Biodiesel production by microalgal biotechnology

Applied Energy 87 (2010) 38–46

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Applied Energy journal homepage: www.elsevier.com/locate/apenergy

Biodiesel production by microalgal biotechnology GuanHua Huang a,*, Feng Chen b,c, Dong Wei c, XueWu Zhang c, Gu Chen c a b c

School of Chemical Engineering and Technology, China University of Mining and Technology, China School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, China

a r t i c l e

i n f o

Article history: Received 27 April 2009 Received in revised form 9 June 2009 Accepted 10 June 2009 Available online 8 July 2009 Keywords: Microalgae Biodiesel Lipids Fatty acids Production

a b s t r a c t Biodiesel has received much attention in recent years. Although numerous reports are available on the production of biodiesel from vegetable oils of terraneous oil-plants, such as soybean, sunflower and palm oils, the production of biodiesel from microalgae is a newly emerging field. Microalgal biotechnology appears to possess high potential for biodiesel production because a significant increase in lipid content of microalgae is now possible through heterotrophic cultivation and genetic engineering approaches. This paper provides an overview of the technologies in the production of biodiesel from microalgae, including the various modes of cultivation for the production of oil-rich microalgal biomass, as well as the subsequent downstream processing for biodiesel production. The advances and prospects of using microalgal biotechnology for biodiesel production are discussed. Ó 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microalgal biotechnology for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Biosynthesis of lipids/fatty acids in microalgae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. The formation of acetyl coenzyme A (acetyl-coA) in cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. The elongation and desaturation of carbon chain of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. The biosynthesis of triglycerides in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Phototrophic cultivation of microalgae for lipids production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Heterotrophic cultivation of microalgae for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Genetic engineering for lipids production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The extraction of oils from microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Pyrolysis technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Liquefaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transesterification technologies in the production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The production of biodiesel has recently received much attention worldwide. Because of the world energy crisis [1], many countries have started to take a series of measures to resolve this problem [2]. Finding alternative energy resources is a pressing mission for many countries, especially for those countries * Corresponding author. E-mail address: [email protected] (G. Huang). 0306-2619/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.apenergy.2009.06.016

38 39 40 40 40 41 41 42 43 43 43 44 44 45 45

lacking conventional fuel resources. In the 1930s and 1940s, vegetable oils has been used as diesel fuels in the emergency situation. With the rapid development of the modern industry, the demand for energy has been greatly increased in recent years, and therefore alternative energy sources are being explored. Thus, the term ‘‘biodiesel” has appeared very frequently in many recent reports [3]. The world total biodiesel production was estimated to be around 1.8 billion liters in 2003 [4]. Although there was no increase in biodiesel production between 1996 and 1998, a sharp

G. Huang et al. / Applied Energy 87 (2010) 38–46

increase in biodiesel production was observed in the past several years. It is speculated that the production of biodiesel will be further tremendously increased because of increasing demand for fuels and ‘‘cleaner” energy globally. Biodiesel is made from biomass oils, mostly from vegetable oils. Biodiesel appears to be an attractive energy resource for several reasons. First, biodiesel is a renewable resource of energy that could be sustainably supplied. It is understood that the petroleum reserves are to be depleted in less than 50 years at the present rate of consumption [5]. Second, biodiesel appears to have several favorable environmental properties resulting in no net increased release of carbon dioxide and very low sulfur content [6,7]. The release of sulfur content and carbon monoxide would be cut down by 30% and 10%, respectively, by using biodiesel as energy source. Using biodiesel as energy source, the gas generated during combustion could be reduced, and the decrease in carbon monoxide is owing to the relatively high oxygen content in biodiesel. Moreover, biodiesel contains no aromatic compounds and other chemical substances which are harmful to the environment. Recent investigation has indicated that the use of biodiesel can decrease 90% of air toxicity and 95% of cancers compared to common diesel source [64]. Third, biodiesel appears to have significant economic potential because as a non-renewable fuel that fossil fuel prices will increase inescapability further in the future [8]. Finally, biodiesel is better than diesel fuel in terms of flash point and biodegradability [9]. Conventional biodiesel mainly comes from soybean and vegetable oils [10], palm oil [11], sunflower oil [6], rapeseed oil [12] as well as restaurant waste oil [13]. The number of carbon in the carbon chain of the diesel oil molecular is about 15, which is similar to that of the plant oil with 14–18 carbons. The structural characteristic of biodiesel determines that biodiesel is a feasible substitute for conventional energy. Nevertheless, the production cost is generally high for biodiesel. The price of biodiesel is approximately twofold that of the conventional diesel at present. The production cost of biodiesel consists of two main components, namely, the cost of raw materials (fats and oil) and the cost of processing. The cost of raw materials accounts for 60% to 75% of the total cost of the biodiesel fuel [14]. Though there might be large amounts of low-cost oil and fats available such as restaurant waste and animal fats [15], the major problem of using these low-cost oils and fats is that they often contain large amounts of free fatty acids (FFA) which is difficult to convert to biodiesel through transesterification [16]. Raw materials that contain large proportions of fatty acid triglycerides are preferred. For example, plant oil is found to contain more fatty

