(6)biodiesel an alternative fuel

Recent Patents on Biotechnology 2008, 2, 25-34 25

1872-2083/08 $100.00+.00 © 2008 Bentham Science Publishers Ltd.

Biodiesel: An Alternative Fuel

Maximino Manzanera*, Maria L. Molina-Muñoz and Jesús González-López

Group of Environmental Microbiology, Institute of Water Research, University of Granada, C/Ramón y Cajal No. 4, Edificio Fray

Luis de Granada, Granada 18071, Spain

Received: July 16, 2007; Accepted: August 16, 2007; Revised: September 14, 2007

Abstract: Biodiesel is an alternative energy source and could be a substitute for petroleum-based diesel fuel. To be a viable alternative, a biofuel should provide a net energy gain, have environmental benefits, be economically competitive, and be producible in large quantities without reducing food supplies.

Most of the sources, methods and apparatus to produce biodiesel are reviewed here. Some of the patents propose the use of oils and fats of animal or vegetal origin and other kind of sources. Many others focus on the methods for the production or oxidation stability of the biofuel in order to make its production economically competitive. Several apparatus comprising reactors and refineries are also presented. This review article summarizes recent and important patents relating to the production of biodiesel to make its production a viable alternative.

Keywords: Alternative fuels, biodiesel, transesterification, FAEEs, FAMEs, lipase, SCF, refinery, NOx, microdiesel.

INTRODUCTION

The biggest challenge modern industrial society is facing today is the decline and exhaustion of the fossil energy resources. The primary sources of energy that power our civilization are those fossil fuels. The International Energy Agency has forecasted that the world's total energy requirements will rise by half in the next 25 years. On the other hand, global oil product demand has been revised to 84.3 million barrels a day (mb/d) in 2006 and 85.8 mb/d in 2007 [1]. Therefore continued use of petroleum-sourced fuels is now widely recognized as unsustainable because of depleting supplies and increasing demand.

Oil prices have been rising steadily over the past 3 years and surged to a record high above $60 a barrel in June 2005, sustaining a rally built on strong demand for gasoline and diesel and on concerns about refiners' ability to keep up. This trend continued into early August 2005, as NYMEX crude oil futures contracts surged past the $65 mark as consumers kept up the demand for gasoline despite its high price. Crude oil futures peaked at a close of over $78.40 per barrel on July 13, 2006 [2].

In addition, the combustion of the fossil fuels used is considered as the major factor responsible for global warming due to large-scale carbon dioxide emissions. The present atmospheric concentration of CO2 is about 383 parts per million (ppm) by volume. Future CO2 levels are expected to rise due to ongoing burning of these fossil fuels and land-use change. The IPCC Special Report on Emissions Scenarios gives a wide range of future CO2 scenarios, ranging from 541 to 970 ppm by the year 2100. Fossil fuel reserves are sufficient to reach this level and continue emissions past 2100, if coal, tar sands or methane clathrates are extensively used [3]. Fossil fuels are also considered as the main source of local environmental pollution. Thus, alternative energy sources based on sustainable, renewable and environmentally friendly processes are urgently needed. One of the most prominent alternative energy resources, attracting more and more interest in recent years with the price for crude oil reaching record heights, is biodiesel, which is a possible substitute for petroleum-based diesel fuel. Biodiesel fuels are the alkyl monoesters of fatty acids from vegetable oils and animal fats. Many studies have shown that the properties of biodiesel are very close to diesel fuel [4-6]. Therefore,

*Address correspondence to this author at the Group of Environmental Microbiology, Institute of Water Research, University of Granada, C/Ramón y Cajal No. 4, Edificio Fray Luis de Granada, Granada 18071, Spain; Tel: +34 958 242981; Fax: +34 958 243094; E-mail: [email protected]

biodiesel fuel can be used in diesel engines with little or no modification. Biodiesel has a higher cetane number than diesel fuel, no aromatics, no sulfur, and contains 10-11% oxygen by weight. These characteristics of biodiesel are responsible for a reduction in the emissions of carbon monoxide (CO), hydrocarbon (HC) and particulate matter (PM) in the exhaust gas compared to diesel fuel [7,8]. However, to be a viable alternative, a biodiesel should provide a net energy gain, be economically competitive, and be producible in large quantities without reducing food supplies.

1. BIODIESEL FEEDSTOCK

1.1. Vegetable Oils

Vegetable oil was exclusively used to run the early diesel engines since diesel engine was invented in 1892. Nevertheless, the feedstock shifted to petroleum distillates refined from crude oil during gasoline production in the 1920s. But while so-called petrodiesel was cheaper and more abundant than vegetable oil, it was also lighter and less viscous. Vehicle manufacturers had to modify engine designs accordingly, and vegetable oil as a fuel source was sidelined for decades. Then in 1973, the Arab oil embargo produced a swift price increase. With gas and diesel suddenly four times more expensive than before, interest in biofuels returned. Since 1973, many different sources have been proposed in order to obtain biodiesel. Virgin oil feedstock rapeseed and soybean oils are most commonly used, with soybean oil alone accounting for about ninety percent of all biofuel stocks in the US. While in 1994 fuels produced from soybean oil amounted to barely a few thousand gallons a year, ten years later, that volume increased to 25 million gallons. Last year (2006) 200 million gallons were sold. Assuming existing and emerging facilities operate at full capacity, USA biodiesel production capacity could reach 1.5 billion gallons in 2007 [9].

If soybean oil is the preferred biodiesel feedstock in North America, rapeseed or canola, is the one preferred in Europe for biodiesel production. Nevertheless, rapeseed oil is a promising source for manufacturing biodiesel in North America as well, evolving into a major cash crop. Canada and the United States produce between 7 and 10 million metric tons (tonnes) of rapeseed per year. This is partly because rapeseed produces more oil per unit of land area as compared with soybean in these areas. Sommerville estimated that the average yield of soybean oil is 450 pounds per acre and the average canola seed oil was 650 pounds per acre in 2005 [10]. Although this is in contrast with the much higher yields of cellulosic biomass that could be grown on the same acres to produce fuel ethanol, alternative feedstocks are needed to obtain

26 Recent Patents on Biotechnology 2008, Vol. 2, No. 1 Manzanera et al.

biodiesel where ethanol feedstock can not be grown or do not render such high yields. In this sense, in 1998 Beer and coworkers developed a variety of transgenic plant considered to be the most disease- and drought-resistant variety of rapeseed to date [11] that allegedly could be translated in an increase in oil production as a result of growing this variety in locations where neither cellulosic biomass for ethanol production nor rapeseed was viable before.

A different feedstock of vegetable origin is palm oil, obtained from the fruit of the oil palm tree. Previously the second-most widely produced edible oil, after soybean it may have now surpassed soybean oil as the most widely produced vegetable oil in the world and certainly is the preferred oil in South East Asia. Thus, some Asian governments are refocusing on the use of palm oil to the production of biodiesel to cater to the huge demand from European countries encouraging the building of biodiesel plants. These plants will start operating in the middle of next year and produce 100,000 tons of biodiesel annually. Strong demand for biodiesel from Europe as well as Colombia, India, South Korea and Turkey has fueled the industry's growth as more countries seek to reduce their reliance on fossil fuels. Malaysia has already begun preparations to change from diesel to bio-fuels by 2008, including drafting legislation that will make the switch mandatory. From 2007, all diesel sold in this country must contain 5% palm oil. Being the world's largest producer of crude palm oil, Malaysia intends to take advantage of the rush to find cleaner fuels. Palm-based biodiesel exhibits a relatively high pour point (approximately 15oC) due to its high content of saturated fatty compounds; hence, its use is only suitable for tropical countries and unsuitable for low temperate or cold climate countries where operational temperature is below its pour point. Nevertheless, palm-based biodiesel formulations with enhanced cold flow properties are provided using new formulation comprising at least 60% vol/vol petroleum-based fuel oil mixed with palm-based fuel oil [12]. Similarly, a novel alkyl-ester composition derived from specific blend of rapeseed and sunflower, in particular from Brassica napus and Helianthus annuus to be used in diesel engines to solve the same kind of problem [13].

