Biodiesel is a renewable biodegradable, atoxic and environment fuel. That is defined as a fuel comprised of mono-alkyl esters of long-chain fatty acids from vegetable oils or animate being fats ( Xiong et al. , 2008 ) . Many get downing stuffs such as soybean oil, sunflower oil, cotton seed oil, rapeseed oil, thenar oil and eating house kitchen waste have been tried for readying of biodiesel by the enzymatic path. For the readying of biodiesel, tranesterification reaction was used. Besides biodiesel is first-class replacing for crude oil Diesel. The cetane figure, energy content, viscousness and stage alterations are besides similar to those of petroleum-based Diesel fuel ( Noureddini et al. , 2005 ) .

Although production of biodiesel by chemical accelerator that are besides have jobs, such as glycerin recovery and remotion of inorganic salts. At present all of biodiesel were utilizing a chemical accelerator like a strong acid or base with used methyl alcohol or ethyl alcohol produce biodiesel and glycerin. The attack is the enzymatic one, which lipase catalyzed tranesterification is carried out in non-aqueous environment ( Shah et al. , 2004 ) .

Furthermore, it is a good manner as the starting oil for production of biodiesel, which could besides biocatalyst of tranesterification reaction ( Staubmann et al. , 1999 ) .

1.2 Biodiesel Production Worldwide

Europe has been the taking part for biodiesel production. The first pilot works for the production of biodiesl from colza seed oil has been installed in Austria in 1987 in Silberberg, ensuing from several old ages of research at the Institute of Chemistry of the University of Graz. The possibility of utilizing pure biodiesel without paying mineral revenue enhancement has led to the installing of several industrial graduated table works in 1991 in Austria and Germany. But vary shortly states like Italy, France and the Czech Republic followed. The period of slow but changeless development of biodiesel activities in Europe for several old ages with the European directive for publicity of biodiesel in the old ages 2003, A market portion of biodiesel of 5.75 % for conveyance fuel in 2010.Since the development of biodiesel production about exploded in all 27 states of the European Union, taking to a entire production of biodiesel of 5.6 million dozenss in the twelvemonth 2006 and a production capacity of about 10 million dozenss.

The production has been increased by approx. 50 % from the twelvemonth 2005 until 2006 the production capacity in 2007 will transcend 10 million dozenss per twelvemonth. The about detonating development of biodiesel production in Europe will shut down by the deficiency of feedstocks and natural stuff. Almost 10 in every state world-wide biodiesel activities have been initiated the taking states are besides those with the largest production of vegetable oils. In 2006 in the US the production of biodiesel exceeded 1 million dozenss per twelvemonth, nevertheless biodiesel works have been installed with and overall production capacity of over 5 million dozenss per twelvemonth. But besides the major thenar oil production states like Malaysia and Indonesia have a series of biodiesel workss already installed with a capacity of over 1 million dozenss per twelvemonth. Similar activities can be found in China and India with their immense demand for conveyance fuel, but besides in the vegetable oil bring forthing states like Brazil and Argentina.

1.3 Feedstocks for Biodiesel Production

Today a production capacity of about 30 million dozenss of biodiesel of biodiesel exists worldwide. On the other manus there is a entire annul production of vegetable oils of approx.110 million dozenss per twelvemonth, which is chiefly used for nutrient intent. As the production of vegetable oil workss like thenar oil has led to extended treatments taking to the hunt for non-edible oil seeds.

