ISSN 20700504, Catalysis in Industry, 2011, Vol. 3, No. 1, pp. 57–61. © Pleiades Publishing, Ltd., 2011. Original Russian Text © V.A. Galynkin, A.V. Garabadzhiu, A.H. Enikeev, M.M. Karasev, G.V. Kozlov, 2011, published in Kataliz v Promyshlennosti.
BIOCATALYSIS
Marine Biological Resources: An Advanced Raw Material Base for Biofuel1 V. A. Galynkin, A. V. Garabadzhiu, A. H. Enikeev, M. M. Karasev, and G. V. Kozlov OAO HyproRybFlot, St. Petersburg, Russia ZAO NII Rosbio, St. Petersburg, 192019 Russia St. Petersburg Institute of Technology (Technical University), St. Petersburg, 190013 Russia Abstract—This work discusses the problems of nontraditional raw materials for biofuel, provides an analysis of studies on the synthesis of biodiesel in various lipids, and discusses the production of lipid material and the contribution from different sources to the overall resource balance. Key questions and problems of lipase catalysis, the sources and range of commercial preparations of lipases and biocatalysis are discussed in detail. Using of algal feedstock for the production of ethanol fuel is considered. Keywords: biocatalysis, enzymes, lipase, biofuel, ethanol, biodiesel, living marine resources, renewable energy. DOI: 10.1134/S2070050411010053 1
INTRODUCTION
then to natural gas, so world production of biodiesel fuel should rise to 23 million tons by 2020 [1]. The main consumers of oil, the United States and Euro pean Union, along with certain other countries that do not have their own oil and gas reserves, are the world leaders in the production of biofuel. Traditional oil exporters also interested in biofuel due to its ecological properties, but not so actively. The production of biodiesel has thus begun in several petroleum exporting countries: Azerbaijan, Nigeria, Norway, Venezuela, and Russia, and even in Saudi Arabia [2], which depends on external supplies of lip ids rather than oil.
One of the most rapidly developing fields of bioen ergy is now the production of biodiesel fuel (biodie sel). The European standard for biodiesel is EN 14214, which is made up of fatty acid monoalkyl esters derived from biologically produced oils or fats, includ ing vegetable oils and animal fats. Biodiesel fuels can also be produced using other alcohols (e.g., using eth anol to produce fatty acid ethyl esters), and various vegetable oils and animal fats (particularly fish oil) can be used as feedstock. Biodiesel contains no molecular sulfur, so it is much more environmentally friendly than traditional hydrocarbon fuels. In addition, its cetane number is 51, or 6–9 units higher than that of petroleum diesel fuel. As a result, there is no need to use additional igni tion boosters to start an engine. The use of biodiesel substantially increases the life cycle of an engine and fuel pump (up to 60% of the nominal resource). An additional advantage of biodiesel is its full bio logical recovery in the natural environment (it is com pletely hydrolyzed in the soil in one month), eliminat ing the disastrous consequences in the event of an oil spill. Switching to biodiesel would help maintain the existing ecological conditions of the environment. Biodiesel itself is biodegradable and does not contam inate natural water systems. Biodiesel is a renewable resource, whereas the formation of fossil fuels (coal, oil) took millions years. Interest in biodiesel is disproportionate to its share of the fuel balance. The conversion to biofuel is natu ral, as was the earlier conversion from coal to oil and
SOURCES OF BIODIESEL FEEDSTOCK Traditional sources of biodiesel in industry are veg etable crops, e.g., canola [4] (or rapeseed, the oil most resistant to low temperatures without additives down to –10°C), soybean [5], sunflower [6], and corn oil [7], along with olive oil [8], oil of saltwort [9], cotton oil [10], borage oil [6], oil of microalgae [11] and other vegetable oils [12]. The selection of raw material depends primarily on the geographical location of future production. For example, jatropha is mainly considered as a source of raw material in India [13]; biodiesel fuel production from palm oil is developing in Africa [14]; and tung oil tree Aleurites fordii [15] and stillingia oil from Sapium sebiferum [16] are used for the production of motor fuel in China. The maximum amount of lipid material that can be produced on the planet in one year is 51 billion liters. Fortyseven billion liters of this could be profitable at current prices for imports. Five countries—Malaysia, Indonesia, Argentina, the United States, and Brazil,
