Pre-requisites: Ester: Esters are chemical compounds derived from an acid (organic or inorganic) in which at least one -OH (hydroxyl) group is replaced by an -O-alkyl (alkoxy) group.

Carboxylic group/acids: The carboxyl group is an organic functional group consisting of a carbon atom double bonded to an oxygen atom and single bonded to a hydroxyl group.

Hydrophilic and Hydrophobic molecules: Polar and Non-Polar molecules are also referred as Hydrophilic and hydrophobic molecule. Fatty Acid: Fatty acids are carboxylic acids with long carbon chains. OR its a hydrophilic carboxylate attached to a hydrophobic carbon chain.

Glycerol/Glycerin: Glycerol has three hydroxyl groups attached to 3 carbon atoms.

Glyceride: When an H from OH of glycerin is replaced by an ester then glyceride is formed.

Fatty Acid Methyl Ester / Bio diesel: Fatty acid methyl esters (FAME) are a type of fatty acid ester that are derived by

transesterification of fats with methanol.

Lipids: Biological molecules soluble in organic solvents. In algae lipids may exist in form of

Fatty acids

Sterols (Alcoholic steroids (Cyclic organic compounds))

Ketones

Pigments (color causing compounds)

Free fatty acids (volatile fatty acids with short carbon chain).

Oxidation Stability in Bio-Diesel:

Fatty wastes in general are more susceptible to oxidation because they vary in level of unsaturation, meaning there are

more carbon-carbon double bonds and fewer hydrogen molecules on the fatty acid chains. When biodiesel made from

unsaturated oil is exposed to oxygen, the oxygen attaches itself to the bis-allylic site directly adjacent to the two double

bonds, which initiates an autoxidation chain reaction sequence. Oxidation stability is not related to the number of

double bonds available but rather the number of bis-allylic sites. The initiation step is the formation of a free radical

that can react directly with oxygen. This leads to the formation of a peroxide or hydroperoxide molecule. The most

reactive site for initial formation is the bis-allylic position. The radials formed at the bis-allylic sites immediately

isomerize to form a more stable conjugated structure, which reacts directly with oxygen to form peroxide. The

existence of these molecules is an early indication of oxidation taking place, and it is measured in terms of peroxide

value. Later, aldehydes and ketones are formed. Finally, during the polymerization process, resins are produced making

the fuel unusable.

Source of FAME: FAME can be obtained by alkaline catalyzed transesterification of triglycerides found in;

Crops with oleaginous seeds.

Algae. There are other sources too, but those cant be scaled up to an industrial scale level.

Comparison of Crops vs Algae for potential to scale up: Crops require large area with large supply of water, which is practically not possible because that will have an

adverse effect on Food crop industry since it is becoming difficult day by day to feed large population.

However Algae doesnt require arable land, it can be cultivated even in saline water and thus doesnt have effects on food crop industry.

Moreover it has a greater photosynthesis rate than fuel crops thus absorb significant amount of CO2 from environment and can accumulate up to 77% of lipids of their dry mass.

Most importantly algae have a tendency to produce 10 times more diesel per unit area than crops do have.

Micro Algal Lipid Composition: Lipid molecules contain fatty acids are designated basically on basis of:

No. of Carbon atoms in HC chain

Number of Double bonds present However they can also be categorized on basis of Polarity:

Neutral Lipids consist of Glycerides mainly; these can be MonoGlycerides, Diglycerides, TriGlycerides. Neutral Lipids in Microalgae acts as energy storage unit.

Glycolipids or phospholipids are polar lipid molecules that have a hydrophilic head attached to a hydrophobic tail. Polar form of lipids forms bilayer cell membranes.

