MMG 445 Basic Biotechnology eJournal

A review of the processes of biodiesel production

Michael Sheedlo
Research on the production of biodiesel has been significant lately due to the increasing price of petroleum. Gaining importance have been the catalysts and possible feedstocks to be used in the production of biodiesel. This reaction can be catalyzed using acid catalysis, alkali catalysis, biocatalysts and nonenzymatic heterogeneous catalysis. For any approach to catalysis, the major issue confronting biodiesel formation is the presence of water and free fatty acids which inactivate catalysis. The most promising method may be biocatalysis with the addition of acyl acceptors because this addresses the problem by eliminating the role these molecules in the reaction. Address Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 Corresponding author: sheedlom@msu.edu MMG445 Basic Biotechnology eJournal, 2008
This review comes from a themed issue based on current advances in the fields of applied microbiology, biotechnology, and pharmacology. It fulfills in part the assignment of the contributing author in MMG 445, Basic Biotechnology, Department of Microbiology and Molecular Genetics, Fall Semester, 2008. Editors - George M. Garrity and Scott H. Harrison

alcohol which results in a biodiesel molecule. An advantage of biodiesel is the ability to ignite in a standard compression ignition engine. This means that the current infrastructure in terms of engine-driven transportation and storage vessels will not need to be modified or overhauled. In fact, biodiesel is a less corrosive substance to transport than conventional petroleum diesel because it does not contain any polycyclic hydrocarbons which can be damaging to a wide variety of substances. This also explains the reason that engines undergo less wear and tear when using biodiesel. Another positive aspect is that it can be mixed with petroleum diesel in any ratio and still function. Because of these advantages, biodiesel may become a future solution to high gas prices [2]. The major issue confronting biodiesel formation today is the presence of water and free fatty acids in most oils. These molecules result in the formation of soap during the transesterification of the triglyceride, which decreases the yield and requires refinement to remove the soap. This is seen especially in large scale operations where an alkali catalyst is utilized. While it is possible to remove these molecules from the oils themselves, it is an expensive process and one which outweighs the benefits of using biodiesel. With current research showing a variety of oils that can be used in this process it becomes apparent that the type of oil used will ultimately dictate the price of biodiesel. Along with the choice of a feedstock, research is also focusing on the selection of a catalyst and the possible use of acceptor molecules [2].

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.

Keywords biodiesel, transesterification, biocatalyst, triglyceride, fatty acid Abbreviations HPA heteropolyacid

Introduction
With petroleum prices skyrocketing and gas reserves waning, there has never been a greater need for an alternative fuel source. Biodiesel is a forerunner in this research. A biodiesel is a high energy, organic compound that can be made from various triglycerides (oils), an alcohol and a catalyst through a transesterification reaction. This reaction is preferable because fatty acids are the most common source of reduced carbon chains [1]. In this reaction, the oil undergoes a transesterification directly from the

Transesterification of oils
To make a biodiesel molecule, an oil must undergo transesterification enabling a fatty acid to undergo esterification [2]. Transesterification and esterification of oils

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2 M. Sheedlo

and fatty acids can be accomplished in the presence of an alcohol and a catalyst. The overall stoichiometric view of this process is shown in Figure 1. Common alcohols used in this process are short chain alcohols, most notably methanol and ethanol [3]. The catalysts commonly used for this process are chemical catalysts, biocatalysts, and nonenzymatic heterogeneous catalysts.

Feedstock analysis
In general, virgin oils tend to have much less water and fewer free fatty acids than those of the unrefined oils but also tend to cost much more to produce. For example, refined soybean oil costs nearly $2.64 per gallon while non-refined soybean oil costs only $2.04 per gallon [5]. Though non-refined oils are much cheaper to produce, further refinement is required throughout the process. Oils suitable for use as a feedstock should have a free fatty acid content lower than 1% and a water content lower than 0.06% [6, 7]. Currently, the most common feedstocks used are rapeseed oil, sunflower oil, soybean oil and animal fats [8, 9]. Animal fats tend to be low in free fatty acids and water, but there is a limited amount of these oils available, meaning these would never be able to meet the fuel needs of the world [10]. There is also ongoing research of the production of suitable lipids produced through microbial growth. This process would utilize the lipids that are produced in molds, yeasts and algae during metabolic stress. In this scenario, 70% of the cellular biomass is lipid [3].

