge of algal cells, thereby promoting their self-aggregation [31]. Under alkaline conditions above pH 9, the changes in the surface charge of algal cells are mainly attributable to significant secretion of protective extracellular polymers [32]. Under acidic conditions, fluctuating dissociations of carboxyl and amine groups in the algal cell wall can cause changes in surface charge. Figure 2: Auto-flocculation harvesting of algal biomass. Three vials contain algal samples cultured in different nitrate concentrations: (**left**) 0.5\(\times\); (**middle**) 1\(\times\) (original); and (**right**) 2\(\times\). Reprinted from Reference [33], distributed under the terms of the Creative Commons Attribution License. It should be noted that auto-flocculation efficiency resulting from pH manipulation is largely species-dependent [32]. There is a sigmoidal relationship between increase in cultural pH and flocculation efficiency for the freshwater alga _Etthia_ sp. YC001. The harvesting efficiency is very low at \(\sim\)11% in the pH range of 6.5-8.5, while it dramatically increases to 83% and 94% with increase in pH to 10.5 and 12.5, respectively [37]. Similarly, a high harvesting efficiency of 94% was reported for _Chlorocoecum_ sp. R-AP13 at pH 12 [36]. However, in the case of _Scenedesmus obliquus_ NRClbr1, a slight improvement in auto-flocculation efficiency from 10.4% to 33.2% was observed despite a pH increase from 7 to 10 [40]. In another study, high flocculation efficiency of \(\sim\)90% was reported for three freshwater algal species, _C. nivale_, _C. ellipsoideum_, and _Scenedesmus_ sp., under acidic conditions (pH 4) [31]. Self-flocculating activity can also be affected by cell density. At pH 11.5, the harvesting efficiency of _S. quadricauda_ was as high as 94.7% for a low cell density of 0.54 g/L, but it was significantly decreased to 71.7% when applied at a high cell density of 2.7 g/L [34]. Aging of algal cultures is usually combined with the release of EOM from algal cells (mainly composed of proteins and polysaccharides) into the aqueous environment [28]. EOM is thought to play an important role in the self-flocculation of algal cells, specifically by forming biofilms and changing the surface charge of cells, presumably via neutralization [41; 42]. The amount and composition of EOM is largely dependent on species, growth stages, and culture conditions (culture duration, pH, light, and temperature) [43]. The auto-flocculation efficiency of _S. obliquus_ did not exceed 5.5% at the early stationary growth-phase under neutral pH, but was considerably increased to 24.4% (for the same culture and pH range) at the late stationary growth-phase [40]. Similarly, by increasing the cell aging time, the auto-flocculation efficiency of _Chlorocoecum_ sp. R-AP13 increased considerably from 62% in one-week-old culture to 75% in the three-week-old culture [36]. The composition and concentration of the nutrients in the medium such as nitrogen sources and minerals can influence auto-flocculation efficiency as well as algal biomass production. In this context, relatively higher auto-flocculation efficiency for _Chlorella vulgaris_ has been reported when nitrate (NO\({}_{3}\)\({}^{-}\)) was used as the nitrogen substrate instead of ammonium (NH\({}_{4}\)\({}^{+}\)). It has been proposed that this enhancement may be attributed mainly to the co-precipitation of Ca\({}^{2+}\) and Mg\({}^{2+}\) ions originally dissolved in the medium with algal cells, under a high pH environment resulting from the nitrate assimilating metabolic activities of algal cells [44].