Factors effecting Lipid content in Microalgae: Lipids in microalgae can range from 5% to 77% of their total dry mass. It varies from specie to specie but there are other factors too which affect greatly amount of lipids in algae. These include Life-cycle, cultivation conditions (indoor or outdoor cultivation), medium composition (medium in which algae is cultivated, proper nutrition effects alga growth), temperature control, illumination, ratio of light/dark cycle and aeration rate. Since indoor cultivation is energy inefficient because of all controls, therefore outdoor cultivation of algae is suitable for large scale production of biodiesel. Most of above mentioned factors cant be controlled for outdoor cultivation but algae life-cycles effect on lipid amount can be controlled.

Above graph shows life-cycle of a culture of microalgae, experiments showed that percentage of lipids from algae harvested during exponential phase are greater than percentage of lipids extracted harvested during stationary phase. And lipid quantity increases greatly in algae harvested while shifting from exponential phase to stationary phase. Therefore setting appropriate life-cycle ultimately increases biodiesel production.

Quality and Quantity of lipids in microalgae for production of Bio-diesel: Not all lipids have similar tendency to be converted into bio-diesel; glycerides ( i.e MG, DG, TG) are desired lipids from algae because other forms of lipids have low efficiency to produce biodiesel through transesterification. And secondly glycerides have a lower degree of unsaturation, which gives FAME more oxidation stability.

Above figure shows amount of different lipids extracted from 3 particular species of algae with logarithmic and stationary phase harvesting. Quantity of lipids decreases for algae harvested during stationary phase but amount of useful lipids increases effectively (20-41% of total lipids). Fatty acids in microalgae can have carbon chain from 12-22 in length which both saturated and unsaturated fatty acid, however number of double bond is limited to 6. Since saturated fatty acids is 27.6 wt% of total fatty acid whereas unsaturated fatty acids comprise of 71.6% of total. Figure below shows proportion of fatty acid w.r.t carbon chain and double bonds.

Therefore bio-diesel from unsaturated fatty acids is cost effective and has a low cloud (temperature at which wax in diesel becomes cloudy) and pour point (temperature at which liquid stops flowing) because of bends due to double bonds which makes it difficult to pack. These advantages are followed by disadvantage of Low-oxidation stability, poor volatility and gum formation tendency after oxidation thus have a lower shelf life.

Overview of Downstream process for production of Bio-diesel: Cultivation of Microalgae: A microalga during cultivation needs aeration, medium (nutrients such as nitrogen, phosphorous, iron). There are two modes of cultivation;

Indoor cultivation system (Photo bioreactors): Control Illumination, temperature, aeration, Protect against local species invasion, but have an high operating cost.

Outdoor Cultivation System (Raceway ponds, Photo bioreactors): Uncontrolled environment and unpredictable growth rates, but cost efficient.

Dewatering / Drying: Harvested microalgae exist as a dilute solution with (0.1 2 gram dried algae / liter culture) thus needed to be concentrated to reduced downstream processes length. Therefore drying can give 10- 450 gram of dried algae /liter of water and any suspension with concentration greater than 200 gram / Liter forms sludge normally referred as paste or pellet. Dewatering / drying can be done by following methods:

Centrifugation: process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge

Filtration: Filtration is mechanical method of separating two mediums by interposing a material during flow.

Flocculation: It is works through sedimentation but utilizes flocculants which adhere to microalgae to form an aggregate which settles down. Flocculants usually are cat-ionic, an-ionic or non-ionic polyelectrolytes (polymer that dissolves).

Pretreatment before lipid extraction: Pretreatment of algae before extraction ensures complete extraction of lipids, however in industrial applications it can be avoided due to an additional cost. Pretreatment can be done in following ways;

Concentrate is exposed to minor cell disruption by high pressure homogenization or ultrasonic cell disruption method.

For small scale concentrate is completely dried and then milled into fine powder for efficient extraction and proper mixing of chemicals with solute.

Lipid Extraction from microalgae: There are number of methods for extraction of lipids from microalgae, however most of them dont have potential to scale up to industrial level because of their high energy consumption. Lipid extraction methods based on cell disruption principle are;

For laboratory scale extraction, chemical method commonly known as Solvent extraction process and mechanical method Supercritical fluids extraction is commonly used.