For all vegetable oils and carnal fat can be used as feedstock for biodiesel production. Most of these oils and fats have similar chemical composing of triglycerides with difference sums of fatty acids. The major fatty acids are those with a concatenation length of 16 and 18 C, whereas the concatenation could be saturated or unsaturated. Methyl esters produce from these fatty acids. The major constituent in fossil Diesel fuel are besides directly concatenation hydrocarbons with a concatenation length of about 16 Cs. The major differences between the methyl ester from feedstocks refer to the sum of unsaturated fatty acid. The best composing feature every bit good as oxidization come from saturated fatty acid, nevertheless cold temperature and the high thaw point of these fatty acids. The major feesstock for biodiesel production today are colza seed oil ( Canola ) , soybean oil and thenar oil. The fuel belongingss of the methyl ester out of these oils are rather similar constructs for the hapless cold temperature behaviour of palm oil because of the high part of concentrated fatty acids. However, depening on the clime conditions of a state, an optimal mix of methyl ester our of these feedstocks can be used. Merely coconut oil and plam meat oil have fatty acid with 12 or 14 Cs as major constituents. Therefore the methyl esters out of these fats have lower boiling points, but could be used absolutely as ad-mixture to common biodiesel. In Asiatic states like India and China the usage of non-edible seed oils for biofuels production is really popular. In that instance there would be no competition with the nutrient production, particularly when these oil workss are grown on fringy countries non suited for nutrient production. Another interesting feedstocks for biodiesel production is oil production of algae, which can turn in unfastened lbs or in close tubings. The productiveness is estimated to be much higher per country than with the nutrient production and besides with the traditional oil seeds and furthermore there is no demand for agricultural land, it can be produced at any topographic point, where H2O and sunshine are bing. However, today an economic production of biodiesel from algae dosage non look to be vary realistic, but farther research in that country will be necessary.

1.4 Chemical Principle of biodiesel production

Fatty acid methyl ester have been known for over 150 old ages. The first description of the readying of the esters was published in 1852. However for a long clip fatty acerb methyl ester were chiefly used as derivative for analysing the fatty acerb distribution of fats and oils, so the readying chiefly was done in analytical graduated table. Since the mid 20 th century fatty acid methyl esters have become a major oleo chemical in intermediate for the production of non-ionic detergents. But merely since the late Seventiess have fatty acerb methyl ester have been tested and used as Diesel fuel replacement.

Chemically biodiesel is tantamount to fatty acerb methyl esters or ethyl ester, produced out of triacylglycerol transesterification or out of fatty acids esterification. Fatty acerb methyl ester today are the most normally used biodiesel species, whereas fatty acerb ethyl esters ( FAEE ) so far have been merely produced in research lab.

For transesterification one mole of the triglyceride reacts with three moles of intoxicant to from one mole of mole of glycerin and three moles of the several fatty acid alkyl ester. The procedure is a sequence of three reversible reactions, which the triglyceride molecule is converted measure by measure into diglyceride, monoglyceride and glycerin. The equilibrium to the right, methyl alcohol is added in an over the stoichiometric sum in most commercial biodiesel production workss. A chief advantage of methanolysis as compared to transesterification with higher intoxicant is the fact that two chief merchandises, glycerin and fatty acid methyl ester ( FAME ) , are barely mixable and therefore organize separate stages an upper ester stage and a lower glycerin stage. This procedure take glycerin from the reaction mixture enables high transition. Ester outputs can even be increased while at the same clip minimizating the extra sum of methyl alcohol. By carry oning methanolysis in two or three measure, and the glycerin stage produced is separate after each procedure phase. Finally, of the type of intoxicant used, some from of accelerator has to be present to accomplish high ester outputs under relatively mild reaction status.

The procedure of vegetable & A ; works oils into biodiesel fuel is a transesterification. And the transesterification of vegetable oils, a triglyceride have consequence with an intoxicant in acid and base, bring forthing a mixture of fatty acids alkyl esters and glycerin. This liquid is so assorted into vegetable oil. The full mixture so settles. Glycerin is left on the underside and methyl esters, or biodiesel, is left on top. The glycerol can be used to do soap ( or any one of 1600 other merchandises ) and the methyl esters is washed and filtered.

All the procedure of three back-to-back and reversible reactions, in which diglycerides and monoglycerides are formed as intermediates. The stoichiometric reaction have 1 mol of a triglyceride and 3 mol of alcohol.For an surplus of the intoxicant is used to increase the outputs of the alkyl esters and to its stage separation from the glycerin formed. However of including the type of accelerator ( alkalic or acid ) , alcohol/vegetable oil grinder ratio, temperature, H2O content ) and free fatso acid content have an influence on the class of the transesterification up to the type of accelerator was used.

the reaction with the product.t ( Marchetti et al. , 2007 ) .

Fig. 1 Biodiesel readying

Beginning: Chisti Al. ( 2007 )

The procedure is usually a sequence of three back-to-back stairss, which reversible reactions. In the first measure, from triglycerides diglyceride is obtained, from diglyceride monoglyceride is produced and in the last measure, from monoglycerides glycerol is obtained. Thisreactions esters are produced. The stoichiometry showed between intoxicant and the oil is 3:1. However, an surplus of intoxicant is more improve

Fig. 2 Tree back-to-back and reversible reactions R1 R2 R3 and Raa‚¬a„? represent alkyl groups.

