Renewable Energy 34 (2009) 15

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Review

Biodiesel production from oleaginous microorganisms

Xin Meng a, Jianming Yang a, Xin Xu a, Lei Zhang a, Qingjuan Nie b, Mo Xian a, *
a b

Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266071, China Foreign Languages School, Qingdao Agricultural University, Qingdao 266109, China

a r t i c l e i n f o
Article history: Received 19 October 2007 Accepted 7 April 2008 Available online 10 July 2008 Keywords: Biodiesel Oleaginous microorganisms Microbial lipid Metabolic regulation

a b s t r a c t
High energy prices, energy and environment security, concerns about petroleum supplies are drawing considerable attention to nd a renewable biofuels. Biodiesel, a mixture of fatty acid methyl esters (FAMEs) derived from animal fats or vegetable oils, is rapidly moving towards the mainstream as an alternative source of energy. However, biodiesel derived from conventional petrol or from oilseeds or animal fat cannot meet realistic need, and can only be used for a small fraction of existing demand for transport fuels. In addition, expensive large acreages for sufcient production of oilseed crops or cost to feed animals are needed for raw oil production. Therefore, oleaginous microorganisms are available for substituting conventional oil in biodiesel production. Most of the oleaginous microorganisms like microalgae, bacillus, fungi and yeast are all available for biodiesel production. Regulation mechanism of oil accumulation in microorganism and approach of making microbial diesel economically competitive with petrodiesel are discussed in this review. 2008 Elsevier Ltd. All rights reserved.

1. Introduction With the rapid rise in the price of crude oil and projected decrease in oil supplies, greater recognition of the environmental consequences of fossil fuels has drawn more and more researchers interest in transportation biofuels in recent years [6,33,50]. To deal with these serious problems, such as deteriorated situation of the whole world energy supply, energy environment and energy security, alternative renewable biofuels are receiving considerable attention [2,23]. One of the most prominent renewable energy resources is biodiesel, which is produced from renewable biomasss by transesterication of triacylglycerols, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols, for example, fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). It contributes no net carbon dioxide or sulfur to the atmosphere and emits less gaseous pollutants than normal diesel [10,36,69]. In the production of worldwide biodiesel, various renewable lipids have been choosen, including vegetable oils, animal fats and wasting oils [4]. In South East Asia, Europe, United States and China, palm oil, rapeseed oil, transgenetic soybeans and wasting oil were used to produce biodiesel, respectively. However, all these plant oil materials require energy and acreage for sufcient production of oilseed crops. Likewise, animal fat oils need to feed these animals. In spite of the favourable impacts that its commercialization could

provide, the economic aspect of biodiesel production has been restricted by the cost of oil raw materials [10]. If plant oil was used for biodiesel production, the cost of source has account to 7085% of the whole production cost [70]. Therefore, taking into account of these inhibition factors, exploring ways to reduce the high cost of biodiesel is of much interest in recent research, especially for those methods concentrating on lowering the cost of oil raw material. Microorganisms have often been considered for the production of oils and fats as an alternative to agricultural and animal sources. This review deals with the property of oleaginous microorganisms, with emphasis on biotechnological strategies for increasing lipid production (such as, mutation techniques, genetic and metabolic engineering). 2. Microorganisms available for biodiesel production To be a viable substitute for a fossil fuel, an alternative fuel should not only have superior environmental benets over the fossil fuel it displaces, be economically competitive with it, and be producible in sufcient quantities to make a meaningful impact on energy demands, but also provide a net energy gain over the energy sources used to produce it. Oleaginous microorganisms are dened as microbial with the content of microbial lipid excess of 20%. Biodiesel production using microbial lipids, which is named as single cell oils (SCO), has attracted great attention in the whole world. Although there are all kinds of microorganism storaging oils, such as microalgae, bacillus, fungi and yeast, not all of them are available for biodiesel production (Table 1). The rst commercial production of an SCO did not begin until 1995 and this only

* Corresponding author. E-mail addresses: mengxin@qibebt.ac.cn (X. Meng), xianmo@qibebt.ac.cn (M. Xian). 0960-1481/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2008.04.014