Isolation of Lipids: Once Lipids are extracted from microalgae, they may contain contaminants such as protein, carbohydrate, water, process fluids and most importantly extraction techniques doesnt attack neutral lipids, it also extracts polar lipids along with neutral one. So fractionation is done before conversion of lipids into bio-diesel for removal of contaminants. Following are three commonly utilized methods for fractionation;

Chromatography: separate fluids by using stationary and mobile fluids

Acid precipitation: forms acid and precipitate it, something like acid rain.

Urea crystallization Transesterification: Chemically transesterification is the conversion of a carboxylic acid ester into a different carboxylic acid ester. It reacts triglycerides as neutral lipids with alcohol (mostly methanol, ethanol, butanol, isopropanol) in presence of an acid(H2SO4) or base (KOH , NaOH) as catalyst to form Fatty acid methyl ester FAME which is commonly referred as bio-diesel and glycerin as by-product. Since using base as catalyst have faster reaction speed up to 4000 times therefore is most commonly used. It is common practice to use alcohol greater than stoichiometric requirement to ensure quantitative transesterification since available lipids are quite few. Following is reaction equation of transesterification process, where FAME is called untransesterified lipid

Presence of water during transesterification process limits amount of FAME recovered because water under alkaline condition reacts with alcylglycol to form free fatty acid as shown below. It is important because an additional amount of KOH is required to process.

Post Transesterification process: Bio-diesel after transesterification process contains glycerol, alkali catalyst and alcohol. Therefore gravity separation is used to separate mixture since diesel is less dense than rest so it comes on top while other settles down. Gravity separation is followed by water-washing, to ensure proper elimination of methanol and catalyst. In order to completely benefit from bio-diesel both economically and in an environment friendly way we should design above mention downstream processes in such a way that carbon-dioxide produced during completely processing of a batch of algae should be equal to amount of carbon-dioxide absorbed by that batch of algae for cultivation.

Lipid Extraction Technique: Out of all processes involved in production of bio-diesel from algae, lipid extraction from algae cell is most energy consuming process. In order to make algae based biodiesel sustainable, scientists are working on energy efficient extraction methods. Extraction techniques are focused on following prospects;

Limiting extraction to alcylglycerol to reduce downstream separation processes.

Extraction technique should be energy efficient even at large scales.

Chemicals used during extraction should be unreactive with lipids.

Dewater algae consumes a lot of energy, therefore technique should work fine without drying algae.

Organic Solvent Extraction: Organic solvent extraction is most commonly laboratory used extraction techniques in order to study quality and quantity of lipids in algae. It works on principle that Like dissolve like. Specifically extraction can be explained by 5 steps;

Organic solvent (hexane / chloroform) penetrates cell membrane and enters cytoplasm (fluid inside cell).

Solvent interact with neutral lipids to (TG) using similar vander wall forces.

Interaction results in formation of organic solvent lipid complex.

Concentration difference of solvent causes it to diffuse out of cell wall.

Solvent lipid complex moves from static solvent film (layer formed due to interaction between organic solvent and cell wall and is undisturbed by flow agitation) to bulk solvent stream.

Not all neutral lipids in microalgae exist in free form, but major neutral lipids forms a complex with polar lipids which is strongly linked to protein in cell membrane via hydrogen bonding. Therefore limiting process to free neutral lipids decreases yield significantly which is undesired. Use of polar solvent (methanol or iso-propanol) in proportion with non-polar solvent (hexane / chloroform) can disrupt these lipid protein associations. Working procedure of this mixture for non-polar is same, however polar organic solvent surrounds polar lipids in complex and forms hydrogen bond with polar lipids in complex. These bonds are strong enough to displace lipid-protein association. Disadvantage of above mentioned addition is that it also pulls out polar lipids which cant be used for production of bio-diesel. However using polar-nonpolar system can increase up to 200% lipid yield. Below figure shows process diagram with 5 steps, where steps shown below are for neutral and polar solvent mixture.