39

acid triglycerides and therefore has been used in the production of biodiesel for some years [17]. Earlier studies on liquid fuel from microalgae had begun in mid1980s. During the world war II, although some German scientists attempted to extract lipids from diatom in order to resolve energy crisis [18], and soon later in the USA, research was conducted by a group of scientists at the Carnegie Institution of Washington, and their experiences had been summarized in a book [65] entitled ‘‘Algal Culture from Laboratory to Pilot Plant”, but the technologies of making microalgae as fuels had not been fully exploited. The reasons could be as follows. First, as a source of lipids, microalgae are less known than plants and animals. Second, the prices for most plant oils are relatively low and animal fats are even cheaper; therefore, processes for the microbial oils production have mainly focused on high-valued products that cannot be produced by plants, such as omega-3 polyunsaturated fatty acids, especially EPA and DHA [19]. In order to resolve the worldwide energy shortage crisis, seeking for lipid-rich biological materials to produce biodiesel effectively has attracted much renewed interest. Oleaginous microorganisms are favorably considered for their short growth cycles, high lipid contents and ease of being modified by biotechnological means (see Table 1). Some microalgae appear to be suitable group of oleaginous microorganism for lipids production [20]. Microalgae have been suggested as potential candidates for fuel production because of a number of advantages including higher photosynthetic efficiency, higher biomass production and higher growth rate compared to other energy crops [21–23]. Moreover, according to biodiesel standard published by the American Society for Testing Materials (ASTM), biodiesel from microalgal oil is similar in properties to the standard biodiesel, and is also more stable according to their flash point values (Table 2). 2. Microalgal biotechnology for lipids production Microalgae have high potentials in biodiesel production compared to other oil crops. First, the cultivation of microalgae dose not need much land as compared to that of terraneous plants [20]. Biodiesel produced from microalgae will not compromise the production of food and other products derived from crops. Second, microalgae grow extremely rapidly and many algal species are rich in oils. For instance, heterotrophic growth of Chlorella protothecoides can accumulate lipids as high as 55% of the cell dry weight after 144 h of cultivation with feeding of corn powder hydrolysate in fermenters [24]. Oil levels of 20–50% are common in microalgae [20]. The whole technical process in the production of biodiesel

Table 1 Comparison of types of sources for the oils production. Type of organism

Advantages

Disadvantages

Microalgal oils

(1) (2) (3) (4)

(1) Most algal lipids have lower fuel value than diesel fuel (2) The cost of cultivation is higher compared to common crop oils currently

Fatty acid constitutions similar to common vegetable oils Under certain condition it may be as high as 85% of the dry weight Short-time growth cycle Composition is relative single in microalgae

Bacteria oils

(1) Fast growth rate

(1) Most of bacteria can not yield lipids but complicated lipoid

Oleaginous yeasts and mildews

(1) Resources are abundant in the nature (2) High oil content in some species (3) Short-time growth cycle

(1) Filtration and cultivation of yeasts and mildews with high-content oils are required (2) Process of oils extracted from oleaginous yeasts and mildew is complex and new technology should be exploited to resolve it (3) the cost of cultivation is also higher compared to common crop oils currently

(4) Strong capability of growth in different cultivation on conditions (5) Conversion and utilization of scrap fiber to yield useful oils and the application for dealing with waste oils in environment Waste oils

(1) The waste oils is cheap compared to crop oils

(1) Containing a lot of saturated fatty acids which is hard to be converted to biodiesel by catalyst