2 Table 1 Oil content of some microorganisms Microorganisms Microalgae Botryococcus braunii Cylindrotheca sp. Nitzschia sp. Schizochytrium sp. Bacterium Arthrobacter sp. Acinetobacter calcoaceticus Rhodococcus opacus Bacillus alcalophilus Oil content (% dry wt) 2575 1637 4547 5077 Microorganisms

X. Meng et al. / Renewable Energy 34 (2009) 15

Oil content (% dry wt) 58 65 64 72

Yeast Candida curvata Cryptococcus albidus Lipomyces starkeyi Rhodotorula glutinis Fungi Aspergillus oryzae Mortierella isabellina Humicola lanuginosa Mortierella vinacea

>40 2738 2425 1824

57 86 75 66

lasted for 6 years before it was closed down as no longer being costeffective [57]. Numerous oleaginous yeasts and microalgaes have been reported to grow and accumulate signicant amounts of lipids similar to vegetable oil [4,5], methyl-esters and soaps [44], used as sole carbon and energy sources. In recent years, a large number of hydrophobic lipid accumulating microorganisms have been studied as substrates for SCO production, especially used in the production of biodiesel. In microorgamisms, the extent of lipid accumulation is determined by the genetic constitution, as maximum attainable lipid contents can vary enormously among species and even among individual strains. According to different microorganisms and different culture conditions (such as temperature, pH, culture time, etc.), oil content and composition are different, see Table 2 [7,51,63]. Microalgaes are sunlight-driven cell factories that convert carbon dioxide to potential biofuels [42,66], and it can provide several different types of renewable biofuels, including methane produced by anaerobic digestion of the algal biomass, biodiesel [12,20], and photobiologically produced biohydrogen [19,32,41]. Study showed [75] that most microalgae biomasses are a rich source of u3 and u6 fatty acids, essential amino acids, like leucine, isoleucine, valine, etc. The accumulated oil in almost all microalgaes is mainly triglyceride (>80%), with a fatty acid prole rich in C16 and C18, showing O9, O12 and O15 desaturation, comparable with plant seed oil, such as Yarrowia lipolytica, containing stearic, oleic, linoleic and palmitic acid (S. Papanikolaou, 2002). Microalgae grow extremely rapidly and many are exceedingly rich in oil, and double their biomass within 24 h. The average lipid content of algal cells varies between 1% and 70%, but can reach 90% of dry weight under certain conditions [9,66]. By now, the microalgal biomass market produces about 5000 t of dry mass/ year and generates a turnover of approximately US$1.25 109/year [26,27,52]. It has been believed as one of good candidates for biodiesel production, based on higher photosynthetic efciency, higher biomass production and faster growth compared to other energy crops. Although microalgae are high lipid microbial, they need a larger acreages to culture algaes and long fermentation period than bacteria. Table 1 tells that nearly all bacteria accumulate lower lipid than microalgaes, the average oil content is about 2040% of dry biomass, such as Arthrobacter sp. and Acinetobacter calcoaceticus,

