Production Biodiesel From The Algae Biology Essay

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Biodiesel is a renewable biodegradable, nontoxic and environment fuel. That is defined as a fuel comprised of mono-alkyl esters of long-chain fatty acids from vegetable oils or animal fats (Xiong et al., 2008). Many starting materials such as soybean oil, sunflower oil, cotton seed oil, rapeseed oil, palm oil and restaurant kitchen waste have been tried for preparation of biodiesel by the enzymatic route. For the preparation of biodiesel, tranesterification reaction was used. Also biodiesel is excellent replacement for petroleum diesel. The cetane number, energy content, viscosity and phase changes are also similar to those of petroleum-based diesel fuel (Noureddini et al., 2005).

Although production of biodiesel by chemical catalyst that are also have problems, such as glycerol recovery and removal of inorganic salts. At present all of biodiesel were using a chemical catalyst like a strong acid or base with used methanol or ethanol produce biodiesel and glycerol. The approach is the enzymatic one, which lipase catalyzed tranesterification is carried out in non-aqueous environment (Shah et al., 2004).

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

1.2 Biodiesel Production Worldwide

Europe has been the leading region for biodiesel production. The first pilot plant for the production of biodiesl from rape seed oil has been installed in Austria in 1987 in Silberberg, resulting from several years of research at the Institute of Chemistry of the University of Graz. The possibility of using pure biodiesel without paying mineral tax has led to the installation of several industrial scale plant in 1991 in Austria and Germany. But vary soon countries like Italy, France and the Czech Republic followed. The period of slow but constant development of biodiesel activities in Europe for several years with the European directive for promotion of biodiesel in the years 2003, A market share of biodiesel of 5.75% for transport fuel in 2010.Since the development of biodiesel production almost exploded in all 27 countries of the European Union, leading to a total production of biodiesel of 5.6 million tons in the year 2006 and a production capacity of almost 10 million tons.

The production has been increased by approx. 50% from the year 2005 until 2006 the production capacity in 2007 will exceed 10 million tons per year. The almost exploding development of biodiesel production in Europe will close down by the lack of feedstocks and raw material. Almost 10 in every country worldwide biodiesel activities have been initiated the leading countries are also those with the largest production of vegetable oils. In 2006 in the US the production of biodiesel exceeded 1 million tons per year, however biodiesel plant have been installed with and overall production capacity of over 5 million tons per year. But also the major palm oil production countries like Malaysia and Indonesia have a series of biodiesel plants already installed with a capacity of over 1 million tons per year. Similar activities can be found in China and India with their huge demand for transport fuel, but also in the vegetable oil producing countries like Brazil and Argentina.

1.3 Feedstocks for Biodiesel Production

Today a production capacity of almost 30 million tons of biodiesel of biodiesel exists worldwide. On the other hand there is a total annul production of vegetable oils of approx.110 million tons per year, which is mainly used for food purpose. As the production of vegetable oil plants like palm oil has led to extensive discussions leading to the search for non-edible oil seeds.

For all vegetable oils and animal fat can be used as feedstock for biodiesel production. Most of these oils and fats have similar chemical composition of triglycerides with difference amounts of fatty acids. The major fatty acids are those with a chain length of 16 and 18 carbon, whereas the chain could be saturated or unsaturated. Methyl esters produce from these fatty acids. The major component in fossil diesel fuel are also straight chain hydrocarbons with a chain length of about 16 carbons. The major differences between the methyl ester from feedstocks refer to the amount of unsaturated fatty acid. The best composition characteristic as well as oxidation come from saturated fatty acid, however cold temperature and the high melting point of these fatty acids. The major feesstock for biodiesel production today are rape seed oil (Canola), soybean oil and palm oil. The fuel properties of the methyl ester out of these oils are quite similar concepts for the poor cold temperature behavior of palm oil because of the high portion of saturated fatty acids. However, depening on the climate conditions of a country, an optimum mix of methyl ester our of these feedstocks can be used. Only coconut oil and plam kernel oil have fatty acid with 12 or 14 carbons as major components. Therefore the methyl esters out of these fats have lower boiling points, but could be used perfectly as ad-mixture to common biodiesel. In Asian countries like India and China the use of non-edible seed oils for biofuels production is very popular. In that case there would be no competition with the food production, especially when these oil plants are grown on marginal areas not suitable for food production. Another interesting feedstocks for biodiesel production is oil production of algae, which can grown in open pounds or in close tubes. The productivity is estimated to be much higher per area than with the food production and also with the traditional oil seeds and furthermore there is no need for agricultural land, it can be produced at any place, where water and sunlight are existing. However, today an economic production of biodiesel from algae dose not seem to be vary realistic, but further research in that area will be necessary.