40

G. Huang et al. / Applied Energy 87 (2010) 38–46

Table 2 Comparison of properties of microalgal oil, conventional diesel fuel, and ASTM biodiesel standard [24]. Properties

Biodiesel from microalgal oil

Diesel fuel

ASTM biodiesel standard

Density (kg L ) Viscosity (mm2 s 1, cSt at 40 °C) Flash point (°C) Solidifying point (°C) Cold filter plugging point (°C)

0.864 5.2 115 12 11

0.838 1.9–4.1 75 50 to 10 3.0 (max

Acid value (mg KOH g 1) Heating value (MJ kg 1) H/C ratio

0.374 41 1.81

Max 0.5 40–45 1.81

0.84–0.90 3.5–5.0 Min 100 – Summer max 0 Winter max < 15 Max 0.5 – –

1

from microalgae has been well investigated in recent years. Third, the entire production process ranging from the cultivation of high-lipid microalgae to the production of biodiesel from the microalgal oils has also been explored. In the laboratory conditions, the ideal oil content could reach 56–60% of total dry biomass by genetic engineering or heterotrophic culture techniques. These technological advances suggest that the industrial production of biodiesel from microalgal oils may be feasible in the near future. 2.1. Biosynthesis of lipids/fatty acids in microalgae It is known that both inorganic carbon (CO2) and organic carbon sources (glucose, acetate, etc.) can be utilized by microalgae for lipids production. The components and contents of lipids in microalgal cells vary from species to species. The lipid classes basically are divided into neutral lipids (e.g., triglycerides, cholesterol) and polar lipids (e.g., phospholipids, galactolipids). Triglycerides as neutral lipids are the main materials in the production of biodiesel. The synthesis routes of triglycerides in microalgae may consist of the following three steps: (a) the formation of acetyl coenzyme A (acetyl-coA) in the cytoplasm; (b) the elongation and desaturation of carbon chain of fatty acids; and (c) the biosynthesis of triglycerides in microalgae. 2.1.1. The formation of acetyl coenzyme A (acetyl-coA) in cytoplasm The metabolism flux route on the utilization of carbon dioxide and glucose for the formation of acetyl-coA in microalgae is described by Yang et al. [25]. It is concluded that glyceraldehyde phosphate (GAP) is a key intermediate both for the two metabolism systems. The formation of acetyl-coA in photosynthetic reactions, including the light reactions, Calvin cycle and synthesis, is located in chloroplast. GAP is withdrawn from Calvin cycle and exported to cytoplasm for consumption. After the export of GAP from chloroplast to cytoplasm, the flow of carbon is directed to the synthesis of sugars or oxidation through the glycolytic pathway to pyruvate. Sugars including sucrose are the major storage products in the cytoplasm of plant cells. Akazawa and Okamoto [26] reported that glucose was easy to be stored as starch without prior conversion to GAP and then uptake by the chloroplast which suggested starch is the main storage formation for carbon source in Chlorella sp. Therefore, one part of the exogenous glucose was directly converted to starch, and the remainder was oxidized through glycolytic pathway. 2.1.2. The elongation and desaturation of carbon chain of fatty acids The elongation of carbon chain of fatty acids is mainly dependent on the reaction of two enzyme systems including acetyl-coA carboxylic enzyme (ACCE) and fatty acid synthase (FAS) in most organisms. In the process of synthesis of fatty acids (Fig. 1), acetyl-coA is the primer. The process of carbon chain elongation needs the cooperation with malonyl-coA, the substrate on which enzyme act are acetyl-ACP and malonyl-ACP. The C16–C18 fatty acid thioes-

6.7)

ter can be formed after several reaction steps. The formation of short carbon chain fatty acids is similar in the cells of advanced plants, animals, fungi, bacteria, and algae. For example, in the cell of green algae, the reaction routes of primer such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid in fatty acid synthesis are similar to that in plant cells and yeast cells [27]. The desaturation of carbon chain of fatty acid occurs from C18 and further elongation of carbon chain takes place to produce long-chain fatty acids which are unusual in normal plant oils (Fig. 2). Long-chain fatty acids (C20–C22) often exist in microalgae and the content varies from species to species [28]. Normally, short-chain fatty acids (C14–C18) which are the main components of biodiesel are majority of fatty acids in Chlorella sp., but high content of long-chain fatty acid and hydrocarbons exist in some specific species of microalgae. So, It is vital to choose proper microalgae species as materials of biodiesel production.