the oil content is 40% and 38%, respectively. Bacteria have a superiority in the production of biodiesel with highest growth rate (reach huge biomass only need 1224 h) and easy culture method. As an alternative, storage-lipid-accumulating bacteria, in particular those of the actinomycete group may be used, and these bacteria are capable of synthesizing remarkably high amounts of fatty acids (up to w70% of the cellular dry weight) from simple carbon sources like glucose under growth-restricted conditions and accumulate them intracellularly as TAGs [7]. However, most bacteria are generally not oil producer, only a few bacteria accumulate complicated lipoid (i.e., polyhydroxyalkanoates [76]). It is difcult to extract because these lipoid are generated in the outer membrane, so there is no industrial signicance in the actual production of biodiesel by using oleaginous bacteria as raw material. Yeasts and fungi (especially molds) are considered as favourable oleaginous microorganisms since 1980s [1,54]. Some yeast strains, such as Rhodosporidium sp., Rhodotorula sp. and Lipomyces sp. can accumulate intracellular lipids as high as 70% of their biomass dry weight. The most efcient oleaginous yeast Crptococcus curvatus can accumulate storage lipid up to >60% on a dry weight basis, while when it grows under N-limiting conditions, these lipids usually consist of SOC-90% w/w triacylglycerol with a percentage of saturated fatty acids (% SFA) of about 44% which is similar to many plant seed oils [53]. Oleaginous yeasts and molds accumulate triacylglycerols rich in polyunsaturated fatty acids or having specic structure [77], somewhat limited: oleic (18:1) and linoleic (18:2) acids together with palmitic (16:0) or palmitoleic acids (C16:1) are the most frequently found fatty acids (See Table 2), and nearly all of them are unsaturated fatty acid. For example, Rhodosporidium toruloides Y4 contained mainly long-chain fatty acids with 16 and 18 carbon atoms, which is a well-known microbial lipid producer and extensive characterization of the oleaginous prole of R. toruloides CBS 14 has been reported [78]. Recently, report showed that it can reach a dry biomass and cellular lipid content of 151.5 g L1 and 48.0% (w/w), respectively, in ask fed-batch cultures run for 25 days [72]. A lamentous fungus Mortierella alliacea Strain YN-15, accumulated arachidonic acid (AA, C20:4n-6) mainly in the form of triglyceride in its mycelia, which yielded 46.1 g/L dry cell weight, 19.5 g/L total fatty acid, and 7.1 g/L AA by 7-d cultivation in a 50-L jar fermenter [60,68]. Based on these data, oleaginous yeasts and fungi are all potential alternative oil resources for biodiesel production. 3. Metabolic regulation of microbial lipid SCO produced by various oleaginous microorganisms has been known for over 100 years, but why microorganisms can produce and accumulate oil, and why some oleaginous species are able to accumulate more lipid than other species? Not all microorganisms have the capability to accumulate lipid, usually in the form of triacylglycerols. Only within the last 20 years or so, any attempt has been made to understand the process of lipid accumulation in the so-called oleaginous species [55]. For example, among the some 600 species of yeasts, less than 30 have so far been identied as being able to accumulate more than 25% of their biomass weight as oil [2,55]. Rhodotorula spp. and Cryptococcus curvatus (also known as Apiotricum curvatum) can accumulate 4070% lipid of their biomass, while Saccharomyces cerevisiae and the food yeast Candida utilis are normally unable to accumulate >510% oil even when grown in the same culture condition. Two main points are described as follows for this phenomenon. Lipid accumulation in an oleaginous microorganism begins when it exhausts a nutrient from the medium (it is usually nitrogen), but an excess of carbon (in the form of glucose) is still assimilated by the cells and is converted into triacylglycerols (TAG), while lipid is synthesized during the balance phase of growth at

Table 2 Lipid composition of some microorganisms Microorganisms Lipid composition (w/total lipid) C16:0 Microalga Yeast Fungi Bacterium 1221 1137 723 810 C16:1 5557 16 16 1011 C18:0 12 110 26 1112 C18:1 5860 2866 1981 2528 C18:2 420 324 840 1417 C18:3 1430 13 442

X. Meng et al. / Renewable Energy 34 (2009) 15

nearly the same rate. As the limitation supply of nitrogen, it means that cell proliferation is prevented and the formed lipid has to be stored within the existing cells which can no longer divide. Therefore, it leads to the accumulation of lipid. Two critical regulated enzymes, including malate enzyme and ATP: citrate lyase (ACL), have effect on lipid accumulation. There is a strong correlation between the presence of ACL activity and the ability to accumulate lipid in yeasts, fungi and other oleaginous microorganisms [56]. However, there is a small amount of yeast that has been found to have ACL activity but still do not accumulate lipid beyond the level of a few percent of their biomass. Consequently, ACL is a prerequisite for lipid accumulation to occur, but its possession is not necessarily crucial for lipid accumulation, it cannot be the sole effecting factories [2]. In fact, other enzymes must be responsible for controlling the extent of lipid biosynthesis in individual organisms. Fatty acids are highly reduced materials and to achieve their synthesis as a ready supply of reductant as NADPH is essential [57]. The synthesis of 1 mol of a C18 fatty acid requires 16 mols NADPH to be provided as 2 mols NADPH are needed to reduce each 3-keto-fattyacyl group arising after every condensation reaction of acetyl-CoA with malonyl-CoA as part of the standard fatty acid synthetase complex into the saturated fattyacyl chain. The total mass of microbial lipid is also regulated by the content of fatty acid. It means that controlling factors of fatty acid synthesis will make a great deal of SCO. There are some different kinds of enzymes measured fatty acid synthesis, such as acetyl-CoA carboxylase (ACCase). It is the rate-limiting enzyme of synthesis, and catalyzes the rst reaction of synthesis of fatty acid in microorganisms. If using modern biotechnology to enhance the overall activity in vivo, it may get a high level of fatty acid [6]. The Escherichia coli ACCase gene were cloned in a single plasmid and expressed in BL21 (DE3), and the results showed a 6-fold increase in the rate of fatty acid synthesis [39].