Beginning: Miguel ( 2001 )

1.5 Catalysis for Tranesterification and Esterification Reaction

Alkali accelerator on transesterification

For a basic accelerator for biodiesel production, either Na hydrated oxide ( NaOH ) or potassium hydrated oxide ( KOH ) should be used with methyl alcohol or ethyl alcohol every bit good as any sort of oils, refine, petroleum. The chief advantage of this from of contact action over acid-catalysted transesterificatios is the high transition rate under mild conditions in relatively short reaction clip. So it was estimated that under the same temperature conditions and accelerator concentrations methanolysis might continue about 4000 times faster in the presence of an alkaline accelerator than in the presence of the same sum of an acidic equivalent. And consequence of wet and free fatso acid sort of alkali-catalyzed tranesterification the acylglycerol of intoxicant of must be well anhydrous because of H2O cause partial reaction to saponification, which merchandise soap. The soup consume and accelerator and cut down the catalytic efficiency every bit good as doing addition in viscousness. The formation of gels and difficultly demobilizing separation of glycerin ( Wright: 1994 ) .The most of alkalic accelerator depends on the quality of the oil, particularly on the content of free fatty acids. Under alkalic contact action free fatty acid are instantly converted in to soaps. Which can forestall the separation of glycerin and eventually can take to entire saponification of all fatty acerb stuff? So the alkalic accelerator is limited to feedstocks up to a content of approx. 3 % of fatty acid.

Table 1 Overview of homogeneous alkaline accelerator

beginning: ( Soetaert et al.,2009 )

Type of accelerator


Sodium hydrated oxide

Cheap, disposal of residuary salts necessary

Potassium hydrated oxide

Reused as fertiliser possible fast reaction rate, better offprint of glycerin

Sodium methoxide

No disintegration of accelerator necessary, disposal of salts necessary

Potassium methoxide

No disintegration of accelerator necessary, usage as fertiliser possible, better separation of glycerin, high monetary value

2. Acid accelerator on transesterification

Acid for transesterification include sulphuric, phosphorous, hydrochloric, and organic sulphuric acid. Although tranesterication by acerb contact action it much slower than alkali contact action and acerb accelerator transesterification is more suited for acylglycerols that have comparative high free fatso acid contents and more H2O ( Aksoy et al. , 1998 ) . And acerb accelerator offers the advantage of besides esterifying free fatty acids contained in the fats and oils and is hence particularly suited for the transesterification of extremely acidic fatty stuffs a farther disadvantage of acerb contact action likely prompted by the higher reaction temperature is an increased formation of unwanted secondary merchandises, such as dialkylehers or glycerin quintessences. Because of the slow reaction rates and high temperatures needed for transesterification acid accelerator are used for esterification reaction. So for vegetable oils or carnal fats with an sum of free fatty acids can over approx. 3 % two schemes are possible. The free fatty acids can either be removed by alkalic intervention. The cheapest accelerator for esterification reactions is concentrated sulfuric acid. The chief disadvantage of this accelerator is the possibility of the formation of lb besides p-toluene sulphonic acid can be used. However, the high monetary value of the compound has prevented broader application.

3. Enzymatic on tranesterification by lipase

Although chemical tranesterification utilizing alkali contact action procedure give high transition degrees of triglyceride to methyl ester in short reaction clip, the reactions have drawback, There is recovery of glycerin, remotion of acid or alkalic contact action. On the other manus, lipases are enzymes used to catalyse some reaction such as hydrolysis of glycerin, alcoholysis. Lipase contact action is important greater than that alkalic one ( Linko et al. , 1998 ) . However, the extracellular and the intracellular lipases are besides able to catalyse the transesterification of triglycerides efficaciously ( Marchett et al. , 2007 ) .And lipase-catalyzed transesterification besides a series of drawbacks. As compared to converional alkaline contact action, reaction efficiency tends to be hapless, so that biodiesel accelerator normally necessitates far longer reaction times and higher accelerator concentrations. The chief application of lipase industrial biodiesel production is their high monetary value, particularly if s are used in the signifier of extremely purified, excess cellular enzyme readyings, which can be recovered from the reaction merchandises. For the immobilisation of lipase on a bearer, so that the enzymes can be removed from the reaction mixture and can theoretically be reused for subsequent transesterification. Immobilization besides offers the advantage that in many instance the fixed lipase tend to be more active and stable than free enzymes.

