**10. Algae oil extraction techniques**

filtration are other possible alternatives to conventional filtration for recovering algal biomass, which are more suitable for fragile cells and small scale production processes. Furthermore these filtration processes are more expensive especially because of the need for membrane

**for microalgae Closed systems (PBRs) Open systems(Ponds)**

Batch or semi-continuous

**Contamination control** Easy Difficult **Contamination risk** Reduced High **Sterility** Achievable None **Process control** Easy Difficult **Species control** Easy Difficult **Mixing** Uniform Very poor

**Space required** A matter of productivity PBRs ≅ Ponds **Area/volume ratio** High (20–200 m-1) Low (5–10 m-1)

**Capital/operating costs ponds** Ponds 3–10 times lower cost PBRs "/> Ponds

**Water losses** Depends upon cooling design PBRs ≅ Ponds

**CO2 losses** Depends on pH, alkalinity, etc. PBRs≅Ponds **O2 inhibition** Greater problem in PBRs PBRs "/> Ponds **Biomass concentration** 3–5 times in PBRs PBRs "/> Ponds

**Scale-up** Difficult Difficult

**Table 11.** A comparison of open and closed large-scale culture systems for microalgae [115].

**Population (algal cell) density** High Low **Investment** High Low **Operation costs** High Low

**Light utilization efficiency** High Poor **Temperature control** More uniform temperature Difficult **Productivity** 3–5 times more productive Low

**Evaporation of growth medium** Low High **Hydrodynamic stress on algae** Low–high Very low **Gas transfer control** High Low

**Operation regime** Batch or semi-continuous

Richmond [96] suggested one main criterion for selecting a proper harvesting procedure, which is the desired product quality. In one hand for low value products, gravity sedimenta‐

replacement and pumping.

**Culture systems**

124 Biofuels - Status and Perspective

The general extraction techniques are mechanical extraction/ cell disruption methods and Solvent extraction coupled with mechanical cell disruption methods. Other novel methods are Supercritical CO2 extraction and direct conversion of algal biomass to biodiesel. Extraction methods such as ultrasound and microwave assisted are also employed for oil extraction from vegetable sources. The results indicate that compared with conventional methods these new methods can greatly improve oil extraction with higher efficiency. Extraction times are reduced and yields increased by 50–500% with low or moderate costs and minimal added toxicity. In the case of marine microalgae Crypthecodinium cohnii, ultrasound worked best as the disruption of the tough algal cell wall considerably improved the extraction yield from 4.8% (in Soxhlet) to 25.9%.

### **10.1. Mechanical extraction/ cell disruption methods**

The first and simple extraction method is mechanical cell disruption of algal cells to extract oil without contamination of other chemicals. Mechanical pressing or French pressing of dry algal lumps involves pressurizing the algal biomass to high-pressure, where the cell walls are ruptured to releases the oil similar to oil extraction from seeds or nuts through mechanical pressing. Homogenization through bead or ball milling is a process to disintegrate the alga cells which takes place in a jacketed chamber or vessel. The shear force created by the high velocity beads which moves radially causes the disruption of cells [121]. Cell disruption in this method depends on factors like residence time, cell concentration, chamber volume, bead volume and umber of rotations. All these mechanical cell disruption are usually combined with the solvent extraction to improve the extraction efficiency. Along with the mechanical methods new pretreatment techniques ultra-sonication, microwave also getting attention. In ultra-sonication & microwave pretreatments, the biomass will be treated in a sonication [96]/ microwave [105] chamber prior to solvent extraction.

### **10.2. Solvent extraction**

Solvent extraction is a common practice used to extract oils from the algal biomass and other biomasses. The solvent should be selected based on efficiency, selectivity towards the different classes of lipids and ability of solvent to prevent any possible degradation of lipids. In order to achieve maximum extraction, the linkages between the lipids and other organelles of the algae cells which are connected with van der Waals interactions, hydrogen bonding and covalent bonding should be broken18. The most common solvents used for extraction are nhexane, chloroform, petroleum ether, methanol, ethanol, isopropanol, dichloromethane and mixture of any of these solvents depending upon method and desired class selection of lipids. The conventional solvent extraction methods are Bligh and dyer, folch [90], Soxhlet extraction. The steps involved in the solvent extraction at micro level were explained by halim et al. When the algal cells interacted with the organic solvents, these solvents penetrate through the cell wall and interact with the selective class of lipids depending upon its dielectric constant to form a solvent- lipid complex. This complex diffuses in to the bulk solvent due to the concen‐ tration gradient continues until this process reaches equilibrium [51]. The solvent extraction methods shows a lot of variability depending upon the organic solvent (dielectric constant) used and biological matrix being used in selection of different class of lipids [20, 51]. The cell wall and its composition and solvents dielectric constant could be the reasons for these variable extraction properties of individual methods [51].