In certain parts of the world, governments and some corporations consider the jatropha plant, common in hot climates, one of the most promising sources of biodiesel. The plant can grow in wastelands, and it yields more than four times as much fuel per unit of area as soybean, and more than ten times that of corn [14]. Another example of crop, recently proposed for biodiesel’s feedstock is the oil extracted from the nuts of the desert plant Balanites aegyptiaca [15]. Other crops such as cottonseed [16], mustard, flax, sunflower, hemp and even algae are considered promising biodiesel feedstocks too.

The advantages of deriving biodiesel from algae include rapid growth rates, a high per-acre yield. Some species of algae are ideally suited to biodiesel production due to their high oil content [17-21]; The National Renewable Energy Laboratory (NREL) in Golden, Colorado had demonstrated oil production from algae rates 200 times greater per acre than achievable with fuel production from soybean farming [22]. Unlike other oil crops, microalgae grow extremely rapid and commonly double their biomass within 24 h. Biomass doubling times during exponential growth are commonly as short as 3.5 h. Oil content in microalgae can exceed 80% by weight of dry biomass [23,24]. Depending on species, microalgae produce many different kinds of lipids, hydrocarbons and other complex oils [20,25,26]. And although not all algal oils are satisfactory for making biodiesel, suitable oils occur commonly. The strains of race A of microalgae Botryococcus braunii have been proposed as a renewable source of liquid fuel via CO2 fixation [27] with hydrocarbon content as high as 30% of biomass. Recent patents comprise a novel variety of Botryococcus (Ninsei) distinct from the previously cultured variety in color, biochemistry, size, shape, and habit in order to facilitate its cultivation [28], or a race

capable of producing different types of hydrocarbons [29] more suitable for biodiesel production. Genetic and metabolic engi-neering are likely to have the greatest impact on improving the economical production of microalgal diesel [17,19,30].

1.2. Non-Vegetable Oils as Biodiesel Feedstock

Although, it has been shown that biodiesel from vegetable feedstocks have environmental benefits there are as well some concerns about its use. In this sense, the locations where oil-producing plants are grown is one of the prime worries being that countries, such as the Philippines and Indonesia are clearing cut large areas of tropical forest in order to grow such lucrative crops, in particular, oil palm [31]. Even more, it has been suggested that biodiesel might increase pressure on world food supplies, therefore urging to look for alternative feedstocks for biodiesel such as waste vegetable oils and waste animal fats. The main sources of waste animal fats are primarily meat animal processing facilities. Recycled grease products are referred to waste grease being generally classified in two categories, yellow grease and brown grease. Yellow grease is produced from vegetable oil or animal fat that has been heated and used for cooking. Renderers filter out the solids and heat the spent cooking oil to drive out moisture until it meets industry specifications for yellow grease. Yellow grease is required to have a free fatty acid level of less than 15%. If the free fatty acid level exceeds 15%, it is called brown grease, sometimes referred to as trap grease. Waste vegetable oil from restaurants and rendered animal fats are inexpensive compared with food-grade vegetable oil hence biodiesel production from waste grease can be expected to benefit from a raw material cost advantage and it will help to reduce overall biodiesel cost [32]. There are patents that provide methods for producing biodiesel fuel from waste vegetable or animal oils and fats, and methanol that does not use a catalyst and does not generate glycerol as a by-product [33], or smaller processing plant that enables site selection closer to the raw material supplies, thereby minimizing or eliminating transportation costs for yellow grease [34]. More important is the method by Hammond Earl G. and Wang Tong of converting free fatty acids in acid oil, and the acid fat into FAMEs (biodiesel) using a small amount of methanol and an acid catalyst. A method especially useful for the generation of biodiesel using animal oils and fats that contain a relatively high level of free fatty acids as starting material [35]. A different application developed in order to use waste oils and fats as biodiesel feedstock focus on reducing the viscosity of these wastes in order to facilitate a filtering process particularly needed for biodiesel synthesis from this kind of oils and fats [36]. Similarly a mobile refinery has been developed in order to separate contaminants of waste fryer oil used for cooking including food particles, and water and emulsified or congealed grease by vacuuming and heating these wastes into a container and passing the heated fluid via a strainer [37]. The use of whole microorganisms producing lipases or the purified lipases themselves has been proposed as an invention in order to use waste lipids and fat to produce fatty acid lower alcohol esters as well [38].

Finally, different waste fats are proposed for lower production cost such as those present in sewage sludge that contains high levels of lipids, in the form of triglycerides, phospholipids, phospho-glycerides, sphinolipids, glycolipids, and fat-soluble vitamins. In this method, sludge produced under wastewater treatment is collected then partially dehydrated by sludge thickeners, filter presses, centrifuges, and driers to a final solid concentration ranging from 60% to 100% solids by weight. After drying, lipids are extracted from the processed material using any of the methods of chemical extraction such as chemical solvents including aliphatics, supercritical gases and liquids, hexane, acetone, or primary alcohols [39]. Alternatively, a different waste material proposed as biodiesel feedstock is the one produced by the diary industry resulting from their wastewater processing plants. In these plants, dissolved air flotation (DAF) systems are commonly used to separate the

Biodiesel Recent Patents on Biotechnology 2008, Vol. 2, No. 1 27

insoluble solids from the liquid by injecting gas bubbles into the wastewater solution. These bubbles adhere to the insoluble particles and form a floating layer of sludge, which is mechanically scraped off producing an emulsion of fat and protein in water in significant quantities whose disposal is both problematic and expensive. The method by Hamilton RB involves adjustment of the DAF sludge pH between 2.5 and 4.5, mixing it with an emulsifying agent and heating the mixture to boiling point. By allowing the mixture to cool down the fat and protein component parts separate out, and fat rich component is then used as biodiesel feedstock [40].

2. METHODS FOR BIODIESEL PRODUCTION

Biodiesel production is mainly performed by the transes-terification of a triglyceride with an alcohol in a chemical reaction known as alcoholysis outlined in Fig. (1). In the transesterification of different types of oils, triglycerides react with an alcohol, generally methanol or ethanol, to produce esters and glycerol. The overall process is normally a sequence of three consecutive reversible reactions [41,42]. During the first reaction, triglycerides are transformed into diglycerides. Then from diglycerides, monoglycerides are produced in the second reaction and in the last reaction glycerol is obtained from monoglycerides. In all these reactions esters are produced which are the products used as biodiesel, that finally as the resulting product has to be purified. The stechiometric relation between alcohol and oil is 3:1. However, an excess of alcohol is usually added to improve the reaction towards the desired product. To make the reaction possible, a catalyst is added as well. This transesterification reaction is affected by alcohol type, molar ratio of alcohol to oil, temperature, purity of the reactants and type and amount of catalyst see [43] for a review.

Different types of catalysts are used, from basic ones such as sodium or potassium hydroxides, acids such as sulfuric acid, ion exchange resins, lipases and supercritical fluids. For a basic catalyst, either sodium hydroxide (NaOH) or potassium hydroxide (KOH) is normally used with methanol or ethanol as well as any kind of oils, although sodium and potassium alkoxides such as sodium methoxide, sodium ethoxide, sodium propoxide, sodium butoxideto have been proposed as alkali catalysts as well. In this process it is better to produce the alcoxide before the reaction to obtain a better global efficiency [44]. The alcohol-oil molar ratio that should be used varies from 1:1-6:1. However, 6:1 is the most used ratio giving an important conversion for the alkali catalyst without using a great amount of alcohol. The types of alcohol are usually methanol and ethanol. The last one has fewer safety problems because it is less toxic, but methanol is most frequently used because of its lower cost and its physical and chemical advantages (polar and shortest chain alcohol), although in some countries such as Brazil, there is a special interest in developing an appropriate technology for the use of ethanol instead of methanol [45] since ethanol is easily available due to the extensive fermentation industry in the country.

2.1. The Alkali-Catalyzed Transesterification

For the alkali-catalyzed transesterification, both the glycerides and alcohol must be substantially anhydrous, and oils should be neutral or weak acids since a too high proportion of fatty acids (higher than 0.5%) would inhibit the reaction by consuming catalyst to form soaps, lowering the reaction yield and making more difficult the separation of the products, in a process known as saponification [46,47]. On the other hand, if absolute alcohol is used, the formed glycerol does not settle spontaneously and the alcohol excess must be evaporated or water added, nevertheless not obtaining a complete conversion. In order to use hydrated alcohol with success Robert and coworkers proposed a process for preparing a composition of fatty acid esters, by acid transesterification where the alcohol could contain up to 60% by weight of water. This method consists of reducing the free acidity of the ester phase, followed by a basic esterification in the presence of a monoalcohol, finished with the recovery of the ester phase [48]. The process is simplified since, in the presence of hydrated alcohol, glycerol is easily settled and the ester phase is purer and the glycerol content lower despite of the high water content of the alcohol phase. Sometimes, there might be special interest in obtaining concentrated glycerol in the process of making biodiesel [49] as added value byproduct for the synthesis of chemicals such as dichloropropanol [50] polyglycerols [51] or glyceryl ethers or syngas [52-56] currently extracted from petrochemical resources that can be alternatively extracted from biological feedstock.