Fig. 3 Flow diagrams comparing biodiesel production utilizing the ( a ) base and ( B ) lipase contact action in procedure.

Beginning: Fukada et Al. ( 2001 )

The figure show the procedure since of none reacted methyl alcohol and waste H2O intervention are unneeded. In add-on, merely is recovery of glycerin. Since the cost of lipase production is the chief to commercialisation of the lipase catalyzed procedure. Several studies have been made to develop cost effectual system ( Fukada et al. , 2001 ) .

Table 2 Comparison of the different engineerings to bring forth biodiesel

Beginning: Marchetti et Al. ( 2007 )

The tabular array showed a sum-up of the advantages and disadvantages of each technological possibility to bring forth biodiesel.

The advantages of utilizing lipases

1. The immobilized residue because it can be left in the reactor if you keep the reactive flow.

2. Use of enzymes in reactors allows usage of high concentration of them and that makes for a longer activation of the lipases.

3. For a bigger thermic stableness of the enzyme due to the native province.

4. Immobilization of lipase could protect it from the dissolver that could be used in the

reaction and that will forestall all the enzyme particles acquiring together.

5. Separation of merchandise will be easier when utilizing this accelerator.

The disadvantages of utilizing lipase

1. Initial of activity can be lost due to volume of the oil molecule.

2. Number of support enzyme is non unvarying.

3. Biocatalyst is more expensive that the natural enzyme.

( Fukada et al. , 2001 )

2. Natural stuffs for biodiesel production

2.1 Microalgae for biodiesel production

Microalgae are procaryotic or eucaryotic photosynthetic micro-organisms that can turn quickly and live in rough conditions due to their unicellular or simple multicellular construction. Microalgae are present in all bing Earth ecosystems, non merely aquatic but besides tellurian, stand foring a large assortment of species populating in a wild scope of environmental conditions. It is estimated that more than 50,000 species exist, but merely a limited figure, of around 30,000 have been studied and analyzed.

During the past decennaries extended aggregations of microalgae have been created by research workers in different states. And illustration is the fresh water microalgae aggregation from Coimbra ( Portugal ) considered one of the universes largest, holding more than 4000 strains and 1000 species. The aggregation to the big assortment of different microalgae available to be selected for in usage in a wide diverseness of applications, such as value added merchandise for pharmaceutical intents, nutrient harvests for human ingestion and energy beginning. And The university of Texas Algal civilizations that was founded in 1953. It include 2300 different strain of fresh water ( Mata et al.,2010 )

2.1.2 Advantage of utilizing microalgae for biodiesel production

Many researcher studies and articles described many advantages for utilizing microalgae for biodiesel production in comparing with other available feedstocks. From a paractical point of position, there are easy to cultivate, can turn with small or even on attending, utilizing H2O unsuitable for human ingestion and easy to obtain nutrients.and microalgae reproduce themselves utilizing photosynthesis to change over Sun energy into chemical energy, finishing and growing every few yearss. Furthermore they can turn about anyplace by sunshine and more simple foods. Algae biodiesel contains no S and performs every bit good as crude oil Diesel. ( Mata et al.,2010 )

2.1.3 Microalgae lipid content and productive

Many microalgae spices can be induced to roll up a measures of lipoids therefore to high oil output. The advantage lipids content varies between 1 and 70 % but under certain conditions some species can make 90 % of dry weight. For Chlorella seem to be good option for biodiesel production. Yet, as other species are so efficient and productive as this one, the choice of the most equal species needs to take into history other factors, such as for illustration the ability of microalgae to develop utilizing the foods available or under specific environment conditions. All these parametric quantity should be considered at the same time in the choice of the most equal species or strains for biodiesel production.

