The sources of petroleum derived fuel resources are on the verge of extension due to exploitation of the non-renewable source, as well as rapid industrialization and boom of the population with increasing demand is troubling the government and many organizations. There have been numerous numbers of sustainable choices but still there is no ultimate use of such choices. Biofuels can be the best and a sustainable choice for the future fuel. The production of the biofuel is done by biomass which gives a promising alternative to the fossil fuel derived energy as well as reduces the effect of greenhouse effect which has been the major part in global warming which poses a threat to the biosphere.
Biodiesel production of microalgae is the most potential source for renewable energy with both the economic and environmental benefits. Microalgae have been major sources in different fields which include food, fish feeds, pharmaceutical products etc. Microalgae is identified and isolated for the production of the biodiesel majorly because of the lipid ratio in it.
Microalgal lipid contains two groups according to their structure: non- polar NLs and polar lipids. Since no consensus have been recorded which can provide us the information about the highest lipid productivity of the algae, different types of species with different environment can have different production of the lipid. With the growing interest in the algal based fuel many algae biorefineries are also there. The project highlights the practical application of the microalgae as well as robust methods for the selection and isolation of the microalgae and the traits which maybe most relevant for the commercial biodiesel production.
Keywords: biodiesel, biofuel, microalgae, lipids, triacyglycerides
Microalgae/ microphytes are the microscopic algae which are majorly found in the water column, sediment and majorly can be found in freshwater and water system. They are capable enough to survive individually or in a cluster. Microalgae are devoid of roots, stem or leaves and it is known for the production of the half atmospheric oxygen due photoautotrophic. Microalgae contains both eukaryotic as well as prokaryotic species, prokaryotic species include cynobacteria and eukaryotic species (nine phyla) includes:
The biodiversity of the microalgae is an enormous one and is a whole dimension but almost 2/3rd of 50,000 species has been identified and preserved in the algal research institutes. The products of microalgae include caretonids, lipids, polymers, toxin, sterols etc. Producing 100 tonnes of algal biomass fixes roughly 183 tonnes of carbon dioxide in the atmosphere.  The advantages of using microalgae for the production of the biofuel are: Use of simple nutrition and no complex nutrition or cultural broth is required It is easy to cultivate in the lab with the suitable conditions The span time for the growth is less so, it is easier to grow generations in short period of time which also saves time. The size of the microalgae can vary from micrometers to hundred micrometers. The chemical composition of microalgae can vary with species and the conditions provided for the growth. Some microalgae have the ability to change their chemical composition in response to the environment variability. They grow in the aqueous suspension so it is easy for them to access CO2, other nutrients required.
The desirable products can be obtained from the microalgae by shifting some changes in the nutrients, illumination, salt, pH etc. Some of the examples of microalgae are: Chlamydomonas sp. ,Dinoflagellate, Cladophora sp., Closterium sp. etc
The algal lipids are most found the in the polar bodies/ lipid bodies. Lipids present in the microalgae are basically neutral lipids (NLs), such lipids are hydrophobic molecules and lack charge group in the structure. Triglycerides (oen molecule of glycerol and 3 FAs) are the major source for the lipid production and with the suitable techniques esterificaton of the lipid can take place. TAG in storage lipids mostly has saturated fatty acids which has high energy content while the polar bodies contain lipids which are mostly long chain polyunsaturated fatty acids.
Microalgal lipids has two types of lipid structurally, non-polar lipids (acrylglycerols,sterols, free fatty acid,steryl esters) and polar lipids (phosphoglycerids, glycosylglycerides and sphingolipids). The major function of the lipids is to keep the metabolism of the algae active as well as keep a check on the growth time. Lipids majorly also helps in the cell signaling pathways which keeps the algae its sensory adaptability to the environment, for such functions ionsitol lipids, sphinolipids but also the oxidative products of polysaturated fatty acids are important. The polar lipids such as phosphglycerides,glycosylglycerids etc. provides an integral support to the membrane of the algae.
Lipids almost take upto 20%-50% of the dry biomass in some species (Chlorella, Crypthecpdinium, Neochloris etc.) or it can even shoot upto 80% of the dry biomass put ultimately different species of the algae can have varied amount of lipids present in it. The major part of the metabolism of the algae is that in the stress condition or high salt concentration it can overproduce the lipid production which can be dominated for the production of the biofuel. Biofuel is mostly produced out of TAG with a suitable with methanol using base-catalyzed transesterification.  Biofuel can be classified into three categories based on their physical characteristics: solid(i.e biochar), liquid(i.e bioethanol, biodiesel) and gaseous( i.e biogas, biosyngas and biphydrogen). Based on the types of feedstock which is being used it can be divided into three generation: first generation which includes food crops such as corn,soyabeen, sunflower etc ,second generation includes the inedible feedstocks such as organic waste, switch grass etc. and third generation includes the fuel production from the microalgae. The review article summaries the techniques and methodologies used for the extraction and the quantification of the lipids and the pretreatment and the post treatment for the lipid extraction and quantification of the fatty acids for the use of the biofuel production.