Figure 1: Stoichiometric reaction of a triglyceride and alcohol yielding a biodiesel molecule. The catalyst is undefined and may be any of those catalysts discussed. Based on Canakci M and Sanli H, 2008, J Ind Microbiol Biotechnol 35:431441.

One of the major issues of producing biodiesel is limiting the presence of water and free fatty acids in solution. This can result in the formation of soap in the presence of some catalysts. This can be directly linked to the quality of the feedstock used in the transesterification process. The discrepancy in the scientific world is the debate of whether non-refined oils or refined (virgin) oils should be utilized. The higher the grade and more refined the oil is, the less water it is likely to contain. Because of this, there is less interaction of water and free fatty acids in the reaction, thus biodiesels can be much more efficiently produced from higher grade oils. The drawback here, though, is the high cost of refining such oils [4]. To overcome this hindrance, solvents and other acceptor molecules are being tested to bind to the reactive ends of these molecules, rendering them inactive [2]. Another issue confronting the production of biodiesel is, during the transesterification of oil, glycerol is also formed in a 1:10 ratio. The presence of glycerol may require additional purification and has been shown to reduce the function of some biocatalysts because it can act as a substrate in some reactions [4].

Chemical catalysts
Chemical catalysts are the most studied and most often used catalysts during biodiesel formation. This can be accredited to the availability and low cost of these compounds. Chemical catalysts generally proceed at a faster rate than other forms of catalysis. In biodiesel production, there are two classes of chemical compounds used, alkali catalysts and acid catalysts. There is a large amount of research being conducted using these two systems independently, but research has just begun on using these two methods together. One such study has found that increased yield and diminished reaction time occurs when using an acid pretreatment on the oil, followed by the addition of an alkali catalyst. This increases the yield because the acid can efficiently lower the amount of free fatty acids in the oil while moderating the transesterification reaction. Once the free fatty acid content has been decreased, an alkali catalyst may be added,

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resulting in a faster reaction time as well as an increased yield [11].

is that it is much slower than other catalysts [12]. Another class of acid that can be used is heteropolyacids (HPA). These acids are generally more effective because they have greater proton mobility. Another benefit of HPAs is that they exist as solids and can mediate the transesterification reaction of liquid oils making them much easier to use than liquid acids. This would also address the problem that water creates as the multiple acidic protons on the HPAs would continue catalysis when bound to a glycerol molecule. Another advantage of this is the fact that these acids have been used in the food industry. This means that their properties have been extensively studied and documented. Though these molecules tend to be tolerant of water presence in oil, a recent study showed that these catalysts exhibited reduced catalytic activity in the presence of greater than 1% of water [12].

Alkali catalysts
The majority of recent research has been centered on the use of alkali catalysts to mediate the transesterification of fatty acids [3]. This process has been shown to be faster than that of the acid catalysts but dictates more moderated reaction conditions. This process requires less alcohol to be used in the reaction than acid catalysis also. These reactions however produce soap more commonly than those of acid catalysts when the content of free fatty acids in the oil is high. This occurs because the free fatty acids and water are neutralized more readily by bases than by acids. These reactions then, in turn, consume the catalyst. Because of these soap forming properties, alkali catalysts are inefficient in large commercial operations, where the levels of water and free fatty acids are difficult to regulate [2, 3].