One of the advantages of the alkali transesterification is that basically any kind of vegetable oil can be used. The amount of catalyst that should be added to the reactor varies from 0.5% to 1% w/w [57], but some authors prefer advice values between 0.005% and 0.35% w/w [43,58]. Other important reason for the great interest in the alkali process is due to its higher reaction efficiency and for showing a lower corrosive effect than that for the acid process, making alkali the preferred catalyst to be used in industries. However, as mentioned before, in the alkali catalyzed transesterification, the presence of water causes a partial reaction change to saponification, which produces soap. The soap consumes the catalyst and reduces catalyst activity, causing an increase in viscosity, formation of gels and difficulty in separation of glycerol. Freedam et al. reported that sodium hydroxide or sodium methoxide reacted with moisture and carbon dioxide in the air leading to lowering of their effectiveness to catalyze the transesterification of oil [47].

Although transesterification processes using alkali catalysts give high conversion of fatty oil glycerides to esters in relatively shorter residence time, besides interference of water and free fatty acids with the reaction, the processes based on alkali catalysts have several drawbacks. These drawbacks are the intensive energy required for the reaction, the difficulty in recovering the glycerol, the fact that the alkaline catalyst has to be removed from the product, and the high production of alkaline waste water that is

Fig. (1). Transesterification of triglyceride with alcohol.

Triglycerides from oils and fats are reacted with an alcohol in a reaction known as transesterification or alcoholisis in presence of a catalyst. The products of

the reaction are esters of fatty acids (biodiesel) and glycerol.


required for the treatment. Additionally, further several steps such as evaporation of methanol, removal of saponified products, neutrali-zation and concentration are needed to recover glycerol.

2.2. The Acid Transesterification

The acid process is the second method most commonly used for producing biodiesel. It consists of using an acid as catalyst, either a homogeneous acidic catalyst or a heterogeneous acidic catalyst. As homogeneous acidic catalysts, we find compounds such as sulfuric acid, hydrochloric acid, para-toluene sulfonic acid, methane sulfonic acid, etc. Although, sulfuric acid is the preferred catalyst for this kind of reaction [47,42,59,60]. Among the heterogeneous acidic catalysts, we can use strong acidic sulfonated ion exchange resins and acidic zeolites.

Similarly to the alkali reaction, if an excess of alcohol is used in the experiment a better conversion of triglycerides is obtained, but recovering glycerol becomes more difficult and that is why optimal relation between alcohol and raw material should be determined experimentally considering each process as a new problem. The possible operation condition is, usually, molar ratio 30:1. The type of alcohol, as well as the oils, is the same as the one that can be used in alkali catalyst reaction. The amount of catalyst supposed to be added to the reactor varies from 0.5 to 1 mol%, and if the typical value is 1%, some authors prefer to use 3.5 mol% [61,62]. Temperature is another important factor for this kind of reaction with a range varying from 55 to 80oC. Thus the acid transesteri-fication is a great way to make biodiesel if the sample has relatively high free fatty acid content. In general, 1 mol% of sulfuric acid is a good amount for a final conversion of 99% in a time around 50 h.

The acid catalyzed reactions give very high yield in esters, though the drawback of that method is the long time required for the reaction to happen since the reaction usually requires longer than 24 hrs to finish. Several groups have proposed the esterification of triglycerides in fats and oils in a two-step process using an acidic catalyst in the first step followed by a second esterification using an alkaline catalyst. The methods can include specifications such as a reaction temperature of 60 to 120oC in order to restrain the interesterification reaction of the fat component [63]; the use of an acidic cation exchange resin as a solid esterification catalyst [64]; or the removal of the glycerol after the first step before the second step is started [65] in order to optimize the reaction. Nevertheless, Basu et al. and Gupta et al. proposed single step processes for producing esters from fat or from oil. The first group presented a process by mixing the feedstock with an alcohol, and a mixture of calcium acetate and barium acetate as catalyst, heating the reaction mixture to 200-250oC for about 3 hours and cooling the mixture rapidly [66], while the second group presented a method based in the use of alkyl Tin oxide as catalyst where glycerol can be separated from the fatty acid alkyl ester, as immiscible phase, by decantation, the excess alcohol can be recovered by evaporation or distillation and, the fatty acid alkyl esters can be purified by washing with water [67].

Due to the slow diffusion of triglycerides through the catalyst pores in heterogeneous catalysts such as supported metals, basic oxides and zeolites this type of transesterification reaction is slow, so higher alcohol to glyceride molar ratio is required to achieve appreciable conversion of above 70%.

In order to accelerate the reaction, the reactants can be exposed to microwaves or radio frequency energy, followed by the transfer of these reactants over a heterogeneous catalyst at sufficiently high velocity to produce high shear conditions. Then these emulsified reactants can be maintained at a pressure above autogeneous pressure for an improvement of the reaction. Using one or more of these steps can result in higher process rates, higher conversion levels, or both [68].

In general, there are two basic disadvantages related to chemical catalyzed biodiesel. The first one is that during catalysis, the process is relatively slow and secondly, these methods require removal of the catalyst. Additionally, and as we have mentioned before, during basic catalysis as well as purification of the catalyst product, the by-products of the saponification reaction must be removed. In conclusion some of the disadvantages of the homo-geneous chemical catalysis such as product purification can be overcome by using heterogeneous catalysts or enzymes as catalysts.

2.3. The Enzymatic Catalyst

In the enzymatic approach, the transesterification is carried out by a protein called lipase overcoming some drawbacks, such as difficulty in the recovery of glycerol and the energy-intensive nature of the process. In contrast to chemically catalysts, biocatalysts allow synthesis of specific alkyl esters, easy recovery of glycerol, and transesterification of glycerides with high free fatty acid content [69]. Lipases are enzymes used to catalyze some reactions such as hydrolysis of glycerol, alcoholysis and acidolysis, but it has been discovered that they can be used as catalyst for transesterification and esterification reactions too. Biocompatibility, biodegradability and environmental acceptability of this biotech-nological procedure are the desired properties in a green technology such as the biodiesel production. The extracellular and the intracellular lipases are also able to catalyze the transesterification of triglycerides effectively. Much work has been done on the lipase-catalyzed transesterification of triglyceride [69-75]. Furthermore, transesterification using a lipase is carried out in a nonaqueous system. When waste oil containing a large amount of water is used, the progress of the reaction would be inhibited. Therefore, facilities for removing water should be necessary, which increases the cost. A method for producing a fatty acid alcohol ester in the presence of water was described in [76] by using lipases. However, in this method, the enzyme has to be purified, and isolation of the enzyme takes time and increases the production costs. Thus, in the transesterification using a lipase, none of the above problems has been solved so that there is a demand for a method for producing a fatty acid ester more efficiently.

Foglia et al. proposed a method that utilizes lipases to transesterify triglyceride-containing substances and to esterify free fatty acids to alkyl esters using short chain alcohols such as 1-butanol and isobutanol. The proposed enzymes included 1,3-specific lipase from Mucor miehei and Rhizopus delemar, the acyl-specific lipase from Geotrichum candidum, and the nonspecific lipase from Candida antarctica and Pseudomonas cepacia, for the production of biodiesel [77]. But still and as mentioned earlier, one common drawback with the use of enzyme-based processes is the high cost of the enzyme. In this respect, immobilization of enzymes has generally been used to obtain reusable enzyme derivatives, enabling in the recycling of the biocatalyst and hence lowering this cost. In the case of biocatalysts in nonaqueous media, immobili-zation is also reported to result in better activity. Thus, many trans-esterification processes employing lipases have used an immobilized form of the enzyme [74,75,78-80].