Production of fatty acids and triglycerides go hand in hand. The production of fatty acids leads to production triacyglycerides; the production of fatty acids takes place in chloroplast. The process starts with the Calvin cycle which utilizes the carbon dioxide for the production of acetyl-CoA. With the help of acetyl-CoA carboxylase (AACase) and some bicarbonate there is production of malonyl-CoA. Species like Chlorella desiccate, chlamydomonas has shown the increase level of production of AACase under stress conditions. Malonyl-CoA is converted into Malonyl-ACP with the help of MACT[Malonyl-CoA: Acyl protein (ACP)] Aryl ACP works as the carbon source for substrate elongation of fat, which is the next step. The fatty elongation is accompanied by the Keto-acycl ACP synthase (KAS) which includes KASIII,KASI and KASII for the conduction, dehydration, reduction and for the second round of reduction to happen. The above step is also cataylsed by the FAS complex enzymes: KAR (beta-ketoacyl-ACR reductase),HAD (hydroxyacycl-ACR dehydrase),EAR(Enoyl-ACP reductase) The de-nova synthesis of FA (16-18 C) uses elogases and desaturses for the addition of the carbon or double bond,FAs are then transported to the cytoplasm for the synthesis of TAG. TAG is considered dominant lipid storage in microalgae due to its energy rich acryl chain.
The further process involves Kennedy pathway, glycerol-3-phosphate (G 3-P) acylation takes place which is converted into lysophosphatidic acid with the help of GPAT (glycerol-3-phosphate acyltransferase). The LPA acylation take place to for phosphatidic acid with the help of LPAAT (lysophosphatidic acid acyltransferase) which is the second step of Kennedy pathway. LPA latter forms diacylglycerol (DAG) with the help of PAP (Phosphatidic acid phosphohydrolase) which acts as the precursor for TAG. For the final step DGAT(acyl-CoA:diacylglycerol acyltransferase) is used for the production of triglycerides. Whole production of TAG is acryl-co dependent pathway on the other hand independent pathway is also available by two types of enzyme: the phospholipid:diacylglycerol acyltransferases (PDAT), which catalyze the formation of TAG using DAG and phosphatidylcholine (PC); and the DAG:DAG transacylases (DGTA) which utilize two molecules of DAG to form TAG and MAG. 
Microalgae grow mostly in its natural habitats which include water, rocks and soil but predominantly grow in marine ecosystems which includes freshwater, brackish etc. The samples are not only collected from marine ecosystem but can be collected from extreme environment such as volcanic waters or salt waters. The local microalgae are the best samples that should be collected because it provides a competitive advantage under the local geographical, climate and ecological conditions and as we know the microalgae which are exposed to the adverse condition or fluctuating conditions provide better lipid accumulating microalgae.
Samples from lakes, ponds, wetlands can be collected by the from the surface or by the surface scum or sometimes you have to collect the water from the bottom because sometimes algae sink itself to the bottom, be sure not be in contact with the water and use latex gloves for safety. Nutrient rich waters have more number of planktons suspended/floating algal cells per ml of water while lakes or wetlands have less nutrient present in it so, the algal cells per ml of water is less. Visualization and identification of algae The visualization of algae is easy to perform take a glass slide and place 2-3 drops of sample on it or two –three pinches of filamentous algae on the glass slide and cover it with a covsimple and place it under a compound microscope. Take down the morphological feature of the algae which can help in identifying the different taxa of the algae.
Isolation is the second step to the protocol and one of the major steps for the selection of the particular algae strain for the synthesis of microalgae. It is an important to obtain the pure culture of microalgae. The most popularly used techniques are : Dilution series Plate cultivation to isolate single colonies Single cell isolation by micromanipulator Cytomeric cell sorting (flow cytometry) Dilution series: The dilution series is a very common used technique. The main principle for this technique is that it reduces the density of the cell and helps in using it in usable concentration. The dilution is done to 10-1 to 10-10 and last dilution is used for the growth of the microalgae with the help of the suitable culture media/broth. The incubation is done for 15-20 days. The most used cultural broth media are Beneck’s broth media and Chu’s media. Beneck’s broth media:
The two most important nutrient sources for microalgae are nitrogen and phosphate and silicon for diatoms. The medium is mostly at the 25 degree Celsius and the growth is accompanied by the cool white LED with continuous period.