2.1.4 Algae cultivation

Microalgae are adapted to scavenge their environments for resource, to storage them, or increase their efficiency in resource use, In general for resource, to storage them, or increase their efficiency in resource use. In general for biomass growing of 40-50 % C microalgae depened on a sufficient supply of a C beginning and visible radiation to transport out photosynthesis. Microalgae may presume many types of metamorphosis ( e.g. autophytic, heterotrophic, mixotrophic, photo heterotrophic ) for illustration some beings can turn are Photoautotrophically, i.e. utilizing light as a exclusive energy beginning that is converted to chemical energy through photosynthetic reaction. And heterotrophiclly, ( i.e. using merely organic compounds as C and energy beginning )

2.1.5 Harvesting and biomass concentration

Algae reaping consists of biomass recovery from the civilization medium that may lend to 20-30 % of the entire biomass production cost. In order to take big measures of H2O and procedure big algal biomass volumes, a sutiable harvest home method may affect one or more measure and be achieved in several physical, chemical, or biological ways.

Most common reaping methods include deposit, centrifugation, filtration, and ultra-filtration. After separation from the civilization medium algal biomass ( 5-15 % dry weigh ) must be rapidly process allow it should acquire spoiled in merely a few hours in a hot clime.

2.2 Lipase immobilized

Immobilization is the most widely used method for accomplishing stableness in lipases and to do them more attractive for industrial usage ( Cowan, 1996 and Clark, 1994 ) Common immobilisation techniques include physical surface assimilation into a solid support ( Bosley and pielow, 1997 ) covalent adhering to a solid support and physical entrapment within a polymer matrix support ( Pizzarro et al. , 1997 ) .The immobilized lipase by entrapment is much more stable than physically adsorbed lipase and unlike the covalent adhering method, this method uses a comparatively simple process and at the same clip the immobilized lipase maintains its activity and stableness ( Kennedy and Melo, 1990 ) . This method which was pioneered by Avnir et Al. ( 1994 ) is based on solaa‚¬ ” gel procedure. The application of the solaa‚¬ ” gel stuff in the immobilisation of lipases is good documented ( Reetz et al. , 1997 ) .

Case survey I

Large-scale Biodiesel Production from Microalga Chlorella protothecoides through Heterotrophic Cultivation in Bioreactors ( Li et al. , 2008 )

The Content and Properties of Lipids in Heterotrophic Chlorella Cells

The lipid content decreased as the cultivation was scaled up. The lipid content reached 46.1 % , 48.7 % , and 43 % of the cell dry weight in samples from 5 L, 750 L, and 11,000L bioreactors, severally. Harmonizing to the biomass concentration, the lipid concentration was 7.15 g L,24 g/ L and 6.36 g /L in medium of 5 L, 750 L, and 11,000 L bioreactors, severally.

Fig.5 Cell growing, glucose concentration, and pH value fluctuation in 11,000 Lbioreactor ( ) Glucose, ( ) biomass concentration, ( ) pH value.

Beginning: ( Li et al. , 2008 )

The Output of Biodiesel by Transesterification

By and large, short concatenation intoxicants have strong denaturing effects on enzymes. As a consequence, their presence tins influence the stableness and activity of immobilized lipase greatly. When the molar ratio of methyl alcohol to oil was set at a stoichiometric measure of 3:1, the highest transition rate was reached

( Fig. 6A ) .

Fig. 6A Consequence of molar ratio ( methyl alcohol to oil ) on transition rate.

Consequently, adding methyl alcohol stepwisely into the reaction system improved the esterification well. When the methyl alcohol was added measure sagely at three different times, the transition rate was further increased ( Fig. 6B ) .

Fig. 2B Consequence of methyl alcohol feeding frequence on transition rate.

Tetrahydrofuran ( log P=49 ) , Tert-Amyl intoxicant ( log =1.15 ) , cyclohexane ( log P=3.0 ) , crude oil esters ( log P=3.4 ) , n-hexane ( log P=3.5 ) were tested as organic dissolvers for the reaction. The highest biodiesel output was observed in n-hexane ( log =3.5 ) , followed by crude oil quintessence and cyclo-hexane ( Fig. 6C ) .

Fig.6C Effectss of the organic dissolvers ( with different log P value ) on transition rate.

The optimal sum of n-hexane was 2.5 times of oil. The optimal sum of n-hexane was 2.5 times of oil ( Fig. 6D ) .

Fig.6D Effect of dissolver sum on transition rate

Such a ratio helped prolong the necessary H2O content and suited substrate concentration for lipase contact action. Non-polar dissolvers were superior because they can deprive off the H2O around the enzyme to make a micro aqueous bed. Such a microenvironment could assist keep the active conformation of the enzyme and continue the catalytic activity of the enzyme. The optimal H2O content was 10 % of oil measure ( mL /mL-1 ) , while with increased free H2O decreases the esterification degree ( Fig. 6E ) .