Wu and Chen described a technique to increase and regenerate the activity of immobilized lipases. The technique deals with the effect caused by physical factors, such as the immiscibility between the alcohol and the fatty acid glycerides. When methanol or ethanol is absorbed into the voids of an immobilized lipase, the entry of fatty acid glyceride into the voids will be blocked, stopping the reaction from taking place. To wash the deactivated immobilized lipase, we need to use a solvent that has to be harmless to the lipase, and with a good solubility to oil, grease, moisture, and methanol or ethanol. The proposed solvents were iso-propanol, 2-butanol and tert-butanol [81-83]. The inventors also discover that the activity of an immobilized lipase can be significantly increased when such an


ideal solvent is used to perform an immersion pretreatment on an immobilized lipase.

The enzymatic method has been proposed to make biodiesel directly from the soapstock generated after the alkali refining processes. The soapstock contains 10-60% water, but also contains 35-85% of fatty derivatives including partial glycerides. The combination of this technology with feedstock availability offers an economic and competitive approach to produce biodiesel, since for each metric ton of alkali refined vegetable produced by this method, approximately 30 kg of soapstock is generated too. In this way, the feedstock (the soaps) can be neutralized and split with strong acids until pH 2-8 is reached, and then they can be subjected to an enzymatic esterification using lipases at a temperature of 30 to 45oC [84]. To reduce the cost of keeping these temperatures, a special type of lipase has been described for the production of biodiesel at low temperature. The lipase is produced by Micrococcus antarc-ticus and has good activity and stability at lower temperatures saving the cost of warming up the reaction [85].

Nevertheless, there are three major difficulties in using lipases to produce biodiesel. Still the first difficulty is that the price of lipase is much higher than the price of alkali even when the lipase is reused. Secondly, the lipase process requires up to 48 hours to complete the reaction, which is significantly longer than that for the base catalysis. The third difficulty with enzyme catalysis is that, for transesterification with methanol which is the preferred alcohol for biodiesel preparation of the reaction is extremely sluggish and proceeds in most cases with only low conversion efficiency, if at all. In this way, the use of intact microorganisms containing lipases has been described as well for the esterification of glycerides where fatty acid esters can be prepared in a simple manner avoiding the cost of enzyme purification [86]. In contrast, synthesizing biodiesel through bio-methods has the following advantages: the reaction conditions are milder, there are no toxic emissions, the reactions are not affected by free fatty acid nor by small amounts of water in the renewable oils used as raw materials. On top of that there is a possibility of regeneration and reuse of the immobilized residue, because it can be left in the reactor if the reactive flow is kept. The use of enzymes in reactors allows use of high concentration of them and that makes for a longer activation of the lipases. Additionally, the 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. Finally, the separation of products is easier using these biocatalysts. Nevertheless further research needs to be made in order to reduce the high cost for biotechnological production of biodiesel fuel, specially the use of lipase, immobilized cells or algae cultivation. Further discussion will be made in this respect on the current and future developments section.

2.4. The Supercritical Method

Chemically catalyzed triglyceride transesterification requires a multiple step process with one or more batch reactors. Initially, the triglycerides and alcohol form two immiscible liquid phases, and as the reaction proceeds, two separate liquid phases are formed. One contains the newly formed alkyl esters of the triglyceride and the other contains the glycerol, with the excess alcohol, catalyst and feed oil being dispersed into both phases. The reaction time for each step takes at least several hours and once completed, the liquid products must be allowed sufficient time to separate phases before additional processing and separation steps can occur to produce the final products. It is important to recycle the excess of alcohol and the unused catalyst must be neutralized.

Even the most efficient of the traditional chemically catalyzed processes require multiple hours to process each batch of feed. Additionally, significant problems arise in the separation steps. Significant quantities of glycerol left in the alkyl esters diminish the quality of the diesel fuel and likewise contaminated glycerol also loses much of its value compared to pure uncontaminated glycerol.

Finally, the separation procedures necessary to adequately clean the two product-streams produce large quantities of wastewater thereby creating additional cost and process complexity therefore affecting the environment.

Such processes are also costly due to the high cost of catalysts, heterogenization of catalysts, which is even more remarkable for the enzymatic transformation. Recently, a non-catalytic transes-terification reaction in supercritical methanol has been proposed [87-94]. A Super Critical Fluid is defined as a substance above its critical temperature and critical pressure. The critical point represents the highest temperature and pressure at which the substance can exist as a vapor and liquid in equilibrium. In order to reach the super critical state the alcohol and vegetable oil are pumped into a reactor with the pressure between 20 Bar and 400 Bar and a temperature between 150oC and 450oC. A mixture of non-reacted alcohol, glycerol, and esters is thereby formed, and is released via a flow and pressure control valve. The mixture of non-reacted alcohol, glycerol, and esters is then cooled in a cooler, and an upper phase of non-reacted alcohol is separated from the intermediate phase of esters and the lower phase of glycerol. The intermediate phase of esters and the lower phase of glycerol are then directed to a separation reservoir where the esters are separated from the glycerol. There are specific protocols for the Super Critical Method in continuous mode such as the described by Dall et al. [95] or where the supercritical state is only applied to the alcohol [96]. However, the main disadvantage of this method is the energy required to obtain the supercritical state that increases greatly the production cost of the chemical catalyzed methods.

A summary of the advantages and disadvantages of each technological possibility to produce biodiesel could be found in Table 1 (from Marchetti et al. [97]).

3. APPARATUS AND REFINERIES

Another area of research of interest is the use of different apparatus and refineries in order to perform reactions easily, quickly, efficiently, and safely. Both batch and continuous-flow technologies have been used to set-up and scale-up the reaction [98-100]. Although in general biodiesel is produced by the batch-type process, the disadvantages of the batch-type process are time consuming for the process, the unreliable production quality and the high labor cost. Alternatively the preferred method is known as the Connemann process [101]. In contrast to batch reaction methods [47], the Connemann process utilizes continuous flow of the reaction mixture through reactor columns, in which the flow rate is lower than the sinking rate of glycerol as shown in Fig. (2). This results in the continuous separation of glycerol from the biodiesel. The reaction mixture may be processed through further reactor columns to complete the transesterification process. Residual alcohol, glycerol, free fatty acids and catalyst may be removed by aqueous extraction.

In 1996, Krawczyk presented a flow diagram for producing biodiesel via transesterification on the industrial scale. The process mainly consisted of a transesterification reactor, an alcohol/glycerol distillation column and a methyl/ethyl ester distillation column. A continuous deglycerolization process to produce biodiesel from refined rapeseed oil by alkali-catalyzed transesterification at ambient pressure and 65-70 °C was introduced by Connemann and Fischer [102]. In this process, a distillation column was also used to separate methanol from biodiesel and glycerol. The alcohol was recycled to the transesterification reactor and multi-stage water washing was employed to purify the biodiesel product. The above industrial manufacturing information on biodiesel production formed the principle basis for the design of the alkali-catalyzed processes.

More recently, researchers have put forward several continuous production processes using catalytic methods, which can increase the production efficiency and quality of biodiesel [103-107].


Fig. (2). Simplified flow scheme of biodiesel production plant as described

by Connemann et al. in [101].

Triglycerides are supplied from the oil tank at a rate of 57 l/h through a heat

exchanger, together with the alcohol (1.4-1.6 times) and catalyst (0.24-

0.36% w/w) into a mixer at a rate of 10 l/h. Reaction mixture is passed into

the top of the first reactor, set up as a standing column at a lower rate of

flow than the glycerol sinking rate, being separated off in the direction of

flow. Transesterification of 85-90% already takes place in this column.

Reaction product is then passed into a second stirring reactor, where further

transesterification -up to 95-97%- takes place. Aqueous extraction solution

and separator are used to extract biodiesel. Further purification steps are

repeated in order to improve reaction efficiency and to remove, traces of

soap, alcohol, water and glycerol.

Both methods, batch and continuous-flow require an efficient blending of triglycerides with the alcohol and catalyst together at the transesterification reactor. Mixing efficiency of the reagents is of crucial importance and is considered as one of the most important parameters affecting the transesterification reaction. In order to guarantee a proper mixing several devices can be used, such as oscillatory baffled reactors [108-110], where a pulsed stream of a heterogenous multiphase mixture is passed through a flow tube. Alternatively, instead of the baffles described above, a metal helix can be present in the flow tube, obtaining in this way a much more thorough mixing of the heterogeneous mixture, increasing in this way the biodiesel yield during the transes-terification process [111]. Intensive mixing can also be achieved using centrifugal pumps, high shear mixers, or motionless mixers [55,112]. The disadvantage of high intensity mixing is that the presence of soap materials, partially reacted triglycerides, or excess alcohol can lead to stable emulsions and very slow separation of the ester and glycerol phases.