1.4 Chemical Principle of biodiesel production

Fatty acid methyl ester have been known for over 150 years. The first description of the preparation of the esters was published in 1852. However for a long time fatty acid methyl ester were mainly used as derivative for analyzing the fatty acid distribution of fats and oils, so the preparation mainly was done in analytical scale. 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 only since the late Seventies have fatty acid methyl ester have been tested and used as diesel fuel substitute.

Chemically biodiesel is equivalent to fatty acid methyl esters or ethyl ester, produced out of triacylglycerol transesterification or out of fatty acids esterification. Fatty acid methyl ester today are the most commonly used biodiesel species, whereas fatty acid ethyl esters (FAEE) so far have been only produced in laboratory.

For transesterification one mole of the triglyceride reacts with three moles of alcohol to from one mole of mole of glycerol and three moles of the respective fatty acid alkyl ester. The process is a sequence of three reversible reactions, which the triglyceride molecule is converted step by step into diglyceride, monoglyceride and glycerol. The equilibrium to the right, methanol is added in an over the stoichiometric amount in most commercial biodiesel production plants. A main advantage of methanolysis as compared to transesterification with higher alcohol is the fact that two main products, glycerol and fatty acid methyl ester (FAME), are hardly miscible and thus form separate phases an upper ester phase and a lower glycerol phase. This process remove glycerol from the reaction mixture enables high conversion. Ester yields can even be increased while at the same time minimizating the excess amount of methanol. By conducting methanolysis in two or three step, and the glycerol phase produced is separate after each process stage. Finally, of the type of alcohol used, some from of catalyst has to be present to achieve high ester yields under comparatively mild reaction condition.

The process of vegetable & plant oils into biodiesel fuel is a transesterification. And the transesterification of vegetable oils, a triglyceride have effect with an alcohol in acid and base, producing a mixture of fatty acids alkyl esters and glycerol. This liquid is then mixed into vegetable oil. The entire mixture then settles. Glycerin is left on the bottom and methyl esters, or biodiesel, is left on top. The glycerin can be used to make soap (or any one of 1600 other products) and the methyl esters is washed and filtered.

All the process of three consecutive 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 excess of the alcohol is used to increase the yields of the alkyl esters and to its phase separation from the glycerol formed. However of including the type of catalyst (alkaline or acid), alcohol/vegetable oil molar ratio, temperature, water content) and free fatty acid content have an influence on the course of the transesterification up to the type of catalyst was used.

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

Fig. 1 Biodiesel preparation

Source: Chisti al. (2007)

The process is normally a sequence of three consecutive steps, which reversible reactions. In the first step, from triglycerides diglyceride is obtained, from diglyceride monoglyceride is produced and in the last step, from monoglycerides glycerin is obtained. Thisreactions esters are produced. The stoichiometry showed between alcohol and the oil is 3:1. However, an excess of alcohol is more improve

Fig. 2 Tree consecutive and reversible reactions R1 R2 R3 and R’ represent alkyl groups.

Source: Miguel (2001)

1.5 Catalysis for Tranesterification and Esterification Reaction

Alkali catalyst on transesterification

For a basic catalyst for biodiesel production, either sodium hydroxide (NaOH) or potassium hydroxide (KOH) should be used with methanol or ethanol as well as any kind of oils, refine, crude. The main advantage of this from of catalysis over acid-catalysted transesterificatios is the high conversion rate under mild conditions in comparatively short reaction time. So it was estimated that under the same temperature conditions and catalyst concentrations methanolysis might proceed about 4000 times faster in the presence of an alkaline catalyst than in the presence of the same amount of an acidic equivalent. And effect of moisture and free fatty acid kind of alkali-catalyzed tranesterification the glyceride of alcohol of must be substantially anhydrous because of water cause partial reaction to saponification, which product soap. The soup consume and catalyst and reduce the catalytic efficiency as well as causing increase in viscosity. The formation of gels and difficultly inactivating separation of glycerol (Wright: 1994).The most of alkaline catalyst depends on the quality of the oil, especially on the content of free fatty acids. Under alkaline catalysis free fatty acid are immediately converted in to soaps. Which can prevent the separation of glycerol and finally can lead to total saponification of all fatty acid material? So the alkaline catalyst is limited to feedstocks up to a content of approx. 3 % of fatty acid.