1 The article was translated by the authors.
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which together produce 80% of the planet’s vegetable lip ids—are the leading producers of the world’s two most popular oilbearing crops: palm and soybeans [3]. On the other hand, the most environmentally friendly and economically feasible strategy is the pro duction of biodiesel fuel from waste oils [7] and the treatment of fat [17] in the food industry and from fish waste [8]. To avoid using agricultural land to produce raw materials, pyrolysis technology is being developed for producing liquid product and fuel from synthesis gas, but these second generation products cannot be called biofuel: they are obtained through chemical engineer ing, and only the raw material is biological. This is also true of first generation biodiesel fuels, but the products from the pyrolysis of biomass or the synthetic fuels produced from synthesis gas lack a major advantage of biodiesel—environmental com patibility. Moreover, this process is expensive and technically complex. We are thus faced with a dilemma: the first genera tion technologies are simple and inexpensive, but they impact the food market, while second generation technologies are more complicated, expensive, and have no environmental benefits. The best choice is to use raw material that does not affect the food market and can be processed using first generation technologies. Waste frying oils (whose share in the production of biodiesel is 5%) meet these requirements, but they contain carcinogens that remain in the hydrophobic phase (i.e., in the fuel) dur ing processing. One source of alternative lipid materials could be a marine bioresource: trash fish. Trash fish make up an appreciable part of the catch of fishing vessels. Ruff predominates on the Yamal coast, accounting for 66.8% of the catch [19]. One option for processing trash fish could be the production of biodiesel in coastal areas. The competi tive advantages of this are the nearly yearround avail ability of raw materials; independence from weather conditions; environmental safety; the preservation of arable land; increased profitability for the fishing fleet; and, most important, security for the food market. We have conducted industrial testing and developed the technology for producing biodiesel from fish oil, using potassium methoxide as a catalyst and a number of microapparatuses to create a compact biodiesel module that can be used on fishing vessels as well. Fish oil is used as a raw material for biodiesel fuel on an industrial scale: in the Canadian city of Halifax, municipal buses run on a unique blend of 80% petro leum diesel and 20% biodiesel [20]. As in the case of vegetable oils, lipids need refining in order to produce highquality biodiesel fuel; with regard to fish oil, there is one more aspect besides the extraction of water and phospholipids: polyunsaturated fatty acids. It would obviously make economic sense to introduce a
stage of isolating this highly valuable fraction into the technological process and to process the depleted oil into biodiesel fuel. Another source of lipid material could be unicellu lar algae, the fastest growing plants on the planet, which contain extremely high volumes of vegetable oil (up to 50 %), but their artificial breeding is already under way and a number of problems have been encountered: first, light penetrates only 4 cm into the interior of the plant mass; second, the water can evap orate and algae weeds can appear if bioreactors are used [21]. ENZYMES: PROMISING CATALYSTS FOR BIODIESEL The world’s present production of biodiesel is based mainly on alkaline catalysis. If the raw oil is more than 0.5% free fatty acids (FFAs), however, they are formed of soap, thus reducing the yield of biodiesel and creating problems in purifying biodiesel, a process that requires a great deal of washing water. In addition, the resulting byproduct (glycerin) is sufficiently con taminated (mainly with alkali) that additional costs are required for its cleanup. The enzymatic method of producing biodiesel using lipase can be considered an alternative. The advantages of lipase catalysis are – the method works under milder conditions (most lipases have an optimum temperature of 20–50°C); – the possibility of converting free fatty acids into biodiesel; – the ease of recycling the immobilized lipases; – it does not require flushing with large quantities of water; – its ability to handle wet materials; – the possibility of improving the catalyst geneti cally and optimizing it for specific processes. Disadvantages of lipase catalysis are – the high cost of enzymes; – its longer reaction time; – the risk of inactivating the lipase with the metha nol/ethanol and glycerol formed during reaction. Despite these problems, lipase catalysis is the most environmentally safe and promising technology for producing biodiesel. It allows us to obtain an environ mentally ideal product without causing damage to the environment. Today there are many commercially available lipases. The most widely used are listed below: – Novozym 435 is a lipase from Candida antarc tica, immobilized on macroporous polymethyl methacrylate. – Lipozyme RM IM is a lipase from Rhizomucor miehei, immobilized on an anionic resin. – Lipozyme TL IM—is a lipase from Thermomy ces lanuginosus, immobilized on silica. Lipase may be regiospecific or nonspecific. The vast majority of the regiospecific lipases act on CATALYSIS IN INDUSTRY
