Microalgal Downstream Processing: Harvesting, Drying, Extraction, Separation, and Purification 93
the sedimentation velocity increases with the size of the particles. Therefore, processes that contribute
to an increase of the size of the particles would effectively increase sedimentation speed. Flocculation
is the coalescence of individual suspended particles into loosely attached conglomerates termed as flocs
which are less buoyant. Flocculation is known to occur naturally, especially in seawater environment, a
phenomenon called autoflocculation. Alternatively, flocculation can be induced by adding a flocculant
or it can be achieved by co-culture of the microalga of interest with another microorganism (termed as
bio-flocculation). Flocculation can also promoted by using ultrasonication or even electro-coagulationflocculation for batch mode harvesting.
Autoflocculation
Autoflocculation has been observed to take place naturally for the alga Phaeodactylum tricornutum when
the pH increases above 9 (Spilling et al. 2011). Autoflocculation is the spontaneous formation of flocs due
to the precipitation of calcium carbonate and magnesium hydroxide with algal cells at high pH (Sukenik
et al. 1985). Hence, autoflocculation often depends on the pH of the culture broth. In algal cultures, high
pH is usually the consequence of photosynthesis resulting in CO 2 consumption by the algae. High pH can
also be achieved by addition of caustic soda or lime (Nurdogan and Oswald 1996) and even though the
flocculation process is induced by addition of chemicals, the process is still referred to as autoflocculation.
Şirin et al. (2012, 2013) investigated the autoflocculation of two marine algae, Nannochloropsis gaditana
and P. tricornutum. These two algae did not show significant sedimentation under natural conditions.
Effect of pH (from 2 to 11) on flocculation of N. gaditana, showed a flocculation efficiency above 90%
in the pH range 9.70 to 11, and sedimentation rate as high as 119 cm/h at pH 9.70. Similar results were
reported by Spilling et al. (2011) for P. tricornutum.
Addition of ammonia has also been reported to be efficient for harvesting marine microalgae (Chen
et al. 2012b). The flocculation efficiency was performed on two marine algae, N. oculata and a Chinese
algal species classified as Dunaliella. The cultures were stirred with a magnetic stirrer and added various
doses of commercial aqueous ammonia. A removal efficiency of 91.2% after 3 hr settling and an optimum
concentration of ammonia of 38.37 mmol/L for Dunaliella were achieved. For N. oculata the optimum
removal efficiency (93%) was performed with 57.31 mmol/L of ammonia measured after 3 hours. This
treatment however caused cell damages as observed by scanning electron microscopy (SEM): cell
morphology was fuzzy due to membrane disruption.
As autoflocculation is caused by the formation of inorganic precipitates, the removal of these from
the biomass has to be envisaged.
Flocculation using flocculants
Where autoflocculation fails, it is possible to induce flocculation of the cultured algae cells. Several types
of flocculants are commercially available for that purpose: polyvalent metal salts, synthetic polymers, and
natural polymers (usually modified). However salinity levels above 5 g/L tend to inhibit flocculation with
polymers used as the sole agent (Bilanovic and Shelef 1988). Flocculation efficiency can be improved
by using inorganic coagulants (ferric ion, alum, lime) in conjunction with the polymers (Knuckey et al.
2006).
Flocculation with polyvalent metal salts. Flocculation of microalgae can be achieved by adding a
flocculating agent releasing multivalent cationic ions, typically polyelectrolyte salts of aluminium (e.g.,
alum: aluminium sulfate, Al 2 (SO 4 ) 3 ) and/or iron (e.g., ferric chloride, ferric sulfate), which reduce the
inter-particular repulsive forces between the negatively charged surface of the cells yielding aggregates
of cells. In the case of aluminium in the range of pH 5.0 to 6.0, flocculation has been explained by
a combined action of electrostatic interactions due to positively charged polyaluminium species and
entrapment mechanism referred to as sweep flocculation due to amorphous aluminium hydroxide
precipitate (Amirtharajah and Mills 1982; Duan and Gregory 2003; Zhang et al. 2004). Aluminium
chloride (AlCl 3 , 30 g/L) was efficient to flocculate the marine algae Isochrysis galbana (Sánchez et al.