Table 1 Overview of homogenous alkaline catalyst

source: (Soetaert et al.,2009)

Type of catalyst


Sodium hydroxide

Cheap, disposal of residual salts necessary

Potassium hydroxide

Reused as fertilizer possible fast reaction rate ,better separate of glycerol

Sodium methoxide

No dissolution of catalyst necessary, disposal of salts necessary

Potassium methoxide

No dissolution of catalyst necessary,use as fertilizer possible, better separation of glycerol, high price

2. Acid catalyst on transesterification

Acid for transesterification include sulfuric, phosphoric, hydrochloric, and organic sulfuric acid. Although tranesterication by acid catalysis it much slower than alkali catalysis and acid catalyst transesterification is more suitable for glycerides that have relative high free fatty acid contents and more water (Aksoy et al., 1998). And acid catalyst offers the advantage of also esterifying free fatty acids contained in the fats and oils and is therefore especially suited for the transesterification of highly acidic fatty materials a further disadvantage of acid catalysis probably prompted by the higher reaction temperature is an increased formation of unwanted secondary products,such as dialkylehers or glycerol ethers. Because of the slow reaction rates and high temperatures needed for transesterification acid catalyst are used for esterification reaction. So for vegetable oils or animal fats with an amount of free fatty acids can over approx. 3% two strategies are possible. The free fatty acids can either be removed by alkaline treatment. The cheapest catalyst for esterification reactions is concentrated sulphuric acid. The main disadvantage of this catalyst is the possibility of the formation of pound also p-toluene sulphonic acid can be used. However, the high price of the compound has prevented broader application.

3. Enzymatic on tranesterification by lipase

Although chemical tranesterification using alkali catalysis process give high conversion levels of triglyceride to methyl ester in short reaction time, the reactions have drawback, There is recovery of glycerol, removal of acid or alkaline catalysis. On the other hand, lipases are enzymes used to catalyze some reaction such as hydrolysis of glycerol, alcoholysis. Lipase catalysis is significant greater than that alkaline one (Linko et al., 1998). However, the extracellular and the intracellular lipases are also able to catalyze the transesterification of triglycerides effectively (Marchett et al., 2007).And lipase-catalyzed transesterification also a series of drawbacks. As compared to converional alkaline catalysis, reaction efficiency tends to be poor, so that biodiesel catalyst usually necessitates far longer reaction times and higher catalyst concentrations. The main application of lipase industrial biodiesel production is their high price, especially if s are used in the form of highly purified, extra cellular enzyme preparations, which can be recovered from the reaction products. For the immobilization of lipase on a carrier ,so that the enzymes can be removed from the reaction mixture and can theoretically be reused for subsequent transesterification. Immobilization also offers the advantage that in many case the fixed lipase tend to be more active and stable than free enzymes.

Fig. 3 Flow diagrams comparing biodiesel production using the (a) alkali and (b) lipase catalysis in process.

Source: Fukada et al. (2001)

The figure show the process since of none reacted methanol and waste water treatment are unnecessary. In addition, only is recovery of glycerol. Since the cost of lipase production is the main to commercialization of the lipase catalyzed process. Several reports have been made to develop cost effective system (Fukada et al ., 2001).

Table 2 Comparison of the different technologies to produce biodiesel

Source: Marchetti et al. (2007)

The table showed a summary of the advantages and disadvantages of each technological possibility to produce biodiesel.

The advantages of using 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 use of high concentration of them and that makes for a longer activation of the lipases.

3. For a bigger thermal stability of the enzyme due to the native state.

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

reaction and that will prevent all the enzyme particles getting together.

5. Separation of product will be easier when using this catalyst.

The disadvantages of using lipase

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

2. Number of support enzyme is not uniform.

3. Biocatalyst is more expensive that the natural enzyme.

(Fukada et al., 2001)

2. Raw materials for biodiesel production

2.1 Microalgae for biodiesel production

Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that can grow rapidly and live in harsh conditions due to their unicellular or simple multicellular structure. Microalgae are present in all existing earth ecosystems, not just aquatic but also terrestrial, representing a big variety of species living in a wild range of environmental conditions. It is estimated that more than 50,000 species exist, but only a limited number, of around 30,000 have been studied and analyzed.