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1,3ester bonds of triglyceride, as it is difficult for lipases to react with the central 2bond. Lipozyme IM is one of the most widely used regiospecific lipases, while Novozym 435 is the most popular nonspecific lipase. There is naturally a disadvantage in using 1,3regiospecific lipases for biodiesel production due to the nonuse of the 2position of triglyceride; i.e., the yield is reduced by one third. This obstacle can be reduced through the phenomenon of acyl migration, however, if we stimulate the migration of acyl groups from the 2position of triglycerides to the free 1 or 3positions followed by a reaction with 1,3regiospe cific lipase. Acyl migration can be induced under cer tain conditions, e.g., if we use a polar carrier such as ion exchange resins (anion resin in the case of Lipozyme IM) for immobilization, or if we add silica to the reaction medium. In [22], the yield of product was thus increased from 66% to 90% using a 1,3regiospecific lipase Lipozyme TL with the addi tion of 6 wt. % silica in the reaction medium. In some studies, the combined use of 1,3regiospecificity (Lipozyme TL IM) and nonspe cific (Novozym 435) lipase was tested. Given that the cost of Novozym 435 is much higher than that of Lipozyme TL IM, the price of biodiesel can also be reduced. In works devoted to transesterification, certain fungi are popular among the producers of lipases: ⎯the fungi Candida: C. antarctica [5] (including commercial Lipozyme CALB L, Lipase SP435, Novozym 435), C. rugosa [24] and C. sp. 99–125 [29]; ⎯Rhizomucor miehei (previously Mucor mihei) [5] (including commercial Lipozyme IM and Lipozyme RMIM); ⎯Thermomyces lanuginosus [22] (including com mercial Lipozyme TL, LipozymeTL IM); ⎯the fungi Rhizopus: R. niveus and R. oryzae [24]; ⎯the fungi Penicillium: P. camembertii [24] and P. expansum [7]; ⎯Aspergillus niger [24]; ⎯Saccharomyces cerevisiae [25]. Lipaseproducing bacteria are used slightly less popular: ⎯Burkholderia cepacia (previously Pseudomonas cepacia) [24] (including commercial Lipase PS30); ⎯Pseudomonas fluorescens [24]; ⎯Alcaligenes sp. [4]; ⎯Bacillus subtilis [26]; ⎯Chromobacterium viscosum [27]; ⎯Enterobacter aerogenes [28]; ⎯Photobacterium lipolyticum [8]. Among animal lipases, pig pancreatic lipase (1,3 regiospecificity) is commonly used [9]. In contrast to other types of catalysis of the transes terification of triglycerides, lipase catalysis can solve the problem with FFA: its rate is many times greater than that of acid catalysis. Part of the raw materials CATALYSIS IN INDUSTRY
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(often the quite substantial portion made up of fatty acids and which must be separated and discarded by alkaline catalysis) is translated into esters using lipases thereby dramatically increasing the output of biodie sel. The closest economically advantageous use of lipases would seem to lie in the processing of acid oils. The works of Y. Watanabe et al. [30] (the researchers conducted twostep lipase catalysis with unrefined rapeseed oil, saturated with free oleic acid (77.9%)), and L. Wang et al. [23] (the researchers used a byprod uct from the refining of soybean oil (28% FFA))are extremely interesting. It should be noted that the phospholipids con tained in unrefined raw materials are substances detri mental to the process. In the methanolysis of crude soybean oil by immobilized Candida antarctica lipase (Novozym 435), it was found [31] that the phospholip ids in the plant gum considerably inhibit the reaction. FLAWLESS FUEL Fuel patented as Ecodiesel100 was obtained in [32]. This product is a mixture of two parts fatty acid ethyl esters and one part monoglycerides (FAEE/MG) with a small amount of diglycerides that have physico chemical properties similar to those of conventional biodiesel and/or petrodiesel, thereby avoiding the pro duction of glycerin as byproduct. In order to obtain Ecodiesel100, sunflower oil was transesterified by 1,3regiospecific pig pancreatic sepiolite immobilized lipase and correlated with the product yield and vari ous alcohols. It turned out that the FAEE yield fell by almost half (from 60.7% to 35.3%) when using 96% ethanol instead of pure ethanol, due to the doubling of the proportion of monoand diglycerides. KEY PROBLEMS OF BIODIESEL PRODUCTION VIA BIOCATALYSIS Immobilization Nowadays the high cost of lipase is a major limiting factor in its use in biodiesel production, and can account for more than 90% of the total cost of fatty acid methyl esters (FAMEs) [4]. The use of free lipase makes no sense from an economic point of view: the catalyst can be used effectively only once, due to the difficulty of isolating it from the reaction mixture as in alkaline catalysis. Lipase must thus be reused for the maximum number of cycles, and this requires its immobilization. We must also be guided by economic considerations in choosing the carrier for immobiliza tion. Relatively lowcost carriers are silica [22], diato maceous earth [27], and kaolinite [33]. Optimization of AcylAcceptor One way to avoid the inactivation of lipase is to conduct the transesterification reaction using acyl acceptors such as methyl acetate [34] and ethyl acetate