O

COO-

CH2C-SCoA

CH2 C O

Econd

SCoA

ACP-SH

O

NADP+

RC-S-ACP NADPH(H+)

O -

COO HSCoA

CH2

RCH=CHC-S-ACP

C O S

O

RC-S-Econd

ACP

CO2 O

HS-Econd

O

OH

O

RCH-CH2C-S-ACP

RC-CH2C-S-ACP NADPH (H+)

NADP+

Acetyl-CoA-ACP acyl transferase malonyl CoA-ACP acyl transferase β-ketoacyl-ACP Condensing enzyme) β-ketoacyl-ACP reductase β-hydroxyacyl-ACP dehydrase enoyl-ACP reductase Fig. 1. Reaction process of the FFA biological synthesis system (Shen and Wang [62]).

G. Huang et al. / Applied Energy 87 (2010) 38–46

C18:0

41

acetyl-coA to form Lysophosphatidic acid and later combines with another acetyl-coA to form phosphatidic acid. These two reactions are catalyzed by glycerol phosphate acyl-transferase. In the following steps, lysophosphatidic acid is hydrolyzed by phosphatidate phosphatase to form diglyceride which is then combined with the third acetyl-coA to complete the biosynthesis of triglycerides. The last reaction step is catalyzed by glyceryl diester transacylase.

9 desaturase C18:1-9

12 desaturase C18:2-9,12

15 desaturase

2.2. Phototrophic cultivation of microalgae for lipids production

C18:3-9,12,15

6 desaturase C18:4-6,9,12,15

Fatty acid elongase C18:4-8,11,14,17

5 desaturase C20:4-8,11,14,17

elongation C22:5-7,10,13,16,19

4 desaturase C22:6-4, 7,10,13,16,19 Fig. 2. The elongation and desaturation of carbon chain of fatty acids (modified from Guschina and Harwood [63]).

2.1.3. The biosynthesis of triglycerides in microalgae Like other higher plant and animal, microalgae are able to biosynthesize triglycerides to store substance and energy. Generally, L-a-phosphoglycerol and acetyl-coA are two major primers in the biosynthesis of triglycerides. The L-a-phosphoglycerol mainly derives from phosphodihydroxyacetone which is the product of the glycolysis process. The reaction steps are shown in Fig. 3. One of the hydroxyl in L-a-phosphoglycerol reacts with

Microalgae can transform carbon dioxide from the air and light energy through photosynthesis to various forms of chemical energies such as polysaccharides, proteins, lipids and hydrocarbons. Compared to higher plants, microalgae have a number of advantages including higher photosynthetic efficiency and growth rate [20]. In phototrophic culture, usually microalgae can be grown in two systems such as open ponds and enclosed photobioreactors. Enclosed photobioreactor system is more suitable for some microalgae which are readily contaminated by other microbes, except for some special microalgae which can survive well in extreme environments such as high pH (e.g., Spirulina) and high salinity (e.g., Dunaliella) or can grow very rapidly (e.g., Chlorella) in the open pond. Because of better environmental control, enclosed photobioreactor system has been suggested for the production of high-value long-chain fatty acids (e.g., DHA, EPA). Nevertheless, due to the high cost in terms of operation and capital investment and the small scale due to the complexity of bioreactor design compared to open pond system, it might not be economical to produce biodiesel on a large scale by enclosed photobioreactors. Open pond system is perhaps more suitable for cultivating microalgae for biodiesel because of its relatively cheap operating cost compared to the enclosed photobioreactors. The basic requirements for microalgal phototrophic growth should include carbon dioxide, other macro- and micro-nutrients, as well as light. Carbon source can be obtained from power plants which release large

Fig. 3. The biosynthesis of triglycerides in microalgae.