Acid catalysts
Acids used in the catalysis of the transesterification of biodiesels are usually either hydrochloric acid or sulfuric acid. Though these two acids are the most common, any Bronsted acid can be used in this reaction [12]. A large amount of commercially produced biodiesel is synthesized using acid catalysis. For this process, an acid-catalyzed esterification processor is used because of the low price and the ability of acids to minimize the amount of water and free fatty acid involved in the reaction. This method is useful commercially, because the mass production of biodiesel usually results in the presence of a relatively large amount of free fatty acids. The use of an acid catalyst is observed to be more effective than alkali catalysts when the concentration of free fatty acids is high, greater than 1%. These reactions require post reaction clean-up though, because the acids involved produce a large amount of salt during the reaction which can be corrosive. Because of the presence of these salts, equipment maintenance also plays a crucial role in this process. Another drawback of this reaction

Biocatalysts
Biocatalysts have been becoming increasingly important in the discussion of biodiesel preparation lately. It is even hypothesized that these catalysts will eventually have the ability to outperform chemical catalysts [13]. Biocatalysts are naturally occurring lipases which have been identified as having the ability to perform the transesterification reactions that are essential to biodiesel production. These lipases have been isolated from a number of bacterial species: Pseudomonas fluorescens, Pseudomonas cepacia, Rhizomucor miehei, Rhizopus oryzae, Candida rugosa, Thermomyces lanuginosus, and Candida antarctica [2]. According to a recent study, the Pseudomonas cepacia bacterium is the most promising organism for producing a lipase that can be used in transesterification [14]. Many of these lipases have since become commercially available; one of the most popular is Novozym 435 [2, 9]. During experimentation, it has also been shown that the pH of solution can increase overall yields. Pseudomonas cepacia has an optimum pH of around 7, making it easier to use than other lipases while still producing a greater yield without requiring pH tuning.

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During the transesterification of triglycerides via biocatalysts, glycerol is produced. The presence of glycerol can then inhibit the reaction by binding to the biocatalyst instead of the triglyceride molecule. To overcome this, an acyl acceptor molecule can be used to bind the glycerol, forming a triglyceride molecule that can no longer bind to the active site of the biocatalyst. When an acyl acceptor is introduced to the reaction, the biocatalyst can exhibit a higher rate of turnover, proving more useful. It has also been shown that the triacetylglycerol molecule does not diminish the properties of biodiesel when used as a fuel [2]. For biocatalyst reactions, the use of an immobilized lipase has also yielded positive results. This process usually involves the use of Celite, an abrasive agent which remains solid in an aqueous solution. When coupled with a biocatalyst that also remains solid in solution, the Celite allows for the dispersal of the enzyme over a larger area, decreasing the mobilization of the enzyme, and thus increasing the catalytic activity [14].

Another compound that is being considered as a catalyst is sulfonized, pyrolized, sucrose. Though the initial research on this compound produced low yields, it is thought that the numerous sulfur bonds it is capable of making will increase the importance of such a molecule in the near future [2].

Conclusion
As of now, the future of biodiesel production is unclear and more research is required. One promising area is the use of biocatalysts to mediate the transester-ification process. Though much more is known about the use of chemical catalysts, most current research points in the direction of these biocatalysts for a couple reasons. First, lipases have much more opportunity for further development in terms of pH optimization, immobilization, and the addition of an acyl acceptor. Second they have recently been commercially produced which translates to a cheaper reaction overall. There is a possibility that nonenzymatic heterogeneous catalysts could further benefit the production of biodiesels, but much more research is needed in order to determine if this process is plausible.

Nonenzymatic heterogeneous catalysts

Another, relatively new, area of study is in the area of nonenzymatic heterogeneous catalysts. These include ZrO2, ZnO, SO4, 2-/SnO2, SO4 2-/ZrO2, KNO3/KL zeolite, and KNO3/ZrO2. The main advantages of these catalysts are that they are less corrosive and are more environmentally friendly. Because of this, they have the distinct possibility of lowering the cost of biodiesel formation. Non-enzymatic heterogeneous catalysts have also shown a reduction in the formation of soap, even in low quality oils, addressing the issue of expense when determining a feedstock for the formation of biodiesels. However, the reaction has to be carried out at 200 degrees Celsius and 50 bar with a 6:1 molar ratio of methanol to oil and 3% by weight catalyst to be used to maximize efficiency. During this study, all catalysts exhibited some degree of activity, but those which were sulfonated produced the highest overall yields [2].