5.1. Screening for potential oleaginous microorganism It is the rst and an essential step limiting the number of strain for further study and practical use. Although several wild-type oleaginous microorganisms are able to synthesize rich oil, these strains have a limited ability to produce biomass. Making use of mutation techniques in microbial lipid production to ltrate better strain will get much more biomass than wild-type. Recently, the Greece researcher [51] reported that Mortierella isabellina cultivated in nitrogen-limited media presented remarkable cell growth (up to 35.9 g/L) and high glucose uptake even with initial sugar concentrations at 100 g/L in media. 5.2. Genetic and metabolic engineering With the development of biochemistry and molecular biology (such as DNA recombination, site-directed mutagenesis), engineer progress in microorganisms has led to speculation that certain oil components could become marketable commodities if the genomes of traditional oleaginous microorganisms were appropriately modied. It is likely to have the greatest impact on improving the economics of production of microalgal diesel [16,21,58,73]. Stability of engineered strains and methods for achieving stable production in industrial microbial processes are known to be important issues [74]. Change in the degree of fatty acid unsaturation and decrease or increase of the chain length of fatty acids are the major challenges in modifying the lipid composition. All these are regulated by enzymes, but most of the enzymes are membrane bound, which has signicantly hindered efforts to purify and study their function [17]. Three most important interdependent genetic technologies are involved, including cloning genes of critical enzymes, transgenic expression of these genes aimed to achieve a ne high-product microbial oil recombination strain, and modication of cloned genes in order to engineer the expressed protein [25,58]. It has been mainly advanced in plants using DNA recombination [22]. Likewise, there are also successful examples in recombination oleaginous microorganisms in recent years. Those recombinantly synthesized fatty acid ester derivatives are wax esters in recombinant Pseudomonas citronellolis [28], fatty acid butyl esters (FABEs) in recombinant E. coli [30,31], and FAME in recombinant S. cerevisiae [29]. By heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecic acyltransferase from Acinetobacter baylyi strain ADP 1, it was indicated that FAEE concentrations of 1.28 g/L and a FAEE content in vivo accounting to 26% of the cellular dry mass [30,31]. Most of the work has, so far, been performed on oleaginous microorganisms mainly because of their ability to accumulate high amounts of intracellular lipid, their relatively high growth rates and the resemblance of their triacylglycerols fraction to plant oils. But when it comes to the production of biodiesel, only those with a high content of stearic acid (C18:0) and oleic acid (C18:1) are potential targets of interest because such microorganisms lipid mimic the properties of higher value oils, improve oxidative stability, and they have more potential adaptability in the industrial production of biodiesel. Changing the lipid composition of oleaginous microorganisms to enable them satisfy in the actual biodiesel production, and modifying the fatty acid by genetic engineering methods have been used successfully. Thus, synthesis of lipids with varying fatty acid composition should be feasible by culturing oleaginous microorganism under different conditions, and by metabolic regulation. In 1990s, temperature-induced changes are reported in the fatty acid composition of the accumulated lipids of various oleaginous microbial cells [67]. For example, at 20  C, the proportion of palmitic acid decreased, while those of palmitoleic and vaccenic acid