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G. Huang et al. / Applied Energy 87 (2010) 38–46

Water Nutrients

Algae oil recovery system

biodiesel production

Open pond

Motorized paddle

Waster CO2

Waste water

Fig. 4. Open pond photosynthesis system (OPSS).

amounts of waste gases (mainly CO2) daily. Typical coal-fired power plants emit fuel gas from their stacks containing up to 13% CO2. This high concentration of CO2 enhances transfer and uptake of CO2 in the pond system. The concept of combining a coal-fired power plant with algae cultivation provides a feasible approach to recycle CO2 from coal combustion into useable liquid fuel (Fig. 4). When grown in large outdoor ponds, microalgae could use CO2 from fuel gas directly injected into the culture [29]. Besides, the wastewater may contain abundant nutrients (e.g., inorganic irons) which are necessary for microalgal growth. Chlorella vulgaris was grown in waste water from a steelmaking plant with the aim of developing an economically feasible system to remove ammonia from wastewater and CO2 from flue gas [30]. 2.3. Heterotrophic cultivation of microalgae for lipids production Although microalgae can utilize light efficiently, phototrophic growth of microalgae is often slow because of light limitation at high cell densities on a large scale [31] or ‘‘photoinhibition” due to excessive light, especially in sunny days [32]. In view of these disadvantages associated with photoautotrophic cultivation, heterotrophic growth of microalgae in conventional fermentors should be favorably considered [33]. Heterotrophic cultivation of microalgae offers several advantages over phototrophic cultivation including elimination of light requirement, good control of the cultivation process, and low-cost for harvesting the biomass because of higher cell density obtained in heterotrophic culture of microalgae [34]. In heterotrophic culture, both cell growth and biosynthesis of products are significantly influenced by medium nutrients and environmental factors. Carbon sources are the most important element for heterotrophic culture of microalgae in the production of lipids. For example, although the green microalgae C. protothecoides can grow photoautotrophically or heterotrophically. heterotrophic growth of C. protothecoides using acetate, glucose, or other organic compounds as carbon source results in much higher biomass as well as lipid content in cells [35]. More-

over, heterotrophic microalgae might utilize other carbon sources such as ethanol, glycerol, and fructose depending on the microalgal species used [36]. Liu et al. [37] compared several carbon sources and concluded that glucose was preferred. In order to lower the production cost of microalgal oils as biodiesel, cheaper carbon sources should be considered. For instance, corn powder hydrolysate (CPH) or molasses instead of glucose may be used as organic carbon source in heterotrophic culture. It was reported occasionally CPH was superior to glucose solution, because CPH contained some beneficial components to Chlorella, and as a result, C. protothecoides produced 55.2% crude lipids in the cells with a cell dry weight concentration of 15.5 g L 1 [24]. The utilization of corn powder hydrolysate instead of glucose in heterotrophic culture greatly reduced the cost of production, which is important for the biodiesel production by microalgae in terms of economical significance. Nitrogen is also an essential macronutrient in lipids production. Complex nitrogen source might be superior to simple nitrogen source in heterotrophic culture of microalgae, because it might provide amino acids, vitamins and growth factors simultaneously. Industrial wastewater rich in nitrogen also can be considered for the cultivation of microalgae. Monosodium glutamate waste after diluted was well treated as a cheap fermentation medium for Rhodotorula glutinis to biosynthesize lipids as the raw material for the production of biodiesel [38]. Many microalgae growing under nitrogen limitation show enhanced lipid content. In the late 1940s, it was noted that nitrogen starvation is most influential on lipid storage and lipid fractions, and as a result of nitrogen starvation, the lipid content as high as 70–85% of dry weight was reported [39]. It was also reported that Prophyridium cruentum might double its total lipid content (mainly neutral lipids) under nitrogen starvation [39]. Nevertheless, nitrogen starvation might not always result in an increase in total lipid content in microalgae but a change in lipid composition. Zhila et al. [40] reported that the alga Botryococcus braunii contained high content (28.4–38.4%) of oleic acid under nitrogen limitation, but the content of total lipids and triacylglycerols did not change.