References and recommended reading

Papers of special significance that have been published within the period of review are highlighted as follows: of significance of special significance 1. Thelen JJ, Ohlrogge JB: Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng 2002, 4:1221. PMID: 11800570 2. Vasudevan PT, Briggs M: Biodiesel productioncurrent state of the art and challenges. J Ind Microbiol Biotechnol 2008, 35:421430. PMID: 18205018 This paper is a review of the many different ways that a biodiesel can be made. It summarizes the many different pathways that catalysts can take and which seem to be the most productive as of now. 3. Li, B., Zongba, Z.: Biodiesel production by direct methanolysis of oleaginous microbial biomass. Journal of Chemical Technology and Biotechnology 2007, 82:775780. DOI:10.1002/jctb.1744 This is a primary research paper that focuses on the transesterification of biologically produced

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oils through the use of an acid catalyst. It focuses on the transesterification of oils produced by oleaginous yeasts. 4. Asad-ur-Rehman, Wijesekara SR, Nomura N. Sato S, Matsumura M: Pre-treatment and utilization of raw glycerol from sunflower o i l b i o d i e s e l f o r g r o w t h a n d 1,3propanediol production by Clostridium butyricum. J Chem Technol Biotechnol 2008, 83:10721080. DOI: 10.1002/jctb. 1917 5. Haas MJ, McAloon AJ, Yee WC, Foglia TA: A process model to estimate biodiesel production costs. Bioresource Technology 2006, 97:671678. PMID: 15935657 6. Canakci M: The potential of restaurant waste lipids as biodiesel feedstocks. Bioresource Technology 2007, 98:183190. PMID: 16412631 7. Kusdiana D, Saka S: Effects of water on biodiesel fuel production by supercritical methanol treatment. Biores Technol 2004, 91:289295. PMID: 14607489 8. Rashid U, Farooq A, Anwar F, Moser BR, Knothe G: Moringa oleifera oil: A possible source of biodiesel. Biores Technol 2006, 99:81758179. PMID: 18474424 9. Lai C, Zullaikah S, Vali SR, Ju Y: Lipasecatalyzed production of biodiesel from rice bran oil. J Chem Technol Biotechnol 2005, 80:331337. DOI: 10.1016/j.biortech. 2008.06.008 10. Zhu LY, Zong MH, Wu H: Efficient lipid production with trichosporon fermentans and its use for biodiesel preparation. Biores Technol 2008, 99:78817885. PMID: 18394882

This paper looks at the process of biodiesel formation, specifically dealing with lipids produced from Trichosporon fermentans. It is also a good paper to get information pertaining to the optimum pH of biodiesel formation. 11. Berchmans HJ,Hirata S: Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Biores Technol 2008, 99:17161721. PMID: 17531473 This paper addresses the issue of free fatty acid content in oils to be used for biodiesel formation from oleaginous microbes. It also addresses the presence of free fatty acids in oils produced by microbes. 12. Cao F, Chen Y, Zhai F, Li J, Wang J, Wang X, Wang S, Zhu W: Biodiesel production from high acid value waste frying oil catalyzed by superacid heteropolyacid. Biotechnol Bioeng 2008, 101:93-100. PMID: 18646228 This paper addresses the use of heteropolyacids during the transesterification of waste frying oil. 13. Sulaiman A, Wei FL, Lim SJ: Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Proc Biochem 2007, 42:951960. DOI: 10.1016/ j.procbio.2007.03.002 14. Shah S, Gupta MN: Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system. Proc Biochem 2007, 42:409414. DOI: 10.1016/j.procbio. 2006.09.024. 15. Canakci M, Sanli H: Biodiesel production from various feedstocks and their effects on the fuel properties. J Ind Microbiol Biotechnol 2008, 35:431441. PMID: 18338187

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