Fig.6E Effect of pH value on transition rate

The optimal pH value was at 7.0 for the immobilized lipase, and the transition rate deceased greatly as the pH value departed ( Fig. 6F ) . Because the pH value and H2O content changed the ionisation status of the enzyme, accordingly, changed the activeconformation of lipase and the interaction between the enzyme and its substrate. The ionisation of enzyme is by and large affected by the pH value of the hydration bed because of the pH memory consequence ( the surface ionisation of the immobilized lipase in the buffer solution during immobilisation can be retained in the organic dissolver system ) . When the optimum pH value was applied, the ionizing environment suitable for the combination between enzyme and substrate was achieved.

Fig.6F Effect of H2O content on transition rate,

The optimal temperature was observed at 38 oC, which resulted in faster reaction rate and the highest biodiesel output ( Fig. 6G ) . Lipase sum straight affected the reaction rate, and the best accelerator sum was 75 % immobilized lipase ( w/w of oil measure, 12,000 Ug-1 ( Fig. 6H ) .

Fig.6H Effect of immobilized lipase measure Fig.6G Effect of temperature on transition rate on transition rate

The transition rate was detected after the reaction conditions were stabilized, and the transition rate achieved the highest after 12 H of reaction ( Fig. 6I ) . Therefore, the best procedure combination of enzymatic transesterification of lipoids was 75 % immobilized lipase ( g g-1, 12,000 Ug-1 ) , 10 % H2O content ( mL mL-1 ) based on lipoids measure, 3:1 molar ratio of methyl alcohol to oil batched in three times, at the temperature of 388C and the pH value of 7.0. With these conditions, a transition of 98.15 % was achieved in approximately 12 h. The constituents of biodiesel estimated by gas chromatography ( GC ) .

Fig.6I The alteration of transition rate with reaction clip

Furthermore, the lipid content was aggressively increased by metabolic technology through heterotrophic growing of Chlorella protothecoides. The transmutation resulted in the petroleum lipid content of up to 55.2 % in heterotrophic Chlorella cells. This lipid degree is three times higher than that in autophytic Chlorella cells ( Miao and Wu, 2006 ) . Lipase catalyzed transesterification has been as a high efficiency option compared with conventional protocols. When the immobilized lipase was adopted as a conversation reached 98.15 % .

Case survey II

High-density agitation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production

Immobilized-lipase-catalyzed biodiesel production

The output of microbio-diesel merchandise as reaction clip went by was shown in Fig. 6. The constituents of reaction mixture were 97.73 % triglycerides, 1.92 % diglyceride, and 0.35 % monoglyceride ab initio. After 12 h reaction, the composing was 98.15 % fatty acerb methyl esters, 0.94 % triglyceride, 0.63 % diglyceride, and 0.28 % monoglyceride. So the transition rate from heterotrophic Chlorella lipoids to microbio-diesel was 98.15 % .

Fig 7. Merchandise composing for transesterification during the reaction clip ( unfastened square, fatty acid methyl esters ; unfastened circle, triglyceride ; unfastened diamond, diglyceride ; unfastened trigon, monoglyceride )

The microalgae biomass production has been achieved by at least two chief attacks: one is photoautotrophic cultivation in unfastened pools or photo-bioreactors by utilizing solar energy and repairing C dioxide, the other is heterotrophic agitation utilizing glucose as energy beginning and C beginning. From the point of view of energy economy, the former option seems to be more economical. First, C.protothecoides accumulates much higher proportion of lipoids and has higher growing rate in heterotrophic civilization manner. Second, in heterotrophic civilizations, production conditions can be easy controlled to high cell denseness. During the immobilized-lipase-catalyzed transesterification, stoichiometric measure methyl alcohol was batch-fed ; the highest microbio-diesel output reached 98.15 % in 12 H.


The production biodiesel from the algae for the algae growing status demand to be carefully controlled and optimal nurturing environment. And production from heterotrophic Chlorella in bioreactors shown that Chlorella accumulates much higer proportion of lipoid and has higer growing rate in heterotrophic civilization manner. During the immobilized- lipase catalyzed transesterification was the highest microbiodiesel output in 98.15 % in 12 H.