Another algal culture broth can be used:
Plating cultivation: The main reason for the plating is for the isolation of the discrete colonies with the suitable medium which is used for the growth. Agar plating recipe:
Single cell isolation: The principle is culturally the large intact living cells with the help of the nutrient media for their growth and development. Single cell isolation by a micropipette (e.g. a glass capillary) is a very effective method.  Cytometric cell sorting: This is one of the popularly automated single cell isolation. It is used in sorting different microalgal strains from the water samples on the basis of chlorophyll autofluorescence (CAF) and green autofluorescence (GAF), it helps in separating algal species such as diatoms, dinoflagellates and prokaryotic phytoplankton.
Microalgal cell disruption methods: Pretreatment Microalgal cell disruption is important and important pre-treatment is beneficial for water biomass because such treatment helps in the disruption of the microalgal cell walls and it allows the lipids to be released into the extracting mixture. Bead beating: Bead beating/ball or bead mill uses high impact speed spinning of fine beads on the biomass slurry. It’s not a time consuming job, only takes few minutes for the disruption. There are two types of bead mill: shaking vessel and agitated vessel. Shaking vessel has multiple plates of containers on a vibrating platform and hence the vibration helps in the disruption of the cells. Agitated vessel has a fixed agitator attached to its machinery with a fixed vessel filled with beads which helps in the cell disruption, cooling jacket is equipped for the better efficiency and helps in reducing heat production which can disturb the disruption process.
Microwave is an electromagnetic wave, the frequency lies between 300MHz and 300GHz. The microalgal cells respond to a specific frequency (2450GHz), the cell rapidly oscillates due to the electric field, the heat generation is there due to the frictional forces from the inter- and intra-movement. The intracellular heating causes the water to evaporate in vapor which causes the disruption of the cell and it opens the cell membrane. The major advantages of this procedure are: short reaction time, low-operating costs, efficient extraction but the vast cooling system makes it difficult for large-scale application
Ultrasonication uses ultrasound waves for the disruption of the cells. The wave produces micro-bubbles and production of micro-bubbles helps in the cavitations which produces pressure against the cells which eventually break it up. This method helps in the high productivity in its short duration. But the only disadvantage which is there is that cell disruption takes place near the ultrasonic probes which restrict its large scale production.
The chemical treatments with acids (HCL, H2SO4), alkali (NaOH) and surfactants help in the chemical degradation of the cell wall and helps in the lysis of the cell. The chemical methods doesn’t require large amount of heat and has high cell disruption efficiency. But such strong acids and bases can lead to the corrosion of the reactor and attacking microalgal lipids.
Specific enzymes are able degrade structural cell components which help in release of desired intercellular compounds. The yield of lipids can be improved when enzymatic hydrolysis is combined with the acid/alkaline pretreatment. Microalgal Lipid Extraction and Quantification Approach Extraction of lipids is basically the separation of valuable neutral lipids and fatty acids from the cellular matrix and water. Quantification of lipids includes different methods such as conventional gravimetric method, Nile red lipid visualization, SPV and TLC etc. Gravimetric method The gravimetric method uses solvent and lipid quantification for lipid extraction. Lipid quantification is achieved by recording the weight of the extracted lipids after the evaporation of the organic solvent. The solvent which is used are organic solvent, CO2-based solvents, ionic liquids (ILs) and switchable solvents.
Organic solvent extraction is widely used because of the main principle of “like dissolving like” A solvent should also be free of toxicity, easy to remove, more selective towards target products. Steps which are used for the lipid extraction mechanism are:
Chloroform :Isopropanol (1:1, v/v) and hexane Add solvent to the frozen pellets, centrifuge and transfer the supernatant, re-extract with the help of hexane and centrifuge to collect the supernatant Dry the combined supernatant in a speed vacuum and record the weight. Percentage of total fresh weight (% w/w) Ethyl ester Ground dry samples to powder and use the Soxhlet extractor Distill the solvent, dry the residue and record the weight Percentage of dry cell weight(% w/w)
Methanol:choloroform :1% NaCl (2:2:1,v/v/v) Extract the biomass with the solvent mixture Evaporate the chloroform layer: Cool the tube and record the weight Percentage of total weight (%,w/w)
Chloroform: methanol (2:1, v/v) Extract lipid from dry biomass with the solvent mixture. Evaporate the solvent and weight Weight difference between the blank flask and flask containing extracted oil
Advantages: Little environmental impact, not highly selective towards the desired neutral lipids and free fatty acid components.
Disadvantages: Requires large amount of biomass CO2-Based Solvent Extraction.