4. Quality of biodiesel To assess the potential of biodiesel as a substitute of diesel fuel, the properties of biodiesel such as density, viscosity, ash point, cold lter plugging point, solidifying point and heating value were determined [70]. An important fuel criterium for biodiesel is bound glycerol, which functions the residual amount of triglycerides and partial glycerides in the biodiesel. Biodiesel fuel, in the form of FAME, is now manufactured in many countries. In the United States, the relevant standard is the ASTM Biodiesel Standard D 6751 [11,34], while in Europe Union, separate standard exists for biodiesel intended for vehicle use (Standard EN 14214) and for use as heating oil (Standard EN 14213). The ASTM D 6751 biodiesel acidnumber limit was harmonized with the European biodiesel value of 0.5. Microorganism oils quite differ from most vegetable oils in being quite rich in polyunsaturated fatty acids [13]. No such limitation exists for biodiesel intended for use as heating oil, but acceptable ones must meet other criteria relating to the extent of total unsaturation of the oil, which is indicated by its iodine value. Standard EN 14214 and EN 14213 require the iodine value of biodiesel to exceed 120 and 130 g iodine/100 g biodiesel, respectively.

5. Improvement of microbial lipid production The extensive research and development of microbial oil production carried out over the past several years are still continuous and are basically aimed at improving the economic competitiveness of microbial lipids compared to plant- and animal-derived oils [17,38]. Three main pathways are mentioned as follows to improve economics of microorganism biodiesel.

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increased [49]. Both bacteria and yeasts have been reported to contain an increasing proportion of unsaturated fatty acids as the growth temperature decreases [14,48]. Microalgal oils differ from most vegetable oils in being quite rich in polyunsaturated fatty acids with four or more double bonds [13], which make it susceptible to oxidation during storage and reduce their acceptability for use in biodiesel production [71]. For example, the extent of unsaturation of microalgal oil and its content of fatty acids with more than four double bonds can be reduced easily by partial catalytic hydrogenation of the oil [79]. Different culture conditions also play an important role in the lipid composition, like C: N ratio, which is the major impact factor [64]. When oleaginous organisms are grown with an excess of carbon and limited quantity of nitrogen (high C:N), they may accumulate up to >60% of their cellular dry weight as intracellular lipid, such as C. curvatus, the concentration of lipid reached its maximal value of about 70 g/L and the main lipid were palmitic (C16:0), stearic (C18:0) and oleic acid (C18:1) [37].A marked difference in the fatty acid composition was observed in the samples obtained from Bacillus subtilis cultured in nutrient broth (C:N 50) compared with original nutrient broth. Branched chain fatty acids, when increase in C:N ratio signicantly improved lipid production from 16.3% to 42.7% [65]. Therefore, how to improve the total fatty acid and change the lipid composition to adapt for the biodiesel production by useful methods is vital in biodiesel production. 5.3. Making full use of byproducts In the course of fatty acid production, there are usually some other byproducts useable in industry. In addition to oils, oleaginous microorganisms contain signicant quantities of proteins, carbohydrates and other nutrient contents [59]. Therefore, how to make use of these byproducts and improve their industrial value is another way to reduce the production cost in biodiesel. For example, the residual biomass from biodiesel production can be used potentially as animal feed [71], and also to produce methane by anaerobic digestion. The waste glycerol from the biodiesel industry could therefore be easily transformed into a precious chemical [24]. Moreover, the price of the nal product could be reduced considerably in this way. 6. Conclusion Negative environmental consequences of fossil fuels and concerns about petroleum supplies have spurred the search for renewable transportation biofuels. Among current shortage of petrodiesel oil and highest production cost of biodiesel using plant oil or animal fat, the major challenge mankind confronts in this century is developing oleaginous microorganisms to obtain large amount of standard biodiesel for industry. At present, however, neither bioethanol nor biodiesel is competitive with conventional fuels in the whole world. Only by improving the quality and reducing the cost of biodiesel, can it compete with conventional fuels. Producing low cost microbial diesel primarily requires improvements of biotechnology as described in this review. Developing high lipid content microorganisms or engineered strains for biodiesel production would be becoming a potential and promising way in the future. The manipulation and regulation of microbial lipid biosyntheses open a possibility for academic research and demonstrate the potential in its industrial application in biodiesel production. References
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Further reading Fatty acid methyl esters: requirements and test methods, EN 134214 (2003).