After extraction is completed cell debris (residue) is filtered out and then biphasic separation (mostly distillation) is carried out which separates crude lipids and organic solvent along with protein, carbohydrates.

Selection Criteria of organic solvent: Table 3 (page 11) summarizes setup used by different researches for extraction of lipids corresponding to yields associated with setup. But maximum yield is not a single consideration for selection. Other factors to be considered include;

Volatility: Solvent must be volatile for low energy distillation for separation of crude lipids.

Non-toxicity: Solvent must be less toxic in nature, because use of toxic chemical to fulfill and environment friendly purpose isnt promising.

Chloroform / methanol (1/2 v/v) is most frequently used with water inside cell acting as 3rd component to ensure complete extraction but its use is undesirable because of chloroforms toxic nature. When chloroform is used for extraction then after removal of cell debris more chloroform / methanol is added to induce biphasic partitioning. Lipids with dense nature settle down with some solvent while aqueous phase comes on top with most solvent, carbohydrate, protein and water. Hexane / iso-propanol (3/2 v/v) is a low toxic alternate but is limited to fewer specie of algae; it has a tendency to attack most neutral lipids reducing downstream process. After cell debris filtration lipids with solvents comes on top while aqueous phase settles down. Drawbacks of using solvent extraction process include;

Limitation of yield because of batch production

Energy inefficiency

Higher solvent consumption

Advanced / Modified organic solvent extraction technique: Soxhlet apparatus: Soxhlet lipid extraction apparatus uses methylene chloride : methanol (2:1 v/v) solvents. It have showed higher lipid yields and process has a tendency to be used for can continuous process.

Main advantage of using soxhlet apparatus is that it provides fresh organic solvent for extraction in each cycle, it does it by distillation and thus reduces solvent consumption. But main drawback of using soxhlet process is that it degrade lipids because it operates continuously at an elevated temperature (boiling temperature of solvent), which is not desirable. Microwave assisted Lipid extraction: In microwave assisted lipid extraction process, electromagnetic radiations are focused on algae causing superheating of fluids inside algae cell thus rising pressure and ultimately rupturing cell wall making cell constituents to mix up in surrounding solvent and making extraction faster and easier (Focused heating through radiation is more rapid than heat transfer through conduction or convection). Accelerated / Sub-critical organic solvent extraction: Solvent extraction when performed at elevated sub-critical pressures allows liquid heating at higher temperatures (higher pressure leads to higher boiling temperatures) thus accelerating process and causing a rapid disintegration of cellular structure. Despite of all modification in solvent extraction process, it cant be scaled up because of its energy inefficiency.

Supercritical Fluid Extraction (SFE): Supercritical state is state in which any substance exists as both liquid and gas. It means that it shows intermediate properties of liquid and gas. (Table 4) shows different properties of supercritical fluid in contrast to liquid and gaseous state.

Solvent power of any Supercritical fluid is a function of density which can be controlled by temperature and pressure control. Supercritical Carbon dioxide is most commonly used extractor, its moderate temperature (31.1 C) and pressure (21.9 atm) for supercritical state and its non-toxicity makes it attractive for industrial scale plant. In addition it gives solvent free crude lipids at end of process which reduces downstream process makes it an appropriate option.