Ultrasonic reactors have been described for chemical activation via sonochemistry, where reaction is induced by ultrasonic sound waves. These waves are provided through the energy derived from the collapse of cavitation bubbles generated within the ultrasonic reactor. Such energy is derived when the cavitation bubbles collapse rapidly and violently and, in doing so, generate apparent temperatures of many thousands of degrees Kelvin and apparent pressures of several thousand atmospheres within the individual bubbles. Thus, the bubbles that are formed serve as individual microreactors operating at high temperature and high pressure while the liquid region immediately adjacent to the bubbles is at temperatures ranging generally from about 70oC to 80oC and at between 1.0 and 5.0 atmospheres of pressure. Through asymmetric collapse of the bubbles, micro jets are created, leading to intensive mixing of the liquids [113]. Microwaves are reported to greatly speed up the hydrolysis and esterification processes, reducing the reaction time to around one quarter of the time required by the conventional process and reducing the quantity of required alcohol to just over the stoichiometric quantity. In order to use microwaves Pyrex bioreactors can be used [114]. Additionally the reactor can be preceded by different devices to precondition the reagents in order to improve the reaction, from vacuum chambers to introduce the alcohol in gaseous form in the reaction mixture and to keep the reaction vessel under vacuum (i.e. 15-50 mm Hg) and at temperatures of 180-200oC [115], or an atomiser (atomization apparatus) to produce the vaporization of the alcohol with the chemical catalyst in a mixing chamber prior to entry into the reaction vessel [116].

Sometimes pretreatment of the feedstock is needed. In order to pretreat the oil feedstock before it is added to the reactor different

Table 1. Comparison of the Different Technologies to Produce Biodiesel (from Marchetti et al., [96])

Variable Alkali catalysis Lipase catalysis Supercritical

Alcohol.

Acid catalysis

Reaction temperature (oC) 60-70 30-40 239-385 55-80

Free fatty acid in raw materials Saponified products Methyl esters Esters Esters

Water in raw materials Interference with reaction No influence Interference with reaction

Yield of methyl esters Normal Higher Good Normal

Recovery of glycerol Difficult Easy Difficult

Purification of methyl esters Repeated washing None Repeated washing

Production cost of catalyst Cheap Relatively expensive Medium Cheap


apparatus have been developed. The design of the apparatus depends on the type and quality of feedstock. In the case of algae feedstock, there are apparatus for separating algae cells from liquid medium, for example based on a tangential flow filter device, [117], or based on a multi-stage loop-flow flotation column where bubbles are used to adsorb the algal cells that can be separated from the aqueous medium [118] or based on whirlpool circulation using special bags [22]. Modified cylindrical screening can be used as filters when waste oils and fats are used as feedstock [119], or the above mentioned filtering devices for waste fryer oils based on vacuuming and heating passing the heated fluid via a strainer [36,37]. Devices containing particles or polymers that include amine or imine moieties for filtering contaminants formed upon heating or cooking with oil are also used [120]. Tysinger et al. described a process in which free fatty acids and other impurities may be removed from mechanically extracted soybean oil by physical refining. In physical refining, the oil is vacuum distilled at high temperatures to separate more volatile components from the oil. However, this physical refining is of limited value in refining solvent extracted soybean oil, due to the higher levels on elemental phosphorous content, of non-hydratable phospholipids, and the high temperatures required for physical refining, may suppose an increase in production cost non affordable for the prerequisites of this product [121]. In order to reduce the production cost a compact processor including a vapor recovery system for removing excess alcohols from the fuel has been developed as well. This vapor recovery system includes a system for conserving and storing the excess alcohol for further use in the processing of biodiesel fuel so the process can be scaled down as well for small refineries [122]. For industrial scale, different technologies are also applied in order to recover the methanol and methoxide vapor from the reaction vessel by condensing it [123]. This selectively continuous process of separating the reaction products eliminates the costly washing step, and those further steps associated with such processing, because continuous separation is more efficient than batch decantation, yielding reaction products of greater purity. No additional water is introduced into the system since water washing has been eliminated and because the vacuum system used to recover the methanol from the FAME and glycerol products is also dry. This permits the use of a molecular sieve column to eliminate the minute amounts of water present in the excess methanol, whether water comes from air leaks into the system or from moisture in the natural oil feed etc., rather than rectification of the excess methanol from the wash water using distillation as in the batch approach. Desiccant columns equipped with molecular sieves are used to dry the recovered excess methanol. Bam and coworker designed the technology to not only recover the excess alcohol but to recover unspent catalyst. Since unspent catalyst is contained in the by-product alcohol, the recycle of the by-product alcohol also recycles unspent catalyst, which reduces the amount of fresh catalyst required. Vapors comprising unreacted alcohol from the extractive distillation are condensed in condenser located just after the reactor [124].

Once the reaction has occurred, separation of the alkyl esters is needed. Stidham et al. [125], Hayafuji [126] and Wimmer [127] disclosed the processes to wash impurities such as soap particles from the product alkyl ester. A more recent approach is based on bringing unpurified biodiesel in contact with cellulose-containing material by mixing the latter in order to eliminate the glycerol and catalyst residues, such as potassium soaps, accumulated during the production of the biodiesel. The system includes an agitated sedimentation tank with a conduit for delivering purified biodiesel into de agitated sedimentation tank [128].

Once the reaction has taken place in the reactor an alcohol/ glycerol distillation device has to be included. Alasti has recently described a refinery for the separation of alcohol, glycerol, esters and salts performed by volatility [129].

4. AVOIDING DRAWBACKS

4.1. NOx (Nitrogen Oxide) Emissions

If biodiesel improves engine emissions in most categories when compared to petroleum based diesel fuel, however it is also responsible for an increase in nitrous oxide emissions. Blends containing higher concentrations of biodiesel, show a proportional increase in emissions of nitrogen oxides (NOx). Presently, NOx emissions could be a significant limitation to the widespread adoption of biodiesel fuels. NOx is a generic term for mono-nitrogen oxides. These oxides are produced during combustion, and are of interest as air pollution. They are believed to aggravate breathing conditions, to produce ozone at surface heights, which is also an irritant, and to react with the oxygen present in the air, and eventually can form nitric acid when dissolved in water [130]. When NOx is dissolved in atmospheric moisture it can result in acid rain that potentially can damage entire forests. Some researchers have sought to address the NOx issue by the addition of certain fuel additives to the biodiesel. For example, Bradin described a fuel additive composition including fatty acid alkyl esters and glyceryl ethers. The additive-containing fuel is made by a multi-step process that includes separation of glycerol from biodiesel, conversion of glycerol to glycerol ether, and then addition of the glycerol ether back into the biodiesel fuel [52]. Other researchers describe controlling engine emission NOx by producing a blending of a synthetic middle distillate derived from Fischer-Tropsch produced synthesis gas (syngas) and biodiesel, which generate very low levels of NOx and sulfur emissions when used in diesel engines [131]. Addition of water to the fuel, to cool down the combustion process reduces the formation of NOx as well. However, that process undesirably lowers fuel BTU (British Thermal Units) value by replacing fuel with water. A large number of materials have been found to be catalytically active for selective catalytic reduction of NOx. By far, the most widely reported in the open literature are metal-exchanged zeolites, such as Cu-ZSM-5, Co-ZSM-5, and Fe-ZSM-5. However, the zeolite-based materials lose much of their activity when water (common in exhaust streams) is added to the exhaust stream. While other catalysts that possess greater water stability have been found, such as metal-supported oxides, these tend to lack the desired activity shown by the metal zeolites, and produce large amounts of N2O (also a major pollutant). Baacke et al. described a zeolite catalyst and a method for reduction of nitrogen oxides by mixing the waste gas with ammonia [132]. However, the use of ammonia in the reduction of nitrogen oxide is less desirable, especially in transportation systems, due to the toxic nature of the ammonia that must be stored locally, presenting a hazard.