During the past decades extensive collections of microalgae have been created by researchers in different countries. And example is the freshwater microalgae collection from Coimbra (Portugal) considered one of the worlds largest, having more than 4000 strains and 1000 species. The collection to the large variety of different microalgae available to be selected for in use in a broad diversity of applications, such as value added product for pharmaceutical purposes, food crops for human consumption and energy source. And The university of Texas Algal cultures that was founded in 1953. It include 2300 different strain of freshwater (Mata et al.,2010)

2.1.2 Advantage of using microalgae for biodiesel production

Many researcher reports and articles described many advantages for using microalgae for biodiesel production in comparison with other available feedstocks. From a paractical point of view, there are easy to cultivate, can grow with little or even on attention, using water unsuitable for human consumption and easy to obtain nutrients.and microalgae reproduce themselves using photosynthesis to convert sun energy into chemical energy, completing and growth every few days. Moreover they can grow almost anywhere by sunlight and more simple nutrients. Algae biodiesel contains no sulfur and performs as well as petroleum diesel. (Mata et al.,2010)

2.1.3 Microalgae lipid content and productive

Many microalgae spices can be induced to accumulate a quantities of lipids thus to high oil yield. The advantage lipids content varies between 1 and 70% but under certain conditions some species can reach 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 selection of the most adequate species needs to take into account other factors, such as for example the ability of microalgae to develop using the nutrients available or under specific environment conditions. All these parameter should be considered simultaneously in the selection of the most adequate 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 utilization, In general for resource ,to storage them, or increase their efficiency in resource utilization. In general for biomass growth of 40-50% carbon microalgae depened on a sufficient supply of a carbon source and light to carry out photosynthesis. Microalgae may assume many types of metabolism (e.g. autotrophic, heterotrophic, mixotrophic, photo heterotrophic) for example some organisms can grow are Photoautotrophically, i.e. using light as a sole energy source that is converted to chemical energy through photosynthetic reaction. And heterotrophiclly, (i.e. utilizing only organic compounds as carbon and energy source)

2.1.5 Harvesting and biomass concentration

Algae harvesting consists of biomass recovery from the culture medium that may contribute to 20-30% of the total biomass production cost. In order to remove large quantities of water and process large algal biomass volumes, a sutiable harvesting method may involve one or more step and be achieved in several physical, chemical, or biological ways.

Most common harvesting methods include sedimentation, centrifugation, filtration, and ultra-filtration. After separation from the culture medium algal biomass (5-15% dry weigh)must be quickly process let it should get spoiled in only a few hours in a hot climate.

2.2 Lipase immobilized

Immobilization is the most widely used method for achieving stability in lipases and to make them more attractive for industrial use (Cowan, 1996 and Clark, 1994) Common immobilization techniques include physical adsorption into a solid support ( Bosley and pielow, 1997) covalent bonding 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 bonding method, this method uses a relatively simple procedure and at the same time the immobilized lipase maintains its activity and stability (Kennedy and Melo, 1990). This method which was pioneered by Avnir et al.(1994) is based on solâ€"gel process. The application of the solâ€"gel material in the immobilization of lipases is well documented (Reetz et al., 1997).

Case study 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, respectively. According 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, respectively.

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

Source: ( Li et al., 2008)

The Yield of Biodiesel by Transesterification

Generally, short chain alcohols have strong denaturing effects on enzymes. As a result, their presence cans influence the stability and activity of immobilized lipase greatly. When the molar ratio of methanol to oil was set at a stoichiometric quantity of 3:1, the highest conversion rate was reached

(Fig. 6A).

Fig. 6A Effect of molar ratio (methanol to oil) on conversion rate.

Consequently, adding methanol stepwisely into the reaction system improved the esterification considerably. When the methanol was added step wisely at three different times, the conversion rate was further increased (Fig. 6B).

Fig. 2B Effect of methanol feeding frequency on conversion rate.

Tetrahydrofuran (log P=49), Tert-Amyl alcohol (log =1.15), cyclohexane (log P=3.0), petroleum esters (log P=3.4), n-hexane (log P=3.5) were tested as organic solvents for the reaction. The highest biodiesel yield was observed in n-hexane (log =3.5), followed by petroleum ether and cyclo-hexane(Fig. 6C).