G. Huang et al. / Applied Energy 87 (2010) 38–46

It is also noticeable that the overall rates of oil production might be lower in the case of nutrient deficiency due to the overall lower biomass achieved. Therefore factors other than nitrogen should be considered altogether. Initial carbon to nitrogen (C/N) ratio in the medium has significant impact on the biosynthesis of lipids in microalgae. With the addition of glucose as organic carbon source to the medium and the tremendous decrease of nitrogen source in the medium, a crude lipid content up to 55.2% was achieved in heterotrophic C. protothecoides, which was about 3.4-fold that in photoautotrophic C. protothecoides [41]. Chen and Johns [34] also investigated the effect of C/N ratio and aeration on the fatty acid composition of heterotrophic Chlorella sorokiniana. When C/N ratio of approximately 20, cell lipid content was at a minimum and increased at both higher and lower C/N values. Environmental factors also cannot be neglected in the growth of microalgae and the formation of fatty acids. The high PUFAs content at low temperature might be explained by the fact that the algae need to produce more PUFAs to maintain cell membrane fluidity. Another reason might be that low temperature could lead to high level of intracellular molecular oxygen and hence improves the activities of the desaturase and elongase involved in the biosynthesis of PUFAs [42]. However, the effect of temperature on cell growth and PUFAs production may not be always the same as mentioned above [31]. Therefore, a specific and careful study of the individual microalgae is required. Salinity, pH, and dissolved O2 are also important factors affecting the heterotrophic cultivation of microalgae [43,34]. Besides, it has been demonstrated that different cultivation modes greatly affect the lipid accumulation in microalgae. Heterotrophically grown microalgae usually accumulate more lipids than those cultivated photoautotrophically [44]. Some phototrophic microorganisms could also be grown on cheap organic substrates heterotrophically [33]. Evidences have been shown that the ‘‘dark metabolism” of photosynthetic plants and microalgae is similar to that of non-photosynthetic organisms such as yeasts. Recently, an obligate photoautotrophic microalga Phaeodactylum tricornutum was grown heterotrophically when a single gene (Glut 1) that encoded the glucose transporter protein (Glut 1) was introduced into this alga [45]. Such studies on the production of fatty acids and lipids should be useful for further investigation of microalgae as bio-fuels by heterotrophic cultivation. 2.4. Genetic engineering for lipids production Genetic engineering of microalgae is an upstream technology in microalgal biotechnology. In 1960s, cyanobacteria were chosen as ideal material for academic research by scientists. Since the genome of Anabaena PCC7120 was successfully cloned, the number of cloned functional genes in cyanobacteria has increased to over 130 [46]. Acc1 is a kind of restriction enzyme which was cloned from oceanic diatom Cylclotella cryptica had been efficiently expressed in C. cryptica for the production of bio-fuel [47]. A new method for biodiesel production from microalgal oils has been developed by the application of genetic engineering recently. National Renewable Energy laboratory in the USA (NREL) has established engineered microalgae which belong mostly to diatom species. The lipid content of the engineered microalgae increased to above 60% in laboratory conditions and above 40% in outdoors cultivation, whereas the lipids content in microalgae is 5–20% in common natural conditions. The improvement of lipid content in engineered microalgal cells is mainly due to the high expression of acetyl-coA carboxylase gene, which plays an important role in the control of the level of lipid accumulation. At present, the research has focused on choosing a proper molecular carrier, making ACC gene full expression in bacteria, yeast, and plant. Furthermore, the modified ACC gene is being introduced into microalgae to ob-

43

tain more efficient expression. The utilization of engineered microalgae for the production of biodiesel has important economic and environmental benefits. Its superiorities include high yield of microalgae; saving agricultural resources by using seawater as natural medium; the cellular content of lipids in microalgae is several times higher than that in terrestrial plants.