On the other hand, Green et al. described a single phase catalyst obtained by intimately mixing the powders of the oxide binder and the metal salts. This patent describes the mixing of particles of a metal exchanged zeolite with an oxide [133,134]. Also Sachtler and Chen described that the metal can be introduced into the zeolite in an entirely different way, such as by metal vapor deposition [135]. Recently advances in the use of cerium-oxide, have been described to hold the potential to nearly eliminate NOx emissions from both petrodiesel and biodiesel, and diesel fuel additives based on cerium oxide that can improve fuel consumption by 11% in unmodified diesel engines [136]. Recently, a catalyst for reducing nitrogen oxide has been developed to remove NOx and particulate matter using platinum as activating element [137].

4.3. Oxidation Stability

A different problem in using biodiesel is that since biodiesel has a higher content of unsaturated fatty acid esters, it easily oxidizes in the presence of oxygen, UV light, heat, or trace metals, such as iron or copper. The products formed from this oxidation give rise to sediment and gum formation within the fuel and lead to corrosion


and plugging in injection pumps and fuel lines in engines, heaters, and machines which utilize this fuel source. Consequently, there is a need for a biodiesel fuel composition with improved oxidation stability. Asbahr and Bomba described the use of phenyl compound, and derivatives as an antioxidant for biodiesel [138]. The list of possible antioxidants was extended by Abou-Nemeh Ibrahim, mainly consisting in aromatic derivatives such as 3,4,5-trihydroxybenzoic acid n-propyl ester, 1,2,3-trihydroxybenzene, butylated hydroxyanisole, 2,6-di-tert-butyl-1-hydroxy-4-methyl-benzene, alpha-tocopherol acetate, alpha-tocopherol, gamma-tocopherol, delta-tocopherol, propyl gallate, dodecyl gallate, gallic acid, octyl gallate, ascorbyl palmitate, pyrogallol, alpha naphthol, natural tocopherol, eugenol, or 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, although non aromatic compounds are included as well such as dilauryl thiodipropionate, isopropyl 2-hydroxy-4-methylthio butanoate, lecithin, stearyl citrate, palmityl citrate, chlorophyll, ascorbic acid, citric acid, etc [139]. The use of aromatic derivatives along with N- and S-containing antioxidants can undermine the environmentally friendly nature of biodiesel as a clean fuel by polluting the environment, an important reason for focusing on the non aromatic and the non N- and S-containing compounds as antioxidants.

4.3. Glycerol Production

The transesterification of oils and fats results in the generation of glycerol as a byproduct of biodiesel. The free glycerol formed must be removed from the mixture of esters, since glycerol can cause damage to the engine [140]. Free and bonded glycerol, are the main parameters for defining the quality of biodiesel. During the transesterification process, free glycerol can easily be removed by washing steps, whereas a low content of glycerides can only be achieved by the use of specific catalysts and reaction conditions or by further distillation of the product. A higher content of free glycerol may cause problems during storage or in the fuel system, due to separation of glycerol, or can lead to injector fouling or the formation of higher aldehyde emissions. A higher content of glycerides, especially triglycerides, may cause formation of deposits at the injection nozzles, at the piston and at the valves [141]. A simple method for the almost complete removal of glycerol from methanol-free biodiesel streams coming out from industrial transesterification reactors has recently been described by Yori et al. The method is posed as a "dry" alternative to the conventional "wet" methods involving water washing. It is based on the use of silica beds and relies on the adsorption at room temperature to retain the small amounts of glycerol dissolved in the solutions of fatty acid methyl esters and adjust their content to the quality standards for biodiesel fuel [142]. Nevertheless, the difficulty for the removal of glycerol as by-product is mainly depending on the technical process of transesterification (see Table 1). Once glycerol surplus is removed it can be used in different ways as described previously.

5. CURRENT & FUTURE DEVELOPMENTS

Energy and climate change are the now main subjects among the scientific community, and modern society. And there is broad agreement that there is no single solution to these problems of meeting future demands for energy and managing the environmental consequences of energy production. The future developments will consist of set of many different technologies. Recent scientific advances focus on basic biology and technology that can be applied to metabolic engineering in order to comply with these needed energy production.

Biodiesel is a good alternative energy source and a substitute for petroleum-based diesel fuel with the available technology. The current scientific and technological knowledge can provide the means to obtain a biofuel with net energy gain, producing environmental benefits and at a cheaper cost than that for the

petroleum-based diesel. However, different and combined proto-cols, apparatus and feedstock would be needed in order to produce biodiesel in large quantities.

The most promising alternatives in order to obtain an environmentally friendly product are based on technology with biological origin especially those methods catalyzed by lipases. This technology works at the lowest reaction temperature; it is not influenced by the presence of water or free fatty acid in the feedstock, hence reducing the production cost, and produces the highest yields of biodiesel. In addition, the lipase technology allows an easy recovery of glycerol, without the need of purifying the methyl esters. It is of paramount importance to reduce the production cost derived from the protein purification though. Therefore, the use of whole microorganisms, as biodiesel cell factories, with the ability to synthesize alcohol esters could save this enzyme purification cost.

Steinbüchel and coworkers have created a fuel-refining Escherichia coli bacterial strain by modifying it with genes taken from two other microorganisms, Zymomonas mobilis and Acinetobacter baylyi [143]. The modified E. coli were cultured in growth medium containing glucose and oleic acid, which the bacteria processed into fatty acid ethyl esters, a diesel-substitute named as "microdiesel". This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach Steinbüchel et al., managed to obtain ethanol formation and to combine it with a subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids. Using this transgenic microorganism the obtained concentration of FAEE was of 1.28 g/L and the FAEE content of the cells was of 26 % of the cellular dry mass, that were achieved by fed-batch fermentation using renewable carbon sources.

This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered microorganisms, enabling a broader use of biodiesel-like fuels in the future.

Unlike regular biofuel, microdiesel is produced without toxic chemicals. Waste oils could allegedly be used as source of fatty acids, reducing the need to grow crops specifically for biodiesel production and therefore reducing the production cost of this fuel.

ABBREVIATIONS

IEA = International energy agency

NYMEX = New York mercantile exchange

IPCC = Intergovernmental panel on climate change

NREL = US National renewable energy laboratory

FAMEs = Fatty acid methyl esters

FAEEs = Fatty acid ethyl esters

DAF = Dissolved air flotation

SCF = Super critical fluid

BTU = British termal units

ACKNOWLEDGEMENTS

Maximino Manzanera was granted by Programa Ramón y Cajal (MEC, Spain and ERDF, Euopean Union).

REFERENCES

[1] International Energy Agency. Oil Market Report. A monthly oil market and

stocks assessment. 13 March 2007. http://omrpublic.iea.org/

[2] http://www.nymex.com

[3] IPCC WGI Fourth Assessment Report. Intergovernmental panel on climate

change. February, 2007.

[4] Mittelbach M, Pokits B, Silberholz A. Production and fuel properties of fatty

acid methyl esters from used frying oil. In: Liquid fuels from renewable


Resources. Proceedings of an alternative energy conference. ASAE

Publication, Nashville, TN, USA. 1992; 74-78.

[5] Peterson CL, Reece DL, Cruz R, Thompson J. A comparison of ethyl and

methyl esters of vegetable oil as diesel fuel substitutes. In: Liquid fuels from

renewable resources. Proceedings of an alternative energy conference. ASAE

Publication, Nashville, TN, USA. 1992; 99-110.

[6] Peterson CL, Reece DL, Hammond BJ, Thompson J, Beck SM. Processing,

characterization and performance of eight fuels from lipids. ASAE Paper

1994: 94-6531.

[7] Vellguth G. Performance of vegetable oils and their monoesters as fuels for

diesel engines. SAE Paper 1983: 831358.

[8] Chang DYZ, Van Gerpen JH, Lee I, Johnson LA, Hammond EG, Marley SJ.

Fuel properties and emissions of soybean oil esters as diesel fuel. JAOCS

1996; 73: 1549-1555.

[9] Schmidt CW. Biodiesel: Cultivating alternative fuels. Environ Health Persp

2007; 115: 86-91.

[10] Sommerville C. Biofuels. Curr Biol 2006; 17:115-119.

[11] Beer, S.V., Qiu, D., Wei, Z.M.: CA2274307 (1998).