2013). In this work, the effect of mechanical mixing during harvest was shown to be important in the
the sedimentation velocity increases with the size of the particles. Therefore, processes that contribute
to an increase of the size of the particles would effectively increase sedimentation speed. Flocculation
is the coalescence of individual suspended particles into loosely attached conglomerates termed as flocs
which are less buoyant. Flocculation is known to occur naturally, especially in seawater environment, a
phenomenon called autoflocculation. Alternatively, flocculation can be induced by adding a flocculant
or it can be achieved by co-culture of the microalga of interest with another microorganism (termed as
bio-flocculation). Flocculation can also promoted by using ultrasonication or even electro-coagulationflocculation for batch mode harvesting.
Autoflocculation
Autoflocculation has been observed to take place naturally for the alga Phaeodactylum tricornutum when
the pH increases above 9 (Spilling et al. 2011). Autoflocculation is the spontaneous formation of flocs due
to the precipitation of calcium carbonate and magnesium hydroxide with algal cells at high pH (Sukenik
et al. 1985). Hence, autoflocculation often depends on the pH of the culture broth. In algal cultures, high
pH is usually the consequence of photosynthesis resulting in CO 2 consumption by the algae. High pH can
also be achieved by addition of caustic soda or lime (Nurdogan and Oswald 1996) and even though the
flocculation process is induced by addition of chemicals, the process is still referred to as autoflocculation.
Şirin et al. (2012, 2013) investigated the autoflocculation of two marine algae, Nannochloropsis gaditana
and P. tricornutum. These two algae did not show significant sedimentation under natural conditions.
Effect of pH (from 2 to 11) on flocculation of N. gaditana, showed a flocculation efficiency above 90%
in the pH range 9.70 to 11, and sedimentation rate as high as 119 cm/h at pH 9.70. Similar results were
reported by Spilling et al. (2011) for P. tricornutum.
Addition of ammonia has also been reported to be efficient for harvesting marine microalgae (Chen
et al. 2012b). The flocculation efficiency was performed on two marine algae, N. oculata and a Chinese
algal species classified as Dunaliella. The cultures were stirred with a magnetic stirrer and added various
doses of commercial aqueous ammonia. A removal efficiency of 91.2% after 3 hr settling and an optimum
concentration of ammonia of 38.37 mmol/L for Dunaliella were achieved. For N. oculata the optimum
removal efficiency (93%) was performed with 57.31 mmol/L of ammonia measured after 3 hours. This
treatment however caused cell damages as observed by scanning electron microscopy (SEM): cell
morphology was fuzzy due to membrane disruption.
As autoflocculation is caused by the formation of inorganic precipitates, the removal of these from
the biomass has to be envisaged.
Flocculation using flocculants
Where autoflocculation fails, it is possible to induce flocculation of the cultured algae cells. Several types
of flocculants are commercially available for that purpose: polyvalent metal salts, synthetic polymers, and
natural polymers (usually modified). However salinity levels above 5 g/L tend to inhibit flocculation with
polymers used as the sole agent (Bilanovic and Shelef 1988). Flocculation efficiency can be improved
by using inorganic coagulants (ferric ion, alum, lime) in conjunction with the polymers (Knuckey et al.
2006).
Flocculation with polyvalent metal salts. Flocculation of microalgae can be achieved by adding a
flocculating agent releasing multivalent cationic ions, typically polyelectrolyte salts of aluminium (e.g.,
alum: aluminium sulfate, Al 2 (SO 4 ) 3 ) and/or iron (e.g., ferric chloride, ferric sulfate), which reduce the
inter-particular repulsive forces between the negatively charged surface of the cells yielding aggregates
of cells. In the case of aluminium in the range of pH 5.0 to 6.0, flocculation has been explained by
a combined action of electrostatic interactions due to positively charged polyaluminium species and
entrapment mechanism referred to as sweep flocculation due to amorphous aluminium hydroxide
precipitate (Amirtharajah and Mills 1982; Duan and Gregory 2003; Zhang et al. 2004). Aluminium
chloride (AlCl 3 , 30 g/L) was efficient to flocculate the marine algae Isochrysis galbana (Sánchez et al.
2013). In this work, the effect of mechanical mixing during harvest was shown to be important in the