Microalgae are a highly heterogeneous group of microorganisms belonging to many different evolutionary lineages. Different species of unrelated microalgae can have very different surface properties (Eldridge et al. 2012). When the functional groups on the cell surface differ between species of microalgae, this will cause differences in the flocculation behavior (Henderson et al. 2010). Cell surface properties may even differ between strains of the same species (Cheng et al. 2011). For example, mutant Chlamydomonas strains that lack a cell wall were found to flocculate more easily than wild type strains (Scholz et al. 2011). In addition, smaller species have a higher surface to volume ratio and require a higher flocculant dose per unit of biomass (Vandamme et al. 2010).

Flocculation of microalgae differs from flocculation of inorganic colloids and particles in that microalgae are living organisms that can modify their surface properties and interact with their environment through uptake or excretion of substances (Pieterse and Cloot 1997). The flocculation behavior of microalgae can depend on the culture conditions. Zhang et al. (2012) showed that the cell surface of stationary-phase Chlorella zofingiensis cells had lower concentrations of carboxylic groups than during exponential phase and required a lower dosage of alum to induce flocculation. Because the microalgal Z potential changes from exponential to stationary growth, the flocculation efficiency is likely to vary with the growth phase (Danquah et al. 2009b).

Microalgae excrete a substantial fraction of the photosynthesis products as extracellular organic matter. Hulatt & Thomas (2010) showed that up to 17.3 % of the organic matter produced during photosynthesis is excreted in the culture medium. Organic matter concentrations in microalgal culture medium are often 10-100 mg C L-1, depending on species and culture conditions. This organic matter consists mainly of polysaccharides and proteins (Henderson et al. 2008a). The secreted material has a strong inhibitory effect on flocculation, as is evident from much higher flocculant dosages in media with microalgal organic matter compared to media without the organic matter (Bernhardt et al. 1989; Vandamme et al. 2012b). Most of the chemical demand required for flocculating microalgae may be due to the microalgal organic matter present in the media rather than the microalgal biomass itself, as well as impurities present in the medium, such as humic matter (de Godos et al. 2011; Beuckels et al. 2013). Chen et al. (2008) suggested that microalgal organic matter may cause complexation of free metal ions and as such prevent flocculation by metal hydroxides. It is also possible that negative charges on the extracellular organic matter compete with the microalgal cell surface for the positive charges of the flocculants (Garzon-Sanabria et al. 2013).