[12] Choo, Y.M., Ma, A.N., Basiron Y., Yung, C.L., Cheng, S.F.: US2006288637

(2006).

[13] Despeghel J.P.: WO06128881 (2006).

[14] Ghosh, P.K., Shethia, B.D., Parmar, D.R., Pandya, J.B., Gandhi, M.R.,

Rathod, M.R., Patel, M.G., Vaghela, N.K.K., Dodia, P.J., Parmar, R.A.,

Patel, S.N., Dimurthy, S.: WO06043281 (2006).

[15] Wiesman, Z., Herskowitz, M., Grinberg, S.: WO06126206 (2006).

[16] Kerkman T.M.: WO06102632 (2006).

[17] Roessler PG, Brown LM, Dunahay TG, et al. Genetic-engineering

approaches for enhanced production of biodiesel fuel from microalgae. ACS

Symp Ser 1994; 566: 255-70.

[18] Sawayama S, Inoue S, Dote Y, Yokoyama S-Y. CO2 fixation and oil

production through microalga. Energy Convers Manag 1995; 36: 729-31.

[19] Dunahay TG, Jarvis EE, Dais SS, Roessler PG. Manipulation of microalgal

lipid production using genetic engineering. Appl Biochem Biotechnol 1996;

57-58: 223-31.

[20] Banerjee A, Sharma R, Chisti Y, Banerjee UC. Botryococcus braunii: a

renewable source of hydrocarbons and other chemicals. Crit Rev Biotechnol

2002; 22: 245-79.

[21] Gavrilescu M, Chisti Y. Biotechnology - a sustainable alternative for

chemical industry. Biotechnol Adv 2005; 23: 471-99.

[22] Sears, J.T.: US2007048848 (2007).

[23] Metting FB. Biodiversity and application of microalgae. J Ind Microbiol Biot

1996; 17: 477-489.

[24] Spolaore P, Joannis-Cassan C, Duran E, Isambert A. Commercial

applications of microalgae. J Biosci Bioeng 2006; 101:87-96.

[25] Metzger P, Largeau C. Botryococcus braunii: a rich source for hydrocarbons

and related ether lipids. Appl Microbiol Biotechnol 2005; 66: 486-96.

[26] Guschina IA, Harwood JL. Lipids and lipid metabolism in eukaryotic algae.

Prog Lipid Res 2006; 45:160-86.

[27] Casadevall E, Dif D, Largeau C, Gudin C, Chaumont D, Desanti O. Studies

on batch and continuous cultures of Botryoccus braunii: hydrocarbon

production in relation to physiological state, cell structure, and phosphate

nutrition. Biotechnol Bioeng 1985; 27: 286-295.

[28] Nonomura, A.M.: US2006265800P (2006).

[29] Murakami, N.: JP9234055 (1997).

[30] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007; 25: 294-306.

[31] Tilman D, Hill J, Lehman C. Carbon-negative biofuels from low-input high-

diversity grassland biomass. Science 2006; 314: 1598-1600.

[32] Canakci M. The potential of restaurant waste lipids as biodiesel feedstocks.

Bioresource Technol 2007; 98: 183-190.

[33] Iijima, W.; Kobayashi, Y.; Taniwaki, K.: US2006288636 (2006).

[34] Lastella, J.P.: WO03022961 (2003).

[35] Hammond, E.G.; Wang T.: US20056965044 (2005).

[36] van Noorden, P.: EP1357277 (2003).

[37] Patten, J.P.: US2005006290 (2005).

[38] Fukuda, H.; Noda, H.: US20066982155 (2006).

[39] Zappi, M.; French, W.T.; Hernandez, R.; Dufreche, S.T.; Sparks, D.:

KR20060081714 (2006).

[40] Hamilton, R.B.: WO06115422 (2006).

[41] Schwab AW, Dykstra GJ, Selke E, Sorenson SC, Pryde EH. Diesel fuel from

thermal decomposition of soybean oil. JAOCS 1988; 65:1781-1786.

[42] Freedman B, Butterfields RO, Pryde EH. Transesterification kinetics of

soybean oil. JAOCS 1986; 63:1375-1380.

[43] Ma F, and Hanna MA. Biodiesel production: a review. Bioresour Technol

1999; 70:1-15.

[44] Trent, W.R.: US2383633 (1945).

[45] Vieira, J.A., Sabba, M., Dias-Bruna, S., Ferreira, C., De Menezes, S.:

WO07020465 (2007).

[46] Wright HJ, Segur JB, Clark HV, Coburn SK, Langdon EE, DuPuis RN. A

report on ester interchange. Oil and Soap 1944; 21: 145-148.

[47] Freedman B, Pryde EH, Mounts TL. Variables affecting the yields of fatty

esters from transesterified vegetable oils. JAOCS 1984; 61: 1638-1643.

[48] Stern, R., Hillion, G., Gateau, P., Guibet, J.C.: US4695411 (1984).

[49] Skopal, F., Komers, K., Machek, J., Koropecky, I.: CZ20050155 (2006).

[50] Kraft, P., Gilbeau, P., Gosselin, B., Claessens, S.: EP1770081 (2007).

[51] Jakobson, G., Siemanowski, W.: US4960953 (1990).

[52] Bradin, D.S.: US5578090 (1996).

[53] Gupta, V.P.: US5476971 (1995).

[54] Kesling, Jr H.S., Karas, L.J., Liotta, Jr J.: US5308365 (1994).

[55] Noureddini, H.: US20006015440 (2000).

[56] Bradin, D.S.: WO07027669 (2007).

[57] Srivastava A, Prasad R. Triglycerides-based diesel fuels. Renew Sust Energy

Rev 2000; 4:111-33.

[58] Wimmer, T.: US5399731 (1995).

[59] Harrington KJ, Darcy-Evans C. Trans-esterification insitu of sunflower seed

oil. Ind Eng Chem Prod Res Dev 1985; 24:314.

[60] Stern R, Perdu O, Hillion G. Replacement gas oils from vegetable oil esters:

an opportunity for some countries. Revue del Institut Francais du Petrole.

1988; 43: 883-893.

[61] Zhang Y, Dube MA, McLean DD, Kates M. Biodiesel production from

waste cooking oil: 1. Process design and technological assessment.

Bioresource Technol 2003; 89: 1-16.

[62] Aksoy HA, Kahraman I, Karaosmanoglu F, Civelekoglu H. Evaluation of

turkish sulfur olive oil as an alternative diesel fuel. JAOCS 1988; 65: 936-8.

[63] Kawahara, Y.; Ono, T.: US4164506 (1979).

[64] Jeromin, L., Peukert, E., Wollmann, G.: US4698186 (1987).

[65] Tanaka, Y., Okabe, A., Ando, S.: US4303590 (1981).

[66] Basu, H.N., Norris, M.E.: US5525126 (1996).

[67] Gupta, A.K., Bhatnagar, A.K., Kaul S.: WO05063954 (2005).

[68] Portnoff, M.A., Purta, D.A., Nasta, M.A., Pourarian, F., Zhang, J.:

EP1765762 (2007).

[69] Nelson LA, Fogolia TA, Marmer WN. Lipase-catalyzed production of

biodiesel. J Am Oil Chem Soc 1996; 73: 1191.

[70] Kaieda M, Samukawa T, Kondo A, Fukuda H. Effect of methanol and water

contents on production of biodiesel fuel from plant oil catalyzed by various

lipases in a solvent-free system. J Biosci Bioeng 2001; 91: 12-15.

[71] Kose O, Tuter M, Aksoy HA. Immobilized Candida antarctica lipase-

catalyzed alcoholysis of cotton seed oil in a solvent-free medium. Bioresour

Technol 2002; 83: 125-129.

[72] Wu WH, Foglia TA, Marmer WN, Phillips JG. Optimizing production of

ethyl esters of grease using 95% ethanol by response surface methodology. J

Am Oil Chem Soc 1999; 76:517-521.

[73] Abigor RD, Uadia PO, Foglia TA, et al. Lipase-catalysed production of

biodiesel fuel from some Nigerian lauric oils. Biochem Soc Trans 2000;

28:979-981.

[74] Belafi-Bako K, Kovacs F, Gubicza L, Hancsok J. Enzymatic biodiesel

production from sunflower oil by candida antarctica lipase in a solvent-free

system. Biocat Biotransf 2002; 20:437-439.