3. The extraction of oils from microalgae 3.1. Pyrolysis technologies Although oils extracted from microalgal cells have been investigated for fuel production of internal-combustion engines by transesterification of fatty acids [48], industrial biodiesel production from microalgal oils is still not well developed. At least, high content of microalgal oils is required for this method to realize economic benefits. Since it is difficult to obtain microalgae with high fatty acid content conventionally, it has been considered that only crude fat (lipid) is used for conversion into substitutes for traditional fuels [49]. Recent research on the production of energy from renewable sources such as bio-oils production by pyrolysis of biomass has received much interest. Pyrolyzing microalgae to produce liquid fuel was first put forward in Germany in 1986. It was reported that the method of catalytic pyrolysis could yield gasoline with high content of aromatic hydrocarbon and octane number [21]. Pyrolysis is a phenomenon related to decomposition of biomass under the condition of oxygen deficiency and high temperature. Pyrolysis previously was first used for the production of bio-oils or bio-gases from lignocellulose. However, such a technology may be more suitable for microalgae because of the lower temperature required for pyrolysis and the higher-quality oils obtained [50]. Moreover, the cost of pyrolysis of lignocellulose is relatively higher than that of microalgae. Compared to lignocellulose, microalgae contain high content of cellular lipids, resolvable polysaccharides and proteins, which are easier to be pyrolyzed to bio-oils and bio-gases. Compared to slow pyrolysis [22,23], fast pyrolysis is a new technology, which produces bio-fuel in the absence of air at atmospheric pressure, with a relatively low temperature (450–550 °C) and high heating rate (103–104 °C s 1) as well as short gas residence time to crack into short chain molecules and be cooled to liquid rapidly [50]. The main products of slow pyrolysis are char and char-oils with a 15–20% yield, whereas, the products of fast pyrolysis are oils and gases with a yield of approximately 70% [51]. Fast pyrolysis has proved to be a promising way to produce bio-oils compared to slow pyrolysis [41] for the following reasons: (1) less bio-oils were produced from slow pyrolysis; (2) the viscous bio-oils from slow pyrolysis is not suitable for liquid fuels; and (3) the fast pyrolysis process is time saving and requires less energy compared to the slow prolysis process. Fast pyrolysis tests of microalgae were performed in the fluid bed reactor [52]. It was reported that the experiment was completed 500 °C with a heating rate of 600 °C s 1, a sweep gas (N2) flow rate of 0.4 m3 h 1 and a vapor residence time of 2–3 s. The fast pyrolysis of C. protothecoides and Microcystis aeruginosa yielded 18% and 24% of liquid products, respectively. Compared to the slow pyrolysis from microalgae in autoclave, a great amount of high quality bio-oils can be directly produced from continuously processing microalgae feeds at a rate of 4 g min 1. Many experiments have demonstrated that fast pyrolysis (Table 3) is an efficient method to produce useful fuels and gases from microalgae. As mentioned above, not only the crude fat, but also other chemical components such as protein and water-soluble carbohydrate, can be converted easily into fuel oil or gas by thermochemical techniques [53].

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Table 3 The application of fast pyrolysis for some microalgal species. Samples

Type of pyrolysis

Optimal pyrolysis temperature (K)

Heating rate

Oil yield (% biomass dry weight)

References

C. protothecoides C. protothecoides Microcystis aeruginosa Heterotrophic C. protothecoides C. protothecoides

Fast Fast Fast Fast Fast

773 773 773 723 773

– 600 °C/s 600 °C/s 600 °C/s 10 K/s

52.0 18 24 57.9 53.3

Peng et al. [53] Miao et al. [52] Miao et al. [52] Miao et al. [41] Demirbas et al. [66]

pyrolysis pyrolysis pyrolysis pyrolysis pyrolysis

3.2. Liquefaction High content of water often exists in microalgae after harvesting which requires a great deal of energy to remove moisture in the algal cells in the period of pretreatment. Liquefaction has been developed to produce bio-fuel directly without the need of drying microalgae [22,23]. Moreover, wet microalgae can provide hydrogen for hydrogenolysis. It was reported that Dunaliella tortiolecta cells with 78.4% water content converts to oils directly. The yield of oils reached 37% of the total organic matters [23]. Dote et al. [22] reported that B. braunii produced liquid oils at 57–64% of dry weight under the conditions of a N2 pressure of 10 MPa at 300 °C in warm water and catalyzed by NaCO3. Sawayama et al. [54] investigated the energy balance and CO2 mitigating effect of a liquid fuel production process from B. braunii using thermochemical liquefaction. The study suggested that microalgae consume low amounts of nutrients and accumulate high caloric materials, and nutrient resources which are produced without energy wasting processes encourage the recovery of oil from microalgae and CO2 mitigation.

(Table 4) may be applied in transesterification [56]. The use of acid catalyst has found to be useful for the conversion of high free fatty acid feedstocks to esters but the reaction rates for converting triglycerides to methyl esters are too slow [57]. Alkali catalysts have higher reaction rate and conversion than acid catalysts for the transesterification of triglyceride. Alkali-catalyzed transesterification is about 4000 times faster than the acid catalyzed reaction [58]. So, alkali-catalyzed transesterification is most frequently used commercially. The free fatty acid (FFA), however, may react with the alkali catalyst to form soap and water (Fig. 5) which results in the loss of alkali catalysts in the process of reaction. Therefore, additional catalysts must be added to compensate for the catalyst loss to soap. When the FFA level is above 5%, the soap will inhibit separation of the methyl esters and glycerol and causes emulsion formation during the water washing. Therefore, it is necessary to first convert FFAs to methyl eaters (Fig. 6) in order to reduce the contents of FFAs, and the low FFAs pretreated oil is transesterified with an alkali catalyst to convert triglycerides to methyl esters. In contrast, enzymes exhibit good tolerance to the FFA level of the feedstock, but the enzymes are expensive and may not be able to provide the degree of reaction required

