# Extraction of Pigments from Microalgae and Cyanobacteria--A Review on Current Methodologies
## 8 Cell Milking  
Products coming from microalgae and cyanobacteria traditionally followed the process of biomass cultivation, harvesting, dewatering, extraction, and purification; thus, alternatives to improve the process have been suggested along the years. The term \"cell milking\" has gained popularity due to the potential of bypassing a portion of the process. Cell milking works on the basis of a two-phase cultivation system that includes an aqueous phase (medium) and an extraction phase with a hydrophobic solvent. The contact of the solvent with the cell creates pores in the membrane, exposing the target compounds that pass to the solvent phase due to their compatibility and solubility in the solvent. The cells are then allowed to recover and produce more of the compound and possibly be milked again in a short time interval [71].  
Cell viability is the main factor that distinguishes milking from in situ extraction. In situ extraction uses the same two-phase solvent system, but the cell is not restored after the extraction. The continuous production of a compound through in situ extraction also requires a high productivity in the cultivation phase to overcome cells' death [11]. Figure 7 shows a schematic representation of cell milking and its effects on the cell.  
The main advantage of cell milking is that this process does not require harvesting and the recovery of the products from the hydrophobic phase is relatively simple. Also, the cultivation phase can be maintained continuously, significantly reducing costs [11]. As disadvantages, the solvent needs to be biocompatible, green labelled, and safe, and the solutes can be only extracted in the hydrophobic phase. Moreover, the need of agitation to provoke contact of the two phases requires energy input, increasing costs, although most microalgae and cyanobacteria cultures already have an agitator [11].  
The most commonly used solvent in milking is dodecane, approved as food flavoring but with restrictions for other uses. An important factor when considering solvent compatibility is its log P (partition coefficient) value, which will determine its solubility in the cultivation phase. [PERSON] et al. [72] used solvents with log P ranging from 3 to 9 in order to extract \(\beta\)-carotene from \(D\). _salina_ and found that solvents with log P > 6 were the most compatible with this microalgae. Also, using a higher log P solvent would allow specifying the extraction for \(\beta\)-carotene only, with less chlorophyll contamination. In this study, the optimal extraction was achieved using dodecane. In another study, [PERSON] et al. [73] tested milking with different \(D\). _salina_ strains; interestingly one of the strains (CCAP 19/25) was not milkable, as no carotenoids were extracted. For the other tested strains it was possible to extract different carotenoids profiles by using different mixing methods. Using gentle mixing, it was possible to extract only secondary carotenoids, while when using vigorous mixing primary carotenoids and chlorophyll were mostly extracted instead, although with vigorous mixing the authors reported an increase in cell death.  
The use of milking was also described for the obtention of astaxanthin from \(H\). _pluvialis_ by using a two-phase solvent for the extraction phase, after the switch from the green to the red phase. The process uses dodecane for extraction and methanol for recovering the astaxanthin from the dodecane. The addition of NaOH to methanol is suggested in order to increase the stability of astaxanthin [18]. As an alternative to dodecane, [PERSON] and [PERSON] [74] suggested vegetable oils for the extraction of astaxanthin, with an increase in recovery yields.  
Milking can be applied for the extraction of other compounds besides pigments, including lipids for biofuel. [PERSON] et al. [11] suggest the milking of the microalgae _Botryococcus braunii_ (Chlorophyta) using hexane, octane or octanol as solvents, due to their biocompatibility, boiling point, extraction effectiveness, cost, and safety.