Above figure shows a typical laboratory scale supercritical fluid extraction setup. A mixture of algae and packing material (diatoms: specific type of unicellular algae and Diatomaceous earth: powdered fossilized diatoms to enhance filtration) is placed inside extraction vessel. Extraction vessel is enclosed between Frits which avoids flow of diatoms and cell debris into cycle, it also reduces water flow into cycle, if concentrate algae were used. However CO2 cant extract neutral lipids associated with polar lipids in form of complex, therefore addition of polar modifier increases affinity towards polar lipids. (Table 5 summarizes results of different researchers along with their experimental setup) As compressed supercritical enters extraction vessel it enters cell wall because of it liquid nature and interacts with lipids to form SCCO2 lipid complex. This moves out of cell through diffusion via concentration difference, it moves out of static boundary layer of CO2 around cell wall and enters into bulk CO2 stream. As these complexes enters micro metering valve CO2 is depressurized and turns into gaseous state leaving behind precipitates of crude lipids. Speciality of this process is its ability to be made continuous, which will results in higher processing rate (a key to industrial setups). Comparison between solvent and super-critical fluid extraction: Table 6 briefly compares different aspects of lipid extraction using solvent and supercritical fluid extraction. Effect of cellular pre-treatment on lipid extraction: Pre-treatments like thermal drying and cell disruption are energy intensive and should only be carried out if enhance the efficiency of lipid yield. However efficiency of micro algal lipid extraction is known to increase with increase in degree of cell disruption particularly. Because solvent dont anymore need to cross cell membrane to interact with lipids thus increasing speed of reaction and reducing process time. Cell disruption methods: Following are some common cell disruption methods utilized as pre-treatment:

Bead milling: Bead mill ruptures cell wall by physically grinding the microalga cells against a solid surface of glass beads in a violent agitation. And is most suitable because of its low cost.

High Pressure homogenization: High-pressure homogenization pumps microalgal concentrate through narrow orifice of a valve under high pressure. It then releases the concentrate into a low-pressure chamber. Cellular disintegration is thus achieved through high-pressure impingement of accelerated cellular jet on the stationary valve surface as well as through pressure-drop induced shear stress that the microalgal concentrate experiences as it passes from the valve to the chamber

Ultrasonication: It works on basis of cavitation; a probe vibrating at ultrasonic frequencies is introduced into wet-biomass. Alternating high and low density region develops due to vibration and some liquid/water evaporates due to flashing, forming micro bubbles. As pressure rises due to surrounding liquid bubble turns into liquid again thus occupying a small volume than vapor which cause surrounding water molecules rush a high speeds to fill space causing algae cell membrane to shear.

Simultaneous extraction and transesterification of microalgal lipids (In-situ transesterification):

As name implies both extraction and transesterification is done in a single step. The method involves simultaneous addition of acid catalayst and pure methanol to microalgal biomass. The methanol extracts the lipids from biomass and then catalyzed by acid concurrently transesterifies the extracted lipids to produce fatty acid methyl esters followed by fractioning and post transesterification processes. Microalgal Bio-refinery: The cost of producing microalgal biodiesel can theoretically be offset by revenues generated from other co-products of the microalgal biomass. Microalgae contain significant quantities of proteins and carbohydrates as well as smaller amounts of high-value functional ingredients (astaxanthin, canthaxanthin, carotenes, chlorophylls, 3 free fatty acids, and -linolenic acid). Each of these cell components can be appropriately utilized to co-generate a useable product in a biorefinery. Recent studies have concluded that industrial-scale production of microalgal biodiesel can only be made economically sustainable if a biorefinery based production strategy is pursued. In a biorefinery, the crude lipids are to be fractionated into high-value functional ingredients and lipids for biodiesel (acylglycerols). Functional ingredients have been linked with the promotion of anti-oxidant, anti-inflammatory, as well as anticarcinogenic activities in the human bodies and are typically used as food supplements. If the microalgal species contains a high level of proteins, the residual biomass from biodiesel production processes can be used as livestock feeds. If the species has high carbohydrate contents, the residual biomass can be fermented to produce bioethanol. As such, microalgal biorefinery will simultaneously produce biodiesel, high-value products, livestock feeds, and bioethanol. Unfortunately, the combination of technologies needed to implement a microalgal biorefinery is still in the early stages of development. Milder cell disruption/lipid extraction process needs to be explored to ensure that the functionalities of different cell components are retained.