Fig.6C Effects of the organic solvents (with different log P value) on conversion rate.

The optimum amount of n-hexane was 2.5 times of oil. The optimum amount of n-hexane was 2.5 times of oil (Fig. 6D).

Fig.6D Effect of solvent amount on conversion rate

Such a ratio helped sustain the necessary water content and suitable substrate concentration for lipase catalysis. Non-polar solvents were superior because they can strip off the water around the enzyme to create a micro aqueous layer. Such a microenvironment could help maintain the active conformation of the enzyme and preserve the catalytic activity of the enzyme. The optimum water content was 10% of oil quantity (mL /mL-1), while with increased free water decreases the esterification level (Fig. 6E).

Fig.6E Effect of pH value on conversion rate

The optimum pH value was at 7.0 for the immobilized lipase, and the conversion rate deceased greatly as the pH value departed (Fig. 6F). Because the pH value and water content changed the ionization condition of the enzyme, consequently, changed the activeconformation of lipase and the interaction between the enzyme and its substrate. The ionization of enzyme is generally affected by the pH value of the hydration layer because of the pH memory effect (the surface ionization of the immobilized lipase in the buffer solution during immobilization can be retained in the organic solvent system). When the optimal pH value was applied, the ionizing environment suitable for the combination between enzyme and substrate was achieved.

Fig.6F Effect of water content on conversion rate,

The optimum temperature was observed at 38 oC, which resulted in faster reaction rate and the highest biodiesel yield (Fig. 6G). Lipase amount directly affected the reaction rate, and the best catalyst amount was 75% immobilized lipase (w/w of oil quantity, 12,000 Ug-1 (Fig. 6H).

Fig.6H Effect of immobilized lipase quantity Fig.6G Effect of temperature on conversion rate on conversion rate

The conversion rate was detected after the reaction conditions were stabilized, and the conversion rate achieved the highest after 12 h of reaction (Fig. 6I). Therefore, the best process combination of enzymatic transesterification of lipids was 75% immobilized lipase (g g-1, 12,000 Ug-1), 10% water content (mL mL-1) based on lipids quantity, 3:1 molar ratio of methanol to oil batched in three times, at the temperature of 388C and the pH value of 7.0. With these conditions, a conversion of 98.15% was achieved in about 12 h. The components of biodiesel estimated by gas chromatography (GC).

Fig.6I The change of conversion rate with reaction time

Moreover, the lipid content was sharply increased by metabolic engineering through heterotrophic growth of Chlorella protothecoides. The transformation resulted in the crude lipid content of up to 55.2% in heterotrophic Chlorella cells. This lipid level is three times higher than that in autotrophic 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 study II

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

Immobilized-lipase-catalyzed biodiesel production

The yield of microbio-diesel product as reaction time went by was shown in Fig. 6. The components of reaction mixture were 97.73% triglycerides, 1.92% diglyceride, and 0.35% monoglyceride initially. After 12 h reaction, the composition was 98.15% fatty acid methyl esters, 0.94% triglyceride, 0.63% diglyceride, and 0.28% monoglyceride. So the conversion rate from heterotrophic Chlorella lipids to microbio-diesel was 98.15%.

Fig 7. Product composition for transesterification during the reaction time (open square, fatty acid methyl esters; open circle, triglyceride;open diamond, diglyceride; open triangle, monoglyceride)

The microalgae biomass production has been achieved by at least two main approaches: one is photoautotrophic cultivation in open ponds or photo-bioreactors by using solar energy and fixing carbon dioxide, the other is heterotrophic fermentation using glucose as energy source and carbon source. From the viewpoint of energy saving, the former alternative seems to be more economical. First, C.protothecoides accumulates much higher proportion of lipids and has higher growth rate in heterotrophic culture mode. Secondly, in heterotrophic cultures, production conditions can be easily controlled to high cell density. During the immobilized-lipase-catalyzed transesterification, stoichiometric quantity methanol was batch-fed; the highest microbio-diesel yield reached 98.15% in 12 h.


The production biodiesel from the algae for the algae growth condition need to be carefully controlled and optimum nurturing environment. And production from heterotrophic Chlorella in bioreactors shown that Chlorella accumulates much higer proportion of lipid and has higer growth rate in heterotrophic culture mode. During the immobilized- lipase catalyzed transesterification was the highest microbiodiesel yield in 98.15% in 12 h.