[75] Shieh CJ, Liao HF, Lee CC. Optimization of lipase-catalyzed biodiesel by

response surface methodology. Bioresour Technol 2003; 88:103-106.

[76] Fukuda, H., Otsuka K., Nomoto, F.: US2000270886 (2000).

[77] Foglia, T.A., Nelson, L.A., Marmer, W.N.: US5713965 (1998).

[78] Hsu AF, Jones K, Foglia TA, Marmer WN. Immobilized lipase-catalysed

production of alkyl esters of restaurant grease as biodiesel. Biotechnol Appl

Biochem 2002; 36:181-186.

[79] Shimada Y, Watanabe Y, Samukawa T, et al. Conversion of vegetable oil to

biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc

1999; 76:789-793.

[80] Iso M, Chen BX, Eguchi M, Kudo T, Shreestha S. Production of biodiesel

fuel from triglycerides and alcohol using immobilized lipase. J Mol Catal B

Enzymatic 2001; 16:53-58.

[81] Wu, W.T., Chen, J.W.: US20026398707 (2002).

[82] Wu, W.T., Chen, J.W.: DE10217607 (2002).

[83] Wu, W.T., Chen, J.W.: TW491890B (2002).

[84] Sato, S., Bueno, A.W., Shigueu, A.A.: WO06050589 (2006).

[85] Liu, H., Ma, Y., Xue, Y.: CN1611600 (2005).

[86] Fukuda, H., Noda H.: US20066982155 (2006).

[87] Saka S, Kusdiana D. Biodiesel fuel from rapeseed oil as prepared in

supercritical methanol. Fuel 2001; 80: 225-231;

[88] Kusdiana D, Saka S. Kinetics of transesterification in rapeseed oil to

biodiesel fuel as treated in supercritical methanol. Fuel 2001; 80: 693-693.


[89] Kusdiana D, Saka S. Effects of water on biodiesel fuel production by

supercritical methanol treatment. Bioresource Technol 2004; 91:289-295.

[90] Ginosar, D.M., Fox, R.V.: WO0005327 (2000).

[91] Saka, S., Fukuzono, T.: WO04108873 (2004).

[92] Boocock, D.G.B.: US20046712867 (2004).

[93] Siegfried, P., Ganswindt, R., Weidner, E.: US20016211390 (2001).

[94] Ijima, W., Kobayashi, Y.; Taniwaki, K.: US2006288636 (2006).

[95] Dall, A.A., Osvaldo,B.A., Dariva, C., Nascimento, S.E., Vladimir de Olivera,

J.: US2007010681 (2007).

[96] Noh, M.J., Yoo, K.P., Choi, Y.H.: WO07058485 (2007).

[97] Marchetti JM, Miguel VU, Errazu AF. Possible methods for biodiesel

production. Renew Sust Energ Rev 2007; 11:1300-1311.

[98] Bouaid A, Diaz Y, Martinez M, Aracil J. Pilot plant studies of biodiesel

production using Brassica carinata as raw material. Catal Today 2005;

106:193-196.

[99] Harvey AP, Mackley MR, Seliger T. Process intensification of biodiesel

production using a continuous oscillatory flow reactor. J Chem Technol

Biotechnol 2003; 78:338-341.

[100] Haupt J, Dimmig T, Dittmar T, Ondruschka B, Heyn B, Lauterbach M.

Production of standard specification biodiesel from rapeseed oil and spent

fats - Process concept. Chem Ing Tech 2003; 75:787-791.

[101] Connemann, J., Krallmann, A., Fischer, E.: US5354878 (1994).

[102] Connemann J, Fischer J. Biodiesel in Europe: Biodiesel processing

technologies. International Liquid Biofuels Congress, Brazil. (1998).

[103] Darnoko D, Cheryan M. Continuous production of palm methyl esters.

JAOCS 2000; 77:1269-1272.

[104] Noureddini H, Harkey D, Medikonduru V. A continuous process for the

conversion of vegetable oils into methyl esters of fatty acid. JAOCS 1998;

75:1775-1783.

[105] Harvey AP, Mackley MR, Seliger T. Process intensification of biodiesel

production using a continuous oscillatory flow reactor. J Chem Technol

Biotechnol 2003; 78:338-341.

[106] Kanit K, Ratchadaporm S. Continuous transmethylation of palm oil in an

organic solvent. JAOCS 1992; 69:166-169.

[107] Zhang Y, Dube MA, Mclean DD. Biodiesel production from waste cooking

oil: process design and technological assessment. Bioresour Technol 2003;

89:1-16.

[108] Colman, D.A., Tallis, W.: EP0631809 (1995).

[109] Xiongwei, N.: WO9955457 (1990).

[110] Colman, D.A., Scotland, J.M., Co BP Chemic, Stewart, D.: EP540180

(1993).

[111] Groen, H., Schuette, R., Drauz, K., Stadtmueller, K., Grayson, J.I.:

WO06092360 (2006).

[112] Noureddini, H.: US20016174501 (2001).

[113] Hooker, J.: WO05016560 (2005).

[114] Breccia, F.A.: WO03014272 (2003).

[115] Nimcevic, D., Gapes, J.R.: WO0188072 (2001).

[116] Farid, M., Behzadi, S.: WO07049979 (2007).

[117] Borodyanski, G., Konstantinov, I.: US2002079270 (2002).

[118] Guelcher, S.A., Kanel, J.S.: US5910254 (1999).

[119] Butler, C.: US2007033863 (2007).

[120] Soane, D., Berg, M.C., Mowers, W.A.: WO07016645 (2007).

[121] Tysinger, J.E., Richmond, J.F., Dawson, R.B., Farr, W.E.: US20036511690

(2003).

[122] Landano, A.: US2006260184 (2006).

[123] McDonald, W.M.: US20016262285 (2001).

[124] Bam, N., Drown, D.C., Korus, R., Hoffman, D.S., Johnson, T.G., Washam,

J.M.: US5424467 (1995).

[125] Stidham, W.D., Seaman, D.W., Danzer, M.F.: US20006127560 (2000).

[126] Hayafuji, S., Shimidzu, T., Oh, S., Zaima, H.: US5972057 (1999).

[127] Wimmer, T.: US5399731 (1995).

[128] Moser, A.: WO07056786 (2007).

[129] Alasti, P.: WO06036836 (2006).

[130] Hurley MD, Ball JC, Wallington TJ, et al. Atmospheric chemistry of a model

biodiesel fuel, CH3C(O)O (CH2)2OC(O)CH3: kinetics, mechanisms, and

products of Cl atom and OH radical initiated oxidation in the presence and

absence of NOx J Phys Chem A 2007; 111: 2547-2554.

[131] Esen, E., Boehman, A.L., Morris, D.P.: US2005210739 (2005).

[132] Baacke, M., Beand, R., Engler, B., Kiss, A., Kleine-Moellhoff, P.,

Kleinschmit, P., Koberstein, E., Siray, M.: US5116586 (1992).

[133] Green, G.J., Shihabi, D.S.: US4962075 (1990).

[134] Greene, H.L., Chatterjee, S.: US5276249 (1994).

[135] Sachtler, W.M.H., Chen, H.: US20006143681 (2000).

[136] Marshall, C.L., Neylon, M.K.: US2005101473 (2005).

[137] Park, J.-S., Lee, Y.-J., Yoon, W.-L., Lee, H.-T., Seo, D.-J., Cho, S.-H., Lee,

S.-K., Choi, S.-H., Yoo, K.-S.: WO07037652 (2007).

[138] Asbahr, H.O., Bomba, T.: KR0106903 (2006).

[139] Abou-Nemeh, I.: US2007113467 (2007).

[140] Goncalves LC, Micke GA. Development and validation of a fast method for

determination of free glycerol in biodiesel by capillary electrophoresis J

Chromatogr A 2007; 1154:477-480.

[141] Mittelbach M. Bioresour Technol 1996; 56:7-11.

[142] Yori JC, D'Ippolito SA, Pieck CL, Vera CR. Deglycerolization of biodiesel

streams by adsorption over silica beds. Energ Fuel 2007; 21:347-353.

[143] Kalscheuer R, Stolting T, Steinbuchel A. Microdiesel: Escherichia coli

engineered for fuel production. Microbiology-SGM 2006; 152: 2529-2536.

(6)biodiesel an alternative fuel

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