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[35] instead of methanol and ethanol, respectively. It should be noted that a more valuable byproduct (tri acetin instead of glycerol) is formed in the process of transesterification. The authors of [34] used methylacetate for the con version of soybean oil with Novozym 435 as a catalyst. It was found that there was no inhibitory effect on lipase: a yield of 92% was obtained, and the activities of lipase were well preserved even after 100 repeated cycles. When the authors used methanol, considerable inhibition of the lipase was observed even when the methanoltooil molar ratio was 1 : 1, while with methyl acetate they used a ratio of 12 : 1 without dam age to the lipase. A reaction yield of only about 30% was obtained with unrefined soybean oil and methanol due to the lipase being deactivated by the phospholip ids [31]. The latter did not affect the reaction of methyl acetate, probably because of its high concentration (the yield was also 92%). In using ethyl acetate to produce biodiesel from jat ropha oil and sunflower seed (catalyst, Novozym 435), the authors of [35] also observed similar advantages over ethanol. The lipase activity lasted for more than 12 cycles upon transesterification and disappeared after just six cycles upon ethanolysis. The cost of these acyl acceptors for transesterifica tion is nevertheless much higher than that of the cor responding alcohols, and the reaction requires a large molar ratio of acyl acceptor to oil. Eliminating the Influence of Reaction Products In some works, it is argued that the lipase lose some of their activity due to the presence of glycerol in the reac tion medium: it accumulates in the reactor and envelops the surface of the enzymes. It was therefore proposed that the glycerol be removed in situ by dialysis [36] or that transesterification be conducted in tertbutanol [23] or isopropanol [37], which dissolve glycerol and simulta neously increase the solubility of alcohol. Reactivation of Biocatalyst Deactivated lipase can be restored by keeping it in alcohols containing three or more carbon atoms, pref erably 2butanol or tertbutanol [38]. In this work, the authors were able to restore about 56% and 75%, respectively, of the initial activity of completely deac tivated lipase Novozym 435 using these alcohols. Marine Raw Material for Bioethanol Marine biological resources are also promising for use in ethanol fuel. Even at the highest level of devel opment, second generation technology based on the use of lignocellulose (agricultural waste and wood pro cessing) has a fundamental drawback: solid waste lig nin. The lignin content of softwood and hardwood is 23–38 and 14–25 wt % respectively [39]. This short
coming generally cannot be eliminated even when using genetically modified crops, since the minimum content of lignin is limited by the requirements for the plants’ mechanical stability. In coastal regions, algae biomass can serve as an alternative raw material. It is more productive than plants that grow on dry land, since because photosyn thesis in these plants occurs only in their leaves or nee dles, while photosynthesis in algae takes place over the surface of the cells. The carbohydrate content in Lam inaria and fucoid is as high as 73–74% [40] (with up to 20% cellulose [41]). The annual productivity of lami narian kelp is 24.1 kg/m2 in dry weight; a field of algae in the Barents Sea can produce approximately 18 mil lion liters of biofuel [42]. A key challenge is to replace environmentally haz ardous acid hydrolysis with enzymatic hydrolysis. The composition of algae cell walls includes cellu lose, hemicellulose, pectins, proteins [43], and the most profound degradation of cellulose and other polysaccharides of plant biomass is thus possible under the influence of complex polyenzyme systems. We have developed a cascade technique for the enzymatic hydrolysis of biomass of algae kelp Laminaria saccha rina sugary using commercial enzyme preparations available on the Russian market (Dysticim BA, Zere mix 6XMG, Ultraflow L, Viscoflow MG, CelloluxA, Ollzime BG). Joint use of the most effective enzymes under optimal conditions enabled us to attain a 70% yield of reducing substances [44]. CONCLUSIONS The rapid development of the biofuel industry involves not only technological and economic aspects but ethical and political considerations as well. We are constantly searching for environmentally friendly cat alysts, technologies, and raw materials that do not impact the food market. One option for the develop ment of the biodiesel industry is the biocatalytic pro cessing of marine raw materials. Efforts in this direc tion will not only allow us to create environmentally friendly and ethically ideal fuel that neither upsets the food market nor damages the environment, but to refrain from the use of highly toxic methanol. The transition to fermentative catalysts will enable us to produce not only diesel but glycerin and a mixture of amino acids and peptides (from the proteins of sea food) as well. These products are biologically active and are widely used both in pharmaceuticals and in medical practice. REFERENCES 1. Fedorenko, V.F., Status and Development of Biofuels: Scientific Analytic Review, Fedorenko, V.F., Kolchin skij, Yu.L., and Shilova, E.P., Eds., M.: FGNU “Ros informagrotekh”, 2007. CATALYSIS IN INDUSTRY
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