4. Transesterification technologies in the production of biodiesel The viscosities of vegetable oils and microalgal oils are usually higher than that of diesel oils [55]. Hence, they cannot be applied to engines directly. The transesterification of microalgal oils will greatly reduce the original viscosity and increase the fluidity. Few reports on the production of biodiesel from microalgal oils are available [20]. Nevertheless the technologies of the biodiesel production for vegetable oils can be applied to the biodiesel production of microalgal oils because of the similar physical and chemical properties. In the process of transesterification, alcohols are key substrates in transesterification. The commonly used alcohols are methanol, ethanol, propanol, butanol, and amyl alcohol but methanol is applied more widely because of its low-cost and physical advantages. Alkali, acid, or enzyme catalyzed processes

O

O

HO-C-R

+

Fatty Acid

+-

K O-C-R + H2O

KOH

Potassium Hydroxide

Potassium soap

Water

Fig. 5. Transesterification by alkali catalyst.

O

O

HO-C-R + Fatty Acid

CH3OH Methanol

H2SO4

CH3-O-C-R + H2O Methyl ester

Water

Fig. 6. Transesterification by acid catalyst.

Table 4 Application of transesterification technologies. Type of transesterification

Advantages

Disadvantages

Chemical catalysis

(a) Reaction condition can be well controlled (b) Large scale production (c) The cost of the production process is cheap (d) The methanol produced in the process can be recycled (e) High conversion of the production

(a) Reaction temperature is relative high and the process is complex (b) The later disposal process is complex (c) The process need much energy (d) Need a installation for methanol recycle

Enzymatic catalysis

(a) Moderate reaction condition (b) The small amount of methanol required in the reaction (c) Have no pollution to natural environment

(a) Limitation of enzyme in the conversion of short chain of fatty acids (b) Chemicals exist in the process of production are poisonous to enzyme

Supercritical fluid techniques

(a) Easy to be controlled (b) It is safe and fast (c) Friendly to environment

(a) High temperature and high pressure in the reaction condition leads to high cost of production and wastes energy

(e) The waste water pollutes the environment

G. Huang et al. / Applied Energy 87 (2010) 38–46

to meet the ASTM fuel specification [59]. Immobilization of the enzyme and multiple enzymes may provide more choices in the future [60]. Besides, the effects of molar ratio of glycerides to alcohol; catalysts, reaction temperature and time, the contents of FFAs and water in oils and fats have been reported in some recent reports [9]. It was reported that biodiesel could be produced by acidic transesterification with 56:1 M ratio of methanol to microalgal oil at temperature at 30 °C and 100% catalyst quantity (based on oil weight) was achieved [44]. The most abundant composition of microalgal oil transesterified with methanol is C19H36O2, which is suggested to accord with the standard of biodiesel [24]. In recent years, there have been many reports about the applications of transesterified technologies for biodiesel [61]. 5. Conclusion Since oil crisis in the mid 1970s, finding new energy resources to replace petroleum has been a hot topic worldwide. Because of the many advantages over the conventional energy resources, the production of biodiesel has attracted much attention in recent years. There have been numerous publications on the production of biodiesel made from vegetable oils and other oil-plants. At present, the high cost of oleaginous materials is the main problem hindering commercial production of biodiesel. Therefore, finding cheaper oleaginous materials and improving transesterification technologies are the key to producing biodiesel successfully. To date, large scale commercial production of biodiesel from microalgal oils has not been reported. The low lipid content and low biomass that can be achieved leading to the high cost of biodiesel from microalgal oils are the bottleneck for industrial production. Heterotrophic cultivation of lipid-rich microalgae with fast pyrolysis may lead to a high yield of bio-oils on a large scale. Research in genetic engineering coupled with advanced cultivation and downstream technologies will benefit the future development of microalgae for biodiesel production.

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