These solvent extraction methods have been slightly modified by many researchers to improve the kinetics of the extraction process often called as accelerated solvent extraction (ASE). Kauffmann and christen reviewed these accelerated solvent extraction techniques involving microwave heating and pressurized solvent extraction. In the microwave assisted extraction (MAE) the acceleration is achieved by faster disruption of weak hydrogen bonds the dipole rotation of the molecules caused by electromagnetic radiation. In pressurized solvent extrac‐ tion (PSE) the higher temperature and pressure accelerates the extraction process as the high temperature accelerates the extraction kinetics, high pressure keeps the solvent in liquid state and forces the solvent to pass through the pores of the matrix thoroughly [68]. When coupled with the cell disruption techniques described earlier the solvent extraction will be very faster and utilizes small amounts of solvents [81]

### *10.2.1. Hexane solvent method*

Supercritical CO2 extraction and direct conversion of algal biomass to biodiesel. Extraction methods such as ultrasound and microwave assisted are also employed for oil extraction from vegetable sources. The results indicate that compared with conventional methods these new methods can greatly improve oil extraction with higher efficiency. Extraction times are reduced and yields increased by 50–500% with low or moderate costs and minimal added toxicity. In the case of marine microalgae Crypthecodinium cohnii, ultrasound worked best as the disruption of the tough algal cell wall considerably improved the extraction yield from 4.8%

The first and simple extraction method is mechanical cell disruption of algal cells to extract oil without contamination of other chemicals. Mechanical pressing or French pressing of dry algal lumps involves pressurizing the algal biomass to high-pressure, where the cell walls are ruptured to releases the oil similar to oil extraction from seeds or nuts through mechanical pressing. Homogenization through bead or ball milling is a process to disintegrate the alga cells which takes place in a jacketed chamber or vessel. The shear force created by the high velocity beads which moves radially causes the disruption of cells [121]. Cell disruption in this method depends on factors like residence time, cell concentration, chamber volume, bead volume and umber of rotations. All these mechanical cell disruption are usually combined with the solvent extraction to improve the extraction efficiency. Along with the mechanical methods new pretreatment techniques ultra-sonication, microwave also getting attention. In ultra-sonication & microwave pretreatments, the biomass will be treated in a sonication [96]/

Solvent extraction is a common practice used to extract oils from the algal biomass and other biomasses. The solvent should be selected based on efficiency, selectivity towards the different classes of lipids and ability of solvent to prevent any possible degradation of lipids. In order to achieve maximum extraction, the linkages between the lipids and other organelles of the algae cells which are connected with van der Waals interactions, hydrogen bonding and covalent bonding should be broken18. The most common solvents used for extraction are nhexane, chloroform, petroleum ether, methanol, ethanol, isopropanol, dichloromethane and mixture of any of these solvents depending upon method and desired class selection of lipids. The conventional solvent extraction methods are Bligh and dyer, folch [90], Soxhlet extraction. The steps involved in the solvent extraction at micro level were explained by halim et al. When the algal cells interacted with the organic solvents, these solvents penetrate through the cell wall and interact with the selective class of lipids depending upon its dielectric constant to form a solvent- lipid complex. This complex diffuses in to the bulk solvent due to the concen‐ tration gradient continues until this process reaches equilibrium [51]. The solvent extraction methods shows a lot of variability depending upon the organic solvent (dielectric constant) used and biological matrix being used in selection of different class of lipids [20, 51]. The cell

(in Soxhlet) to 25.9%.

126 Biofuels - Status and Perspective

**10.2. Solvent extraction**

**10.1. Mechanical extraction/ cell disruption methods**

microwave [105] chamber prior to solvent extraction.

Algae oil extraction can be done thorough various techniques, hexane extraction is one of them. Hexane, Benzene and ether chemical used as a solvent extraction, in which Benzene and ether is widely used in food industry because of low cost factor. Isolation and oil press/express method are method in which hexane solvent extraction can be used for oil extraction. After oil has been extracted through expeller, remaining pulp can be mixed with cyclohexane chemical to further extraction of the remaining oil content in pulp. When the oil dissolved in the cyclohexane chemical, again pulp s filtered out from the solution and using distillation process oil and cyclohexane can be separated. Using this process more than 95 % of the total oil by the algae can be obtained.

## **10.3. Supercritical CO2 extraction**

Commercial applications of supercritical CO2 extraction dates back to early 1990's. Supercrit‐ ical extraction is being used in food and pharmaceutical industries due to its range of selectivity of compounds, non-toxic nature, and easy separation [87]. The principle behind this technol‐ ogy is, when fluids crosses both critical temperature and critical pressure they attains prop‐ erties of both gases and liquids. This state of the fluid is called supercritical state of fluid, and it exhibits mass transfer properties of gas and solvent properties of liquid with greater diffusion coefficients [100]. Because of the lower critical point at 31.1o C and 72.9 atm carbon dioxide became preferred fluid for extraction applications. The solvent properties of supercritical fluid can be modified by altering extraction pressure and the extraction temperature. As an example target compounds like pigments, proteins and neutral lipids can be extracted at their respective extraction temperature and pressure, where they interact with the solvents [51, 80]. Due to its high selectivity, lower toxicity, chemical inertness and high purity of the extracted compounds, supercritical CO2 extraction is being used in many pharmaceutical, nutraceutical and food industries worldwide [80].
